icp0 dismantles microtubule: Topics by Science.gov

Sample records for icp0 dismantles microtubule

ICP27-dependent resistance of herpes simplex virus type 1 to leptomycin B is associated with enhanced nuclear localization of ICP4 and ICP0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lengyel, Joy; Strain, Anna K.; Perkins, Keith D.

2006-09-01

It was previously shown that herpes simplex virus type 1 (HSV-1) is sensitive to leptomycin B (LMB), an inhibitor of nuclear export factor CRM1, and that a single methionine to threonine change at residue 50 (M50T) of viral immediate-early (IE) protein ICP27 can confer LMB resistance. In this work, we show that deletion of residues 21-63 from ICP27 can also confer LMB resistance. We further show that neither the M50T mutation nor the presence of LMB affects the nuclear shuttling activity of ICP27, suggesting that another function of ICP27 determines LMB resistance. A possible clue to this function emerged whenmore » it was discovered that LMB treatment of HSV-1-infected cells dramatically enhances the cytoplasmic accumulation of two other IE proteins, ICP0 and ICP4. This effect is completely dependent on ICP27 and is reversed in cells infected with LMB-resistant mutants. Moreover, LMB-resistant mutations in ICP27 enhance the nuclear localization of ICP0 and ICP4 even in the absence of LMB, and this effect can be discerned in transfected cells. Thus, the same amino (N)-terminal region of ICP27 that determines sensitivity to LMB also enhances ICP27's previously documented ability to promote the cytoplasmic accumulation of ICP4 and ICP0. We speculate that ICP27's effects on ICP4 and ICP0 may contribute to HSV-1 LMB sensitivity.« less
Regulation of Innate Immune Responses by Bovine Herpesvirus 1 and Infected Cell Protein 0 (bICP0)

PubMed Central

Jones, Clinton

2009-01-01

Bovine herpesvirus 1 (BoHV-1) infected cell protein 0 (bICP0) is an important transcriptional regulatory protein that stimulates productive infection. In transient transfection assays, bICP0 also inhibits interferon dependent transcription. bICP0 can induce degradation of interferon stimulatory factor 3 (IRF3), a cellular transcription factor that is crucial for activating beta interferon (IFN-β) promoter activity. Recent studies also concluded that interactions between bICP0 and IRF7 inhibit trans-activation of IFN-β promoter activity. The C3HC4 zinc RING (really important new gene) finger located near the amino terminus of bICP0 is important for all known functions of bICP0. A recombinant virus that contains a single amino acid change in a well conserved cysteine residue of the C3HC4 zinc RING finger of bICP0 grows poorly in cultured cells, and does not reactivate from latency in cattle confirming that the C3HC4 zinc RING finger is crucial for viral growth and pathogenesis. A bICP0 deletion mutant does not induce plaques in permissive cells, but induces autophagy in a cell type dependent manner. In summary, the ability of bICP0 to stimulate productive infection, and repress IFN dependent transcription plays a crucial role in the BoHV-1 infection cycle. PMID:21994549
Alphaherpesvirus Proteins Related to Herpes Simplex Virus Type 1 ICP0 Affect Cellular Structures and Proteins

PubMed Central

Parkinson, Jane; Everett, Roger D.

2000-01-01

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 interacts with several cellular proteins and induces the proteasome-dependent degradation of others during infection. In this study we show that ICP0 is required for the proteasome-dependent degradation of the ND10 protein Sp100 and, as with the other target proteins, the ICP0 RING finger domain is essential. Further, comparison of the kinetics and ICP0 domain requirements for the degradation of PMI and Sp100 suggests that a common mechanism is involved. Homologues of ICP0 are encoded by other members of the alphaherpesvirus family. These proteins show strong sequence homology to ICP0 within the RING finger domain but limited similarity elsewhere. Using transfection assays, we have shown that all the ICP0 homologues that we tested have significant effects on the immunofluorescence staining character of at least one of the proteins destabilized by ICP0, and by using a recombinant virus, we found that the equine herpesvirus ICP0 homologue induced the proteasome-dependent degradation of endogenous CENP-C and modified forms of PML and Sp100. However, in contrast to ICP0, the homologue proteins had no effect on the distribution of the ubiquitin-specific protease USP7 within the cell, consistent with their lack of a USP7 binding domain. We also found that ICP0 by itself could induce the abrogation of SUMO-1 conjugation and then the proteasome-dependent degradation of unmodified exogenous PML in transfected cells, thus demonstrating that other HSV-1 proteins are not required. Surprisingly, the ICP0 homologues were unable to cause these effects. Overall, these data suggest that the members of the ICP0 family of proteins may act via a similar mechanism or pathway involving their RING finger domain but that their intrinsic activities and effects on endogenous and exogenous proteins differ in detail. PMID:11024129
Identification of TRIM27 as a novel degradation target of herpes simplex virus 1 ICP0.

PubMed

Conwell, Sara E; White, Anne E; Harper, J Wade; Knipe, David M

2015-01-01

The herpes simplex virus 1 (HSV-1) immediate early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification and eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBN and MRE11A, regulators of apoptosis, including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal coimmunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions. During productive infection, a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To
Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.

PubMed

Samrat, Subodh Kumar; Ha, Binh L; Zheng, Yi; Gu, Haidong

2018-01-15

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus early during infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of the ICP0 nuclear-to-cytoplasmic translocation are not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in human embryonic lung fibroblast (HEL) cells, ICP0 C-terminal residues 741 to 775 were necessary but not sufficient for the nuclear-to-cytoplasmic translocation; (ii) the loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than that of wild-type ICP0, suggesting that nuclear retention of ICP0 occurs in an ICP0 E3 ligase-dependent manner; and (iv) the ICP0 C terminus and late viral proteins cooperate in order to overcome nuclear retention and stimulate ICP0 cytoplasmic translocation. Taken together, less ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of functional ICP0. IMPORTANCE A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity of cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus, but it is translocated to the cytoplasm late during HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in
Association between ICP pulse waveform morphology and ICP B waves.

PubMed

Kasprowicz, Magdalena; Bergsneider, Marvin; Czosnyka, Marek; Hu, Xiao

2012-01-01

The study aimed to investigate changes in the shape of ICP pulses associated with different patterns of the ICP slow waves (0.5-2.0 cycles/min) during ICP overnight monitoring in hydrocephalus. Four patterns of ICP slow waves were characterized in 44 overnight ICP recordings (no waves - NW, slow symmetrical waves - SW, slow asymmetrical waves - AS, slow waves with plateau phase - PW). The morphological clustering and analysis of ICP pulse (MOCAIP) algorithm was utilized to calculate a set of metrics describing ICP pulse morphology based on the location of three sub-peaks in an ICP pulse: systolic peak (P(1)), tidal peak (P(2)) and dicrotic peak (P(3)). Step-wise discriminant analysis was applied to select the most characteristic morphological features to distinguish between different ICP slow waves. Based on relative changes in variability of amplitudes of P(2) and P(3) we were able to distinguish between the combined groups NW + SW and AS + PW (p < 0.000001). The AS pattern can be differentiated from PW based on respective changes in the mean curvature of P(2) and P(3) (p < 0.000001); however, none of the MOCAIP feature separates between NW and SW. The investigation of ICP pulse morphology associated with different ICP B waves may provide additional information for analysing recordings of overnight ICP.
Actin–microtubule coordination at growing microtubule ends

PubMed Central

López, Magdalena Preciado; Huber, Florian; Grigoriev, Ilya; Steinmetz, Michel O.; Akhmanova, Anna; Koenderink, Gijsje H.; Dogterom, Marileen

2014-01-01

To power dynamic processes in cells, the actin and microtubule cytoskeletons organize into complex structures. Although it is known that cytoskeletal coordination is vital for cell function, the mechanisms by which cross-linking proteins coordinate actin and microtubule activities remain poorly understood. In particular, it is unknown how the distinct mechanical properties of different actin architectures modulate the outcome of actin–microtubule interactions. To address this question, we engineered the protein TipAct, which links growing microtubule ends via end-binding proteins to actin filaments. We show that growing microtubules can be captured and guided by stiff actin bundles, leading to global actin–microtubule alignment. Conversely, growing microtubule ends can transport, stretch and bundle individual actin filaments, thereby globally defining actin filament organization. Our results provide a physical basis to understand actin–microtubule cross-talk, and reveal that a simple cross-linker can enable a mechanical feedback between actin and microtubule organization that is relevant to diverse biological contexts. PMID:25159196
Persistence length measurements from stochastic single-microtubule trajectories.

PubMed

van den Heuvel, M G L; Bolhuis, S; Dekker, C

2007-10-01

We present a simple method to determine the persistence length of short submicrometer microtubule ends from their stochastic trajectories on kinesin-coated surfaces. The tangent angle of a microtubule trajectory is similar to a random walk, which is solely determined by the stiffness of the leading tip and the velocity of the microtubule. We demonstrate that even a single-microtubule trajectory suffices to obtain a reliable value of the persistence length. We do this by calculating the variance in the tangent trajectory angle of an individual microtubule. By averaging over many individual microtubule trajectories, we find that the persistence length of microtubule tips is 0.24 +/- 0.03 mm.
Glutamine Deprivation Causes Enhanced Plating Efficiency of a Herpes Simplex Virus Type 1 ICP0-Null Mutant ▿

PubMed Central

Bringhurst, Ryan M.; Dominguez, Antonia A.; Schaffer, Priscilla A.

2008-01-01

Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0−) mutant. We now report that glutamine deprivation alone is able to enhance the plating efficiency of an ICP0− virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0− virus. PMID:18768961
Functional Interaction between Class II Histone Deacetylases and ICP0 of Herpes Simplex Virus Type 1

PubMed Central

Lomonte, Patrick; Thomas, Joëlle; Texier, Pascale; Caron, Cécile; Khochbin, Saadi; Epstein, Alberto L.

2004-01-01

This study describes the physical and functional interactions between ICP0 of herpes simplex virus type 1 and class II histone deacetylases (HDACs) 4, 5, and 7. Class II HDACs are mainly known for their participation in the control of cell differentiation through the regulation of the activity of the transcription factor MEF2 (myocyte enhancer factor 2), implicated in muscle development and neuronal survival. Immunofluorescence experiments performed on transfected cells showed that ICP0 colocalizes with and reorganizes the nuclear distribution of ectopically expressed class I and II HDACs. In addition, endogenous HDAC4 and at least one of its binding partners, the corepressor protein SMRT (for silencing mediator of retinoid and thyroid receptor), undergo changes in their nuclear distribution in ICP0-transfected cells. As a result, during infection endogenous HDAC4 colocalizes with ICP0. Coimmunoprecipitation and glutathione S-transferase pull-down assays confirmed that class II but not class I HDACs specifically interacted with ICP0 through their amino-terminal regions. This region, which is not conserved in class I HDACs but homologous to the MITR (MEF2-interacting transcription repressor) protein, is responsible for the repression, in a deacetylase-independent manner, of MEF2 by sequestering it under an inactive form in the nucleus. Consequently, we show that ICP0 is able to overcome the HDAC5 amino-terminal- and MITR-induced MEF2A repression in gene reporter assays. This is the first report of a viral protein interacting with and controlling the repressor activity of class II HDACs. We discuss the putative consequences of such an interaction for the biology of the virus both during lytic infection and reactivation from latency. PMID:15194749
Microtubule heterogeneity of Ornithogalum umbellatum ovary epidermal cells: non-stable cortical microtubules and stable lipotubuloid microtubules.

PubMed

Kwiatkowska, Maria; Stępiński, Dariusz; Polit, Justyna T; Popłońska, Katarzyna; Wojtczak, Agnieszka

2011-01-01

Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of β-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.
Identification of three redundant segments responsible for herpes simplex virus 1 ICP0 to fuse with ND10 nuclear bodies.

PubMed

Zheng, Yi; Gu, Haidong

2015-04-01

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a key regulator in both lytic and latent infections. In lytic infection, an important early event is the colocalization of ICP0 to nuclear domain 10 (ND10), the discrete nuclear bodies that impose restrictions on viral expression. ICP0 contains an E3 ubiquitin ligase that degrades promyelocytic leukemia protein (PML) and Sp100, two major components of ND10, and disperses ND10 to alleviate repression. We previously reported that the association between ICP0 and ND10 is a dynamic process that includes three steps: adhesion, fusion, and retention. ICP0 residues 245 to 474, defined as ND10 entry signal (ND10-ES), is a region required for the fusion step. Without ND10-ES, ICP0 adheres at the ND10 surface but fails to enter. In the present study, we focus on characterizing ND10-ES. Here we report the following. (i) Fusion of ICP0 with ND10 relies on specific sequences located within ND10-ES. Replacement of ND10-ES by the corresponding region from ORF61 of varicella-zoster virus did not rescue ND10 fusion. (ii) Three tandem ND10 fusion segments (ND10-FS1, ND10-FS2, and ND10-FS3), encompassing 200 amino acids within ND10-ES, redundantly facilitate fusion. Each of the three segments is sufficient to independently drive the fusion process, but none of the segments by themselves are necessary for ND10 fusion. Only when all three segments are deleted is fusion blocked. (iii) The SUMO interaction motif located within ND10-FS2 is not required for ND10 fusion but is required for the complete degradation of PML, suggesting that PML degradation and ND10 fusion are regulated by different molecular mechanisms. ND10 nuclear bodies are part of the cell-intrinsic antiviral defenses that restrict viral gene expression upon virus infection. As a countermeasure, infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) localizes to ND10s, degrades the ND10 organizer, and disperses ND10 components in order to
SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing

PubMed Central

Wightman, Raymond; Chomicki, Guillaume; Kumar, Manoj; Carr, Paul; Turner, Simon R.

2013-01-01

Summary One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtubule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1–6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7–10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pavement cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mechanism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs. PMID:24055158
Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus.

PubMed

Deschamps, Thibaut; Kalamvoki, Maria

2017-05-01

Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2'3'-cyclic GAMP (2'3'-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway. IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets
Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus

PubMed Central

Deschamps, Thibaut

2017-01-01

ABSTRACT Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2′3′-cyclic GAMP (2′3′-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway. IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this
Microtubule catastrophe and rescue.

PubMed

Gardner, Melissa K; Zanic, Marija; Howard, Jonathon

2013-02-01

Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed 'dynamic instability'. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule 'catastrophe'), and then by switching back from shrinkage to growth (called microtubule 'rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells. Copyright © 2012 Elsevier Ltd. All rights reserved.
Herpes simplex virus regulatory proteins VP16 and ICP0 counteract an innate intranuclear barrier to viral gene expression.

PubMed

Hancock, Meaghan H; Corcoran, Jennifer A; Smiley, James R

2006-08-15

HSV regulatory proteins VP16 and ICP0 play key roles in launching the lytic program of viral gene expression in most cell types. However, these activation functions are dispensable in U2OS osteosarcoma cells, suggesting that this cell line either expresses an endogenous activator of HSV gene expression or lacks inhibitory mechanisms that are inactivated by VP16 and ICP0 in other cells. To distinguish between these possibilities, we examined the phenotypes of somatic cell hybrids formed between U2OS cells and highly restrictive HEL fibroblasts. The U2OS-HEL heterokarya were as non-permissive as HEL cells, a phenotype that could be overcome by providing either VP16 or ICP0 in trans. Our data indicate that human fibroblasts contain one or more inhibitory factors that act within the nucleus to limit HSV gene expression and argue that VP16 and ICP0 stimulate viral gene expression at least in part by counteracting this innate antiviral defence mechanism.
Microtubule Actin Cross-linking Factor 1 regulates cardiomyocyte microtubule distribution and adaptation to hemodynamic overload.

PubMed

Fassett, John T; Xu, Xin; Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J; Chen, Yingjie

2013-01-01

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r(2) = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.
Submembraneous microtubule cytoskeleton: regulation of microtubule assembly by heterotrimeric G proteins

PubMed Central

Roychowdhury, Sukla; Rasenick, Mark. M

2009-01-01

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. G proteins also interact with microtubules and participate in microtubule-dependent centrosome/chromosome movement during cell division, as well as neuronal differentiation. In recent years, significant progress has been made in our understanding of the biochemical/functional interactions between G protein subunits (α and βγ) and microtubules, and the molecular details emerging from these studies suggest that α and βγ subunits of G proteins interact with tubulin/microtubules to regulate assembly/dynamics of microtubules, providing a novel mechanism for hormone or neurotransmitter induced rapid remodeling of cytoskeleton, regulation of mitotic spindle for centrosome/chromosome movements in cell division, and neuronal differentiation where structural plasticity mediated by microtubules is important for appropriate synaptic connections and signal transmission. PMID:18754776
A microtubule bestiary: structural diversity in tubulin polymers.

PubMed

Chaaban, Sami; Brouhard, Gary J

2017-11-01

Microtubules are long, slender polymers of αβ-tubulin found in all eukaryotic cells. Tubulins associate longitudinally to form protofilaments, and adjacent protofilaments associate laterally to form the microtubule. In the textbook view, microtubules are 1) composed of 13 protofilaments, 2) arranged in a radial array by the centrosome, and 3) built into the 9+2 axoneme. Although these canonical structures predominate in eukaryotes, microtubules with divergent protofilament numbers and higher-order microtubule assemblies have been discovered throughout the last century. Here we survey these noncanonical structures, from the 4-protofilament microtubules of Prosthecobacter to the 40-protofilament accessory microtubules of mantidfly sperm. We review the variety of protofilament numbers observed in different species, in different cells within the same species, and in different stages within the same cell. We describe the determinants of protofilament number, namely nucleation factors, tubulin isoforms, and posttranslational modifications. Finally, we speculate on the functional significance of these diverse polymers. Equipped with novel tubulin-purification tools, the field is now prepared to tackle the long-standing question of the evolutionary basis of microtubule structure. © 2017 Chaaban and Brouhard. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Microtubule Actin Cross-Linking Factor 1 Regulates Cardiomyocyte Microtubule Distribution and Adaptation to Hemodynamic Overload

PubMed Central

Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J.; Chen, Yingjie

2013-01-01

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r2 = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload. PMID:24086300
Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria.

PubMed

Oropesa, Manuel; de la Mata, Mario; Maraver, Juan Garrido; Cordero, Mario D; Cotán, David; Rodríguez-Hernández, Angeles; Domínguez-Moñino, Irene; de Miguel, Manuel; Navas, Plácido; Sánchez-Alcázar, José A

2011-04-01

Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in "reverse" mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.
GTP regeneration influences interactions of microtubules, neurofilaments, and microtubule-associated proteins in vitro.

PubMed

Flynn, G; Purich, D L

1987-11-15

Interactions of microtubules, neurofilaments, and microtubule-associated proteins were investigated by turbidity and falling-ball viscometry measurements. We found evidence of endogenous GTPase activity in neurofilaments and microtubule-associated proteins (MAPs) in preparations that do not include urea or heat treatment, respectively. The absence or presence of either adenyl-5'-yl imidodiphosphonic acid or a GTP-regenerating system markedly influenced observed polymerization and gelation characteristics. Most significantly, the apparent viscosity of neurofilament and microtubule samples did not display a biphasic optimal MAP concentration profile when a GTP-regenerating system was operant. Likewise, GTP regeneration promoted the recovery of gelation following mechanical disruption of neurofilament/MAP/microtubule mixtures. These and other observations require some reassessment of proposed roles for microtubule-associated proteins in modulating neurofilament-microtubule interactions in vitro.
Microtubules in root hairs.

PubMed

Traas, J A; Braat, P; Emons, A M; Meekes, H; Derksen, J

1985-06-01

The microtubules of root hairs of Raphanus sativus, Lepidium sativum, Equisetum hyemale, Limnobium stoloniferum, Ceratopteris thalictroides, Allium sativum and Urtica dioica were investigated using immunofluorescence and electron microscopy. Arrays of cortical microtubules were observed in all hairs. The microtubules in the hairs show net axial orientations, but in Allium and Urtica helical microtubule patterns are also present. Numerical parameters of microtubules in Raphanus, Equisetum and Limnobium were determined from dry-cleave preparations. The results are discussed with respect to cell wall deposition and cell morphogenesis.
Dendrites In Vitro and In Vivo Contain Microtubules of Opposite Polarity and Axon Formation Correlates with Uniform Plus-End-Out Microtubule Orientation.

PubMed

Yau, Kah Wai; Schätzle, Philipp; Tortosa, Elena; Pagès, Stéphane; Holtmaat, Anthony; Kapitein, Lukas C; Hoogenraad, Casper C

2016-01-27

dendrites have mixed orientations. Interestingly, newly formed neurites of nonpolarized neurons already contain mixed microtubules, and the specific organization of uniform plus-end-out microtubules only occurs during axon formation. Based on these findings, the authors propose a model in which the selective formation of uniform plus-end-out microtubules in the axon is a critical process underlying neuronal polarization. Copyright © 2016 the authors 0270-6474/16/361072-15$15.00/0.
Identification of a novel higher molecular weight isoform of USP7/HAUSP that interacts with the Herpes simplex virus type-1 immediate early protein ICP0.

PubMed

Antrobus, Robin; Boutell, Chris

2008-10-01

The Herpes simplex virus type-1 (HSV-1) regulatory protein ICP0, a RING-finger E3 ubiquitin ligase, stimulates the onset of viral lytic replication and the reactivation of quiescent viral genomes from latency. Like many ubiquitin ligases ICP0 induces its own ubiquitination, a process that can lead to its proteasome-dependent degradation. ICP0 counteracts this activity by recruiting the cellular ubiquitin-specific protease USP7/HAUSP. Here we show that ICP0 can also interact with a previously unidentified isoform of USP7 (termed here USP7(beta)). This isoform is not a predominantly ubiquitinated, SUMO-modified, or phosphorylated species of USP7 but is constitutively expressed in a number of different cell types. Like USP7, USP7(beta) binds specifically to an electrophilic ubiquitin probe, indicating that it contains an accessible catalytic core with potential ubiquitin-protease activity. The interaction formed between ICP0 and USP7(beta) requires ICP0 to have an intact USP7-binding domain and results in its susceptibility to ICP0-mediated degradation during HSV-1 infection.
Nanomechanics of Microtubules

NASA Astrophysics Data System (ADS)

Kis, A.; Kasas, S.; Babić, B.; Kulik, A. J.; Benoît, W.; Briggs, G. A.; Schönenberger, C.; Catsicas, S.; Forró, L.

2002-11-01

We have determined the mechanical anisotropy of a single microtubule by simultaneously measuring the Young's and the shear moduli in vitro. This was achieved by elastically deforming the microtubule deposited on a substrate tailored by electron-beam lithography with a tip of an atomic force microscope. The shear modulus is 2orders of magnitude lower than the Young's, giving rise to a length-dependent flexural rigidity of microtubules. The temperature dependence of the microtubule's bending stiffness in the (5-40) °C range shows a strong variation upon cooling coming from the increasing interaction between the protofilaments.
Recruitment of activated IRF-3 and CBP/p300 to herpes simplex virus ICP0 nuclear foci: Potential role in blocking IFN-{beta} induction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melroe, Gregory T.; Silva, Lindsey; Schaffer, Priscilla A.

2007-04-10

The host innate response to viral infection includes the production of interferons, which is dependent on the coordinated activity of multiple transcription factors. Herpes simplex virus 1 (HSV-1) has been shown to block efficient interferon expression by multiple mechanisms. We and others have demonstrated that HSV-1 can inhibit the transcription of genes promoted by interferon regulatory factor-3 (IRF-3), including interferon beta (IFN-{beta}), and that the immediate-early ICP0 protein is sufficient for this function. However, the exact mechanism by which ICP0 blocks IRF-3 activity has yet to be determined. Unlike some other viral proteins that inhibit IRF-3 activity, ICP0 does notmore » appear to affect phosphorylation and dimerization of IRF-3. Here, we show that a portion of activated IRF-3 co-localizes with nuclear foci containing ICP0 at early times after virus infection. Co-localization to ICP0-containing foci is also seen with the IRF-3-binding partners and transcriptional co-activators, CBP and p300. In addition, using immunoprecipitation of infected cell lysates, we can immunoprecipitate a complex containing ICP0, IRF-3, and CBP. Thus we hypothesize that ICP0 recruits activated IRF-3 and CBP/p300 to nuclear structures, away from the host chromatin. This leads to the inactivation and accelerated degradation of IRF-3, resulting in reduced transcription of IFN-{beta} and an inhibition of the host response. Therefore, ICP0 provides an example of how viruses can block IFN-{beta} induction by sequestration of important transcription factors essential for the host response.« less
Microtubule bundling plays a role in ethylene-mediated cortical microtubule reorientation in etiolated Arabidopsis hypocotyls.

PubMed

Ma, Qianqian; Sun, Jingbo; Mao, Tonglin

2016-05-15

The gaseous hormone ethylene is known to regulate plant growth under etiolated conditions (the 'triple response'). Although organization of cortical microtubules is essential for cell elongation, the underlying mechanisms that regulate microtubule organization by hormone signaling, including ethylene, are ambiguous. In the present study, we demonstrate that ethylene signaling participates in regulation of cortical microtubule reorientation. In particular, regulation of microtubule bundling is important for this process in etiolated hypocotyls. Time-lapse analysis indicated that selective stabilization of microtubule-bundling structures formed in various arrays is related to ethylene-mediated microtubule orientation. Bundling events and bundle growth lifetimes were significantly increased in oblique and longitudinal arrays, but decreased in transverse arrays in wild-type cells in response to ethylene. However, the effects of ethylene on microtubule bundling were partially suppressed in a microtubule-bundling protein WDL5 knockout mutant (wdl5-1). This study suggests that modulation of microtubule bundles that have formed in certain orientations plays a role in reorienting microtubule arrays in response to ethylene-mediated etiolated hypocotyl cell elongation. © 2016. Published by The Company of Biologists Ltd.
Multianalytical determination of trace elements in atmospheric biomonitors by k0-INAA, ICP-MS and AAS

NASA Astrophysics Data System (ADS)

Freitas, M. C.; Pacheco, A. M. G.; Dionísio, I.; Sarmento, S.; Baptista, M. S.; Vasconcelos, M. T. S. D.; Cabral, J. P.

2006-08-01

Elemental contents of atmospheric biomonitors—epiphytic lichens and tree bark, exposed in continuous and discontinuous modes—have been assessed through k0-standardised instrumental neutron activation analysis ( k0-INAA) (two different institutions), inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS). Certified reference materials—ISE-921 (river clay), NIST-1547 (peach leaves), ICHTJ-INCT-TL-1 (tea leaves; TL-1 hereinafter) and IAEA-336 (lichen material), and nonparametric statistics—rank-order correlations (Spearman RS) and enhanced-sign tests (Wilcoxon T)—were used for analytical control and data comparison, respectively. In general, quality of procedures was deemed good, except for k0-INAA in determining Br, Cu and Na, all likely affected by high counting statistics, and/or contamination issues (the latter). Results for Cu, Ni, Pb and Sr (by both ICP-MS and AAS) revealed that, despite an outstanding correlation (asymptotic p=0.000), they could be viewed as statistically equal for Cu only: AAS tended to yield higher values for Pb and Ni, and lower ones for Sr. The comparison between ICP-MS and k0-INAA data from TUDelft, for Al, Ca, Cu, Mg, Mn, Na, Ti and V, showed an excellent correlation (as above) and random (relative) magnitude for Cu, Mg, Mn and Ti only: ICP-MS tended to yield higher values for Al, Na and V, and lower ones for Ca, whereas between k0-INAA data from TUDelft and ITN, for Br, Ca and Na, resulted in systematically higher [Br] and [Ca] variates from TUDelft, even if all corresponding data sets were found to correlate at stringent significance levels. In a few cases, though—Ca, Sr in lichens; Pb in bark—matrix effects did appear to interfere in the outcome of matched-pairs, signed-rank tests, since random hierarchy of variates could be asserted just when lichen and bark data sets were processed separately.
Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

PubMed Central

Volkov, Vladimir A.; Zaytsev, Anatoly V.; Grishchuk, Ekaterina L.

2014-01-01

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion. PMID:24686554
Microtubule Severing Stymied by Free Tubulin

NASA Astrophysics Data System (ADS)

Ross, Jennifer; Bailey, Megan

2015-03-01

Proper organization of the microtubule cytoskeletal network is required to perform many necessary cellular functions including mitosis, cell development, and cell motility. Network organization is achieved through filament remodeling by microtubule-associated proteins (MAPs) that control microtubule dynamics. MAPs that stabilize are relatively well understood, while less is known about destabilizing MAPs, such as severing enzymes. Katanin, the first-discovered microtubule-severing enzyme, is a AAA + enzyme that oligomerizes into hexamers and uses ATP hydrolysis to sever microtubules. Using quantitative fluorescence imaging on reconstituted microtubule severing assays in vitro we investigate how katanin can regulate microtubule dynamics. Interestingly, we find microtubule dynamics inhibits katanin severing activity; dynamic microtubules are not severed. Using systematic experiments introducing free tubulin into the assays we find that free tubulin can compete for microtubule filaments for the katanin proteins. Our work indicates that katanin could function best on stabile microtubules or stabile regions of microtubules in cells in regions where free tubulin is sequesters, low, or depleted.
Adenosine regulation of microtubule dynamics in cardiac hypertrophy.

PubMed

Fassett, John T; Xu, Xin; Hu, Xinli; Zhu, Guangshuo; French, Joel; Chen, Yingjie; Bache, Robert J

2009-08-01

There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics.
Role of microtubules in the contractile dysfunction of hypertrophied myocardium

NASA Technical Reports Server (NTRS)

Zile, M. R.; Koide, M.; Sato, H.; Ishiguro, Y.; Conrad, C. H.; Buckley, J. M.; Morgan, J. P.; Cooper, G. 4th

1999-01-01

OBJECTIVES: We sought to determine whether the ameliorative effects of microtubule depolymerization on cellular contractile dysfunction in pressure overload cardiac hypertrophy apply at the tissue level. BACKGROUND: A selective and persistent increase in microtubule density causes decreased contractile function of cardiocytes from cats with hypertrophy produced by chronic right ventricular (RV) pressure overloading. Microtubule depolymerization by colchicine normalizes contractility in these isolated cardiocytes. However, whether these changes in cellular function might contribute to changes in function at the more highly integrated and complex cardiac tissue level was unknown. METHODS: Accordingly, RV papillary muscles were isolated from 25 cats with RV pressure overload hypertrophy induced by pulmonary artery banding (PAB) for 4 weeks and 25 control cats. Contractile state was measured using physiologically sequenced contractions before and 90 min after treatment with 10(-5) mol/liter colchicine. RESULTS: The PAB significantly increased RV systolic pressure and the RV weight/body weight ratio in PAB; it significantly decreased developed tension from 59+/-3 mN/mm2 in control to 25+/-4 mN/mm2 in PAB, shortening extent from 0.21+/-0.01 muscle lengths (ML) in control to 0.12+/-0.01 ML in PAB, and shortening rate from 1.12+/-0.07 ML/s in control to 0.55+/-0.03 ML/s in PAB. Indirect immunofluorescence confocal microscopy showed that PAB muscles had a selective increase in microtubule density and that colchicine caused complete microtubule depolymerization in both control and PAB papillary muscles. Microtubule depolymerization normalized myocardial contractility in papillary muscles of PAB cats but did not alter contractility in control muscles. CONCLUSIONS: Excess microtubule density, therefore, is equally important to both cellular and to myocardial contractile dysfunction caused by chronic, severe pressure-overload cardiac hypertrophy.
Boron detection from blood samples by ICP-AES and ICP-MS during boron neutron capture therapy.

PubMed

Linko, S; Revitzer, H; Zilliacus, R; Kortesniemi, M; Kouri, M; Savolainen, S

2008-01-01

The concept of boron neutron capture therapy (BNCT) involves infusion of a (10)B containing tracer into the patient's bloodstream followed by local neutron irradiation(s). Accurate estimation of the blood boron level for the treatment field before irradiation is required. Boron concentration can be quantified by inductively coupled plasma atomic emission spectrometry (ICP-AES), mass spectrometry (ICP-MS), spectrofluorometric and direct current atomic emission spectrometry (DCP-AES) or by prompt gamma photon detection methods. The blood boron concentrations were analysed and compared using ICP-AES and ICP-MS to ensure congruency of the results if the analysis had to be changed during the treatment, e.g. for technical reasons. The effect of wet-ashing on the results was studied in addition. The mean of all samples analysed with ICP-MS was 5.8 % lower than with ICP-AES coupled to wet-ashing (R (2) = 0.88). Without wet-ashing, the mean of all samples analysed with ICP-MS was 9.1 % higher than with ICP-AES (R (2) = 0.99). Boron concentration analysed from whole blood samples with ICP-AES correlated well with the values of ICP-MS with wet-ashing of the sample matrix, which is generally considered the reference method. When using these methods in parallel at certain intervals during the treatments, reliability of the blood boron concentration values remains satisfactory, taking into account the required accuracy of dose determination in the irradiation of cancer patients.
First time description of dismantling phenomenon

PubMed Central

Barrer, Laurence; Gimenez, Guy

2015-01-01

Dismantling is a complex psychic phenomenon, which is not easy to define, and little interest has been shown in the subject. The authors of this paper want to demonstrate that dismantling is the main defense mechanism in autism, bringing about de-consensus of senses. The effects perceived in a child with autistic disorder are passivity and lack of thought. The authors’ purpose here is to define the dismantled state and reveal its underlying process. This paper will therefore describe for the first time in literature, the dismantling phenomenon and will submit a metapsychological approach of this defense mechanism. PMID:25999871
Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesin-dependent microtubule motility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gearhart, Debra A.; Sickles, Dale W.; Buccafusco, Jerry J.

2007-01-01

Diisopropylfluorophosphate, originally developed as a chemical warfare agent, is structurally similar to nerve agents, and chlorpyrifos has extensive worldwide use as an agricultural pesticide. While inhibition of cholinesterases underlies the acute toxicity of these organophosphates, we previously reported impaired axonal transport in the sciatic nerves from rats treated chronically with subthreshold doses of chlorpyrifos. Those data indicate that chlorpyrifos (and/or its active metabolite, chlorpyrifos-oxon) might directly affect the function of kinesin and/or microtubules-the principal proteins that mediate anterograde axonal transport. The current report describes in vitro assays to assess the concentration-dependent effects of chlorpyrifos (0-10 {mu}M), chlorpyrifos-oxon (0-10 {mu}M), andmore » diisopropylfluorophosphate (0-0.59 nM) on kinesin-dependent microtubule motility. Preincubating bovine brain microtubules with the organophosphates did not alter kinesin-mediated microtubule motility. In contrast, preincubation of bovine brain kinesin with diisopropylfluorophosphate, chlorpyrifos, or chlorpyrifos-oxon produced a concentration-dependent increase in the number of locomoting microtubules that detached from the kinesin-coated glass cover slip. Our data suggest that the organophosphates-chlorpyrifos-oxon, chlorpyrifos, and diisopropylfluorophosphate-directly affect kinesin, thereby disrupting kinesin-dependent transport on microtubules. Kinesin-dependent movement of vesicles, organelles, and other cellular components along microtubules is fundamental to the organization of all eukaryotic cells, especially in neurons where organelles and proteins synthesized in the cell body must move down long axons to pre-synaptic sites in nerve terminals. We postulate that disruption of kinesin-dependent intracellular transport could account for some of the long-term effects of organophosphates on the peripheral and central nervous system.« less
Electric field generated by longitudinal axial microtubule vibration modes with high spatial resolution microtubule model

NASA Astrophysics Data System (ADS)

Cifra, M.; Havelka, D.; Deriu, M. A.

2011-12-01

Microtubules are electrically polar structures fulfilling prerequisites for generation of oscillatory electric field in the kHz to GHz region. Energy supply for excitation of elasto-electrical vibrations in microtubules may be provided from GTP-hydrolysis; motor protein-microtubule interactions; and energy efflux from mitochondria. It recently was determined from anisotropic elastic network modeling of entire microtubules that the frequencies of microtubule longitudinal axial eigenmodes lie in the region of tens of GHz for the physiologically common microtubule lengths. We calculated electric field generated by axial longitudinal vibration modes of microtubule, which model is based on subnanometer precision of charge distribution. Due to elastoelectric nature of the vibrations, the vibration wavelength is million-fold shorter than that of the electromagnetic field in free space and the electric field around the microtubule manifests rich spatial structure with multiple minima. The dielectrophoretic force exerted by electric field on the surrounding molecules will influence the kinetics of reactions via change in the probability of the transport of charge and mass particles. The electric field generated by vibrations of electrically polar cellular structures is expected to play a role in biological self-organization.
Microtubules, Tubulins and Associated Proteins.

ERIC Educational Resources Information Center

Raxworthy, Michael J.

1988-01-01

Reviews much of what is known about microtubules, which are biopolymers consisting predominantly of subunits of the globular protein, tubulin. Describes the functions of microtubules, their structure and assembly, microtube associated proteins, and microtubule-disrupting agents. (TW)
Anomalous Flexural Behaviors of Microtubules

PubMed Central

Liu, Xiaojing; Zhou, Youhe; Gao, Huajian; Wang, Jizeng

2012-01-01

Apparent controversies exist on whether the persistence length of microtubules depends on its contour length. This issue is particularly challenging from a theoretical point of view due to the tubular structure and strongly anisotropic material property of microtubules. Here we adopt a higher order continuum orthotropic thin shell model to study the flexural behavior of microtubules. Our model overcomes some key limitations of a recent study based on a simplified anisotropic shell model and results in a closed-form solution for the contour-length-dependent persistence length of microtubules, with predictions in excellent agreement with experimental measurements. By studying the ratio between their contour and persistence lengths, we find that microtubules with length at ∼1.5 μm show the lowest flexural rigidity, whereas those with length at ∼15 μm show the highest flexural rigidity. This finding may provide an important theoretical basis for understanding the mechanical structure of mitotic spindles during cell division. Further analysis on the buckling of microtubules indicates that the critical buckling load becomes insensitive to the tube length for relatively short microtubules, in drastic contrast to the classical Euler buckling. These rich flexural behaviors of microtubules are of profound implication for many biological functions and biomimetic molecular devices. PMID:22768935

ICP measurement accuracy: the effect of temperature drift. Design of a laboratory test for assessment of ICP transducers.

PubMed

Morgalla, M H; Mettenleiter, H; Katzenberger, T

1999-01-01

Intracranial pressure (ICP) monitoring has become the mainstay of multimodal neuromonitoring of comatous patients after head injury. In the presence of rising ICP and faced with pressures, difficult to control, aggressive measures, such as hypothermia may be used. The ICP readings should not be influenced by temperature changes. A laboratory test was designed to simulate temperature variations between 20 degrees C and 45 degrees C at different pressure levels under physiological conditions. Five types of transducers were examined: Epidyn Braun Melsungen, ICT/B-Titan Gaeltec, Camino-OLM-110-4B, Codman MicroSensor ICP-Transducer, Neurovent ICP transducer Rehau Ag+Co. Tests were performed at 6 different pressure levels between 0 mmHg and 50 mmHg. The results show very low drifts of less than 0.15 mmHg degree C-1 for Codman, Epidyn and Neurovent. Gaeltec and Camino exhibited higher drifts of 0.18 mmHg and 0.2 mmHg degree C-1 respectively. Within the temperature range from 35 degrees C to 42 degrees C all probes tested show insignificant temperature drift. Whether these results also apply to other types of transducers needs further evaluation. Problems and requirements related to the design of a laboratory test for the in vitro assessment of ICP transducers are discussed in detail.
An ELMO2-RhoG-ILK network modulates microtubule dynamics.

PubMed

Jackson, Bradley C; Ivanova, Iordanka A; Dagnino, Lina

2015-07-15

ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin-dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca(2+)-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes. © 2015 Jackson et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy

PubMed Central

Sato, Hiroshi; Nagai, Toshio; Kuppuswamy, Dhandapani; Narishige, Takahiro; Koide, Masaaki; Menick, Donald R.; IV, George Cooper

1997-01-01

Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, α-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin ↔ Glu-tubulin) and then irreversible deglutamination (Glu-tubulin → Δ2-tubulin), such that Glu- and Δ2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Δ2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Δ2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Δ2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy. PMID:9362514
Microtubule nucleation and organization in dendrites

PubMed Central

Delandre, Caroline; Amikura, Reiko; Moore, Adrian W.

2016-01-01

ABSTRACT Dendrite branching is an essential process for building complex nervous systems. It determines the number, distribution and integration of inputs into a neuron, and is regulated to create the diverse dendrite arbor branching patterns characteristic of different neuron types. The microtubule cytoskeleton is critical to provide structure and exert force during dendrite branching. It also supports the functional requirements of dendrites, reflected by differential microtubule architectural organization between neuron types, illustrated here for sensory neurons. Both anterograde and retrograde microtubule polymerization occur within growing dendrites, and recent studies indicate that branching is enhanced by anterograde microtubule polymerization events in nascent branches. The polarities of microtubule polymerization events are regulated by the position and orientation of microtubule nucleation events in the dendrite arbor. Golgi outposts are a primary microtubule nucleation center in dendrites and share common nucleation machinery with the centrosome. In addition, pre-existing dendrite microtubules may act as nucleation sites. We discuss how balancing the activities of distinct nucleation machineries within the growing dendrite can alter microtubule polymerization polarity and dendrite branching, and how regulating this balance can generate neuron type-specific morphologies. PMID:27097122
Urinary heavy metal levels and relevant factors among people exposed to e-waste dismantling.

PubMed

Wang, Hongmei; Han, Mei; Yang, Suwen; Chen, Yanqing; Liu, Qian; Ke, Shen

2011-01-01

Primitive electronic waste (e-waste) recycling has become a growing environmental concern, and toxic heavy metals released from e-waste activities may continue to threaten the health of local people. To study the impact of heavy metals in people around e-waste sites, 349 people from e-waste recycling sites (exposure group) and 118 people from a green plantation (control group) were surveyed, and their urinary levels of lead (UPb), cadmium (UCd), manganese (UMn), copper (UCu), and Zinc (UZn) were assayed. Questionnaire surveys for risk factors were also performed and analyzed by using the Pearson correlation analysis. Results indicated that the levels of urinary Cd in both occupational dismantling people {GM(GSD) 0.72(0.71) ug/L} and non-occupational dismantling people {GM(GSD) 0.50(0.79) ug/L} were higher than the control group {GM(GSD) 0.27(0.85) ug/L}. Further analyses of correlations between urinary heavy metal levels and exposure factors in the exposed group revealed positive relationship between the duration of dismantling and the level of UPb (p < 0.05). Meanwhile, rice sources from local village have a positive correlation with the level of UPb and UCd (p < 0.01). Other factors, however, may also have influences on heavy metal burden, and not all urinary heavy metal levels can be contributed to e-waste dismantling exposure levels. Primitive e-waste recycling activities may contribute to the changes of urinary heavy metal levels and increase the health risk for those chronically working on e-waste dismantling. Copyright © 2010 Elsevier Ltd. All rights reserved.
Microtubules as mechanical force sensors.

PubMed

Karafyllidis, Ioannis G; Lagoudas, Dimitris C

2007-03-01

Microtubules are polymers of tubulin subunits (dimers) arranged on a hexagonal lattice. Each tubulin dimer comprises two monomers, the alpha-tubulin and beta-tubulin, and can be found in two states. In the first state a mobile negative charge is located into the alpha-tubulin monomer and in the second into the beta-tubulin monomer. Each tubulin dimer is modeled as an electrical dipole coupled to its neighbors by electrostatic forces. The location of the mobile charge in each dimer depends on the location of the charges in the dimer's neighborhood. Mechanical forces that act on the microtubule affect the distances between the dimers and alter the electrostatic potential. Changes in this potential affect the mobile negative charge location in each dimer and the charge distribution in the microtubule. The net effect is that mechanical forces affect the charge distribution in microtubules. We propose to exploit this effect and use microtubules as mechanical force sensors. We model each dimer as a two-state quantum system and, following the quantum computation paradigm, we use discrete quantum random walk on the hexagonal microtubule lattice to determine the charge distribution. Different forces applied on the microtubule are modeled as different coin biases leading to different probability distributions of the quantum walker location, which are directly connected to different charge distributions. Simulation results show that there is a strong indication that microtubules can be used as mechanical force sensors and that they can also detect the force directions and magnitudes.
The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation.

PubMed

Goldspink, Deborah A; Gadsby, Jonathan R; Bellett, Gemma; Keynton, Jennifer; Tyrrell, Benjamin J; Lund, Elizabeth K; Powell, Penny P; Thomas, Paul; Mogensen, Mette M

2013-09-01

Microtubule end-binding (EB) proteins influence microtubule dynamic instability, a process that is essential for microtubule reorganisation during apico-basal epithelial differentiation. Here, we establish for the first time that expression of EB2, but not that of EB1, is crucial for initial microtubule reorganisation during apico-basal epithelial differentiation, and that EB2 downregulation promotes bundle formation. EB2 siRNA knockdown during early stages of apico-basal differentiation prevented microtubule reorganisation, whereas its downregulation at later stages promoted microtubule stability and bundle formation. Interestingly, although EB1 is not essential for microtubule reorganisation, its knockdown prevented apico-basal bundle formation and epithelial elongation. siRNA depletion of EB2 in undifferentiated epithelial cells induced the formation of straight, less dynamic microtubules with EB1 and ACF7 lattice association and co-alignment with actin filaments, a phenotype that could be rescued by inhibition with formin. Importantly, in situ inner ear and intestinal crypt epithelial tissue revealed direct correlations between a low level of EB2 expression and the presence of apico-basal microtubule bundles, which were absent where EB2 was elevated. EB2 is evidently important for initial microtubule reorganisation during epithelial polarisation, whereas its downregulation facilitates EB1 and ACF7 microtubule lattice association, microtubule-actin filament co-alignment and bundle formation. The spatiotemporal expression of EB2 thus dramatically influences microtubule organisation, EB1 and ACF7 deployment and epithelial differentiation.
Biological Information Processing in Single Microtubules

DTIC Science & Technology

2014-03-05

single Microtubule Google Mountain view campus, workshop on quantum biology 22 October 2010 3. Paul Davies Beyond Center at Arizona State University...Phoenix) Phoenix, workshop on quantum biology and cancer research, Experimental studies on single microtubule, 25-27 October 2010, Tempe, Arizona...State University, USA 4. Quantum aspects of microtubule: Direct experimental evidence for the existence of quantum states in microtubule, Towards a
Microtubule array reorientation in response to hormones does not involve changes in microtubule nucleation modes at the periclinal cell surface

PubMed Central

Atkinson, Samantha; Kirik, Angela; Kirik, Viktor

2014-01-01

Aligned microtubule arrays spatially organize cell division, trafficking, and determine the direction of cell expansion in plant cells. In response to changes in environmental and developmental signals, cells reorganize their microtubule arrays into new configurations. Here, we tested the role of microtubule nucleation during hormone-induced microtubule array reorientation. We have found that in the process of microtubule array reorientation the ratios between branching, parallel, and de-novo nucleations remained constant, suggesting that the microtubule reorientation mechanism does not involve changes in nucleation modes. In the ton2/fass mutant, which has reduced microtubule branching nucleation frequency and decreased nucleation activity of the γ-tubulin complexes, microtubule arrays were able to reorient. Presented data suggest that reorientation of microtubules into transverse arrays in response to hormones does not involve changes in microtubule nucleation at the periclinal cell surface PMID:25135522
Dismantling institutional racism: theory and action.

PubMed

Griffith, Derek M; Mason, Mondi; Yonas, Michael; Eng, Eugenia; Jeffries, Vanessa; Plihcik, Suzanne; Parks, Barton

2007-06-01

Despite a strong commitment to promoting social change and liberation, there are few community psychology models for creating systems change to address oppression. Given how embedded racism is in institutions such as healthcare, a significant shift in the system's policies, practices, and procedures is required to address institutional racism and create organizational and institutional change. This paper describes a systemic intervention to address racial inequities in healthcare quality called dismantling racism. The dismantling racism approach assumes healthcare disparities are the result of the intersection of a complex system (healthcare) and a complex problem (racism). Thus, dismantling racism is a systemic and systematic intervention designed to illuminate where and how to intervene in a given healthcare system to address proximal and distal factors associated with healthcare disparities. This paper describes the theory behind dismantling racism, the elements of the intervention strategy, and the strengths and limitations of this systems change approach.
Inhibition of Microtubule Depolymerization by Osmolytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bachand, George D.; Jain, Rishi; Ko, Randy

Microtubule dynamics play a critical role in the normal physiology of eukaryotic cells as well as a number of cancers and neurodegenerative disorders. The polymerization/depolymerization of microtubules is regulated by a variety of stabilizing and destabilizing factors, including microtubule-associated proteins and therapeutic agents (e.g., paclitaxel, nocodazole). Here in this paper, we describe the ability of the osmolytes polyethylene glycol (PEG) and trimethylamine-N-oxide (TMAO) to inhibit the depolymerization of individual microtubule filaments for extended periods of time (up to 30 days). We further show that PEG stabilizes microtubules against both temperature- and calcium-induced depolymerization. Our results collectively suggest that the observedmore » inhibition may be related to combination of the kosmotropic behavior and excluded volume/osmotic pressure effects associated with PEG and TMAO. Lastly, taken together with prior studies, our data suggest that the physiochemical properties of the local environment can regulate microtubule depolymerization and may potentially play an important role in in vivo microtubule dynamics.« less
Inhibition of Microtubule Depolymerization by Osmolytes

DOE PAGES

Bachand, George D.; Jain, Rishi; Ko, Randy; ...

2018-04-24

Microtubule dynamics play a critical role in the normal physiology of eukaryotic cells as well as a number of cancers and neurodegenerative disorders. The polymerization/depolymerization of microtubules is regulated by a variety of stabilizing and destabilizing factors, including microtubule-associated proteins and therapeutic agents (e.g., paclitaxel, nocodazole). Here in this paper, we describe the ability of the osmolytes polyethylene glycol (PEG) and trimethylamine-N-oxide (TMAO) to inhibit the depolymerization of individual microtubule filaments for extended periods of time (up to 30 days). We further show that PEG stabilizes microtubules against both temperature- and calcium-induced depolymerization. Our results collectively suggest that the observedmore » inhibition may be related to combination of the kosmotropic behavior and excluded volume/osmotic pressure effects associated with PEG and TMAO. Lastly, taken together with prior studies, our data suggest that the physiochemical properties of the local environment can regulate microtubule depolymerization and may potentially play an important role in in vivo microtubule dynamics.« less
Mutations Inactivating Herpes Simplex Virus 1 MicroRNA miR-H2 Do Not Detectably Increase ICP0 Gene Expression in Infected Cultured Cells or Mouse Trigeminal Ganglia.

PubMed

Pan, Dongli; Pesola, Jean M; Li, Gang; McCarron, Seamus; Coen, Donald M

2017-01-15

Herpes simplex virus 1 (HSV-1) latency entails the repression of productive ("lytic") gene expression. An attractive hypothesis to explain some of this repression involves inhibition of the expression of ICP0, a lytic gene activator, by a viral microRNA, miR-H2, which is completely complementary to ICP0 mRNA. To test this hypothesis, we engineered mutations that disrupt miR-H2 without affecting ICP0 in HSV-1. The mutant virus exhibited drastically reduced expression of miR-H2 but showed wild-type levels of infectious virus production and no increase in ICP0 expression in lytically infected cells, which is consistent with the weak expression of miR-H2 relative to the level of ICP0 mRNA in that setting. Following corneal inoculation of mice, the mutant was not significantly different from wild-type virus in terms of infectious virus production in the trigeminal ganglia during acute infection, mouse mortality, or the rate of reactivation from explanted latently infected ganglia. Critically, the mutant was indistinguishable from wild-type virus for the expression of ICP0 and other lytic genes in acutely and latently infected mouse trigeminal ganglia. The latter result may be related to miR-H2 being less effective in inhibiting ICP0 expression in transfection assays than a host microRNA, miR-138, which has previously been shown to inhibit lytic gene expression in infected ganglia by targeting ICP0 mRNA. Additionally, transfected miR-138 reduced lytic gene expression in infected cells more effectively than miR-H2. While this study provides little support for the hypothesis that miR-H2 promotes latency by inhibiting ICP0 expression, the possibility remains that miR-H2 might target other genes during latency. Herpes simplex virus 1 (HSV-1), which causes a variety of diseases, can establish lifelong latent infections from which virus can reactivate to cause recurrent disease. Latency is the most biologically interesting and clinically vexing feature of the virus. Ever since
Physical Modeling of Microtubules Network

NASA Astrophysics Data System (ADS)

Allain, Pierre; Kervrann, Charles

2014-10-01

Microtubules (MT) are highly dynamic tubulin polymers that are involved in many cellular processes such as mitosis, intracellular cell organization and vesicular transport. Nevertheless, the modeling of cytoskeleton and MT dynamics based on physical properties is difficult to achieve. Using the Euler-Bernoulli beam theory, we propose to model the rigidity of microtubules on a physical basis using forces, mass and acceleration. In addition, we link microtubules growth and shrinkage to the presence of molecules (e.g. GTP-tubulin) in the cytosol. The overall model enables linking cytosol to microtubules dynamics in a constant state space thus allowing usage of data assimilation techniques.
Arabidopsis phospholipase D alpha 1-derived phosphatidic acid regulates microtubule organization and cell development under microtubule-interacting drugs treatment.

PubMed

Zhang, Qun; Qu, Yana; Wang, Qing; Song, Ping; Wang, Peipei; Jia, Qianru; Guo, Jinhe

2017-01-01

Phospholipase D (PLD) and its product phosphatidic acid (PA) are emerging as essential regulators of cytoskeleton organization in plants. However, the underlying molecular mechanisms of PA-mediated microtubule reorganization in plants remain largely unknown. In this study, we used pharmacological and genetic approaches to analyze the function of Arabidopsis thaliana PLDα1 in the regulation of microtubule organization and cell development in response to microtubule-affecting drugs. Treatment with the microtubule-stabilizing drug paclitaxel resulted in less growth inhibition and decreased rightward slant of roots, longitudinal alignment of microtubules, and enhanced length of hypocotyl epidermal cells in the pldα1 mutant, the phenotype of which was rescued by exogenous application of PA. Moreover, the pldα1 mutant was sensitive to the microtubule-disrupting drugs oryzalin and propyzamide in terms of seedling survival ratio, left-skewing angle of roots and microtubule organization. In addition, both disruption and stabilization of microtubules induced by drugs activated PLDα1 activity. Our findings demonstrate that in A. thaliana, PLDα1/PA might regulate cell development by modulating microtubule organization in an activity-dependent manner.
Synchronous Oscillations in Microtubule Polymerization

NASA Astrophysics Data System (ADS)

Carlier, M. F.; Melki, R.; Pantaloni, D.; Hill, T. L.; Chen, Y.

1987-08-01

Under conditions where microtubule nucleation and growth are fast (i.e., high magnesium ion and tubulin concentrations and absence of glycerol), microtubule assembly in vitro exhibits an oscillatory regime preceding the establishment of steady state. The amplitude of the oscillations can represent >50% of the maximum turbidity change and oscillations persist for up to 20 periods of 80 s each. Oscillations are accompanied by extensive length redistribution of microtubules. Preliminary work suggests that the oscillatory kinetics can be simulated using a model in which many microtubules undergo synchronous transitions between growing and rapidly depolymerizing phases, complicated by the kinetically limiting rate of nucleotide exchange on free tubulin.
Site-specific microtubule-associated protein 4 dephosphorylation causes microtubule network densification in pressure overload cardiac hypertrophy.

PubMed

Chinnakkannu, Panneerselvam; Samanna, Venkatesababa; Cheng, Guangmao; Ablonczy, Zsolt; Baicu, Catalin F; Bethard, Jennifer R; Menick, Donald R; Kuppuswamy, Dhandapani; Cooper, George

2010-07-09

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac alpha- and beta-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 --> Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal
Site-specific Microtubule-associated Protein 4 Dephosphorylation Causes Microtubule Network Densification in Pressure Overload Cardiac Hypertrophy*

PubMed Central

Chinnakkannu, Panneerselvam; Samanna, Venkatesababa; Cheng, Guangmao; Ablonczy, Zsolt; Baicu, Catalin F.; Bethard, Jennifer R.; Menick, Donald R.; Kuppuswamy, Dhandapani; Cooper, George

2010-01-01

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac α- and β-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 → Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal
Assembly and control of large microtubule complexes

NASA Astrophysics Data System (ADS)

Korolev, Kirill; Ishihara, Keisuke; Mitchison, Timothy

Motility, division, and other cellular processes require rapid assembly and disassembly of microtubule structures. We report a new mechanism for the formation of asters, radial microtubule complexes found in very large cells. The standard model of aster growth assumes elongation of a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we found evidence for microtubule nucleation away from centrosomes. By combining polymerization dynamics and auto-catalytic nucleation of microtubules, we developed a new biophysical model of aster growth. The model predicts an explosive transition from an aster with a steady-state radius to one that expands as a travelling wave. At the transition, microtubule density increases continuously, but aster growth rate discontinuously jumps to a nonzero value. We tested our model with biochemical perturbations in egg extract and confirmed main theoretical predictions including the jump in the growth rate. Our results show that asters can grow even though individual microtubules are short and unstable. The dynamic balance between microtubule collapse and nucleation could be a general framework for the assembly and control of large microtubule complexes. NIH GM39565; Simons Foundation 409704; Honjo International 486 Scholarship Foundation.
Microtubules self-repair in response to mechanical stress

PubMed Central

Schaedel, Laura; John, Karin; Gaillard, Jérémie; Nachury, Maxence V.; Blanchoin, Laurent; Théry, Manuel

2015-01-01

Microtubules - which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport - can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of larger damages, which further decrease microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses. PMID:26343914

Microtubules self-repair in response to mechanical stress

NASA Astrophysics Data System (ADS)

Schaedel, Laura; John, Karin; Gaillard, Jérémie; Nachury, Maxence V.; Blanchoin, Laurent; Théry, Manuel

2015-11-01

Microtubules--which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport--can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses.
Microtubules self-repair in response to mechanical stress.

PubMed

Schaedel, Laura; John, Karin; Gaillard, Jérémie; Nachury, Maxence V; Blanchoin, Laurent; Théry, Manuel

2015-11-01

Microtubules--which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport--can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses.
Optomechanical proposal for monitoring microtubule mechanical vibrations

NASA Astrophysics Data System (ADS)

Barzanjeh, Sh.; Salari, V.; Tuszynski, J. A.; Cifra, M.; Simon, C.

2017-07-01

Microtubules provide the mechanical force required for chromosome separation during mitosis. However, little is known about the dynamic (high-frequency) mechanical properties of microtubules. Here, we theoretically propose to control the vibrations of a doubly clamped microtubule by tip electrodes and to detect its motion via the optomechanical coupling between the vibrational modes of the microtubule and an optical cavity. In the presence of a red-detuned strong pump laser, this coupling leads to optomechanical-induced transparency of an optical probe field, which can be detected with state-of-the art technology. The center frequency and line width of the transparency peak give the resonance frequency and damping rate of the microtubule, respectively, while the height of the peak reveals information about the microtubule-cavity field coupling. Our method opens the new possibilities to gain information about the physical properties of microtubules, which will enhance our capability to design physical cancer treatment protocols as alternatives to chemotherapeutic drugs.
Mechanics of microtubules: effects of protofilament orientation.

PubMed

Donhauser, Zachary J; Jobs, William B; Binka, Edem C

2010-09-08

Microtubules are hollow cylindrical polymers of the protein tubulin that play a number of important dynamic and structural roles in eukaryotic cells. Both in vivo and in vitro microtubules can exist in several possible configurations, differing in the number of protofilaments, helical rise of tubulin dimers, and protofilament skew angle with respect to the main tube axis. Here, finite element modeling is applied to examine the mechanical response of several known microtubule types when subjected to radial deformation. The data presented here provide an important insight into microtubule stiffness and reveal that protofilament orientation does not affect radial stiffness. Rather, stiffness is primarily dependent on the effective Young's modulus of the polymerized material and the effective radius of the microtubule. These results are also directly correlated to atomic force microscopy nanoindentation measurements to allow a more detailed interpretation of previous experiments. When combined with experimental data that show a significant difference between microtubules stabilized with a slowly hydrolyzable GTP analog and microtubules stabilized with paclitaxel, the finite element data suggest that paclitaxel increases the overall radial flexibility of the microtubule wall. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Mechanics of Microtubules: Effects of Protofilament Orientation

PubMed Central

Donhauser, Zachary J.; Jobs, William B.; Binka, Edem C.

2010-01-01

Microtubules are hollow cylindrical polymers of the protein tubulin that play a number of important dynamic and structural roles in eukaryotic cells. Both in vivo and in vitro microtubules can exist in several possible configurations, differing in the number of protofilaments, helical rise of tubulin dimers, and protofilament skew angle with respect to the main tube axis. Here, finite element modeling is applied to examine the mechanical response of several known microtubule types when subjected to radial deformation. The data presented here provide an important insight into microtubule stiffness and reveal that protofilament orientation does not affect radial stiffness. Rather, stiffness is primarily dependent on the effective Young's modulus of the polymerized material and the effective radius of the microtubule. These results are also directly correlated to atomic force microscopy nanoindentation measurements to allow a more detailed interpretation of previous experiments. When combined with experimental data that show a significant difference between microtubules stabilized with a slowly hydrolyzable GTP analog and microtubules stabilized with paclitaxel, the finite element data suggest that paclitaxel increases the overall radial flexibility of the microtubule wall. PMID:20816081
Non-invasively estimated ICP pulse amplitude strongly correlates with outcome after TBI.

PubMed

Budohoski, Karol P; Schmidt, Bernhard; Smielewski, Peter; Kasprowicz, Magdalena; Plontke, Ronny; Pickard, John D; Klingelhöfer, Jurgen; Czosnyka, Marek

2012-01-01

An existing monitoring database of brain signal recordings in patients with head injury has been re-evaluated with regard to the accuracy of estimation of non-invasive ICP (nICP) and its components, with a particular interest in the implications for outcome after head injury. Middle cerebral artery blood flow velocity (FV), ICP and arterial blood pressure (ABP) were recorded. Non-invasive ICP (nICP) was calculated using a mathematical model. Other signals analysed included components of ICP (n" indicates non-invasive): ICP pulse amplitude (Amp, nAmp), amplitude of the respiratory component (Resp, nResp), amplitude of slow vasogenic waves of ICP (Slow, nSlow) and index of compensatory reserve (RAP, nRAP). Mean values of analysed signals were compared against each other and between patients who died and survived. The correlation between ICP and nICP was moderately strong, R = 0.51 (95% prediction interval [PI] 17 mm Hg). The components of nICP and ICP were also moderately correlated with each other: the strongest correlation was observed for Resp vs. nResp (r = 0.66), while weaker for Amp vs. nAmp (r = 0.41). Non-invasive pulse amplitude of ICP showed the strongest association with outcome, with the -difference between those who survived and those who died reaching a significance level of p < 0.000001. When compared between patients who died and who survived mean nAmp showed the greatest difference, suggesting its potential to predict mortality after TBI.
Dismantling of Loop-Type Channel Equipment of MR Reactor in NRC 'Kurchatov Institute' - 13040

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volkov, Victor; Danilovich, Alexey; Zverkov, Yuri

2013-07-01

. 66, 66A, 66B, 72, 64, 63) - as well as from water and gas loop corridors - was dismantled, with the total radwaste weight of 53 tons and the total removed activity of 5,0 x 10{sup 10} Bq; - loop-type channel equipment from underground reactor hall premises was dismantled; - 93 loop-type channels were characterized, chopped and removed, with radwaste of 2.6 x 10{sup 13} Bq ({sup 60}Co) and 1.5 x 10{sup 13} Bq ({sup 137}Cs) total activity removed from the reactor pool, fragmented and packaged. Some of this waste was placed into the high-level waste (HLW) repository of the Center. Dismantling works were executed with application of remotely operated mechanisms, which promoted decrease of radiation impact on the personnel. The average individual dose for the personnel was 1.9 mSv/year in 2011, and the collective dose is estimated as 0.0605 man x Sv/year. (authors)« less
A novel mechanism important for the alignment of microtubules.

PubMed

Wightman, Raymond; Turner, Simon R

2008-04-01

Using a live-cell imaging approach to study individual micro-tubules, we have compared microtubule behavior between net-like and aligned cortical arrays. In contrast to previous studies, a steep angled collision between the growing end of a microtubule and a preexisting microtubule was found to favor crossover. Frequencies of microtubule crossovers, bundling and catastrophes are similar regardless of whether the cell exhibited a net-like or aligned microtubule array. In the predominantly aligned array of petiole cells, severing occurs at the sites of microtubule crossovers and serves to remove unaligned microtubules and to increase microtubule density. Severing was observed to be rare in net-like arrays. Microtubule severing is carried out by the katanin enzyme. In this addendum, we present new insights into the possible mechanism of crossing over and preliminary data looking at organization of the array in a katanin mutant.
GDP-tubulin incorporation into growing microtubules modulates polymer stability.

PubMed

Valiron, Odile; Arnal, Isabelle; Caudron, Nicolas; Job, Didier

2010-06-04

Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule "structural plasticity."
Method validation for simultaneous determination of chromium, molybdenum and selenium in infant formulas by ICP-OES and ICP-MS.

PubMed

Khan, Naeem; Jeong, In Seon; Hwang, In Min; Kim, Jae Sung; Choi, Sung Hwa; Nho, Eun Yeong; Choi, Ji Yeon; Kwak, Byung-Man; Ahn, Jang-Hyuk; Yoon, Taehyung; Kim, Kyong Su

2013-12-15

This study aimed to validate the analytical method for simultaneous determination of chromium (Cr), molybdenum (Mo), and selenium (Se) in infant formulas available in South Korea. Various digestion methods of dry-ashing, wet-digestion and microwave were evaluated for samples preparation and both inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS) were compared for analysis. The analytical techniques were validated by detection limits, precision, accuracy and recovery experiments. Results showed that wet-digestion and microwave methods were giving satisfactory results for sample preparation, while ICP-MS was found more sensitive and effective technique than ICP-OES. The recovery (%) of Se, Mo and Cr by ICP-OES were 40.9, 109.4 and 0, compared to 99.1, 98.7 and 98.4, respectively by ICP-MS. The contents of Cr, Mo and Se in infant formulas by ICP-MS were found in good nutritional values in accordance to nutrient standards for infant formulas CODEX values. Copyright © 2013 Elsevier Ltd. All rights reserved.
Precise and accurate isotope ratio measurements by ICP-MS.

PubMed

Becker, J S; Dietze, H J

2000-09-01

The precise and accurate determination of isotope ratios by inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) is important for quite different application fields (e.g. for isotope ratio measurements of stable isotopes in nature, especially for the investigation of isotope variation in nature or age dating, for determining isotope ratios of radiogenic elements in the nuclear industry, quality assurance of fuel material, for reprocessing plants, nuclear material accounting and radioactive waste control, for tracer experiments using stable isotopes or long-lived radionuclides in biological or medical studies). Thermal ionization mass spectrometry (TIMS), which used to be the dominant analytical technique for precise isotope ratio measurements, is being increasingly replaced for isotope ratio measurements by ICP-MS due to its excellent sensitivity, precision and good accuracy. Instrumental progress in ICP-MS was achieved by the introduction of the collision cell interface in order to dissociate many disturbing argon-based molecular ions, thermalize the ions and neutralize the disturbing argon ions of plasma gas (Ar+). The application of the collision cell in ICP-QMS results in a higher ion transmission, improved sensitivity and better precision of isotope ratio measurements compared to quadrupole ICP-MS without the collision cell [e.g., for 235U/238U approximately 1 (10 microg x L(-1) uranium) 0.07% relative standard deviation (RSD) vs. 0.2% RSD in short-term measurements (n = 5)]. A significant instrumental improvement for ICP-MS is the multicollector device (MC-ICP-MS) in order to obtain a better precision of isotope ratio measurements (with a precision of up to 0.002%, RSD). CE- and HPLC-ICP-MS are used for the separation of isobaric interferences of long-lived radionuclides and stable isotopes by determination of spallation nuclide abundances in an irradiated tantalum target.
An assay to image neuronal microtubule dynamics in mice.

PubMed

Kleele, Tatjana; Marinković, Petar; Williams, Philip R; Stern, Sina; Weigand, Emily E; Engerer, Peter; Naumann, Ronald; Hartmann, Jana; Karl, Rosa M; Bradke, Frank; Bishop, Derron; Herms, Jochen; Konnerth, Arthur; Kerschensteiner, Martin; Godinho, Leanne; Misgeld, Thomas

2014-09-12

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.
Do prokaryotes contain microtubules?

NASA Technical Reports Server (NTRS)

Bermudes, D.; Hinkle, G.; Margulis, L.

1994-01-01

In eukaryotic cells, microtubules are 24-nm-diameter tubular structures composed of a class of conserved proteins called tubulin. They are involved in numerous cell functions including ciliary motility, nerve cell elongation, pigment migration, centrosome formation, and chromosome movement. Although cytoplasmic tubules and fibers have been observed in bacteria, some with diameters similar to those of eukaryotes, no homologies to eukaryotic microtubules have been established. Certain groups of bacteria including azotobacters, cyanobacteria, enteric bacteria, and spirochetes have been frequently observed to possess microtubule-like structures, and others, including archaebacteria, have been shown to be sensitive to drugs that inhibit the polymerization of microtubules. Although little biochemical or molecular biological information is available, the differences observed among these prokaryotic structures suggest that their composition generally differs among themselves as well as from that of eukaryotes. We review the distribution of cytoplasmic tubules in prokaryotes, even though, in all cases, their functions remain unknown. At least some tend to occur in cells that are large, elongate, and motile, suggesting that they may be involved in cytoskeletal functions, intracellular motility, or transport activities comparable to those performed by eukaryotic microtubules. In Escherichia coli, the FtsZ protein is associated with the formation of a ring in the division zone between the newly forming offspring cells. Like tubulin, FtsZ is a GTPase and shares with tubulin a 7-amino-acid motif, making it a promising candidate in which to seek the origin of tubulins.
The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics.

PubMed

Currie, Joshua D; Stewman, Shannon; Schimizzi, Gregory; Slep, Kevin C; Ma, Ao; Rogers, Stephen L

2011-11-01

Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, we conducted an in vivo structure-function analysis of the Drosophila homologue Mini spindles (Msps). Msps exhibits EB1-dependent and spatially regulated MT localization, targeting to microtubule plus ends in the cell interior and decorating the lattice of growing and shrinking microtubules in the cell periphery. RNA interference rescue experiments revealed that the NH(2)-terminal four TOG domains of Msps function as paired units and were sufficient to promote microtubule dynamics and EB1 comet formation. We also identified TOG5 and novel inter-TOG linker motifs that are required for targeting Msps to the microtubule lattice. These novel microtubule contact sites are necessary for the interplay between the conserved TOG domains and inter-TOG MT binding that underlies the ability of Msps to promote MT dynamic instability.
An epidermal plakin that integrates actin and microtubule networks at cellular junctions.

PubMed

Karakesisoglou, I; Yang, Y; Fuchs, E

2000-04-03

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.
Loop formation of microtubules during gliding at high density

NASA Astrophysics Data System (ADS)

Liu, Lynn; Tüzel, Erkan; Ross, Jennifer L.

2011-09-01

The microtubule cytoskeleton, including the associated proteins, forms a complex network essential to multiple cellular processes. Microtubule-associated motor proteins, such as kinesin-1, travel on microtubules to transport membrane bound vesicles across the crowded cell. Other motors, such as cytoplasmic dynein and kinesin-5, are used to organize the cytoskeleton during mitosis. In order to understand the self-organization processes of motors on microtubules, we performed filament-gliding assays with kinesin-1 motors bound to the cover glass with a high density of microtubules on the surface. To observe microtubule organization, 3% of the microtubules were fluorescently labeled to serve as tracers. We find that microtubules in these assays are not confined to two dimensions and can cross one other. This causes microtubules to align locally with a relatively short correlation length. At high density, this local alignment is enough to create 'intersections' of perpendicularly oriented groups of microtubules. These intersections create vortices that cause microtubules to form loops. We characterize the radius of curvature and time duration of the loops. These different behaviors give insight into how crowded conditions, such as those in the cell, might affect motor behavior and cytoskeleton organization.
Targeting and transport: How microtubules control focal adhesion dynamics

PubMed Central

Stehbens, Samantha

2012-01-01

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge. PMID:22908306
The replication defect of ICP0-null mutant herpes simplex virus 1 can be largely complemented by the combined activities of human cytomegalovirus proteins IE1 and pp71.

PubMed

Everett, Roger D; Bell, Adam J; Lu, Yongxu; Orr, Anne

2013-01-01

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 is required for efficient lytic infection and productive reactivation from latency and induces derepression of quiescent viral genomes. Despite being unrelated at the sequence level, ICP0 and human cytomegalovirus proteins IE1 and pp71 share some functional similarities in their abilities to counteract antiviral restriction mediated by components of cellular nuclear structures known as ND10. To investigate the extent to which IE1 and pp71 might substitute for ICP0, cell lines were developed that express either IE1 or pp71, or both together, in an inducible manner. We found that pp71 dissociated the hDaxx-ATRX complex and inhibited accumulation of these proteins at sites juxtaposed to HSV-1 genomes but had no effect on the promyelocytic leukemia protein (PML) or Sp100. IE1 caused loss of the small ubiquitin-like modifier (SUMO)-conjugated forms of PML and Sp100 and inhibited the recruitment of these proteins to HSV-1 genome foci but had little effect on hDaxx or ATRX in these assays. Both IE1 and pp71 stimulated ICP0-null mutant plaque formation, but neither to the extent achieved by ICP0. The combination of IE1 and pp71, however, inhibited recruitment of all ND10 proteins to viral genome foci, stimulated ICP0-null mutant HSV-1 plaque formation to near wild-type levels, and efficiently induced derepression of quiescent HSV-1 genomes. These results suggest that ND10-related intrinsic resistance results from the additive effects of several ND10 components and that the effects of IE1 and pp71 on subsets of these components combine to mirror the overall activities of ICP0.
The Replication Defect of ICP0-Null Mutant Herpes Simplex Virus 1 Can Be Largely Complemented by the Combined Activities of Human Cytomegalovirus Proteins IE1 and pp71

PubMed Central

Bell, Adam J.; Lu, Yongxu; Orr, Anne

2013-01-01

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 is required for efficient lytic infection and productive reactivation from latency and induces derepression of quiescent viral genomes. Despite being unrelated at the sequence level, ICP0 and human cytomegalovirus proteins IE1 and pp71 share some functional similarities in their abilities to counteract antiviral restriction mediated by components of cellular nuclear structures known as ND10. To investigate the extent to which IE1 and pp71 might substitute for ICP0, cell lines were developed that express either IE1 or pp71, or both together, in an inducible manner. We found that pp71 dissociated the hDaxx-ATRX complex and inhibited accumulation of these proteins at sites juxtaposed to HSV-1 genomes but had no effect on the promyelocytic leukemia protein (PML) or Sp100. IE1 caused loss of the small ubiquitin-like modifier (SUMO)-conjugated forms of PML and Sp100 and inhibited the recruitment of these proteins to HSV-1 genome foci but had little effect on hDaxx or ATRX in these assays. Both IE1 and pp71 stimulated ICP0-null mutant plaque formation, but neither to the extent achieved by ICP0. The combination of IE1 and pp71, however, inhibited recruitment of all ND10 proteins to viral genome foci, stimulated ICP0-null mutant HSV-1 plaque formation to near wild-type levels, and efficiently induced derepression of quiescent HSV-1 genomes. These results suggest that ND10-related intrinsic resistance results from the additive effects of several ND10 components and that the effects of IE1 and pp71 on subsets of these components combine to mirror the overall activities of ICP0. PMID:23135716
Effects of the KIF2C neck peptide on microtubules: lateral disintegration of microtubules and β-structure formation.

PubMed

Shimizu, Youské; Shimizu, Takashi; Nara, Masayuki; Kikumoto, Mahito; Kojima, Hiroaki; Morii, Hisayuki

2013-04-01

Members of the kinesin-13 sub-family, including KIF2C, depolymerize microtubules. The positive charge-rich 'neck' region extending from the N-terminus of the catalytic head is considered to be important in the depolymerization activity. Chemically synthesized peptides, covering the basic region (A182-E200), induced a sigmoidal increase in the turbidity of a microtubule suspension. The increase was suppressed by salt addition or by reduction of basicity by amino acid substitutions. Electron microscopic observations revealed ring structures surrounding the microtubules at high peptide concentrations. Using the peptide A182-D218, we also detected free thin straight filaments, probably protofilaments disintegrated from microtubules. Therefore, the neck region, even without the catalytic head domain, may induce lateral disintegration of microtubules. With microtubules lacking anion-rich C-termini as a result of subtilisin treatment, addition of the peptide induced only a moderate increase in turbidity, and rings and protofilaments were rarely detected, while aggregations, also thought to be caused by lateral disintegration, were often observed in electron micrographs. Thus, the C-termini are not crucial for the action of the peptides in lateral disintegration but contribute to structural stabilization of the protofilaments. Previous structural studies indicated that the neck region of KIF2C is flexible, but our IR analysis suggests that the cation-rich region (K190-A204) forms β-structure in the presence of microtubules, which may be of significance with regard to the action of the neck region. Therefore, the neck region of KIF2C is sufficient to cause disintegration of microtubules into protofilaments, and this may contribute to the ability of KIF2C to cause depolymerization of microtubules. © 2013 The Authors Journal compilation © 2013 FEBS.

Microtubules negatively regulate insulin secretion in pancreatic β cells

PubMed Central

Zhu, Xiaodong; Hu, Ruiying; Brissova, Marcela; Stein, Roland W.; Powers, Alvin C.; Gu, Guoqiang; Kaverina, Irina

2015-01-01

Summary For glucose-stimulated insulin secretion (GSIS) insulin granules have to be localized close to the plasma membrane. The role of microtubule-dependent transport in granule positioning and GSIS has been debated. Here, we report that microtubules, counterintuitively, restrict granule availability for secretion. In β cells, microtubules originate at the Golgi and form a dense non-radial meshwork. Non-directional transport along these microtubules limits granule dwelling at the cell periphery, restricting granule availability for secretion. High glucose destabilizes microtubules, decreasing their density; such local microtubule depolymerization is necessary for GSIS, likely because granule withdrawal from the cell periphery becomes inefficient. Consistently, microtubule depolymerization by nocodazole blocks granule withdrawal, increases their concentration at exocytic sites, and dramatically enhances GSIS in vitro and in mice. Furthermore, glucose-driven MT destabilization is balanced by new microtubule formation, which likely prevents over-secretion. Importantly, microtubule density is greater in dysfunctional β cells of diabetic mice. PMID:26418295
Microtubule stabilization with paclitaxel does not protect against infarction in isolated rat hearts.

PubMed

Rodríguez-Sinovas, Antonio; Abad, Elena; Sánchez, Jose A; Fernández-Sanz, Celia; Inserte, Javier; Ruiz-Meana, Marisol; Alburquerque-Béjar, Juan José; García-Dorado, David

2015-01-01

What is the central question of this study? The microtubule network is disrupted during myocardial ischaemia-reperfusion injury. It was suggested that prevention of microtubule disruption with paclitaxel might reduce cardiac infarct size; however, the effects on infarction have not been studied. What is the main finding and its importance? Paclitaxel caused a reduction in microtubule disruption and cardiomyocyte hypercontracture during ischaemia-reperfusion. However, it induced a greater increase in cytosolic calcium, which may explain the lack of effect against infarction that we have seen in isolated rat hearts. The large increase in perfusion pressure induced by paclitaxel in this model may have clinical implications, because detrimental effects of the drug were reported after its clinical application. Microtubules play a major role in the transmission of mechanical forces within the myocardium and in maintenance of organelle function. However, this intracellular network is disrupted during myocardial ischaemia-reperfusion. We assessed the effects of prevention of microtubule disruption with paclitaxel on ischaemia-reperfusion injury in isolated rat cardiomyocytes and hearts. Isolated rat cardiomyocytes were submitted to normoxia (1 h) or 45 min of simulated ischaemia (pH 6.4, 0% O2 , 37 °C) and reoxygenation, without or with treatment with the microtubule stabilizer, paclitaxel (10(-5) M), or the inhibitor of microtubule polymerization, colchicine (5 × 10(-6) M). Simulated ischaemia leads to microtubule disruption before the onset of ischaemic contracture. Paclitaxel attenuated both microtubule disruption and the incidence of hypercontracture, whereas treatment with colchicine mimicked the effects of simulated ischaemia and reoxygenation. In isolated normoxic rat hearts, treatment with paclitaxel induced concentration-dependent decreases in heart rate and left ventricular developed pressure and increases in perfusion pressure. Despite protection against
Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins).

PubMed

Alves-Silva, Juliana; Sánchez-Soriano, Natalia; Beaven, Robin; Klein, Melanie; Parkin, Jill; Millard, Thomas H; Bellen, Hugo J; Venken, Koen J T; Ballestrem, Christoph; Kammerer, Richard A; Prokop, Andreas

2012-07-04

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.
ACF7: an essential integrator of microtubule dynamics.

PubMed

Kodama, Atsuko; Karakesisoglou, Iakowos; Wong, Ellen; Vaezi, Alec; Fuchs, Elaine

2003-10-31

ACF7 is a member of the spectraplakin family of cytoskeletal crosslinking proteins possessing actin and microtubule binding domains. Here, we show that ACF7 is an essential integrator of MT-actin dynamics. In endodermal cells, ACF7 binds along microtubules but concentrates at their distal ends and at cell borders when polarized. In ACF7's absence, microtubules still bind EB1 and CLIP170, but they no longer grow along polarized actin bundles, nor do they pause and tether to actin-rich cortical sites. The consequences are less stable, long microtubules with skewed cytoplasmic trajectories and altered dynamic instability. In response to wounding, ACF7 null cultures activate polarizing signals, but fail to maintain them and coordinate migration. Rescue of these defects requires ACF7's actin and microtubule binding domains. Thus, spectraplakins are important for controlling microtubule dynamics and reinforcing links between microtubules and polarized F-actin, so that cellular polarization and coordinated cell movements can be sustained.
Herpes simplex virus VP16, but not ICP0, is required to reduce histone occupancy and enhance histone acetylation on viral genomes in U2OS osteosarcoma cells.

PubMed

Hancock, Meaghan H; Cliffe, Anna R; Knipe, David M; Smiley, James R

2010-02-01

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.
Assessing the economics of processing end-of-life vehicles through manual dismantling.

PubMed

Tian, Jin; Chen, Ming

2016-10-01

Most dismantling enterprises in a number of developing countries, such as China, usually adopt the "manual+mechanical" dismantling approach to process end-of-life vehicles. However, the automobile industry does not have a clear indicator to reasonably and effectively determine the manual dismantling degree for end-of-life vehicles. In this study, five different dismantling scenarios and an economic system for end-of-life vehicles were developed based on the actual situation of end-of-life vehicles. The fuzzy analytic hierarchy process was applied to set the weights of direct costs, indirect costs, and sales and to obtain an optimal manual dismantling scenario. Results showed that although the traditional method of "dismantling to the end" can guarantee the highest recycling rate, this method is not the best among all the scenarios. The profit gained in the optimal scenario is 100.6% higher than that in the traditional scenario. The optimal manual dismantling scenario showed that enterprises are required to select suitable parts to process through manual dismantling. Selecting suitable parts maximizes economic profit and improves dismantling speed. Copyright © 2016 Elsevier Ltd. All rights reserved.
Visualizing individual microtubules by bright field microscopy

NASA Astrophysics Data System (ADS)

Gutiérrez-Medina, Braulio; Block, Steven M.

2010-11-01

Microtubules are slender (˜25 nm diameter), filamentous polymers involved in cellular structure and organization. Individual microtubules have been visualized via fluorescence imaging of dye-labeled tubulin subunits and by video-enhanced, differential interference-contrast microscopy of unlabeled polymers using sensitive CCD cameras. We demonstrate the imaging of unstained microtubules using a microscope with conventional bright field optics in conjunction with a webcam-type camera and a light-emitting diode illuminator. The light scattered by microtubules is image-processed to remove the background, reduce noise, and enhance contrast. The setup is based on a commercial microscope with a minimal set of inexpensive components, suitable for implementation in a student laboratory. We show how this approach can be used in a demonstration motility assay, tracking the gliding motions of microtubules driven by the motor protein kinesin.
Regulation of microtubule nucleation mediated by γ-tubulin complexes.

PubMed

Sulimenko, Vadym; Hájková, Zuzana; Klebanovych, Anastasiya; Dráber, Pavel

2017-05-01

The microtubule cytoskeleton is critically important for spatio-temporal organization of eukaryotic cells. The nucleation of new microtubules is typically restricted to microtubule organizing centers (MTOCs) and requires γ-tubulin that assembles into multisubunit complexes of various sizes. γ-Tubulin ring complexes (TuRCs) are efficient microtubule nucleators and are associated with large number of targeting, activating and modulating proteins. γ-Tubulin-dependent nucleation of microtubules occurs both from canonical MTOCs, such as spindle pole bodies and centrosomes, and additional sites such as Golgi apparatus, nuclear envelope, plasma membrane-associated sites, chromatin and surface of pre-existing microtubules. Despite many advances in structure of γ-tubulin complexes and characterization of γTuRC interacting factors, regulatory mechanisms of microtubule nucleation are not fully understood. Here, we review recent work on the factors and regulatory mechanisms that are involved in centrosomal and non-centrosomal microtubule nucleation.
The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila

PubMed Central

Duncan, Jason E.; Lytle, Nikki K.; Zuniga, Alfredo; Goldstein, Lawrence S. B.

2013-01-01

Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila . The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila , which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila , we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport. PMID:23840848
Measuring the number and spacing of molecular motors propelling a gliding microtubule

NASA Astrophysics Data System (ADS)

Fallesen, Todd L.; Macosko, Jed C.; Holzwarth, G.

2011-01-01

The molecular motor gliding assay, in which a microtubule or other filament moves across a surface coated with motors, has provided much insight into how molecular motors work. The kinesin-microtubule system is also a strong candidate for the job of nanoparticle transporter in nanotechnology devices. In most cases, several motors transport each filament. Each motor serves both to bind the microtubule to a stationary surface and to propel the microtubule along the surface. By applying a uniform transverse force of 4-19 pN to a superparamagnetic bead attached to the trailing end of the microtubule, we have measured the distance d between binding points (motors). The average value of d was determined as a function of motor surface density σ. The measurements agree well with the scaling model of Duke, Holy, and Liebler, which predicts that ~σ-2/5 if 0.05⩽σ⩽20μm-2 [Phys. Rev. Lett.PRLTAO0031-900710.1103/PhysRevLett.74.330 74, 330 (1995)]. The distribution of d fits an extension of the model. The radius of curvature of a microtubule bent at a binding point by the force of the magnetic bead was ≈1 μm, 5000-fold smaller than the radius of curvature of microtubules subjected only to thermal forces. This is evidence that at these points of high bending stress, generated by the force on the magnetic bead, the microtubule is in the more flexible state of a two-state model of microtubule bending proposed by Heussinger, Schüller, and Frey [Phys. Rev. EPLEEE81063-651X10.1103/PhysRevE.81.021904 81, 021904 (2010)].
AMPK attenuates microtubule proliferation in cardiac hypertrophy.

PubMed

Fassett, John T; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie; Bache, Robert J

2013-03-01

Cell hypertrophy requires increased protein synthesis and expansion of the cytoskeletal networks that support cell enlargement. AMPK limits anabolic processes, such as protein synthesis, when energy supply is insufficient, but its role in cytoskeletal remodeling is not known. Here, we examined the influence of AMPK in cytoskeletal remodeling during cardiomyocyte hypertrophy, a clinically relevant condition in which cardiomyocytes enlarge but do not divide. In neonatal cardiomyocytes, activation of AMPK with 5-aminoimidazole carboxamide ribonucleotide (AICAR) or expression of constitutively active AMPK (CA-AMPK) attenuated cell area increase by hypertrophic stimuli (phenylephrine). AMPK activation had little effect on intermediate filaments or myofilaments but dramatically reduced microtubule stability, as measured by detyrosinated tubulin levels and cytoskeletal tubulin accumulation. Importantly, low-level AMPK activation limited cell expansion and microtubule growth independent of mTORC1 or protein synthesis repression, identifying a new mechanism by which AMPK regulates cell growth. Mechanistically, AICAR treatment increased Ser-915 phosphorylation of microtubule-associated protein 4 (MAP4), which reduces affinity for tubulin and prevents stabilization of microtubules (MTs). RNAi knockdown of MAP4 confirmed its critical role in cardiomyocyte MT stabilization. In support of a pathophysiological role for AMPK regulation of cardiac microtubules, AMPK α2 KO mice exposed to pressure overload (transverse aortic constriction; TAC) demonstrated reduced MAP4 phosphorylation and increased microtubule accumulation that correlated with the severity of contractile dysfunction. Together, our data identify the microtubule cytoskeleton as a sensitive target of AMPK activity, and the data suggest a novel role for AMPK in limiting accumulation and densification of microtubules that occurs in response to hypertrophic stress.
Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins.

PubMed

Sun, D; Leung, C L; Liem, R K

2001-01-01

MACF (microtubule actin cross-linking factor) is a large, 608-kDa protein that can associate with both actin microfilaments and microtubules (MTs). Structurally, MACF can be divided into 3 domains: an N-terminal domain that contains both a calponin type actin-binding domain and a plakin domain; a rod domain that is composed of 23 dystrophin-like spectrin repeats; and a C-terminal domain that includes two EF-hand calcium-binding motifs, as well as a region that is homologous to two related proteins, GAR22 and Gas2. We have previously demonstrated that the C-terminal domain of MACF binds to MTs, although no homology was observed between this domain and other known microtubule-binding proteins. In this report, we describe the characterization of this microtubule-binding domain of MACF by transient transfection studies and in vitro binding assays. We found that the C-terminus of MACF contains at least two microtubule-binding regions, a GAR domain and a domain containing glycine-serine-arginine (GSR) repeats. In transfected cells, the GAR domain bound to and partially stabilized MTs to depolymerization by nocodazole. The GSR-containing domain caused MTs to form bundles that are still sensitive to nocodazole-induced depolymerization. When present together, these two domains acted in concert to bundle MTs and render them stable to nocodazole treatment. Recently, a study has shown that the N-terminal half of the plakin domain (called the M1 domain) of MACF also binds MTs. We therefore examined the microtubule binding ability of the M1 domain in the context of the entire plakin domain with and without the remaining N-terminal regions of two different MACF isoforms. Interestingly, in the presence of the surrounding sequences, the M1 domain did not bind MTs. In addition to MACF, cDNA sequences encoding the GAR and GSR-containing domains are also found in the partial human EST clone KIAA0728, which has high sequence homology to the 3' end of the MACF cDNA; hence, we refer to
Understanding force-generating microtubule systems through in vitro reconstitution

PubMed Central

Kok, Maurits; Dogterom, Marileen

2016-01-01

ABSTRACT Microtubules switch between growing and shrinking states, a feature known as dynamic instability. The biochemical parameters underlying dynamic instability are modulated by a wide variety of microtubule-associated proteins that enable the strict control of microtubule dynamics in cells. The forces generated by controlled growth and shrinkage of microtubules drive a large range of processes, including organelle positioning, mitotic spindle assembly, and chromosome segregation. In the past decade, our understanding of microtubule dynamics and microtubule force generation has progressed significantly. Here, we review the microtubule-intrinsic process of dynamic instability, the effect of external factors on this process, and how the resulting forces act on various biological systems. Recently, reconstitution-based approaches have strongly benefited from extensive biochemical and biophysical characterization of individual components that are involved in regulating or transmitting microtubule-driven forces. We will focus on the current state of reconstituting increasingly complex biological systems and provide new directions for future developments. PMID:27715396
Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation

PubMed Central

Winans, Amy M; Collins, Sean R; Meyer, Tobias

2016-01-01

Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon. DOI: http://dx.doi.org/10.7554/eLife.12387.001 PMID:26836307
Dynamics of microtubules: highlights of recent computational and experimental investigations

NASA Astrophysics Data System (ADS)

Barsegov, Valeri; Ross, Jennifer L.; Dima, Ruxandra I.

2017-11-01

Microtubules are found in most eukaryotic cells, with homologs in eubacteria and archea, and they have functional roles in mitosis, cell motility, intracellular transport, and the maintenance of cell shape. Numerous efforts have been expended over the last two decades to characterize the interactions between microtubules and the wide variety of microtubule associated proteins that control their dynamic behavior in cells resulting in microtubules being assembled and disassembled where and when they are required by the cell. We present the main findings regarding microtubule polymerization and depolymerization and review recent work about the molecular motors that modulate microtubule dynamics by inducing either microtubule depolymerization or severing. We also discuss the main experimental and computational approaches used to quantify the thermodynamics and mechanics of microtubule filaments.
AMPK attenuates microtubule proliferation in cardiac hypertrophy

PubMed Central

Fassett, John T.; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie

2013-01-01

Cell hypertrophy requires increased protein synthesis and expansion of the cytoskeletal networks that support cell enlargement. AMPK limits anabolic processes, such as protein synthesis, when energy supply is insufficient, but its role in cytoskeletal remodeling is not known. Here, we examined the influence of AMPK in cytoskeletal remodeling during cardiomyocyte hypertrophy, a clinically relevant condition in which cardiomyocytes enlarge but do not divide. In neonatal cardiomyocytes, activation of AMPK with 5-aminoimidazole carboxamide ribonucleotide (AICAR) or expression of constitutively active AMPK (CA-AMPK) attenuated cell area increase by hypertrophic stimuli (phenylephrine). AMPK activation had little effect on intermediate filaments or myofilaments but dramatically reduced microtubule stability, as measured by detyrosinated tubulin levels and cytoskeletal tubulin accumulation. Importantly, low-level AMPK activation limited cell expansion and microtubule growth independent of mTORC1 or protein synthesis repression, identifying a new mechanism by which AMPK regulates cell growth. Mechanistically, AICAR treatment increased Ser-915 phosphorylation of microtubule-associated protein 4 (MAP4), which reduces affinity for tubulin and prevents stabilization of microtubules (MTs). RNAi knockdown of MAP4 confirmed its critical role in cardiomyocyte MT stabilization. In support of a pathophysiological role for AMPK regulation of cardiac microtubules, AMPK α2 KO mice exposed to pressure overload (transverse aortic constriction; TAC) demonstrated reduced MAP4 phosphorylation and increased microtubule accumulation that correlated with the severity of contractile dysfunction. Together, our data identify the microtubule cytoskeleton as a sensitive target of AMPK activity, and the data suggest a novel role for AMPK in limiting accumulation and densification of microtubules that occurs in response to hypertrophic stress. PMID:23316058
Herpes Simplex Virus VP16, but Not ICP0, Is Required To Reduce Histone Occupancy and Enhance Histone Acetylation on Viral Genomes in U2OS Osteosarcoma Cells▿ †

PubMed Central

Hancock, Meaghan H.; Cliffe, Anna R.; Knipe, David M.; Smiley, James R.

2010-01-01

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure. PMID:19939931
Pollution profiles and health risk assessment of VOCs emitted during e-waste dismantling processes associated with different dismantling methods.

PubMed

An, Taicheng; Huang, Yong; Li, Guiying; He, Zhigui; Chen, Jiangyao; Zhang, Chaosheng

2014-12-01

Pollution profiles of typical volatile organic compounds (VOCs) emitted during dismantling of various printed circuit board assemblies (PCBAs) of e-wastes using different methods were comparatively investigated in the real e-waste dismantling workshops in South China in April 2013. Similar pollution profiles and concentrations of VOCs were observed between dismantling mobile phone and hard disk PCBAs by using electric blowers and between dismantling television and power supplier PCBAs using electric heating furnaces. Aromatic hydrocarbons (accounting for >60% of the sum of VOCs) were the dominant group during using electric blowers, while aromatic (accounting for >44% of the sum of VOCs) and halogenated hydrocarbons (accounting for >48% of the sum of VOCs) were the two dominant groups which contributed equally using electric heating furnaces. However, the distribution profiles of VOCs emitted during dismantling of televisions, hard disks and micro motors using rotary incinerators varied greatly, though aromatic hydrocarbons were still the dominant group. The combustion of e-wastes led to the most severe contamination of VOCs, with total VOCs (3.3×10(4) μg m(-3)) using rotary incinerators about 190, 180, 139, and 40 times higher than those using mechanical cutting, electric soldering iron, electric blower, and electric heating furnace, respectively. Both cancer and non-cancer risks existed for workers due to exposure to on-site emitted VOCs in all workshops especially in those using rotary incinerators according to the USEPA methodology, whereas only cancer risks existed in rotary incinerator workshops according to the American Conference of Industrial Hygienists methodology. Copyright © 2014 Elsevier Ltd. All rights reserved.
Microtubules and epithem-cell morphogenesis in hydathodes of Pilea cadierei.

PubMed

Galatis, B

1988-12-01

When cell divisions have ceased, the epithem of the hydathodes of Pilea cadierei Gagnep. et Guill. consists of small polyhedral cells exhibiting a meristematic appearance, and completely lacks intercellular spaces. The cortical microtubules in epithem cells exhibit a unique organization: they are not scattered along the whole wall surface but form groups lying at some distance from each other. In sections, from two to eight groups of microtubules can be observed, each lining a wall region averaging between 0.5 and 1.5 μm in length. These groups represent sections of microtubule bundles girdling a major part or the whole of the cell periphery. They are connected to one another by anastomoses, forming a microtubular reticulum. The assembly of microtubule bundles is followed by the appearance of distinct local thickenings in the adjacent wall areas. The cellulose microfibrils in the thickenings are deposited in parallel to the underlying microtubules. Gradually, the vacuolating epithem cells undergo swelling, except for the areas bounded by the wall thickenings. Since the latter, and actually their constituent bundles of cellulose microfibrils, cannot extend in length the differential cell growth results in schizogenous formation of intercellular spaces between contiguous cell walls at their thickened regions. The spaces then broaden and merge to become an extensive intercellular space system. As a result of the above processes, the epithem cells become constricted and finally deeply lobed. The observations show that (i) the cortical microtubules are intimately involved in the morphogenesis of the epithem cells and (ii) the initiation and development of the epithem intercellular spaces is a phenomenon directly related to cell morphogenesis and therefore to the cortical microtubule cytoskeleton. The sites of initiation of these spaces are highly predictable.
LA-ICP-MS of magnetite: Methods and reference materials

USGS Publications Warehouse

Nadoll, P.; Koenig, A.E.

2011-01-01

Magnetite (Fe3O4) is a common accessory mineral in many geologic settings. Its variable geochemistry makes it a powerful petrogenetic indicator. Electron microprobe (EMPA) analyses are commonly used to examine major and minor element contents in magnetite. Laser ablation ICP-MS (LA-ICP-MS) is applicable to trace element analyses of magnetite but has not been widely employed to examine compositional variations. We tested the applicability of the NIST SRM 610, the USGS GSE-1G, and the NIST SRM 2782 reference materials (RMs) as external standards and developed a reliable method for LA-ICP-MS analysis of magnetite. LA-ICP-MS analyses were carried out on well characterized magnetite samples with a 193 nm, Excimer, ArF LA system. Although matrix-matched RMs are sometimes important for calibration and normalization of LA-ICP-MS data, we demonstrate that glass RMs can produce accurate results for LA-ICP-MS analyses of magnetite. Cross-comparison between the NIST SRM 610 and USGS GSE-1G indicates good agreement for magnetite minor and trace element data calibrated with either of these RMs. Many elements show a sufficiently good match between the LA-ICP-MS and the EMPA data; for example, Ti and V show a close to linear relationship with correlation coefficients, R2 of 0.79 and 0.85 respectively. ?? 2011 The Royal Society of Chemistry.

GDP-to-GTP exchange on the microtubule end can contribute to the frequency of catastrophe.

PubMed

Piedra, Felipe-Andrés; Kim, Tae; Garza, Emily S; Geyer, Elisabeth A; Burns, Alexander; Ye, Xuecheng; Rice, Luke M

2016-11-07

Microtubules are dynamic polymers of αβ-tubulin that have essential roles in chromosome segregation and organization of the cytoplasm. Catastrophe-the switch from growing to shrinking-occurs when a microtubule loses its stabilizing GTP cap. Recent evidence indicates that the nucleotide on the microtubule end controls how tightly an incoming subunit will be bound (trans-acting GTP), but most current models do not incorporate this information. We implemented trans-acting GTP into a computational model for microtubule dynamics. In simulations, growing microtubules often exposed terminal GDP-bound subunits without undergoing catastrophe. Transient GDP exposure on the growing plus end slowed elongation by reducing the number of favorable binding sites on the microtubule end. Slower elongation led to erosion of the GTP cap and an increase in the frequency of catastrophe. Allowing GDP-to-GTP exchange on terminal subunits in simulations mitigated these effects. Using mutant αβ-tubulin or modified GTP, we showed experimentally that a more readily exchangeable nucleotide led to less frequent catastrophe. Current models for microtubule dynamics do not account for GDP-to-GTP exchange on the growing microtubule end, so our findings provide a new way of thinking about the molecular events that initiate catastrophe. © 2016 Piedra et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
EB-Family Proteins: Functions and Microtubule Interaction Mechanisms.

PubMed

Mustyatsa, V V; Boyakhchyan, A V; Ataullakhanov, F I; Gudimchuk, N B

2017-07-01

Microtubules are polymers of tubulin protein, one of the key components of cytoskeleton. They are polar filaments whose plus-ends usually oriented toward the cell periphery are more dynamic than their minus-ends, which face the center of the cell. In cells, microtubules are organized into a network that is being constantly rebuilt and renovated due to stochastic switching of its individual filaments from growth to shrinkage and back. Because of these dynamics and their mechanical properties, microtubules take part in various essential processes, from intracellular transport to search and capture of chromosomes during mitosis. Microtubule dynamics are regulated by many proteins that are located on the plus-ends of these filaments. One of the most important and abundant groups of plus-end-interacting proteins are EB-family proteins, which autonomously recognize structures of the microtubule growing plus-ends, modulate their dynamics, and recruit multiple partner proteins with diverse functions onto the microtubule plus-ends. In this review, we summarize the published data about the properties and functions of EB-proteins, focusing on analysis of their mechanism of interaction with the microtubule growing ends.
In Vitro Motility of Liver Connexin Vesicles along Microtubules Utilizes Kinesin Motors*

PubMed Central

Fort, Alfredo G.; Murray, John W.; Dandachi, Nadine; Davidson, Michael W.; Dermietzel, Rolf; Wolkoff, Allan W.; Spray, David C.

2011-01-01

Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μm of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μm ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4–0.5 μm/s, which was inhibited with 1 mm of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μm vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32. PMID:21536677
Role of the microtubule cytoskeleton in gravisensing Chara rhizoids.

PubMed

Braun, M; Sievers, A

1994-04-01

The arrangement of the microtubule cytoskeleton in tip-growing and gravisensing Chara rhizoids has been documented by immunofluorescence microscopy. Predominantly axially oriented undulating bundles of cortical microtubules were found in the basal zone of the rhizoids and colocalized with the microfilament bundles underlying the cytoplasmic streaming. Microtubules penetrate the subapical zone, forming a three-dimensional network that envelops the nucleus and organelles. Microtubules are present up to 5 to 10 microns basal from the apical cytoplasmic region containing the statoliths. No microtubules were found in the apical zone of the rhizoid which is the site of tip growth and gravitropism. Depolymerization of microtubules by application of oryzalin does not affect cytoplasmic streaming and gravitropic growth until the relatively stationary and polarly organized apical and subapical cytoplasm is converted into streaming cytoplasm. When the statoliths and the apical cytoplasm are included in the cytoplasmic streaming, tip growth and gravitropism are stopped. Oryzalin-induced disruption of the microtubule cytoskeleton also results in a rearrangement of the dense network of apical and subapical microfilaments into thicker bundles, whereas disruption of the microfilament cytoskeleton by cytochalasin D had no effect on the organization of the microtubule cytoskeleton. It is, therefore, concluded that the arrangement of microtubules is essential for the polar cytoplasmic zonation and the functionally polar organization of the actin cytoskeleton which is responsible for the motile processes in rhizoids. Microtubules are not involved in the primary events of gravitropism in Chara rhizoids.
Microtubule-dependent distribution of mRNA in adult cardiocytes.

PubMed

Scholz, Dimitri; Baicu, Catalin F; Tuxworth, William J; Xu, Lin; Kasiganesan, Harinath; Menick, Donald R; Cooper, George

2008-03-01

Synthesis of myofibrillar proteins in the diffusion-restricted adult cardiocyte requires microtubule-based active transport of mRNAs as part of messenger ribonucleoprotein particles (mRNPs) to translation sites adjacent to nascent myofibrils. This is especially important for compensatory hypertrophy in response to hemodynamic overloading. The hypothesis tested here is that excessive microtubule decoration by microtubule-associated protein 4 (MAP4) after cardiac pressure overloading could disrupt mRNP transport and thus hypertrophic growth. MAP4-overexpressing and pressure-overload hypertrophied adult feline cardiocytes were infected with an adenovirus encoding zipcode-binding protein 1-enhanced yellow fluorescent protein fusion protein, which is incorporated into mRNPs, to allow imaging of these particles. Speed and distance of particle movement were measured via time-lapse microscopy. Microtubule depolymerization was used to study microtubule-based transport and distribution of mRNPs. Protein synthesis was assessed as radioautographic incorporation of [3H]phenylalanine. After microtubule depolymerization, mRNPs persist only perinuclearly and apparent mRNP production and protein synthesis decrease. Reestablishing microtubules restores mRNP production and transport as well as protein synthesis. MAP4 overdecoration of microtubules via adenovirus infection in vitro or following pressure overloading in vivo reduces the speed and average distance of mRNP movement. Thus cardiocyte microtubules are required for mRNP transport and structural protein synthesis, and MAP4 decoration of microtubules, whether directly imposed or accompanying pressure-overload hypertrophy, causes disruption of mRNP transport and protein synthesis. The dense, highly MAP4-decorated microtubule network seen in severe pressure-overload hypertrophy both may cause contractile dysfunction and, perhaps even more importantly, may prevent a fully compensatory growth response to hemodynamic overloading.
Microtubules soften due to cross-sectional flattening

DOE PAGES

Memet, Edvin; Hilitsk, Feodor; Morris, Margaret A.; ...

2018-06-01

We use optical trapping to continuously bend an isolated microtubule while simultaneously measuring the applied force and the resulting filament strain, thus allowing us to determine its elastic properties over a wide range of applied strains. We find that, while in the low-strain regime, microtubules may be quantitatively described in terms of the classical Euler-Bernoulli elastic filament, above a critical strain they deviate from this simple elastic model, showing a softening response with increasing deformations. A three-dimensional thin-shell model, in which the increased mechanical compliance is caused by flattening and eventual buckling of the filament cross-section, captures this softening effectmore » in the high strain regime and yields quantitative values of the effective mechanical properties of microtubules. Our results demonstrate that properties of microtubules are highly dependent on the magnitude of the applied strain and offer a new interpretation for the large variety in microtubule mechanical data measured by different methods.« less
Microtubules soften due to cross-sectional flattening

DOE Office of Scientific and Technical Information (OSTI.GOV)

Memet, Edvin; Hilitsk, Feodor; Morris, Margaret A.

We use optical trapping to continuously bend an isolated microtubule while simultaneously measuring the applied force and the resulting filament strain, thus allowing us to determine its elastic properties over a wide range of applied strains. We find that, while in the low-strain regime, microtubules may be quantitatively described in terms of the classical Euler-Bernoulli elastic filament, above a critical strain they deviate from this simple elastic model, showing a softening response with increasing deformations. A three-dimensional thin-shell model, in which the increased mechanical compliance is caused by flattening and eventual buckling of the filament cross-section, captures this softening effectmore » in the high strain regime and yields quantitative values of the effective mechanical properties of microtubules. Our results demonstrate that properties of microtubules are highly dependent on the magnitude of the applied strain and offer a new interpretation for the large variety in microtubule mechanical data measured by different methods.« less
Push-me-pull-you: how microtubules organize the cell interior

PubMed Central

2008-01-01

Dynamic organization of the cell interior, which is crucial for cell function, largely depends on the microtubule cytoskeleton. Microtubules move and position organelles by pushing, pulling, or sliding. Pushing forces can be generated by microtubule polymerization, whereas pulling typically involves microtubule depolymerization or molecular motors, or both. Sliding between a microtubule and another microtubule, an organelle, or the cell cortex is also powered by molecular motors. Although numerous examples of microtubule-based pushing and pulling in living cells have been observed, it is not clear why different cell types and processes employ different mechanisms. This review introduces a classification of microtubule-based positioning strategies and discusses the efficacy of pushing and pulling. The positioning mechanisms based on microtubule pushing are efficient for movements over small distances, and for centering of organelles in symmetric geometries. Mechanisms based on pulling, on the other hand, are typically more elaborate, but are necessary when the distances to be covered by the organelles are large, and when the geometry is asymmetric and complex. Thus, taking into account cell geometry and the length scale of the movements helps to identify general principles of the intracellular layout based on microtubule forces. PMID:18404264
Tensile stress stimulates microtubule outgrowth in living cells

NASA Technical Reports Server (NTRS)

Kaverina, Irina; Krylyshkina, Olga; Beningo, Karen; Anderson, Kurt; Wang, Yu-Li; Small, J. Victor

2002-01-01

Cell motility is driven by the sum of asymmetric traction forces exerted on the substrate through adhesion foci that interface with the actin cytoskeleton. Establishment of this asymmetry involves microtubules, which exert a destabilising effect on adhesion foci via targeting events. Here, we demonstrate the existence of a mechano-sensing mechanism that signals microtubule polymerisation and guidance of the microtubules towards adhesion sites under increased stress. Stress was applied either by manipulating the body of cells moving on glass with a microneedle or by stretching a flexible substrate that cells were migrating on. We propose a model for this mechano-sensing phenomenon whereby microtubule polymerisation is stimulated and guided through the interaction of a microtubule tip complex with actin filaments under tension.
The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus Ends.

PubMed

Fan, Yuanwei; Burkart, Graham M; Dixit, Ram

2018-03-19

The contribution of microtubule tip dynamics to the assembly and function of plant microtubule arrays remains poorly understood. Here, we report that the Arabidopsis SPIRAL2 (SPR2) protein modulates the dynamics of the acentrosomal cortical microtubule plus and minus ends in an opposing manner. Live imaging of a functional SPR2-mRuby fusion protein revealed that SPR2 shows both microtubule plus- and minus-end tracking activity in addition to localization at microtubule intersections and along the lattice. Analysis of microtubule dynamics showed that cortical microtubule plus ends rarely undergo catastrophe in the spr2-2 knockout mutant compared to wild-type. In contrast, cortical microtubule minus ends in spr2-2 depolymerized at a much faster rate than in wild-type. Destabilization of the minus ends in spr2-2 caused a significant decrease in the lifetime of microtubule crossovers, which dramatically reduced the microtubule-severing frequency and inhibited light-induced microtubule array reorientation. Using in vitro reconstitution experiments combined with single-molecule imaging, we found that recombinant SPR2-GFP intrinsically localizes to microtubule minus ends, where it binds stably and inhibits their dynamics. Together, our data establish SPR2 as a new type of microtubule tip regulator that governs the length and lifetime of microtubules. Copyright © 2018 Elsevier Ltd. All rights reserved.
Ion Permeability of a Microtubule in Neuron Environment.

PubMed

Shen, Chun; Guo, Wanlin

2018-04-19

Microtubules, constituted by end-to-end negatively charged α- and β-tubulin dimers, are long, hollow, pseudohelical cylinders with internal and external diameters of about 16 and 26 nm, respectively, and widely exist in cell cytoplasm, neuron axons, and dendrites. Although their structural functions in physiological processes, such as cell mitosis, cell motility, and motor protein transport, have been widely accepted, their role in neuron activity remains attractively elusive. Here we show a new function of microtubules: they can generate instant response to a calcium pulse because of their specific permeability for ions. Our comprehensive simulations from all-atom molecular dynamics to potential of mean force and continuum modeling reveal that K + and Na + ions can permeate through the nanopores in the microtubule wall easily, while Ca 2+ ions are blocked by the wall with a much higher free energy barrier. These cations are adsorbed to the surfaces of the wall with affinity decreasing in the sequence Ca 2+ , Na + , and K + . As a result, when the concentration of Ca 2+ ions increases outside the microtubule during neuronal excitation, K + and Na + ions will be driven into the microtubule, triggering subsequent axial ion redistribution within the microtubule. The results shed light on the possibility of the ion-permeable microtubules being involved in neural signal processing.
Control of neuronal polarity and plasticity--a renaissance for microtubules?

PubMed

Hoogenraad, Casper C; Bradke, Frank

2009-12-01

Microtubules have been regarded as essential structures for stable neuronal morphology but new studies are highlighting their role in dynamic neuronal processes. Recent work demonstrates that the microtubule cytoskeleton has an active role during different phases of neuronal polarization - microtubules and their stability determine axon formation, they maintain the identity of axons and they regulate the dynamics of dendritic spines, the major sites of excitatory synaptic input. Although microtubules fulfill distinct cellular functions at different developmental stages, the underlying molecular mechanisms are remarkably similar. Reccurring themes are that microtubules direct specific membrane traffic and affect actin dynamics to locally organize axon growth and spine dynamics. We review the novel role of microtubules during neuronal development and discuss models for microtubule-dependent signaling in neuronal plasticity.
Protein Kinase C Activation Promotes Microtubule Advance in Neuronal Growth Cones by Increasing Average Microtubule Growth Lifetimes

PubMed Central

Kabir, Nurul; Schaefer, Andrew W.; Nakhost, Arash; Sossin, Wayne S.; Forscher, Paul

2001-01-01

We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow—a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance. PMID:11238458
Microtubule organization during human parthenogenesis.

PubMed

Terada, Yukihiro; Hasegawa, Hisataka; Ugajin, Tomohisa; Murakami, Takashi; Yaegashi, Nobuo; Okamura, Kunihiro

2009-04-01

In human fertilization, the sperm centrosome plays a crucial role as a microtubule organizing center (MTOC). We studied microtubule organization during human parthenogenesis, which occurs when a human egg undergoes cleavage without a sperm centrosome. Multiple cytoplasmic asters were organized in the human oocyte after parthenogenetic activation, indicating that multiple MTOC are present in the human oocyte cytoplasm and function like a human sperm centrosome during parthenogenesis.
Centrosome and microtubule instability in aging Drosophila cells

NASA Technical Reports Server (NTRS)

Schatten, H.; Chakrabarti, A.; Hedrick, J.

1999-01-01

Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.
Molecular mechanisms of microtubule-dependent kinetochore transport toward spindle poles

PubMed Central

Tanaka, Kozo; Kitamura, Etsushi; Kitamura, Yoko; Tanaka, Tomoyuki U.

2007-01-01

In mitosis, kinetochores are initially captured by the lateral sides of single microtubules and are subsequently transported toward spindle poles. Mechanisms for kinetochore transport are not yet known. We present two mechanisms involved in microtubule-dependent poleward kinetochore transport in Saccharomyces cerevisiae. First, kinetochores slide along the microtubule lateral surface, which is mainly and probably exclusively driven by Kar3, a kinesin-14 family member that localizes at kinetochores. Second, kinetochores are tethered at the microtubule distal ends and pulled poleward as microtubules shrink (end-on pulling). Kinetochore sliding is often converted to end-on pulling, enabling more processive transport, but the opposite conversion is rare. The establishment of end-on pulling is partly hindered by Kar3, and its progression requires the Dam1 complex. We suggest that the Dam1 complexes, which probably encircle a single microtubule, can convert microtubule depolymerization into the poleward kinetochore-pulling force. Thus, microtubule-dependent poleward kinetochore transport is ensured by at least two distinct mechanisms. PMID:17620411
TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types

PubMed Central

Nwagbara, Belinda U.; Faris, Anna E.; Bearce, Elizabeth A.; Erdogan, Burcu; Ebbert, Patrick T.; Evans, Matthew F.; Rutherford, Erin L.; Enzenbacher, Tiffany B.; Lowery, Laura Anne

2014-01-01

Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end–tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics. PMID:25187649
Physical basis of large microtubule aster growth

PubMed Central

Ishihara, Keisuke; Korolev, Kirill S; Mitchison, Timothy J

2016-01-01

Microtubule asters - radial arrays of microtubules organized by centrosomes - play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks. DOI: http://dx.doi.org/10.7554/eLife.19145.001 PMID:27892852
The parallel lives of microtubules and cellulose microfibrils.

PubMed

Lloyd, Clive; Chan, Jordi

2008-12-01

A major breakthrough was the recent discovery that cellulose synthases really do move along the plasma membrane upon tracks provided by the underlying cortical microtubules. It emphasized the cytoplasmic contribution to cell wall organization. A growing number of microtubule-associated proteins has been identified and shown to affect the way that microtubules are ordered, with downstream effects on the pattern of growth. The dynamic properties of microtubules turn out to be key in understanding the behaviour of the global array and good progress has been made in deciphering the rules by which the array is self-organized.
Microtubule defects influence kinesin-based transport in vitro.

NASA Astrophysics Data System (ADS)

Xu, Jing

Microtubules are protein polymers that form ``molecular highways'' for long-range transport within living cells. Molecular motors actively step along microtubules to shuttle cellular materials between the nucleus and the cell periphery; this transport is critical for the survival and health of all eukaryotic cells. Structural defects in microtubules exist, but whether these defects impact molecular motor-based transport remains unknown. Here, we report a new, to our knowledge, approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo. Supported by the UC Merced (to J.X.), NIH (NS048501 to S.J.K.), NSF (EF-1038697 to A.G.), and the James S. McDonnell Foundation (to A.G.). Work carried out at the Aspen Center for Physics was supported by NSF Grant PHY-1066293.

Subnanometre-resolution structure of the doublet microtubule reveals new classes of microtubule-associated proteins

PubMed Central

Ichikawa, Muneyoshi; Liu, Dinan; Kastritis, Panagiotis L.; Basu, Kaustuv; Hsu, Tzu Chin; Yang, Shunkai; Bui, Khanh Huy

2017-01-01

Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the axoneme that consists of nine doublet microtubules radially interlinked and longitudinally organized in multiple specific repeat units. Little is known, however, about how the axoneme allows cilia to be both actively bendable and sturdy or how it is assembled. To answer these questions, we used cryo-electron microscopy to structurally analyse several of the repeating units of the doublet at sub-nanometre resolution. This structural detail enables us to unambiguously assign α- and β-tubulins in the doublet microtubule lattice. Our study demonstrates the existence of an inner sheath composed of different kinds of microtubule inner proteins inside the doublet that likely stabilizes the structure and facilitates the specific building of the B-tubule. PMID:28462916
Kinesin-microtubule interactions during gliding assays under magnetic force

NASA Astrophysics Data System (ADS)

Fallesen, Todd L.

Conventional kinesin is a motor protein capable of converting the chemical energy of ATP into mechanical work. In the cell, this is used to actively transport vesicles through the intracellular matrix. The relationship between the velocity of a single kinesin, as it works against an increasing opposing load, has been well studied. The relationship between the velocity of a cargo being moved by multiple kinesin motors against an opposing load has not been established. A major difficulty in determining the force-velocity relationship for multiple motors is determining the number of motors that are moving a cargo against an opposing load. Here I report on a novel method for detaching microtubules bound to a superparamagnetic bead from kinesin anchor points in an upside down gliding assay using a uniform magnetic field perpendicular to the direction of microtubule travel. The anchor points are presumably kinesin motors bound to the surface which microtubules are gliding over. Determining the distance between anchor points, d, allows the calculation of the average number of kinesins, n, that are moving a microtubule. It is possible to calculate the fraction of motors able to move microtubules as well, which is determined to be ˜ 5%. Using a uniform magnetic field parallel to the direction of microtubule travel, it is possible to impart a uniform magnetic field on a microtubule bound to a superparamagnetic bead. We are able to decrease the average velocity of microtubules driven by multiple kinesin motors moving against an opposing force. Using the average number of kinesins on a microtubule, we estimate that there are an average 2-7 kinesins acting against the opposing force. By fitting Gaussians to the smoothed distributions of microtubule velocities acting against an opposing force, multiple velocities are seen, presumably for n, n-1, n-2, etc motors acting together. When these velocities are scaled for the average number of motors on a microtubule, the force
EML proteins in microtubule regulation and human disease.

PubMed

Fry, Andrew M; O'Regan, Laura; Montgomery, Jessica; Adib, Rozita; Bayliss, Richard

2016-10-15

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.
Cargos Rotate at Microtubule Intersections during Intracellular Trafficking.

PubMed

Gao, Yuan; Anthony, Stephen M; Yu, Yanqi; Yi, Yi; Yu, Yan

2018-06-19

Intracellular cargos are transported by molecular motors along actin and microtubules, but how their dynamics depends on the complex structure of the cytoskeletal network remains unclear. In this study, we investigated this longstanding question by measuring simultaneously the rotational and translational dynamics of cargos at microtubule intersections in living cells. We engineered two-faced particles that are fluorescent on one hemisphere and opaque on the other and used their optical anisotropy to report the rotation of cargos. We show that cargos undergo brief episodes of unidirectional and rapid rotation while pausing at microtubule intersections. Probability and amplitude of the cargo rotation depend on the geometry of the intersecting filaments. The cargo rotation is not random motion due to detachment from microtubules, as revealed by statistical analyses of the translational and rotational dynamics. Instead, it is an active rotation driven by motor proteins. Although cargos are known to pause at microtubule intersections, this study reveals a different dimension of dynamics at this seemingly static state and, more importantly, provides direct evidence showing the correlation between cargo rotation and the geometry of underlying microtubule intersections. Copyright © 2018 Biophysical Society. All rights reserved.
A coarse-grained model of microtubule self-assembly

NASA Astrophysics Data System (ADS)

Regmi, Chola; Cheng, Shengfeng

Microtubules play critical roles in cell structures and functions. They also serve as a model system to stimulate the next-generation smart, dynamic materials. A deep understanding of their self-assembly process and biomechanical properties will not only help elucidate how microtubules perform biological functions, but also lead to exciting insight on how microtubule dynamics can be altered or even controlled for specific purposes such as suppressing the division of cancer cells. Combining all-atom molecular dynamics (MD) simulations and the essential dynamics coarse-graining method, we construct a coarse-grained (CG) model of the tubulin protein, which is the building block of microtubules. In the CG model a tubulin dimer is represented as an elastic network of CG sites, the locations of which are determined by examining the protein dynamics of the tubulin and identifying the essential dynamic domains. Atomistic MD modeling is employed to directly compute the tubulin bond energies in the surface lattice of a microtubule, which are used to parameterize the interactions between CG building blocks. The CG model is then used to study the self-assembly pathways, kinetics, dynamics, and nanomechanics of microtubules.
Microtubule distribution in gravitropic protonemata of the moss Ceratodon

NASA Technical Reports Server (NTRS)

Schwuchow, J.; Sack, F. D.; Hartmann, E.

1990-01-01

Tip cells of dark-grown protonemata of the moss Ceratodon purpureus are negatively gravitropic (grow upward). They possess a unique longitudinal zonation: (1) a tip group of amylochloroplasts in the apical dome, (2) a plastid-free zone, (3) a zone of significant plastid sedimentation, and (4) a zone of mostly non-sedimenting plastids. Immunofluorescence of vertical cells showed microtubules distributed throughout the cytoplasm in a mostly axial orientation extending through all zones. Optical sectioning revealed a close spatial association between microtubules and plastids. A majority (two thirds) of protonemata gravistimulated for > 20 min had a higher density of microtubules near the lower flank compared to the upper flank in the plastid-free zone. This apparent enrichment of microtubules occurred just proximal to sedimented plastids and near the part of the tip that presumably elongates more to produce curvature. Fewer than 5% of gravistimulated protonemata had an enrichment in microtubules near the upper flank, whereas 14% of vertical protonemata were enriched near one of the side walls. Oryzalin and amiprophos-methyl (APM) disrupted microtubules, gravitropism, and normal tip growth and zonation, but did not prevent plastid sedimentation. We hypothesize that a microtubule redistribution plays a role in gravitropism in this protonema. This appears to be the first report of an effect of gravity on microtubule distribution in plants.
Sliding of microtubules by a team of dynein motors: Understanding the effect of spatial distribution of motor tails and mutual exclusion of motor heads on microtubules

NASA Astrophysics Data System (ADS)

Singh, Hanumant Pratap; Takshak, Anjneya; Mall, Utkarsh; Kunwar, Ambarish

2016-06-01

Molecular motors are natural nanomachines that use the free energy released from ATP hydrolysis to generate mechanical forces. Cytoplasmic dynein motors often work collectively as a team to drive important processes such as axonal growth, proplatelet formation and mitosis, as forces generated by single motors are insufficient. A large team of dynein motors is used to slide cytoskeletal microtubules with respect to one another during the process of proplatelet formation and axonal growth. These motors attach to a cargo microtubule via their tail domains, undergo the process of detachment and reattachment of their head domains on another track microtubule, while sliding the cargo microtubule along the track. Traditional continuum/mean-field approaches used in the past are not ideal for studying the sliding mechanism of microtubules, as they ignore spatial and temporal fluctuations due to different possible distributions of motor tails on cargo filament, as well as binding/unbinding of motors from their track. Therefore, these models cannot be used to address important questions such as how the distribution of motor tails on microtubules, or how the mutual exclusion of motor heads on microtubule tracks affects the sliding velocity of cargo microtubule. To answer these, here we use a computational stochastic model where we model each dynein motor explicitly. In our model, we use both random as well as uniform distributions of dynein motors on cargo microtubule, as well as mutual exclusion of motors on microtubule tracks. We find that sliding velocities are least affected by the distribution of motor tails on microtubules, whereas they are greatly affected by mutual exclusion of motor heads on microtubule tracks. We also find that sliding velocity depends on the length of cargo microtubule if mutual exclusion among motor heads is considered.
Drugs That Target Dynamic Microtubules: A New Molecular Perspective

PubMed Central

Stanton, Richard A.; Gernert, Kim M.; Nettles, James H.; Aneja, Ritu

2011-01-01

Microtubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. As such, there is a wide variety of compounds currently in clinical use and in development that act as antimitotic agents by altering microtubule dynamics. Although these diverse molecules are known to affect microtubule dynamics upon binding to one of the three established drug domains (taxane, vinca alkaloid, or colchicine site), the exact mechanism by which each drug works is still an area of intense speculation and research. In this study, we review the effects of microtubule-binding chemotherapeutic agents from a new perspective, considering how their mode of binding induces conformational changes and alters biological function relative to the molecular vectors of microtubule assembly or disassembly. These “biological vectors” can thus be used as a spatiotemporal context to describe molecular mechanisms by which microtubule-targeting drugs work. PMID:21381049
Dimer model for Tau proteins bound in microtubule bundles

NASA Astrophysics Data System (ADS)

Hall, Natalie; Kluber, Alexander; Hayre, N. Robert; Singh, Rajiv; Cox, Daniel

2013-03-01

The microtubule associated protein tau is important in nucleating and maintaining microtubule spacing and structure in neuronal axons. Modification of tau is implicated as a later stage process in Alzheimer's disease, but little is known about the structure of tau in microtubule bundles. We present preliminary work on a proposed model for tau dimers in microtubule bundles (dimers are the minimal units since there is one microtubule binding domain per tau). First, a model of tau monomer was created and its characteristics explored using implicit solvent molecular dynamics simulation. Multiple simulations yield a partially collapsed form with separate positively/negatively charged clumps, but which are a factor of two smaller than required by observed microtubule spacing. We argue that this will elongate in dimer form to lower electrostatic energy at a cost of entropic ``spring'' energy. We will present preliminary results on steered molecular dynamics runs on tau dimers to estimate the actual force constant. Supported by US NSF Grant DMR 1207624.
A role for microtubules in sorting endocytic vesicles in rat hepatocytes.

PubMed Central

Goltz, J S; Wolkoff, A W; Novikoff, P M; Stockert, R J; Satir, P

1992-01-01

The vectorial nature of hepatocyte receptor-mediated endocytosis (RME) and its susceptibility to cytoskeletal disruptors has suggested that a polarized network of microtubules plays a vital role in directed movement during sorting. Using as markers a well-known ligand, asialoorosomucoid, and its receptor, we have isolated endocytic vesicles that bind directly to and interact with stabilized endogenous hepatocyte microtubules at specific times during a synchronous, experimentally initiated, single wave of RME. Both ligand- and receptor-containing vesicles copelleted with microtubules in the absence of ATP but did not pellet under similar conditions when microtubules were not polymerized. When 5 mM ATP was added to preparations of microtubule-bound vesicles, ligand-containing vesicles were released into the supernatant, while receptor-containing vesicles remained immobilized on the microtubules. Release of ligand-containing vesicles from microtubules was prevented by monensin treatment during the endocytic wave. Several proteins, including the microtubule motor protein cytoplasmic dynein, were present in these preparations and were released from microtubule pellets by ATP addition concomitantly with ligand. These results suggest that receptor domains within the endosome can be immobilized by attachment to microtubules so that, following monensin-sensitive dissociation of ligand from receptor, ligand-containing vesicles can be pulled along microtubules away from the receptor domains by a motor molecule, such as cytoplasmic dynein, thereby delineating sorting. Images PMID:1353884
Herpes Simplex Virus Selectively Induces Expression of the CC Chemokine RANTES/CCL5 in Macrophages through a Mechanism Dependent on PKR and ICP0

PubMed Central

Melchjorsen, Jesper; Pedersen, Finn S.; Mogensen, Søren C.; Paludan, Søren R.

2002-01-01

Recruitment of leukocytes is essential for eventual control of virus infections. Macrophages represent a leukocyte population involved in the first line of defense against many infections, including herpes simplex virus (HSV) infection. Through presentation of antigens to T cells and production of cytokines and chemokines, macrophages also constitute an important link between the innate and adaptive immune systems. Here, we have investigated the chemokine expression profile of macrophages after HSV infection and the virus-cell interactions involved. By reverse transcription-PCR and cDNA arrays, we found that HSV type 1 (HSV-1) and HSV-2 induced expression of the CC chemokine RANTES/CCL5 in murine macrophage cell lines and peritoneal cells. The CXC chemokine BCA-1/CXCL13 was also induced in peritoneal cells. Twenty-six other chemokines tested were not affected. Accumulation of RANTES mRNA was detectable after 5 h of infection, was sensitive to UV irradiation of the virus, and was preceded by accumulation of viral immediate-early mRNA and proteins. The viral components responsible for initiation of RANTES expression were examined with virus mutants and RAW 264.7 macrophage-like cells expressing a dominant negative mutant of the double-stranded-RNA-activated protein kinase (PKR). The PKR mutant cell line displayed reduced constitutive and HSV-inducible RANTES expression compared to the control cell line. HSV-1 mutants deficient in genes encoding the immediate-early proteins ICP4, ICP22, and ICP27 remained fully capable of inducing RANTES expression in macrophages. By contrast, the ability of an ICP0-deficient HSV-1 mutant to induce RANTES expression was compromised. Thus, HSV selectively induces expression of RANTES in macrophages through a mechanism dependent on cellular PKR and viral ICP0. PMID:11861845
Proteins from disassembled microtubules characterized by oligospecific antisera.

PubMed

Meier, E; Jorgensen, O S

1977-10-26

The immunochemical properties of in vitro reassembled microtubules were investigated by immunoelectrophoretic techniques. The tubulin dimer gave no measurable immunochemical response, but the tubulin oligomer, the tau-factor and an antigen of about 135 000 daltons all gave precipitating antibodies. Those four proteins were investigated in reassembled microtubules, in DEAE-cellulose purified tubulin, and after molecular sieve chromatography of disassembled and NaCl-dissociated microtubules. Reconstitution of tubulin oligomer from tubulin dimer and tau-factor was also performed. The presence of a unique antigenic structure on tubulin oligomer which was not found in the dissociated components and the role of this aggregate as a nucleation center or intermediate in the assembly of microtubules is discussed.
Mechanism and Dynamics of Breakage of Fluorescent Microtubules

PubMed Central

Guo, Honglian; Xu, Chunhua; Liu, Chunxiang; Qu, E.; Yuan, Ming; Li, Zhaolin; Cheng, Bingying; Zhang, Daozhong

2006-01-01

The breakage of fluorescence-labeled microtubules under irradiation of excitation light is found in our experiments. Its mechanism is studied. The results indicate that free radicals are the main reason for the photosensitive breakage. Furthermore, the mechanical properties of the microtubules are probed with a dual-optical tweezers system. It is found that the fluorescence-labeled microtubules are much easier to extend compared with those without fluorescence. Such microtubules can be extended by 30%, and the force for breaking them up is only several piconewtons. In addition, we find that the breakup of the protofilaments is not simultaneous but step-by-step, which further confirms that the interaction between protofilaments is fairly weak. PMID:16387782
MTCL1 crosslinks and stabilizes non-centrosomal microtubules on the Golgi membrane.

PubMed

Sato, Yoshinori; Hayashi, Kenji; Amano, Yoshiko; Takahashi, Mikiko; Yonemura, Shigenobu; Hayashi, Ikuko; Hirose, Hiroko; Ohno, Shigeo; Suzuki, Atsushi

2014-11-04

Recent studies have revealed the presence of a microtubule subpopulation called Golgi-derived microtubules that support Golgi ribbon formation, which is required for maintaining polarized cell migration. CLASPs and AKAP450/CG-NAP are involved in their formation, but the underlying molecular mechanisms remain unclear. Here, we find that the microtubule-crosslinking protein, MTCL1, is recruited to the Golgi membranes through interactions with CLASPs and AKAP450/CG-NAP, and promotes microtubule growth from the Golgi membrane. Correspondingly, MTCL1 knockdown specifically impairs the formation of the stable perinuclear microtubule network to which the Golgi ribbon tethers and extends. Rescue experiments demonstrate that besides its crosslinking activity mediated by the N-terminal microtubule-binding region, the C-terminal microtubule-binding region plays essential roles in these MTCL1 functions through a novel microtubule-stabilizing activity. These results suggest that MTCL1 cooperates with CLASPs and AKAP450/CG-NAP in the formation of the Golgi-derived microtubules, and mediates their development into a stable microtubule network.
Extracting microtubule networks from superresolution single-molecule localization microscopy data

PubMed Central

Zhang, Zhen; Nishimura, Yukako; Kanchanawong, Pakorn

2017-01-01

Microtubule filaments form ubiquitous networks that specify spatial organization in cells. However, quantitative analysis of microtubule networks is hampered by their complex architecture, limiting insights into the interplay between their organization and cellular functions. Although superresolution microscopy has greatly facilitated high-resolution imaging of microtubule filaments, extraction of complete filament networks from such data sets is challenging. Here we describe a computational tool for automated retrieval of microtubule filaments from single-molecule-localization–based superresolution microscopy images. We present a user-friendly, graphically interfaced implementation and a quantitative analysis of microtubule network architecture phenotypes in fibroblasts. PMID:27852898
Depletion force induced collective motion of microtubules driven by kinesin

NASA Astrophysics Data System (ADS)

Inoue, Daisuke; Mahmot, Bulbul; Kabir, Arif Md. Rashedul; Farhana, Tamanna Ishrat; Tokuraku, Kiyotaka; Sada, Kazuki; Konagaya, Akihiko; Kakugo, Akira

2015-10-01

Collective motion is a fascinating example of coordinated behavior of self-propelled objects, which is often associated with the formation of large scale patterns. Nowadays, the in vitro gliding assay is being considered a model system to experimentally investigate various aspects of group behavior and pattern formation by self-propelled objects. In the in vitro gliding assay, cytoskeletal filaments F-actin or microtubules are driven by the surface immobilized associated biomolecular motors myosin or dynein respectively. Although the F-actin/myosin or microtubule/dynein system was found to be promising in understanding the collective motion and pattern formation by self-propelled objects, the most widely used biomolecular motor system microtubule/kinesin could not be successfully employed so far in this regard. Failure in exhibiting collective motion by kinesin driven microtubules is attributed to the intrinsic properties of kinesin, which was speculated to affect the behavior of individual gliding microtubules and mutual interactions among them. In this work, for the first time, we have demonstrated the collective motion of kinesin driven microtubules by regulating the mutual interaction among the gliding microtubules, by employing a depletion force among them. Proper regulation of the mutual interaction among the gliding microtubules through the employment of the depletion force was found to allow the exhibition of collective motion and stream pattern formation by the microtubules. This work offers a universal means for demonstrating the collective motion using the in vitro gliding assay of biomolecular motor systems and will help obtain a meticulous understanding of the fascinating coordinated behavior and pattern formation by self-propelled objects.Collective motion is a fascinating example of coordinated behavior of self-propelled objects, which is often associated with the formation of large scale patterns. Nowadays, the in vitro gliding assay is being
Feedback Microtubule Control and Microtubule-Actin Cross-talk in Arabidopsis Revealed by Integrative Proteomic and Cell Biology Analysis of KATANIN 1 Mutants.

PubMed

Takáč, Tomáš; Šamajová, Olga; Pechan, Tibor; Luptovčiak, Ivan; Šamaj, Jozef

2017-09-01

Microtubule organization and dynamics are critical for key developmental processes such as cell division, elongation, and morphogenesis. Microtubule severing is an essential regulator of microtubules and is exclusively executed by KATANIN 1 in Arabidopsis In this study, we comparatively studied the proteome-wide effects in two KATANIN 1 mutants. Thus, shotgun proteomic analysis of roots and aerial parts of single nucleotide mutant fra2 and T-DNA insertion mutant ktn1-2 was carried out. We have detected 42 proteins differentially abundant in both fra2 and ktn1-2 KATANIN 1 dysfunction altered the abundance of proteins involved in development, metabolism, and stress responses. The differential regulation of tubulins and microtubule-destabilizing protein MDP25 implied a feedback microtubule control in KATANIN 1 mutants. Furthermore, deregulation of profilin 1, actin-depolymerizing factor 3, and actin 7 was observed. These findings were confirmed by immunoblotting analysis of actin and by microscopic observation of actin filaments using fluorescently labeled phalloidin. Results obtained by quantitative RT-PCR analysis revealed that changed protein abundances were not a consequence of altered expression levels of corresponding genes in the mutants. In conclusion, we show that abundances of several cytoskeletal proteins as well as organization of microtubules and the actin cytoskeleton are amended in accordance with defective microtubule severing. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Nonlinear dynamics of C-terminal tails in cellular microtubules

NASA Astrophysics Data System (ADS)

Sekulic, Dalibor L.; Sataric, Bogdan M.; Zdravkovic, Slobodan; Bugay, Aleksandr N.; Sataric, Miljko V.

2016-07-01

The mechanical and electrical properties, and information processing capabilities of microtubules are the permanent subject of interest for carrying out experiments in vitro and in silico, as well as for theoretical attempts to elucidate the underlying processes. In this paper, we developed a new model of the mechano-electrical waves elicited in the rows of very flexible C-terminal tails which decorate the outer surface of each microtubule. The fact that C-terminal tails play very diverse roles in many cellular functions, such as recruitment of motor proteins and microtubule-associated proteins, motivated us to consider their collective dynamics as the source of localized waves aimed for communication between microtubule and associated proteins. Our approach is based on the ferroelectric liquid crystal model and it leads to the effective asymmetric double-well potential which brings about the conditions for the appearance of kink-waves conducted by intrinsic electric fields embedded in microtubules. These kinks can serve as the signals for control and regulation of intracellular traffic along microtubules performed by processive motions of motor proteins, primarly from kinesin and dynein families. On the other hand, they can be precursors for initiation of dynamical instability of microtubules by recruiting the proper proteins responsible for the depolymerization process.
Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2

PubMed Central

Petry, Sabine; Groen, Aaron C.; Ishihara, Keisuke; Mitchison, Timothy J.; Vale, Ronald D.

2013-01-01

Summary The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence also has suggested that microtubules might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires gamma-tubulin and augmin and is stimulated by GTP-bound Ran and its effector TPX2, factors previously implicated in chromatin-stimulated nucleation. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance. PMID:23415226
Biomimetic Phases of Microtubule-Motor Mixtures

NASA Astrophysics Data System (ADS)

Ross, Jennifer

2014-03-01

We try to determine the universal principles of organization from the molecular scale that gives rise to architecture on the cellular scale. We are specifically interested in the organization of the microtubule cytoskeleton, a rigid, yet versatile network in most cell types. Microtubules in the cell are organized by motor proteins and crosslinkers. This work applies the ideas of statistical mechanics and condensed matter physics to the non-equilibrium pattern formation behind intracellular organization using the microtubule cytoskeleton as the building blocks. We examine these processes in a bottom-up manner by adding increasingly complex protein actors into the system. Our systematic experiments expose nature's laws for organization and has large impacts on biology as well as illuminating new frontiers of non-equilibrium physics.

An ELMO2-RhoG-ILK network modulates microtubule dynamics

PubMed Central

Jackson, Bradley C.; Ivanova, Iordanka A.; Dagnino, Lina

2015-01-01

ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin–dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca2+-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes. PMID:25995380
Acetylated microtubules are required for fusion of autophagosomes with lysosomes.

PubMed

Xie, Rui; Nguyen, Susan; McKeehan, Wallace L; Liu, Leyuan

2010-11-22

Autophagy is a dynamic process during which isolation membranes package substrates to form autophagosomes that are fused with lysosomes to form autolysosomes for degradation. Although it is agreed that the LC3II-associated mature autophagosomes move along microtubular tracks, it is still in dispute if the conversion of LC3I to LC3II before autophagosomes are fully mature and subsequent fusion of mature autophagosomes with lysosomes require microtubules. We use biochemical markers of autophagy and a collection of microtubule interfering reagents to test the question. Results show that interruption of microtubules with either microtubule stabilizing paclitaxel or destabilizing nocodazole similarly impairs the conversion of LC3I to LC3II, but does not block the degradation of LC3II-associated autophagosomes. Acetylation of microtubules renders them resistant to nocodazole treatment. Treatment with vinblastine that causes depolymerization of both non-acetylated and acetylated microtubules results in impairment of both LC3I-LC3II conversion and LC3II-associated autophagosome fusion with lysosomes. Acetylated microtubules are required for fusion of autophagosomes with lysosomes to form autolysosomes.
HOW MICROTUBULE PATTERNS ARE GENERATED

PubMed Central

Tilney, Lewis G.

1971-01-01

The axonemes of Raphidiophrys converge near the center of the cell in an electron-opaque material, the centroplast. In order to establish whether this material acts not only to nucleate the microtubules which form the axonemes but also to give the axoneme its characteristic pattern, the microtubules were disassembled with low temperature and stages in their reformation were studied. It was shown that even though the microtubules appear to be nucleated from the centroplast, pattern formation first appeared at a distance from the centroplast. Thus, the axonemal pattern could not be attributed to any prepattern in the centroplast. Rather, the pattern appears to arise by specific interactions between tubules brought about by bridges. It was concluded that each tubule could bind to a maximum of four other tubules and that once one bridge attached to a tubule it specified the binding positions of the others, thus giving the characteristic axonemal pattern of Raphidiophrys. PMID:5128354
Kinesin expands and stabilizes the GDP-microtubule lattice

NASA Astrophysics Data System (ADS)

Peet, Daniel R.; Burroughs, Nigel J.; Cross, Robert A.

2018-05-01

Kinesin-1 is a nanoscale molecular motor that walks towards the fast-growing (plus) ends of microtubules, hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organization of eukaryotic cells and shows great promise as a tool for nano-engineering1. Recent work hints that kinesin may also play a role in modulating the stability of its microtubule track, both in vitro2,3 and in vivo4, but the results are conflicting5-7 and the mechanisms are unclear. Here, we report a new dimension to the kinesin-microtubule interaction, whereby strong-binding state (adenosine triphosphate (ATP)-bound and apo) kinesin-1 motor domains inhibit the shrinkage of guanosine diphosphate (GDP) microtubules by up to two orders of magnitude and expand their lattice spacing by 1.6%. Our data reveal an unexpected mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, and so allow motile kinesins to influence the structure, stability and mechanics of their microtubule track.
Molecular crowding at microtubule plus-ends acts as a physical barrier to microtubule sliding for the organization of stable anti-parallel overlaps by PRC1 and Kif4A

NASA Astrophysics Data System (ADS)

Wijeratne, Sitara; Subramanian, Radhika

The relative sliding of microtubules by motor proteins is important for the organization of specialized cellular microtubule networks. In cells, sliding filaments are likely to encounter crowded regions of microtubules, such as the plus-ends, which are densely occupied by motor and non-motor proteins. How molecular crowding impacts microtubule sliding is not well understood. Here, we reconstitute the collective activities of the non-motor protein PRC1 and the motor protein Kif4A on anti-parallel microtubules to address this question. We find that the accumulation of PRC1 and Kif4A at microtubule-plus ends (`end-tags') can act as a physical barrier to Kif4A-mediated microtubule sliding. This enables the formation of stable microtubule overlaps that persist even after the deactivation of the motor protein. Our data suggest that while end-tags stabilize anti-parallel overlaps by inhibiting relative sliding, they permit the remodeling of the microtubule bundles by external forces, as may be required for the reorganization of microtubule networks during dynamic cellular processes.
[Influence of microtubule depolymerization of myocardial cells on mitochondria distribution and energy metabolism in adult rats].

PubMed

Dang, Yong-ming; Fang, Ya-dong; Hu, Jiong-yu; Zhang, Jia-ping; Song, Hua-pei; Zhang, Yi-ming; Zhang, Qiong; Huang, Yue-sheng

2010-02-01

To investigate the influence of microtubule depolymerization of myocardial cells on distribution and activity of mitochondria, and energy metabolism of cells in adult rats. Myocardial cells of SD adult rats and SD suckling rats were isolated and cultured. They were divided into adult and suckling rats control groups (AC and SC, normally cultured without any stimulating factor), adult and suckling rats microtubule depolymerization agent groups (AMDA and SMDA, cultured with 8 micromol/L colchicine containing nutrient solution for 30 minutes) according to the random number table. (1) The expression of polymerized beta tubulin in myocardial cells of adult and suckling rats was detected with Western blot. (2) Myocardial cells of rats in AC and AMDA groups were collected. The expression of cytochrome c was detected with Western blot. Distribution of voltage-dependent anion channels (VDAC) and polymerized beta tubulin in myocardial cells were observed with immunofluorescent staining. Mitochondrial inner membrane potential was determined with immunocytochemical method. Activity of myocardial cells was detected with MTT method. Contents of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) and energy charge of cells were determined with high performance liquid chromatography. (1) The expression of polymerized beta tubulin:in AMDA group it was 0.52 + or - 0.07, which was obviously lower than that (1.25 + or - 0.12) in AC group (F = 31.002, P = 0.000); in SMDA group it was 0.76 + or - 0.12, which was significantly lower than that (1.11 + or - 0.24) in SC group (F = 31.002, P = 0.000), but was obviously higher than that in AMDA group (F = 31.002, P = 0.009). (2) The expression of cytochrome c in AC group was 0.26 + or - 0.03, which was obviously lower than that (1.55 + or - 0.13) in AMDA group (t = -24.056, P = 0.000). (3) Immunofluorescent staining result: in AC group, microtubules of myocardial cells were in linear tubiform, distributed in parallel with
Multiscale modeling and simulation of microtubule-motor-protein assemblies

NASA Astrophysics Data System (ADS)

Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, M. D.; Shelley, Michael J.

2015-12-01

Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.
Multiscale modeling and simulation of microtubule-motor-protein assemblies.

PubMed

Gao, Tong; Blackwell, Robert; Glaser, Matthew A; Betterton, M D; Shelley, Michael J

2015-01-01

Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.
Microtubules (tau) as an emerging therapeutic target: NAP (davunetide).

PubMed

Gozes, Illana

2011-01-01

This review focuses on the discovery of activity-dependent neuroprotective protein (ADNP) and the ensuing discovery of NAP (davunetide) toward clinical development with emphasis on microtubule protection. ADNP immunoreactivity was shown to occasionally decorate microtubules and ADNP silencing inhibited neurite outgrowth as measured by microtubule associated protein 2 (MAP2) labeling. ADNP knockout is lethal, while 50% reduction in ADNP (ADNP haploinsufficiency) resulted in the microtubule associated protein tau pathology coupled to cognitive dysfunction and neurodegeneration. NAP (davunetide), an eight amino acid peptide derived from ADNP partly ameliorated deficits associated with ADNP deficiency. NAP (davunetide) interacted with microtubules, protected against microtubule toxicity associated with zinc, nocodazole and oxidative stress in vitro and against tau pathology and MAP6 (stable tubuleonly polypeptide - STOP) pathology in vivo. NAP (davunetide) provided neurotrophic functions promoting neurite outgrowth as measured by increases in MAP2 immunoreactivity and synapse formation by increasing synaptophysin expression. NAP (davunetide) protection against neurodegeneration has recently been shown to extend to katanin-related microtubule disruption under conditions of tau deficiencies. In conclusion, NAP (davunetide) provided potent neuroprotection in a broad range of neurodegenerative models, protecting the neuroglial cytoskeleton in vitro and inhibiting tau pathology (tauopathy) in vivo. Based on these extensive preclinical results, davunetide (NAP) is now being evaluated in a Phase II/III study of the tauopathy, progressive supranuclear palsy (PSP); (Allon Therapeutics Inc.).
ICPS Removal from Shipping Container

NASA Image and Video Library

2017-03-09

Inside the United Launch Alliance (ULA) Horizontal Integration Facility at Cape Canaveral Air Force Station in Florida, a crane lifts the shipping container cover away from the Interim Cryogenic Propulsion Stage (ICPS) for NASA's Space Launch System rocket, followed by the ICPS bring removed and placed on a work stand for processing. The ICPS is the first integrated piece of flight hardware to arrive for the SLS. The ICPS arrived from the ULA facility in Decatur, Alabama. The ICPS is the in-space stage that is located toward the top of the rocket, between the Launch Vehicle Stage Adapter and the Orion Spacecraft Adapter. It will provide some of the in-space propulsion during Orion's first flight test atop the SLS on Exploration Mission 1.
Optical Tweezers-Based Measurements of Forces and Dynamics at Microtubule Ends.

PubMed

Baclayon, Marian; Kalisch, Svenja-Marei; Hendel, Ed; Laan, Liedewij; Husson, Julien; Munteanu, E Laura; Dogterom, Marileen

2017-01-01

Microtubules are dynamic cytoskeletal polymers that polymerize and depolymerize while interacting with different proteins and structures within the cell. The highly regulated dynamic properties as well as the pushing and pulling forces generated by dynamic microtubule ends play important roles in processes such as in cell division. For instance, microtubule end-binding proteins are known to affect dramatically the dynamic properties of microtubules, and cortical dyneins are known to mediate pulling forces on microtubule ends. We discuss in this chapter our efforts to reconstitute these systems in vitro and mimic their interactions with structures within the cell using micro-fabricated barriers. Using an optical tweezers setup, we investigate the dynamics and forces of microtubules growing against functionalized barriers in the absence and presence of end-binding proteins and barrier-attached motor proteins. This setup allows high-speed as well as nanometer and piconewton resolution measurements on dynamic microtubules.
Electric field-induced reversible trapping of microtubules along metallic glass microwire electrodes

NASA Astrophysics Data System (ADS)

Kim, Kyongwan; Sikora, Aurélien; Nakayama, Koji S.; Umetsu, Mitsuo; Hwang, Wonmuk; Teizer, Winfried

2015-04-01

Microtubules are among bio-polymers providing vital functions in dynamic cellular processes. Artificial organization of these bio-polymers is a requirement for transferring their native functions into device applications. Using electrophoresis, we achieve an accumulation of microtubules along a metallic glass (Pd42.5Cu30Ni7.5P20) microwire in solution. According to an estimate based on migration velocities of microtubules approaching the wire, the electrophoretic mobility of microtubules is around 10-12 m2/Vs. This value is four orders of magnitude smaller than the typical mobility reported previously. Fluorescence microscopy at the individual-microtubule level shows microtubules aligning along the wire axis during the electric field-induced migration. Casein-treated electrodes are effective to reversibly release trapped microtubules upon removal of the external field. An additional result is the condensation of secondary filamentous structures from oriented microtubules.
The peroxisomal multifunctional protein interacts with cortical microtubules in plant cells

PubMed Central

2005-01-01

Background The plant peroxisomal multifunctional protein (MFP) possesses up to four enzymatic activities that are involved in catalyzing different reactions of fatty acid β-oxidation in the peroxisome matrix. In addition to these peroxisomal activities, in vitro assays revealed that rice MFP possesses microtubule- and RNA-binding activities suggesting that this protein also has important functions in the cytosol. Results We demonstrate that MFP is an authentic microtubule-binding protein, as it localized to the cortical microtubule array in vivo, in addition to its expected targeting to the peroxisome matrix. MFP does not, however, interact with the three mitotic microtubule arrays. Microtubule co-sedimentation assays of truncated versions of MFP revealed that multiple microtubule-binding domains are present on the MFP polypeptide. This indicates that these regions function together to achieve high-affinity binding of the full-length protein. Real-time imaging of a transiently expressed green fluorescent protein-MFP chimera in living plant cells illustrated that a dynamic, spatial interaction exits between peroxisomes and cortical microtubules as peroxisomes move along actin filaments or oscillate at fixed locations. Conclusion Plant MFP is associated with the cortical microtubule array, in addition to its expected localization in the peroxisome. This observation, coupled with apparent interactions that frequently occur between microtubules and peroxisomes in the cell cortex, supports the hypothesis that MFP is concentrated on microtubules in order to facilitate the regulated import of MFP into peroxisomes. PMID:16313672
Motor-mediated Cortical versus Astral Microtubule Organization in Lipid-monolayered Droplets

PubMed Central

Baumann, Hella; Surrey, Thomas

2014-01-01

The correct spatial organization of microtubules is of crucial importance for determining the internal architecture of eukaryotic cells. Microtubules are arranged in space by a multitude of biochemical activities and by spatial constraints imposed by the cell boundary. The principles underlying the establishment of distinct intracellular architectures are only poorly understood. Here, we studied the effect of spatial confinement on the self-organization of purified motors and microtubules that are encapsulated in lipid-monolayered droplets in oil, varying in diameter from 5–100 μm, which covers the size range of typical cell bodies. We found that droplet size alone had a major organizing influence. The presence of a microtubule-crosslinking motor protein decreased the number of accessible types of microtubule organizations. Depending on the degree of spatial confinement, the presence of the motor caused either the formation of a cortical array of bent microtubule bundles or the generation of single microtubule asters in the droplets. These are two of the most prominent forms of microtubule arrangements in plant and metazoan cells. Our results provide insights into the combined organizing influence of spatial constraints and cross-linking motor activities determining distinct microtubule architectures in a minimal biomimetic system. In the future, this simple lipid-monolayered droplet system characterized here can be expanded readily to include further biochemical activities or used as the starting point for the investigation of motor-mediated microtubule organization inside liposomes surrounded by a deformable lipid bilayer. PMID:24966327
The baseline pressure of intracranial pressure (ICP) sensors can be altered by electrostatic discharges.

PubMed

Eide, Per K; Bakken, André

2011-08-22

The monitoring of intracranial pressure (ICP) has a crucial role in the surveillance of patients with brain injury. During long-term monitoring of ICP, we have seen spontaneous shifts in baseline pressure (ICP sensor zero point), which are of technical and not physiological origin. The aim of the present study was to explore whether or not baseline pressures of ICP sensors can be affected by electrostatics discharges (ESD's), when ESD's are delivered at clinically relevant magnitudes. We performed bench-testing of a set of commercial ICP sensors. In our experimental setup, the ICP sensor was placed in a container with 0.9% NaCl solution. A test person was charged 0.5-10 kV, and then delivered ESD's to the sensor by touching a metal rod that was located in the container. The continuous pressure signals were recorded continuously before/after the ESD's, and the pressure readings were stored digitally using a computerized system A total of 57 sensors were tested, including 25 Codman ICP sensors and 32 Raumedic sensors. When charging the test person in the range 0.5-10 kV, typically ESD's in the range 0.5-5 kV peak pulse were delivered to the ICP sensor. Alterations in baseline pressure ≥ 2 mmHg was seen in 24 of 25 (96%) Codman sensors and in 17 of 32 (53%) Raumedic sensors. Lasting changes in baseline pressure > 10 mmHg that in the clinical setting would affect patient management, were seen frequently for both sensor types. The changes in baseline pressure were either characterized by sudden shifts or gradual drifts in baseline pressure. The baseline pressures of commercial solid ICP sensors can be altered by ESD's at discharge magnitudes that are clinically relevant. Shifts in baseline pressure change the ICP levels visualised to the physician on the monitor screen, and thereby reveal wrong ICP values, which likely represent a severe risk to the patient.
The baseline pressure of intracranial pressure (ICP) sensors can be altered by electrostatic discharges

PubMed Central

2011-01-01

Background The monitoring of intracranial pressure (ICP) has a crucial role in the surveillance of patients with brain injury. During long-term monitoring of ICP, we have seen spontaneous shifts in baseline pressure (ICP sensor zero point), which are of technical and not physiological origin. The aim of the present study was to explore whether or not baseline pressures of ICP sensors can be affected by electrostatics discharges (ESD's), when ESD's are delivered at clinically relevant magnitudes. Methods We performed bench-testing of a set of commercial ICP sensors. In our experimental setup, the ICP sensor was placed in a container with 0.9% NaCl solution. A test person was charged 0.5 - 10 kV, and then delivered ESD's to the sensor by touching a metal rod that was located in the container. The continuous pressure signals were recorded continuously before/after the ESD's, and the pressure readings were stored digitally using a computerized system Results A total of 57 sensors were tested, including 25 Codman ICP sensors and 32 Raumedic sensors. When charging the test person in the range 0.5-10 kV, typically ESD's in the range 0.5 - 5 kV peak pulse were delivered to the ICP sensor. Alterations in baseline pressure ≥ 2 mmHg was seen in 24 of 25 (96%) Codman sensors and in 17 of 32 (53%) Raumedic sensors. Lasting changes in baseline pressure > 10 mmHg that in the clinical setting would affect patient management, were seen frequently for both sensor types. The changes in baseline pressure were either characterized by sudden shifts or gradual drifts in baseline pressure. Conclusions The baseline pressures of commercial solid ICP sensors can be altered by ESD's at discharge magnitudes that are clinically relevant. Shifts in baseline pressure change the ICP levels visualised to the physician on the monitor screen, and thereby reveal wrong ICP values, which likely represent a severe risk to the patient. PMID:21859487
Model for the orientational ordering of the plant microtubule cortical array

NASA Astrophysics Data System (ADS)

Hawkins, Rhoda J.; Tindemans, Simon H.; Mulder, Bela M.

2010-07-01

The plant microtubule cortical array is a striking feature of all growing plant cells. It consists of a more or less homogeneously distributed array of highly aligned microtubules connected to the inner side of the plasma membrane and oriented transversely to the cell growth axis. Here, we formulate a continuum model to describe the origin of orientational order in such confined arrays of dynamical microtubules. The model is based on recent experimental observations that show that a growing cortical microtubule can interact through angle dependent collisions with pre-existing microtubules that can lead either to co-alignment of the growth, retraction through catastrophe induction or crossing over the encountered microtubule. We identify a single control parameter, which is fully determined by the nucleation rate and intrinsic dynamics of individual microtubules. We solve the model analytically in the stationary isotropic phase, discuss the limits of stability of this isotropic phase, and explicitly solve for the ordered stationary states in a simplified version of the model.
Structural differences between yeast and mammalian microtubules revealed by cryo-EM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howes, Stuart C.; Geyer, Elisabeth A.; LaFrance, Benjamin

Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrationsmore » used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. In conclusion, our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.« less
Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scaife, R.M.; Wilson, L.; Purich, D.L.

1992-01-14

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extentmore » of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.« less
Ophthalmodynamometry for ICP prediction and pilot test on Mt. Everest.

PubMed

Querfurth, Henry W; Lieberman, Philip; Arms, Steve; Mundell, Steve; Bennett, Michael; van Horne, Craig

2010-11-01

A recent development in non-invasive techniques to predict intracranial pressure (ICP) termed venous ophthalmodynamometry (vODM) has made measurements in absolute units possible. However, there has been little progress to show utility in the clinic or field. One important application would be to predict changes in actual ICP during adaptive responses to physiologic stress such as hypoxia. A causal relationship between raised intracranial pressure and acute mountain sickness (AMS) is suspected. Several MRI studies report that modest physiologic increases in cerebral volume, from swelling, normally accompany subacute ascent to simulated high altitudes. 1) Validate and calibrate an advanced, portable vODM instrument on intensive patients with raised intracranial pressure and 2) make pilot, non-invasive ICP estimations of normal subjects at increasing altitudes. The vODM was calibrated against actual ICP in 12 neurosurgical patients, most affected with acute hydrocephalus and monitored using ventriculostomy/pressure transducers. The operator was blinded to the transducer read-out. A clinical field test was then conducted on a variable data set of 42 volunteer trekkers and climbers scaling Mt. Everest, Nepal. Mean ICPs were estimated at several altitudes on the ascent both across and within subjects. Portable vODM measurements increased directly and linearly with ICP resulting in good predictability (r = 0.85). We also found that estimated ICP increases normally with altitude (10 ± 3 mm Hg; sea level to 20 ± 2 mm Hg; 6553 m) and that AMS symptoms did not correlate with raised ICP. vODM technology has potential to reliably estimate absolute ICP and is portable. Physiologic increases in ICP and mild-mod AMS are separate responses to high altitude, possibly reflecting swelling and vasoactive instability, respectively.

Biological Information Processing in Single Microtubules

DTIC Science & Technology

2012-02-15

flux moves in the brain at a speed of 400km/hr, when electrons move only a few cm in years, we have found that through microtubule, solitons propagate... soliton ”. Introduction: We started working on the brain microtubule way back in 2008, since, I understood that in the brain, neurons separated by...6 inches, synchronize, get phase and frequency locked and that is the source of enormous computability of the brain. However, no experimental
Four-stranded mini microtubules formed by Prosthecobacter BtubAB show dynamic instability.

PubMed

Deng, Xian; Fink, Gero; Bharat, Tanmay A M; He, Shaoda; Kureisaite-Ciziene, Danguole; Löwe, Jan

2017-07-18

Microtubules, the dynamic, yet stiff hollow tubes built from αβ-tubulin protein heterodimers, are thought to be present only in eukaryotic cells. Here, we report a 3.6-Å helical reconstruction electron cryomicroscopy structure of four-stranded mini microtubules formed by bacterial tubulin-like Prosthecobacter dejongeii BtubAB proteins. Despite their much smaller diameter, mini microtubules share many key structural features with eukaryotic microtubules, such as an M-loop, alternating subunits, and a seam that breaks overall helical symmetry. Using in vitro total internal reflection fluorescence microscopy, we show that bacterial mini microtubules treadmill and display dynamic instability, another hallmark of eukaryotic microtubules. The third protein in the btub gene cluster, BtubC, previously known as "bacterial kinesin light chain," binds along protofilaments every 8 nm, inhibits BtubAB mini microtubule catastrophe, and increases rescue. Our work reveals that some bacteria contain regulated and dynamic cytomotive microtubule systems that were once thought to be only useful in much larger and sophisticated eukaryotic cells.
Quantification of asymmetric microtubule nucleation at sub-cellular structures

PubMed Central

Zhu, Xiaodong; Kaverina, Irina

2012-01-01

Cell polarization is important for multiple physiological processes. In polarized cells, microtubules (MTs) are organized into a spatially polarized array. Generally, in non-differentiated cells, it is assumed that MTs are symmetrically nucleated exclusively from centrosome (microtubule organizing center, MTOC) and then reorganized into the asymmetric array. We have recently identified the Golgi complex as an additional MTOC that asymmetrically nucleates MTs toward one side of the cell. Methods used for alternative MTOC identification include microtubule re-growth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescence labeled MT +TIP binding proteins in living cells. These approaches can be used for quantification of MT nucleation sites at diverse sub-cellular structures. PMID:21773933
The influence of room temperature on Mg isotope measurements by MC-ICP-MS.

PubMed

Zhang, Xing-Chao; Zhang, An-Yu; Zhang, Zhao-Feng; Huang, Fang; Yu, Hui-Min

2018-03-24

We observed that the accuracy and precision of magnesium (Mg) isotope analyses could be affected if the room temperature oscillated during measurements. To achieve high quality Mg isotopic data, it is critical to evaluate how the unstable room temperature affects Mg isotope measurements by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). We measured the Mg isotopes for the reference material DSM-3 using MC-ICP-MS under oscillating room temperatures in spring. For a comparison, we also measured the Mg isotopes under stable room temperatures, which was achieved by the installation of an improved temperature control system in the laboratory. The δ 26 Mg values measured under oscillating room temperatures have a larger deviation (δ 26 Mg from -0.09 to 0.08‰, with average δ 26 Mg = 0.00 ± 0.08 ‰) than those measured under a stable room temperature (δ 26 Mg from -0.03 to 0.03‰, with average δ 26 Mg = 0.00 ± 0.02 ‰) using the same MC-ICP-MS system. The room temperature variation can influence the stability of MC-ICP-MS. Therefore, it is critical to keep the room temperature stable to acquire high precise and accurate isotopic data when using MC-ICP-MS, especially when using the sample-standard bracketing (SSB) correction method. This article is protected by copyright. All rights reserved.
Nonlinear dynamics of C–terminal tails in cellular microtubules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sekulic, Dalibor L., E-mail: dalsek@uns.ac.rs; Sataric, Bogdan M.; Sataric, Miljko V.

2016-07-15

The mechanical and electrical properties, and information processing capabilities of microtubules are the permanent subject of interest for carrying out experiments in vitro and in silico, as well as for theoretical attempts to elucidate the underlying processes. In this paper, we developed a new model of the mechano–electrical waves elicited in the rows of very flexible C–terminal tails which decorate the outer surface of each microtubule. The fact that C–terminal tails play very diverse roles in many cellular functions, such as recruitment of motor proteins and microtubule–associated proteins, motivated us to consider their collective dynamics as the source of localizedmore » waves aimed for communication between microtubule and associated proteins. Our approach is based on the ferroelectric liquid crystal model and it leads to the effective asymmetric double-well potential which brings about the conditions for the appearance of kink–waves conducted by intrinsic electric fields embedded in microtubules. These kinks can serve as the signals for control and regulation of intracellular traffic along microtubules performed by processive motions of motor proteins, primarly from kinesin and dynein families. On the other hand, they can be precursors for initiation of dynamical instability of microtubules by recruiting the proper proteins responsible for the depolymerization process.« less
Direct interaction of microtubule- and actin-based transport motors

NASA Technical Reports Server (NTRS)

Huang, J. D.; Brady, S. T.; Richards, B. W.; Stenolen, D.; Resau, J. H.; Copeland, N. G.; Jenkins, N. A.

1999-01-01

The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.
Beyond taxol: microtubule-based treatment of disease and injury of the nervous system

PubMed Central

Ahmad, Fridoon J.

2013-01-01

Contemporary research has revealed a great deal of information on the behaviours of microtubules that underlie critical events in the lives of neurons. Microtubules in the neuron undergo dynamic assembly and disassembly, bundling and splaying, severing, and rapid transport as well as integration with other cytoskeletal elements such as actin filaments. These various behaviours are regulated by signalling pathways that affect microtubule-related proteins such as molecular motor proteins and microtubule severing enzymes, as well as a variety of proteins that promote the assembly, stabilization and bundling of microtubules. In recent years, translational neuroscientists have earmarked microtubules as a promising target for therapy of injury and disease of the nervous system. Proof-of-principle has come mainly from studies using taxol and related drugs to pharmacologically stabilize microtubules in animal models of nerve injury and disease. However, concerns persist that the negative consequences of abnormal microtubule stabilization may outweigh the positive effects. Other potential approaches include microtubule-active drugs with somewhat different properties, but also expanding the therapeutic toolkit to include intervention at the level of microtubule regulatory proteins. PMID:23811322
Oscillatory fluid flow influences primary cilia and microtubule mechanics.

PubMed

Espinha, Lina C; Hoey, David A; Fernandes, Paulo R; Rodrigues, Hélder C; Jacobs, Christopher R

2014-07-01

Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues, such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity. Copyright © 2014 Wiley Periodicals, Inc.
Automated Stitching of Microtubule Centerlines across Serial Electron Tomograms

PubMed Central

Weber, Britta; Tranfield, Erin M.; Höög, Johanna L.; Baum, Daniel; Antony, Claude; Hyman, Tony; Verbavatz, Jean-Marc; Prohaska, Steffen

2014-01-01

Tracing microtubule centerlines in serial section electron tomography requires microtubules to be stitched across sections, that is lines from different sections need to be aligned, endpoints need to be matched at section boundaries to establish a correspondence between neighboring sections, and corresponding lines need to be connected across multiple sections. We present computational methods for these tasks: 1) An initial alignment is computed using a distance compatibility graph. 2) A fine alignment is then computed with a probabilistic variant of the iterative closest points algorithm, which we extended to handle the orientation of lines by introducing a periodic random variable to the probabilistic formulation. 3) Endpoint correspondence is established by formulating a matching problem in terms of a Markov random field and computing the best matching with belief propagation. Belief propagation is not generally guaranteed to converge to a minimum. We show how convergence can be achieved, nonetheless, with minimal manual input. In addition to stitching microtubule centerlines, the correspondence is also applied to transform and merge the electron tomograms. We applied the proposed methods to samples from the mitotic spindle in C. elegans, the meiotic spindle in X. laevis, and sub-pellicular microtubule arrays in T. brucei. The methods were able to stitch microtubules across section boundaries in good agreement with experts' opinions for the spindle samples. Results, however, were not satisfactory for the microtubule arrays. For certain experiments, such as an analysis of the spindle, the proposed methods can replace manual expert tracing and thus enable the analysis of microtubules over long distances with reasonable manual effort. PMID:25438148
Automated stitching of microtubule centerlines across serial electron tomograms.

PubMed

Weber, Britta; Tranfield, Erin M; Höög, Johanna L; Baum, Daniel; Antony, Claude; Hyman, Tony; Verbavatz, Jean-Marc; Prohaska, Steffen

2014-01-01

Tracing microtubule centerlines in serial section electron tomography requires microtubules to be stitched across sections, that is lines from different sections need to be aligned, endpoints need to be matched at section boundaries to establish a correspondence between neighboring sections, and corresponding lines need to be connected across multiple sections. We present computational methods for these tasks: 1) An initial alignment is computed using a distance compatibility graph. 2) A fine alignment is then computed with a probabilistic variant of the iterative closest points algorithm, which we extended to handle the orientation of lines by introducing a periodic random variable to the probabilistic formulation. 3) Endpoint correspondence is established by formulating a matching problem in terms of a Markov random field and computing the best matching with belief propagation. Belief propagation is not generally guaranteed to converge to a minimum. We show how convergence can be achieved, nonetheless, with minimal manual input. In addition to stitching microtubule centerlines, the correspondence is also applied to transform and merge the electron tomograms. We applied the proposed methods to samples from the mitotic spindle in C. elegans, the meiotic spindle in X. laevis, and sub-pellicular microtubule arrays in T. brucei. The methods were able to stitch microtubules across section boundaries in good agreement with experts' opinions for the spindle samples. Results, however, were not satisfactory for the microtubule arrays. For certain experiments, such as an analysis of the spindle, the proposed methods can replace manual expert tracing and thus enable the analysis of microtubules over long distances with reasonable manual effort.
Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport

PubMed Central

1985-01-01

Native microtubules prepared from extruded and dissociated axoplasm have been observed to transport organelles and vesicles unidirectionally in fresh preparations and more slowly and bidirectionally in older preparations. Both endogenous and exogenous (fluorescent polystyrene) particles in rapid Brownian motion alight on and adhere to microtubules and are transported along them. Particles can switch from one intersecting microtubule to another and move in either direction. Microtubular segments 1 to 30 microns long, produced by gentle homogenization, glide over glass surfaces for hundreds of micrometers in straight lines unless acted upon by obstacles. While gliding they transport particles either in the same (forward) direction and/or in the backward direction. Particle movement and gliding of microtubule segments require ATP and are insensitive to taxol (30 microM). It appears, therefore, that the mechanisms producing the motive force are very closely associated with the native microtubule itself or with its associated proteins. Although these movements appear irreconcilable with several current theories of fast axoplasmic transport, in this article we propose two models that might explain the observed phenomena and, by extension, the process of fast axoplasmic transport itself. The findings presented and the possible mechanisms proposed for fast axoplasmic transport have potential applications across the spectrum of microtubule-based motility processes. PMID:2580845
Microtubules move the nucleus to quiescence.

PubMed

Laporte, Damien; Sagot, Isabelle

2014-01-01

The nucleus is a cellular compartment that hosts several macro-molecular machines displaying a highly complex spatial organization. This tight architectural orchestration determines not only DNA replication and repair but also regulates gene expression. In budding yeast microtubules play a key role in structuring the nucleus since they condition the Rabl arrangement in G1 and chromosome partitioning during mitosis through their attachment to centromeres via the kinetochore proteins. Recently, we have shown that upon quiescence entry, intranuclear microtubules emanating from the spindle pole body elongate to form a highly stable bundle that spans the entire nucleus. Here, we examine some molecular mechanisms that may underlie the formation of this structure. As the intranuclear microtubule bundle causes a profound re-organization of the yeast nucleus and is required for cell survival during quiescence, we discuss the possibility that the assembly of such a structure participates in quiescence establishment.
Processive movement of single kinesins on crowded microtubules visualized using quantum dots

PubMed Central

Seitz, Arne; Surrey, Thomas

2006-01-01

Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions. PMID:16407972
Simulation studies of self-organization of microtubules and molecular motors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jian, Z.; Karpeev, D.; Aranson, I. S.

We perform Monte Carlo type simulation studies of self-organization of microtubules interacting with molecular motors. We model microtubules as stiff polar rods of equal length exhibiting anisotropic diffusion in the plane. The molecular motors are implicitly introduced by specifying certain probabilistic collision rules resulting in realignment of the rods. This approximation of the complicated microtubule-motor interaction by a simple instant collision allows us to bypass the 'computational bottlenecks' associated with the details of the diffusion and the dynamics of motors and the reorientation of microtubules. Consequently, we are able to perform simulations of large ensembles of microtubules and motors onmore » a very large time scale. This simple model reproduces all important phenomenology observed in in vitro experiments: Formation of vortices for low motor density and raylike asters and bundles for higher motor density.« less
Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells

PubMed Central

Akita, Kae; Higaki, Takumi; Kutsuna, Natsumaro; Hasezawa, Seiichiro

2015-01-01

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells. PMID:26039484
Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells.

PubMed

Akita, Kae; Higaki, Takumi; Kutsuna, Natsumaro; Hasezawa, Seiichiro

2015-01-01

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.
Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers.

PubMed

Wang, Richard; Wang, Harris; Wang, Zhixiang

2017-02-20

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged α-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.
Application of ICP-MS and HPLC-ICP-MS for diagnosis and therapy of a severe intoxication with hexavalent chromium and inorganic arsenic.

PubMed

Heitland, Peter; Blohm, Martin; Breuer, Christian; Brinkert, Florian; Achilles, Eike Gert; Pukite, Ieva; Köster, Helmut Dietrich

2017-05-01

ICP-MS and HPLC-ICP-MS were applied for diagnosis and therapeutic monitoring in a severe intoxication with a liquid containing hexavalent chromium (Cr(VI)) and inorganic arsenic (iAs). In this rare case a liver transplantation of was considered as the only chance of survival. We developed and applied methods for the determination of Cr(VI) in erythrocytes and total chromium (Cr) and arsenic (As) in blood, plasma, urine and liver tissue by ICP-MS. Exposure to iAs was diagnosed by determination of iAs species and their metabolites in urine by anion exchange HPLC-ICP-MS. Three days after ingestion of the liquid the total Cr concentrations were 2180 and 1070μg/L in whole blood and plasma, respectively, and 4540μg/L Cr(VI) in erythrocytes. The arsenic concentration in blood was 206μg/L. The urinary As species concentrations were <0.5, 109, 115, 154 and 126μg/L for arsenobetaine, As(III), As(V), methylarsonate (V) and dimethylarsinate (V), respectively. Total Cr and As concentrations in the explanted liver were 11.7 and 0.9mg/kg, respectively. Further analytical results of this case study are tabulated and provide valuable data for physicians and toxicologists. Copyright © 2017. Published by Elsevier GmbH.
Metallic Glass Wire Based Localization of Kinesin/Microtubule Bio-molecular Motility System

NASA Astrophysics Data System (ADS)

Kim, K.; Sikora, A.; Yaginuma, S.; Nakayama, K. S.; Nakazawa, H.; Umetsu, M.; Hwang, W.; Teizer, W.

2014-03-01

We report electrophoretic accumulation of microtubules along metallic glass (Pd42.5Cu30Ni7.5P20) wires free-standing in solution. Microtubules are dynamic cytoskeletal filaments. Kinesin is a cytoskeletal motor protein. Functions of these bio-molecules are central to various dynamic cellular processes. Functional artificial organization of bio-molecules is a prerequisite for transferring their native functions into device applications. Fluorescence microscopy at the individual-microtubule level reveals microtubules aligning along the wire axis during the electrophoretic migration. Casein-treated electrodes are effective for releasing trapped microtubules upon removal of the external field. Furthermore, we demonstrate gliding motion of microtubules on kinesin-treated metallic glass wires. The reversible manner in the local adsorption of microtubules, the flexibility of wire electrodes, and the compatibility between the wire electrode and the bio-molecules are beneficial for spatio-temporal manipulation of the motility machinery in 3 dimensions.
On the nature and shape of tubulin trails: implications on microtubule self-organization.

PubMed

Glade, Nicolas

2012-06-01

Microtubules, major elements of the cell skeleton are, most of the time, well organized in vivo, but they can also show self-organizing behaviors in time and/or space in purified solutions in vitro. Theoretical studies and models based on the concepts of collective dynamics in complex systems, reaction-diffusion processes and emergent phenomena were proposed to explain some of these behaviors. In the particular case of microtubule spatial self-organization, it has been advanced that microtubules could behave like ants, self-organizing by 'talking to each other' by way of hypothetic (because never observed) concentrated chemical trails of tubulin that are expected to be released by their disassembling ends. Deterministic models based on this idea yielded indeed like-looking spatio-temporal self-organizing behaviors. Nevertheless the question remains of whether microscopic tubulin trails produced by individual or bundles of several microtubules are intense enough to allow microtubule self-organization at a macroscopic level. In the present work, by simulating the diffusion of tubulin in microtubule solutions at the microscopic scale, we measure the shape and intensity of tubulin trails and discuss about the assumption of microtubule self-organization due to the production of chemical trails by disassembling microtubules. We show that the tubulin trails produced by individual microtubules or small microtubule arrays are very weak and not elongated even at very high reactive rates. Although the variations of concentration due to such trails are not significant compared to natural fluctuations of the concentration of tubuline in the chemical environment, the study shows that heterogeneities of biochemical composition can form due to microtubule disassembly. They could become significant when produced by numerous microtubule ends located in the same place. Their possible formation could play a role in certain conditions of reaction. In particular, it gives a mesoscopic

Electrostatically Biased Binding of Kinesin to Microtubules

PubMed Central

Zheng, Wenjun; Alonso, Maria; Huber, Gary; Dlugosz, Maciej; McCammon, J. Andrew; Cross, Robert A.

2011-01-01

The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules. PMID:22140358
Contributions of Microtubule Dynamic Instability and Rotational Diffusion to Kinetochore Capture.

PubMed

Blackwell, Robert; Sweezy-Schindler, Oliver; Edelmaier, Christopher; Gergely, Zachary R; Flynn, Patrick J; Montes, Salvador; Crapo, Ammon; Doostan, Alireza; McIntosh, J Richard; Glaser, Matthew A; Betterton, Meredith D

2017-02-07

Microtubule dynamic instability allows search and capture of kinetochores during spindle formation, an important process for accurate chromosome segregation during cell division. Recent work has found that microtubule rotational diffusion about minus-end attachment points contributes to kinetochore capture in fission yeast, but the relative contributions of dynamic instability and rotational diffusion are not well understood. We have developed a biophysical model of kinetochore capture in small fission-yeast nuclei using hybrid Brownian dynamics/kinetic Monte Carlo simulation techniques. With this model, we have studied the importance of dynamic instability and microtubule rotational diffusion for kinetochore capture, both to the lateral surface of a microtubule and at or near its end. Over a range of biologically relevant parameters, microtubule rotational diffusion decreased capture time, but made a relatively small contribution compared to dynamic instability. At most, rotational diffusion reduced capture time by 25%. Our results suggest that while microtubule rotational diffusion can speed up kinetochore capture, it is unlikely to be the dominant physical mechanism for typical conditions in fission yeast. In addition, we found that when microtubules undergo dynamic instability, lateral captures predominate even in the absence of rotational diffusion. Counterintuitively, adding rotational diffusion to a dynamic microtubule increases the probability of end-on capture. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

NASA Technical Reports Server (NTRS)

Granger, C. L.; Cyr, R. J.

2000-01-01

Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.
Variational Principles for Buckling of Microtubules Modeled as Nonlocal Orthotropic Shells

PubMed Central

2014-01-01

A variational principle for microtubules subject to a buckling load is derived by semi-inverse method. The microtubule is modeled as an orthotropic shell with the constitutive equations based on nonlocal elastic theory and the effect of filament network taken into account as an elastic surrounding. Microtubules can carry large compressive forces by virtue of the mechanical coupling between the microtubules and the surrounding elastic filament network. The equations governing the buckling of the microtubule are given by a system of three partial differential equations. The problem studied in the present work involves the derivation of the variational formulation for microtubule buckling. The Rayleigh quotient for the buckling load as well as the natural and geometric boundary conditions of the problem is obtained from this variational formulation. It is observed that the boundary conditions are coupled as a result of nonlocal formulation. It is noted that the analytic solution of the buckling problem for microtubules is usually a difficult task. The variational formulation of the problem provides the basis for a number of approximate and numerical methods of solutions and furthermore variational principles can provide physical insight into the problem. PMID:25214886
Microtubule and cellulose microfibril orientation during plant cell and organ growth.

PubMed

Chan, J

2012-07-01

In this review, I ask the question of what is the relationship between growth and the orientations of microtubules and cellulose microfibrils in plant cells. This should be a relatively simple question to answer considering that text books commonly describe microtubules and cellulose microfibrils as hoops that drive expansion perpendicular to their orientation. However, recent live imaging techniques, which allow microtubules and cellulose synthase dynamics to be imaged simultaneously with cell elongation, show that cells can elongate with nonperpendicular microtubule arrays. In this review, I look at the significance of these different microtubule arrangements for growth and cell wall architecture and how these resultant walls differ from those derived from perpendicular arrays. I also discuss how these divergent arrays in stems may be important for coordinating growth between the different cell layers. This role reveals some general features of microtubule alignment that can be used to predict the growth status of organs. In conclusion, nonperpendicular arrays demonstrate alternative ways of cell elongation that do not require hooped arrays of microtubules and cellulose microfibrils. Such nonperpendicular arrays may be required for optimal growth and strengthening of tissues. © 2011 The Author Journal of Microscopy © 2011 Royal Microscopical Society.
Protein Arms in the Kinetochore-Microtubule Interface of the Yeast DASH Complex

PubMed Central

Miranda, JJ L.; King, David S.

2007-01-01

The yeast DASH complex is a heterodecameric component of the kinetochore necessary for accurate chromosome segregation. DASH forms closed rings around microtubules with a large gap between the DASH ring and the microtubule cylinder. We characterized the microtubule-binding properties of limited proteolysis products and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic C-terminal extensions of tubulin subunits are not essential for DASH binding. We also measured the molecular mass of DASH rings on microtubules with scanning transmission electron microscopy and found that approximately 25 DASH heterodecamers assemble to form each ring. Dynamic association and relocation of multiple flexible appendages of DASH may allow the kinetochore to translate along the microtubule surface. PMID:17460120
Selected problems with boron determination in water treatment processes. Part I: comparison of the reference methods for ICP-MS and ICP-OES determinations.

PubMed

Kmiecik, Ewa; Tomaszewska, Barbara; Wątor, Katarzyna; Bodzek, Michał

2016-06-01

The aim of the study was to compare the two reference methods for the determination of boron in water samples and further assess the impact of the method of preparation of samples for analysis on the results obtained. Samples were collected during different desalination processes, ultrafiltration and the double reverse osmosis system, connected in series. From each point, samples were prepared in four different ways: the first was filtered (through a membrane filter of 0.45 μm) and acidified (using 1 mL ultrapure nitric acid for each 100 mL of samples) (FA), the second was unfiltered and not acidified (UFNA), the third was filtered but not acidified (FNA), and finally, the fourth was unfiltered but acidified (UFA). All samples were analysed using two analytical methods: inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES). The results obtained were compared and correlated, and the differences between them were studied. The results show that there are statistically significant differences between the concentrations obtained using the ICP-MS and ICP-OES techniques regardless of the methods of sampling preparation (sample filtration and preservation). Finally, both the ICP-MS and ICP-OES methods can be used for determination of the boron concentration in water. The differences in the boron concentrations obtained using these two methods can be caused by several high-level concentrations in selected whole-water digestates and some matrix effects. Higher concentrations of iron (from 1 to 20 mg/L) than chromium (0.02-1 mg/L) in the samples analysed can influence boron determination. When iron concentrations are high, we can observe the emission spectrum as a double joined and overlapping peak.
End-of-Life Vehicle Dismantling and Recycling Enterprises: Developing Directions in China

NASA Astrophysics Data System (ADS)

Wang, Lu; Chen, Ming

2013-08-01

End-of-life vehicle (ELV) dismantling and recycling enterprises are the final disposer of the life-cycle process of vehicles. ELV collecting, dismantling technology, and waste disposal directly affect the recovery rate and the friendliness of vehicles toward the environment. China law stipulates that, by 2017, the recovery rate of vehicles should not be less than 95%, and the recycling rate of materials should not be less than 85%. Therefore, knowing the practical running state of such enterprises is needed. This study investigated four ELV dismantling and recycling enterprises in the Yangzi delta district in China and surveyed the ELV collecting, dismantling technology, policy implementation, and running difficulties. After the comparison with the developed countries, the relevant experiences were drawn, and effective measures were put forward to meet the aims stipulated in the law based on the current practical ELV market in China.
AR on the move; boarding the microtubule expressway to the nucleus

PubMed Central

Thadani-Mulero, Maria; Nanus, David M.; Giannakakou, Paraskevi

2012-01-01

Recent studies have shown that the microtubule-stabilizing drug, paclitaxel, which is commonly used for the treatment of prostate cancer inhibits signaling from the androgen receptor (AR) by inhibiting its nuclear accumulation downstream of microtubule stabilization. This mechanism is independent of paclitaxel-induced mitotic arrest and could provide an alternative mechanism of drug action that can explain its clinical activity. In this review, we highlight the importance of signaling and trafficking pathways that depend on intact and dynamic microtubules and as such they represent downstream targets of microtubule inhibitors. We showcase prostate cancer, which is driven by the activity of the androgen receptor (AR), as recent reports have revealed a connection between the microtubule-dependent trafficking of AR and the clinical efficacy of taxanes. Identification and further elucidation of microtubule-dependent tumor-specific pathways will help us better understand the molecular basis of clinical taxane resistance as well as identify individual patients more likely to respond to treatment. PMID:22987486
Connecting macroscopic dynamics with microscopic properties in active microtubule network contraction

NASA Astrophysics Data System (ADS)

Foster, Peter J.; Yan, Wen; Fürthauer, Sebastian; Shelley, Michael J.; Needleman, Daniel J.

2017-12-01

The cellular cytoskeleton is an active material, driven out of equilibrium by molecular motor proteins. It is not understood how the collective behaviors of cytoskeletal networks emerge from the properties of the network’s constituent motor proteins and filaments. Here we present experimental results on networks of stabilized microtubules in Xenopus oocyte extracts, which undergo spontaneous bulk contraction driven by the motor protein dynein, and investigate the effects of varying the initial microtubule density and length distribution. We find that networks contract to a similar final density, irrespective of the length of microtubules or their initial density, but that the contraction timescale varies with the average microtubule length. To gain insight into why this microscopic property influences the macroscopic network contraction time, we developed simulations where microtubules and motors are explicitly represented. The simulations qualitatively recapitulate the variation of contraction timescale with microtubule length, and allowed stress contributions from different sources to be estimated and decoupled.
In vitro assembled plant microtubules exhibit a high state of dynamic instability.

PubMed

Moore, R C; Zhang, M; Cassimeris, L; Cyr, R J

1997-01-01

Higher plants possess four distinct microtubule arrays. One of these, the cortical array, is involved in orienting the deposition of cellulose microfibrils. This plant interphase array is also notable because it contains exceptionally dynamic microtubules. Although the primary sequence of plant and animal tubulin is similar (79-87% amino acid identity overall) there are some regions of divergence. Thus, one possible explanation for the high state of polymer assembly and turnover that is observed in plant interphase arrays is that the tubulins have evolved differently and possess a higher intrinsic dynamic character than their animal counterparts. This hypothesis was tested using highly purified plant tubulin assembled in vitro. Using high-resolution DIC video-enhanced microscopy, we quantified the four characteristic parameters of dynamic instability of plant microtubules and compared them with animal microtubules. The elongation velocities between plant and animal microtubules are similar, but plant microtubules undergo catastrophes more frequently, do not exhibit any rescues, and have an average shortening velocity of 195 microm/min (compared with 21 microm/min for animal microtubules). These data support the hypothesis that plant tubulin forms microtubules that are intrinsically more dynamic than those of animals.
Determination of phosphorus in small amounts of protein samples by ICP-MS.

PubMed

Becker, J Sabine; Boulyga, Sergei F; Pickhardt, Carola; Becker, J; Buddrus, Stefan; Przybylski, Michael

2003-02-01

Inductively coupled plasma mass spectrometry (ICP-MS) is used for phosphorus determination in protein samples. A small amount of solid protein sample (down to 1 micro g) or digest (1-10 micro L) protein solution was denatured in nitric acid and hydrogen peroxide by closed-microvessel microwave digestion. Phosphorus determination was performed with an optimized analytical method using a double-focusing sector field inductively coupled plasma mass spectrometer (ICP-SFMS) and quadrupole-based ICP-MS (ICP-QMS). For quality control of phosphorus determination a certified reference material (CRM), single cell proteins (BCR 273) with a high phosphorus content of 26.8+/-0.4 mg g(-1), was analyzed. For studies on phosphorus determination in proteins while reducing the sample amount as low as possible the homogeneity of CRM BCR 273 was investigated. Relative standard deviation and measurement accuracy in ICP-QMS was within 2%, 3.5%, 11% and 12% when using CRM BCR 273 sample weights of 40 mg, 5 mg, 1 mg and 0.3 mg, respectively. The lowest possible sample weight for an accurate phosphorus analysis in protein samples by ICP-MS is discussed. The analytical method developed was applied for the analysis of homogeneous protein samples in very low amounts [1-100 micro g of solid protein sample, e.g. beta-casein or down to 1 micro L of protein or digest in solution (e.g., tau protein)]. A further reduction of the diluted protein solution volume was achieved by the application of flow injection in ICP-SFMS, which is discussed with reference to real protein digests after protein separation using 2D gel electrophoresis.The detection limits for phosphorus in biological samples were determined by ICP-SFMS down to the ng g(-1) level. The present work discusses the figure of merit for the determination of phosphorus in a small amount of protein sample with ICP-SFMS in comparison to ICP-QMS.
Tau mediates microtubule bundle architectures mimicking fascicles of microtubules found in the axon initial segment

DOE PAGES

Chung, Peter J.; Song, Chaeyeon; Deek, Joanna; ...

2016-07-25

Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent ‘active’ microtubule bundles at 37°C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance D w–w≈25–41nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (D w–w≈16–23nm) is revealedmore » under osmotic pressure. The wide spacing results from a balance between repulsive forces, due to Tau’s projection domain (PD), and a stabilizing sum of transient sub-k BT cationic/anionic charge–charge attractions mediated by weakly penetrating opposing PDs. In the end, we find that this landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration.« less
Microtubules and cellulose biosynthesis: the emergence of new players.

PubMed

Li, Shundai; Lei, Lei; Yingling, Yaroslava G; Gu, Ying

2015-12-01

Microtubules determine the orientation of newly formed cellulose microfibrils in expanding cells. There are many hypotheses regarding how the information is transduced across the plasma membrane from microtubules to cellulose microfibrils. However, the molecular mechanisms underlying the co-alignment between microtubules and cellulose microfibrils were not revealed until the recent discovery of cellulose synthase interacting (CSI) proteins. Characterization of CSIs and additional cellulose synthase-associated proteins will greatly advance the knowledge of how cellulose microfibrils are organized. Copyright © 2015 Elsevier Ltd. All rights reserved.
The feasibility of coherent energy transfer in microtubules.

PubMed

Craddock, Travis John Adrian; Friesen, Douglas; Mane, Jonathan; Hameroff, Stuart; Tuszynski, Jack A

2014-11-06

It was once purported that biological systems were far too 'warm and wet' to support quantum phenomena mainly owing to thermal effects disrupting quantum coherence. However, recent experimental results and theoretical analyses have shown that thermal energy may assist, rather than disrupt, quantum coherent transport, especially in the 'dry' hydrophobic interiors of biomolecules. Specifically, evidence has been accumulating for the necessary involvement of quantum coherent energy transfer between uniquely arranged chromophores in light harvesting photosynthetic complexes. The 'tubulin' subunit proteins, which comprise microtubules, also possess a distinct architecture of chromophores, namely aromatic amino acids, including tryptophan. The geometry and dipolar properties of these aromatics are similar to those found in photosynthetic units indicating that tubulin may support coherent energy transfer. Tubulin aggregated into microtubule geometric lattices may support such energy transfer, which could be important for biological signalling and communication essential to living processes. Here, we perform a computational investigation of energy transfer between chromophoric amino acids in tubulin via dipole excitations coupled to the surrounding thermal environment. We present the spatial structure and energetic properties of the tryptophan residues in the microtubule constituent protein tubulin. Plausibility arguments for the conditions favouring a quantum mechanism of signal propagation along a microtubule are provided. Overall, we find that coherent energy transfer in tubulin and microtubules is biologically feasible. © 2014 The Author(s) Published by the Royal Society. All rights reserved.
Effect of Bizhongxiao decoction and its dismantled formulae on IL-1 and TNF levels in collagen-induced arthritis in rat synovial joints

PubMed Central

2012-01-01

Background Rheumatoid arthritis (RA), a chronic autoimmune disease, affects sufferers in many different ways. Treatment of this chronic condition is particularly challenging. Traditional Chinese Medicine (TCM) provides alternatives. Bizhongxiao decoction (BZX) is a TCM complex, which has been used clinically for many years to treat RA. The purpose of this study is to compare the effects of BZX decoction and its dismantled formulae on IL-1 and TNF-1 levels in rats with RA, and to elucidate its mechanism of action. Methods Ninety healthy normal female SD rats were randomly divided into six groups: normal (control), model, BZX decoction, and the three dismantled formulae (I: heat-clearing and detoxication, II: dissipating dampness, and III: blood circulation promotion). Apart from the normal (control) group, the rats in each group were injected subcutaneously with bovine type II collagen and complete Freund adjuvant to establish a collagen-induced arthritis model, so that inhibition of foot swelling in the rats by BZX decoction and its dismantled formulae could be observed. Immunohistochemistry was used to assess the levels of the inflammatory cytokines IL-1 and TNF in synovial joints at various time points. Results Twenty-one days after the model was established, the levels of TNF and IL-1 were significantly higher in the model group, BZX decoction group and dismantled formula groups I, II and III than in the normal controls (P < 0.05). The levels of these cytokines were significantly higher in the model group than the BZX decoction or the three dismantled formula groups (P <0.01). At longer times, the TNF and IL-1 levels in model group rose gradually; those in the BZX decoction and dismantled formula groups were gradually reduced. The cytokine levels in the BZX decoction group were lower than in the three dismantled formula groups and continued to decline. Conclusions BZX decoction and the three dismantled formulae examined down-regulated the inflammatory
Microtubules are an intracellular target of the plant terpene citral.

PubMed

Chaimovitsh, David; Abu-Abied, Mohamad; Belausov, Eduard; Rubin, Baruch; Dudai, Nativ; Sadot, Einat

2010-02-01

Citral is a component of plant essential oils that possesses several biological activities. It has known medicinal traits, and is used as a food additive and in cosmetics. Citral has been suggested to have potential in weed management, but its precise mode of action at the cellular level is unknown. Here we investigated the immediate response of plant cells to citral at micromolar concentrations. It was found that microtubules of Arabidopsis seedlings were disrupted within minutes after exposure to citral in the gaseous phase, whereas actin filaments remained intact. The effect of citral on plant microtubules was both time- and dose-dependent, and recovery only occurred many hours after a short exposure of several minutes to citral. Citral was also able to disrupt animal microtubules, albeit less efficiently. In addition, polymerization of microtubules in vitro was inhibited in the presence of citral. Taken together, our results suggest that citral is a potent, volatile, anti-microtubule compound.
Single molecule studies reveal new mechanisms for microtubule severing

NASA Astrophysics Data System (ADS)

Ross, Jennifer; Diaz-Valencia, Juan Daniel; Morelli, Margaret; Zhang, Dong; Sharp, David

2011-03-01

Microtubule-severing enzymes are hexameric complexes made from monomeric enzyme subunits that remove tubulin dimers from the microtubule lattice. Severing proteins are known to remodel the cytoskeleton during interphase and mitosis, and are required in proper axon morphology and mammalian bone and cartilage development. We have performed the first single molecule imaging to determine where and how severing enzymes act to cut microtubules. We have focused on the original member of the group, katanin, and the newest member, fidgetin to compare their biophysical activities in vitro. We find that, as expected, severing proteins localize to areas of activity. Interestingly, the association is very brief: they do not stay bound nor do they bind cooperatively at active sites. The association duration changes with the nucleotide content, implying that the state in the catalytic cycle dictates binding affinity with the microtubule. We also discovered that, at lower concentrations, both katanin and fidgetin can depolymerize taxol-stabilized microtubules by removing terminal dimers. These studies reveal the physical regulation schemes to control severing activity in cells, and ultimately regulate cytoskeletal architecture. This work is supported by the March of Dimes Grant #5-FY09-46.
Catastrophic depolymerization of microtubules driven by subunit shape change

DOE PAGES

Bollinger, Jonathan A.; Stevens, Mark J.

2018-01-17

We report that microtubules exhibit a dynamic instability between growth and catastrophic depolymerization. GTP-tubulin (αβ-dimer bound to GTP) self-assembles, but dephosphorylation of GTP- to GDP-tubulin within the tubule results in destabilization. While the mechanical basis for destabilization is not fully understood, one hypothesis is that dephosphorylation causes tubulin to change shape, frustrating bonds and generating stress. To test this idea, we perform molecular dynamics simulations of microtubules built from coarse-grained models of tubulin, incorporating a small compression of α-subunits associated with dephosphorylation in experiments. We find that this shape change induces depolymerization of otherwise stable systems via unpeeling “ram's horns”more » characteristic of microtubules. Depolymerization can be averted by caps with uncompressed α-subunits, i.e., GTP-rich end regions. Thus, the shape change is sufficient to yield microtubule behavior.« less
Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

NASA Astrophysics Data System (ADS)

Gramlich, Michael W.; Conway, Leslie; Liang, Winnie H.; Labastide, Joelle A.; King, Stephen J.; Xu, Jing; Ross, Jennifer L.

2017-03-01

The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell’s control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo.

The taccalonolides and paclitaxel cause distinct effects on microtubule dynamics and aster formation

PubMed Central

2014-01-01

Background Microtubule stabilizers suppress microtubule dynamics and, at the lowest antiproliferative concentrations, disrupt the function of mitotic spindles, leading to mitotic arrest and apoptosis. At slightly higher concentrations, these agents cause the formation of multiple mitotic asters with distinct morphologies elicited by different microtubule stabilizers. Results We tested the hypothesis that two classes of microtubule stabilizing drugs, the taxanes and the taccalonolides, cause the formation of distinct aster structures due, in part, to differential effects on microtubule dynamics. Paclitaxel and the taccalonolides suppressed the dynamics of microtubules formed from purified tubulin as well as in live cells. Both agents suppressed microtubule dynamic instability, with the taccalonolides having a more pronounced inhibition of microtubule catastrophe, suggesting that they stabilize the plus ends of microtubules more effectively than paclitaxel. Live cell microscopy was also used to evaluate the formation and resolution of asters after drug treatment. While each drug had similar effects on initial formation, substantial differences were observed in aster resolution. Paclitaxel-induced asters often coalesced over time resulting in fewer, larger asters whereas numerous compact asters persisted once they were formed in the presence of the taccalonolides. Conclusions We conclude that the increased resistance of microtubule plus ends to catastrophe may play a role in the observed inability of taccalonolide-induced asters to coalesce during mitosis, giving rise to the distinct morphologies observed after exposure to these agents. PMID:24576146
Changes in microtubule stability and density in myelin-deficient shiverer mouse CNS axons

NASA Technical Reports Server (NTRS)

Kirkpatrick, L. L.; Witt, A. S.; Payne, H. R.; Shine, H. D.; Brady, S. T.

2001-01-01

Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelin-deficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.
Miro-Working beyond Mitochondria and Microtubules.

PubMed

Tang, Bor Luen

2018-03-04

The small GTPase Miro is best known for its regulation of mitochondrial movement by engaging with the microtubule-based motor proteins kinesin and dynein. Very recent findings have now showed that Miro also targets peroxisomes and regulates microtubule-dependent peroxisome motility. Moreover, Miro recruits and stabilizes the myosin motor Myo19 at the mitochondria to enable actin-based mitochondria movement, which is important for mitochondrial segregation during mitosis. Miro thus has much broader functions that previously known, and these new findings may have important implications on disease pathology.
Comparative evaluation of ICP sample introduction systems to be used in the metabolite profiling of chlorine-containing pharmaceuticals via HPLC-ICP-MS.

PubMed

Klencsár, Balázs; Sánchez, Carlos; Balcaen, Lieve; Todolí, José; Lynen, Frederic; Vanhaecke, Frank

2018-05-10

A systematic evaluation of four different ICP sample introduction systems to be used in the context of metabolite profiling of chlorine-containing pharmaceuticals via HPLC-ICP-MS was carried out using diclofenac and its major metabolite, 4'-hydroxy-diclofenac, as model compounds. The strict requirements for GMP validation of chromatographic methods in the pharmaceutical industry were adhered to in this context. The final aim of this investigation is an extension of the applicability and validatability of HPLC-ICP-MS in the field of pharmaceutical R&D. Five different gradient programmes were tested while the baseline peak width (w b ), peak capacity (P), USP tailing factor (A s ) and USP signal-to-noise ratio (USP S/N) were determined as major indicators of the chromatographic performance and the values obtained were compared to the corresponding FDA recommendations (if applicable). Four different ICP-MS sample introductions systems were investigated involving two units typically working at higher flow rates (∼1.0 mL min -1 ) and another two systems working at lower flow rates (∼0.1 mL min -1 ). Optimal conditions with potential for applicability under GMP conditions were found at a mobile phase flow rate of 1.0 mL min -1 by using a pneumatic micro-flow LC nebulizer mounted onto a Peltier-cooled cyclonic spray chamber cooled to -1 °C for sample introduction. Under these conditions, HPLC-ICP-MS provided a chromatographic performance similar to that of HPLC with UV detection. The peak shape (USP tailing factor = 1.1-1.4) was significantly improved compared to that obtained with the Peltier-cooled Scott-type spray chamber. Two alternative sample introduction systems - a POINT ® and a High-Temperature Torch-Integrated Sample Introduction System (hTISIS) - were also tested at a flow rate of 0.1 mL min -1 using a chromatographic column with 1.0 mm ID. Although these systems allowed the peak shape to be improved compared to that obtained with
The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis.

PubMed

Sánchez-Alcázar, José A; Rodríguez-Hernández, Angeles; Cordero, Mario D; Fernández-Ayala, Daniel J M; Brea-Calvo, Gloria; Garcia, Katherina; Navas, Plácido

2007-07-01

It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional gamma-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular "cocoon". Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis.
48 CFR 46.313 - Contracts for dismantling, demolition, or removal of improvements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Contracts for dismantling, demolition, or removal of improvements. 46.313 Section 46.313 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION CONTRACT MANAGEMENT QUALITY ASSURANCE Contract Clauses 46.313 Contracts for dismantling...
The influence of laser pulse duration and energy on ICP-MS signal intensity, elemental fractionation, and particle size distribution in NIR fs-LA-ICP-MS

PubMed Central

Diwakar, Prasoon K.; Harilal, Sivanandan S.; LaHaye, Nicole L.; Hassanein, Ahmed; Kulkarni, Pramod

2015-01-01

Laser parameters, typically wavelength, pulse width, irradiance, repetition rate, and pulse energy, are critical parameters which influence the laser ablation process and thereby influence the LA-ICP-MS signal. In recent times, femtosecond laser ablation has gained popularity owing to the reduction in fractionation related issues and improved analytical performance which can provide matrix-independent sampling. The advantage offered by fs-LA is due to shorter pulse duration of the laser as compared to the phonon relaxation time and heat diffusion time. Hence the thermal effects are minimized in fs-LA. Recently, fs-LA-ICP-MS demonstrated improved analytical performance as compared to ns-LA-ICP-MS, but detailed mechanisms and processes are still not clearly understood. Improvement of fs-LA-ICP-MS over ns-LA-ICP-MS elucidates the importance of laser pulse duration and related effects on the ablation process. In this study, we have investigated the influence of laser pulse width (40 fs to 0.3 ns) and energy on LA-ICP-MS signal intensity and repeatability using a brass sample. Experiments were performed in single spot ablation mode as well as rastering ablation mode to monitor the Cu/Zn ratio. The recorded ICP-MS signal was correlated with total particle counts generated during laser ablation as well as particle size distribution. Our results show the importance of pulse width effects in the fs regime that becomes more pronounced when moving from femtosecond to picosecond and nanosecond regimes. PMID:26664120
Spatial organization of xylem cell walls by ROP GTPases and microtubule-associated proteins.

PubMed

Oda, Yoshihisa; Fukuda, Hiroo

2013-12-01

Proper patterning of cellulosic cell walls is critical for cell shaping and differentiation of plant cells. Cortical microtubule arrays regulate the deposition patterns of cellulose microfibrils by controlling the targeting and trajectory of cellulose synthase complexes. Although some microtubule-associated proteins (MAPs) regulate the arrangement of cortical microtubules, knowledge about the overall mechanism governing the spacing of cortical microtubules is still limited. Recent studies reveal that ROP GTPases and MAPs spatially regulate the assembly and disassembly of cortical microtubules in developing xylem cells, in which localized secondary cell walls are deposited. Here, we review recent insights into the regulation of xylem cell wall patterning by cortical microtubules, ROP GTPases, and MAPs. Copyright © 2013 Elsevier Ltd. All rights reserved.
Multiscale polar theory of microtubule and motor-protein assemblies

DOE PAGES

Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; ...

2015-01-27

Microtubules and motor proteins are building blocks of self-organized subcellular biological structures such as the mitotic spindle and the centrosomal microtubule array. These same ingredients can form new “bioactive” liquid-crystalline fluids that are intrinsically out of equilibrium and which display complex flows and defect dynamics. It is not yet well understood how microscopic activity, which involves polarity-dependent interactions between motor proteins and microtubules, yields such larger-scale dynamical structures. In our multiscale theory, Brownian dynamics simulations of polar microtubule ensembles driven by cross-linking motors allow us to study microscopic organization and stresses. Polarity sorting and cross-link relaxation emerge as two polar-specificmore » sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. Finally, the results connect local polar structure to flow structures and defect dynamics.« less
Electrostatic differences: A possible source for the functional differences between MCF7 and brain microtubules.

PubMed

Feizabadi, Mitra Shojania; Rosario, Brandon; Hernandez, Marcos A V

2017-11-04

Recent studies suggested a link between diversity of beta tubulin isotypes in microtubule structures and the regulatory roles that they play not only on microtubules' intrinsic dynamic, but also on the translocation characteristics of some of the molecular motors along microtubules. Remarkably, unlike porcine brain microtubules, MCF7 microtubules are structured from a different beta tubulin distribution. These types of cancer microtubules show a relatively stable and slow dynamic. In addition, the translocation parameters of some molecular motors are distinctly different along MCF7 as compared to those parameters on brain microtubules. It is known that the diversity of beta tubulin isotypes differ predominantly in the specifications and the electric charge of their carboxy-terminal tails. A key question is to identify whether the negative electrostatic charge of tubulin isotypes and, consequently, microtubules, can potentially be considered as one of the sources of functional differences in MCF7 vs. brain microtubules. We tested this possibility experimentally by monitoring the electro-orientation of these two types of microtubules inside a uniform electric field. Through this evaluation, we quantified and compared the average normalized polarization coefficient of MCF7 vs. Porcine brain microtubules. The higher value obtained for the polarization of MCF7 microtubules, which is associated to the higher negative charge of these types of microtubules, is significant as it can further explain the slow intrinsic dynamic that has been recently reported for single MCF7 microtubules in vitro. Furthermore, it can be potentially considered as a factor that can directly impact the translocation parameters of some molecular motors along MCF7 microtubules, by altering the mutual electrostatic interactions between microtubules and molecular motors. Copyright © 2017 Elsevier Inc. All rights reserved.
[Myonuclear domain and microtubule proteome during skeletal muscle maturation].

PubMed

Couturier, Nathalie; Gache, Vincent

2017-11-01

In the normal course of muscle fiber development, myonuclei actively position and adapt a precise localization in mature fibers, shaping MyoNuclear Domains (MNDs). Myonuclei positioning in fibers appears to be essential for muscle function as defects in MNDs settings are always associated with dysfunction (i.e., centronuclear myopathy, sarcopenia). Previous studies have shown that myonuclei positioning in fibers is reversible, suggesting that in pathologies presenting MNDs impairment, myonuclei could be re-addressed to the "correct" position in fibers and this could benefit to muscle function. Cytoskeleton networks, and particularly microtubules, have been implicated in early nuclei localization in myotubes. As the microtubule network is completely redesigned during muscle maturation, we hypothesized that "microtubules associated proteomes" would change between immature and mature fibers and contribute to a microtubule-dependent process resulting in MNDs setting and maintenance in mature fibers. We performed an in vitro biochemical approach to isolate microtubules partners in immature (myotubes) and mature myofibers. Using mass-spectrometry identification, we selected 244 candidates, differentially associated/expressed with microtubules during myofiber maturation and potentially controlling MNDs settings. We are currently conducting a siRNA screen approach on these candidates to decipher their respective implication in early and late phases of MNDs establishment, using an unbiased assay developed by our team allowing statistical analysis of MNDs regarding myonuclei content. This approach will lead to the identification of new pathways related to nuclear positioning and MNDs setting in normal condition and in myopathies associated to MNDs impairment such as CNMs. © 2017 médecine/sciences – Inserm.
Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy

PubMed Central

Wang, Yan; Zheng, Xiyin; Yu, Bingjie; Han, Shaojie; Guo, Jiangbo; Tang, Haiping; Yu, Alice Yunzi L; Deng, Haiteng; Hong, Yiguo; Liu, Yule

2015-01-01

Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation. PMID:26566764
Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles

PubMed Central

Decker, Franziska; Oriola, David; Dalton, Benjamin

2018-01-01

Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting. PMID:29323637
The relative effect of citral on mitotic microtubules in wheat roots and BY2 cells.

PubMed

Chaimovitsh, D; Rogovoy Stelmakh, O; Altshuler, O; Belausov, E; Abu-Abied, M; Rubin, B; Sadot, E; Dudai, N

2012-03-01

The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with γ-tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral's effect on microtubules was both dose- and time-dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of γ-tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP-Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral's effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.
Microtubule behavior in the growth cones of living neurons during axon elongation

PubMed Central

1991-01-01

To understand how microtubules are generated in the growth cone, we have imaged fluorescently tagged microtubules in living frog embryonic neurons. The neurons were labeled by injecting rhodamine-labeled tubulin into the fertilized egg and explanting the neurons from the neural tube. Microtubules extend deep into the growth cone periphery and adopt three characteristic distributions: (a) dispersed and splayed throughout much of the growth cone; (b) looped and apparently contorted by compression; and (c) bundled into tight arrays. These distributions interconvert on a time scale of several minutes and these interconversions are correlated with the behavior of the growth cone. We observed microtubule growth and shrinkage in growth cones, but are unable to determine their contribution to net assembly. However, translocation of polymer form the axon appears to be a major mechanism of generating new polymer in the growth cone, while bundling of microtubules in the growth cone appears to be the critical step in generating new axon. Neurons that were about to turn spontaneously generated microtubules in the future direction of growth, suggesting that orientation of microtubules might be an important early step in neuronal pathfinding. PMID:1918145
Tetrahymena Poc1 ensures proper intertriplet microtubule linkages to maintain basal body integrity

PubMed Central

Meehl, Janet B.; Bayless, Brian A.; Giddings, Thomas H.; Pearson, Chad G.; Winey, Mark

2016-01-01

Basal bodies comprise nine symmetric triplet microtubules that anchor forces produced by the asymmetric beat pattern of motile cilia. The ciliopathy protein Poc1 stabilizes basal bodies through an unknown mechanism. In poc1∆ cells, electron tomography reveals subtle defects in the organization of intertriplet linkers (A-C linkers) that connect adjacent triplet microtubules. Complete triplet microtubules are lost preferentially near the posterior face of the basal body. Basal bodies that are missing triplets likely remain competent to assemble new basal bodies with nine triplet microtubules, suggesting that the mother basal body microtubule structure does not template the daughter. Our data indicate that Poc1 stabilizes basal body triplet microtubules through linkers between neighboring triplets. Without this stabilization, specific triplet microtubules within the basal body are more susceptible to loss, probably due to force distribution within the basal body during ciliary beating. This work provides insights into how the ciliopathy protein Poc1 maintains basal body integrity. PMID:27251062
Screening of TiO2 and Au nanoparticles in cosmetics and determination of elemental impurities by multiple techniques (DLS, SP-ICP-MS, ICP-MS and ICP-OES).

PubMed

de la Calle, Inmaculada; Menta, Mathieu; Klein, Marlène; Séby, Fabienne

2017-08-15

Cosmetics are part of the daily life of most of the people. Thus, a complete characterization of the products we applied in our skin is necessary. In this work, an analytical investigation of a wide variety of cosmetics from the point of view of total element content and metallic nanoparticles (NPs) has been performed. Firstly, we analyzed the total element content by ICP-MS and ICP-OES after acid digestion as an assessment of the presence of metal impurities. Prohibited elements in cosmetics, according to the European Commission regulation No 1223/2009, were not detected, and only elements mentioned in the label were found (e.g. Al, Fe, Ti and Si). Secondly, a screening of the presence of NPs has been performed by Dynamic Light Scattering (DLS) and Single Particle Inductively-Coupled Plasma Mass Spectrometry (SP-ICP-MS). Two sample preparation procedures were applied. The first protocol consisted in the preparation of suspensions in 0.1% w/v SDS and the second based on defatting with hexane followed by resuspension in water. DLS was employed as a routine method for a fast analysis of NPs, but this technique showed limitations due to the lack of specificity. SP-ICP-MS analyses were then performed, first as a screening technique to evaluate the presence of TiO 2 and Au NPs in cosmetics suspensions prepared in SDS; and second, when a positive answer was obtained about the presence of NPs from the screening, SP-ICP-MS was used for particle size determination. Results showed that only TiO 2 NPs were present in two sunscreens, one anti-wrinkle day cream, one lip balm protector labeled as 'nano' and in one brand of toothpaste not labeled as 'nano'. Sizes obtained for both sample preparations were compared and ranged from 30 to 120nm in most of the samples. Copyright © 2017 Elsevier B.V. All rights reserved.
Control of endothelial cell polarity and sprouting angiogenesis by non-centrosomal microtubules

PubMed Central

Martin, Maud; Veloso, Alexandra; Wu, Jingchao; Katrukha, Eugene A

2018-01-01

Microtubules control different aspects of cell polarization. In cells with a radial microtubule system, a pivotal role in setting up asymmetry is attributed to the relative positioning of the centrosome and the nucleus. Here, we show that centrosome loss had no effect on the ability of endothelial cells to polarize and move in 2D and 3D environments. In contrast, non-centrosomal microtubules stabilized by the microtubule minus-end-binding protein CAMSAP2 were required for directional migration on 2D substrates and for the establishment of polarized cell morphology in soft 3D matrices. CAMSAP2 was also important for persistent endothelial cell sprouting during in vivo zebrafish vessel development. In the absence of CAMSAP2, cell polarization in 3D could be partly rescued by centrosome depletion, indicating that in these conditions the centrosome inhibited cell polarity. We propose that CAMSAP2-protected non-centrosomal microtubules are needed for establishing cell asymmetry by enabling microtubule enrichment in a single-cell protrusion. PMID:29547120
A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends

PubMed Central

Kern, David M.; Nicholls, Peter K.; Page, David C.

2016-01-01

The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning. PMID:27138257
Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles.

PubMed

Decker, Franziska; Oriola, David; Dalton, Benjamin; Brugués, Jan

2018-01-11

Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting. © 2018, Decker et al.

Chemical oxygen-iodine laser (COIL) for the dismantlement of nuclear facilities

NASA Astrophysics Data System (ADS)

Hallada, Marc R.; Seiffert, Stephan L.; Walter, Robert F.; Vetrovec, John

2000-05-01

The dismantlement of obsolete nuclear facilities is a major challenge for both the US Department of Energy and nuclear power utilities. Recent demonstrations have shown that lasers can be highly effective for size reduction cutting, especially for the efficient storage and recycling of materials. However, the full benefits of lasers can only be realized with high average power beams that can be conveniently delivered, via fiber optics, to remote and/or confined areas. Industrial lasers that can meet these requirements are not available now or for the foreseeable future. However, a military weapon laser, a Chemical Oxygen Iodine Laser (COIL), which has been demonstrated at over a hundred kilo Watts, could be adapted to meet these needs and enable entirely new industrial applications. An 'industrialized' COIL would enable rapid sectioning of thick and complex structures, such as glove boxes, reactor vessels, and steam generators, accelerating dismantlement schedules and reducing worker hazards. The full advantages of lasers in dismantlement could finally be realized with a portable COIL which is integrated with sophisticated robotics. It could be built and deployed in less than two years, breaking the paradigm of labor-intensive dismantlement operations and cutting processing times and costs dramatically.
GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

PubMed

Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

2016-06-01

Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. Copyright © 2016 Elsevier B.V. All rights reserved.
Measuring and modeling polymer concentration profiles near spindle boundaries argues that spindle microtubules regulate their own nucleation

NASA Astrophysics Data System (ADS)

Kaye, Bryan; Stiehl, Olivia; Foster, Peter J.; Shelley, Michael J.; Needleman, Daniel J.; Fürthauer, Sebastian

2018-05-01

Spindles are self-organized microtubule-based structures that segregate chromosomes during cell division. The mass of the spindle is controlled by the balance between microtubule turnover and nucleation. The mechanisms that control the spatial regulation of microtubule nucleation remain poorly understood. While previous work found that microtubule nucleators bind to pre-existing microtubules in the spindle, it is still unclear whether this binding regulates the activity of those nucleators. Here we use a combination of experiments and mathematical modeling to investigate this issue. We measured the concentration of microtubules and soluble tubulin in and around the spindle. We found a very sharp decay in the concentration of microtubules at the spindle interface. This is inconsistent with a model in which the activity of nucleators is independent of their association with microtubules but consistent with a model in which microtubule nucleators are only active when bound to pre-existing microtubules. This argues that the activity of microtubule nucleators is greatly enhanced when bound to pre-existing microtubules. Thus, microtubule nucleators are both localized and activated by the microtubules they generate.
Polymerization of the tubulin-colchicine complex: relation to microtubule assembly.

PubMed

Andreu, J M; Wagenknecht, T; Timasheff, S N

1983-03-29

The polymerization of purified tubulin-colchicine complex, which results in polymers different from microtubules under microtubule-promoting conditions, has been characterized. It proceeds as a nucleated condensation polymerization, requires Mg2+, and is inhibited by small concentrations of Ca2+. Polymerization requires GTP binding, but GDP is inhibitory. The GTPase activity proceeds, but it is unlinked to polymerization. The thermodynamic characteristics of the growth reaction, namely, the apparent changes of free energy, enthalpy, entropy, heat capacity, and preferential interaction with H+ and Mg2+, are very similar to those of microtubule assembly. It is proposed that the interactions responsible for the two types of polymerization are very similar and that the molecular mechanism of microtubule inhibition by colchicine may consist in a drug-induced distortion of the normal protomer bonding geometry.
ICPS Turnover GSDO Employee Event

NASA Image and Video Library

2017-11-07

Kennedy Space Center Associate Director Kelvin Manning, right, speaks with a guest during a ceremony marking NASA's Spacecraft/Payload Integration and Evolution (SPIE) organization formally turning over processing of the Space Launch System (SLS) rocket's Interim Cryogenic Propulsion Stage (ICPS) to the center's Ground Systems Development and Operations (GSDO) Directorate. The ICPS is the first integrated piece of flight hardware to arrive in preparation for the uncrewed Exploration Mission-1. With the Orion attached, the ICPS sits atop the SLS rocket and will provide the spacecraft with the additional thrust needed to travel tens of thousands of miles beyond the Moon.
Brassinosteroids regulate pavement cell growth by mediating BIN2-induced microtubule stabilization.

PubMed

Liu, Xiaolei; Yang, Qin; Wang, Yuan; Wang, Linhai; Fu, Ying; Wang, Xuelu

2018-02-23

Brassinosteroids (BRs), a group of plant steroid hormones, play important roles in regulating plant development. The cytoskeleton also affects key developmental processes and a deficiency in BR biosynthesis or signaling leads to abnormal phenotypes similar to those of microtubule-defective mutants. However, how BRs regulate microtubule and cell morphology remains unknown. Here, using liquid chromatography-tandem mass spectrometry, we identified tubulin proteins that interact with Arabidopsis BRASSINOSTEROID INSENSITIVE2 (BIN2), a negative regulator of BR responses in plants. In vitro and in vivo pull-down assays confirmed that BIN2 interacts with tubulin proteins. High-speed co-sedimentation assays demonstrated that BIN2 also binds microtubules. The Arabidopsis genome also encodes two BIN2 homologs, BIN2-LIKE 1 (BIL1) and BIL2, which function redundantly with BIN2. In the bin2-3 bil1 bil2 triple mutant, cortical microtubules were more sensitive to treatment with the microtubule-disrupting drug oryzalin than in wild-type, whereas in the BIN2 gain-of-function mutant bin2-1, cortical microtubules were insensitive to oryzalin treatment. These results provide important insight into how BR regulates plant pavement cell and leaf growth by mediating the stabilization of microtubules by BIN2.
Know History, Know Self: Art Therapists' Responsibility to Dismantle White Supremacy

ERIC Educational Resources Information Center

Hamrick, Cassie; Byma, Christine

2017-01-01

In this article, we call on white art therapists to assume responsibility for dismantling white supremacy in the field of art therapy, in personal as well as political-structural arenas. We respond to calls from scholars and writers of color for white people to assume increased responsibility for dismantling white supremacy in white communities…
Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

PubMed Central

Roostalu, Johanna; Cade, Nicholas I.; Surrey, Thomas

2016-01-01

Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilises growing microtubule ends and stimulates microtubule nucleation by stabilising early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homolog) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking TPX2’s interaction with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells. PMID:26414402
Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module.

PubMed

Roostalu, Johanna; Cade, Nicholas I; Surrey, Thomas

2015-11-01

Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilizes growing microtubule ends and stimulates microtubule nucleation by stabilizing early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homologue) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking the interaction of TPX2 with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells.
Regulation of microtubule dynamic instability by the carboxy-terminal tail of β-tubulin

PubMed Central

Fees, Colby P; Moore, Jeffrey K

2018-01-01

Dynamic instability is an intrinsic property of microtubules; however, we do not understand what domains of αβ-tubulins regulate this activity or how these regulate microtubule networks in cells. Here, we define a role for the negatively charged carboxy-terminal tail (CTT) domain of β-tubulin in regulating dynamic instability. By combining in vitro studies with purified mammalian tubulin and in vivo studies with tubulin mutants in budding yeast, we demonstrate that β-tubulin CTT inhibits microtubule stability and regulates the structure and stability of microtubule plus ends. Tubulin that lacks β-tubulin CTT polymerizes faster and depolymerizes slower in vitro and forms microtubules that are more prone to catastrophe. The ends of these microtubules exhibit a more blunted morphology and rapidly switch to disassembly after tubulin depletion. In addition, we show that β-tubulin CTT is required for magnesium cations to promote depolymerization. We propose that β-tubulin CTT regulates the assembly of stable microtubule ends and provides a tunable mechanism to coordinate dynamic instability with ionic strength in the cell.
Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation

PubMed Central

Foe, Victoria E.; von Dassow, George

2008-01-01

The cytokinetic furrow arises from spatial and temporal regulation of cortical contractility. To test the role microtubules play in furrow specification, we studied myosin II activation in echinoderm zygotes by assessing serine19-phosphorylated regulatory light chain (pRLC) localization after precisely timed drug treatments. Cortical pRLC was globally depressed before cytokinesis, then elevated only at the equator. We implicated cell cycle biochemistry (not microtubules) in pRLC depression, and differential microtubule stability in localizing the subsequent myosin activation. With no microtubules, pRLC accumulation occurred globally instead of equatorially, and loss of just dynamic microtubules increased equatorial pRLC recruitment. Nocodazole treatment revealed a population of stable astral microtubules that formed during anaphase; among these, those aimed toward the equator grew longer, and their tips coincided with cortical pRLC accumulation. Shrinking the mitotic apparatus with colchicine revealed pRLC suppression near dynamic microtubule arrays. We conclude that opposite effects of stable versus dynamic microtubules focuses myosin activation to the cell equator during cytokinesis. PMID:18955555
SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

PubMed Central

Raff, Rudolf A.; Greenhouse, Gerald; Gross, Kenneth W.; Gross, Paul R.

1971-01-01

Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-3H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified. PMID:5165266
Molecular sorting by electrical steering of microtubules in kinesin-coated channels.

PubMed

van den Heuvel, Martin G L; de Graaff, Martijn P; Dekker, Cees

2006-05-12

Integration of biomolecular motors in nanoengineered structures raises the intriguing possibility of manipulating materials on nanometer scales. We have managed to integrate kinesin motor proteins in closed submicron channels and to realize active electrical control of the direction of individual kinesin-propelled microtubule filaments at Y junctions. Using this technique, we demonstrate molecular sorting of differently labeled microtubules. We attribute the steering of microtubules to electric field-induced bending of the leading tip. From measurements of the orientation-dependent electrophoretic motion of individual, freely suspended microtubules, we estimate the net applied force on the tip to be in the picoNewton range and we infer an effective charge of 12 e- per tubulin dimer under physiological conditions.
48 CFR 52.249-3 - Termination for Convenience of the Government (Dismantling, Demolition, or Removal of Improvements).

Code of Federal Regulations, 2010 CFR

2010-10-01

... Convenience of the Government (Dismantling, Demolition, or Removal of Improvements). 52.249-3 Section 52.249-3... Convenience of the Government (Dismantling, Demolition, or Removal of Improvements). As prescribed in 49.502(b)(2), insert the following clause: Termination for Convenience of the Government (Dismantling...
A Case for Microtubule Vulnerability in Amyotrophic Lateral Sclerosis: Altered Dynamics During Disease.

PubMed

Clark, Jayden A; Yeaman, Elise J; Blizzard, Catherine A; Chuckowree, Jyoti A; Dickson, Tracey C

2016-01-01

Amyotrophic lateral sclerosis (ALS) is an aggressive multifactorial disease converging on a common pathology: the degeneration of motor neurons (MNs), their axons and neuromuscular synapses. This vulnerability and dysfunction of MNs highlights the dependency of these large cells on their intracellular machinery. Neuronal microtubules (MTs) are intracellular structures that facilitate a myriad of vital neuronal functions, including activity dependent axonal transport. In ALS, it is becoming increasingly apparent that MTs are likely to be a critical component of this disease. Not only are disruptions in this intracellular machinery present in the vast majority of seemingly sporadic cases, recent research has revealed that mutation to a microtubule protein, the tubulin isoform TUBA4A, is sufficient to cause a familial, albeit rare, form of disease. In both sporadic and familial disease, studies have provided evidence that microtubule mediated deficits in axonal transport are the tipping point for MN survivability. Axonal transport deficits would lead to abnormal mitochondrial recycling, decreased vesicle and mRNA transport and limited signaling of key survival factors from the neurons peripheral synapses, causing the characteristic peripheral "die back". This disruption to microtubule dependant transport in ALS has been shown to result from alterations in the phenomenon of microtubule dynamic instability: the rapid growth and shrinkage of microtubule polymers. This is accomplished primarily due to aberrant alterations to microtubule associated proteins (MAPs) that regulate microtubule stability. Indeed, the current literature would argue that microtubule stability, particularly alterations in their dynamics, may be the initial driving force behind many familial and sporadic insults in ALS. Pharmacological stabilization of the microtubule network offers an attractive therapeutic strategy in ALS; indeed it has shown promise in many neurological disorders, ALS included
Titanium dioxide nanoparticles alter cellular morphology via disturbing the microtubule dynamics

NASA Astrophysics Data System (ADS)

Mao, Zhilei; Xu, Bo; Ji, Xiaoli; Zhou, Kun; Zhang, Xuemei; Chen, Minjian; Han, Xiumei; Tang, Qiusha; Wang, Xinru; Xia, Yankai

2015-04-01

Titanium dioxide (TiO2) nanoparticles (NPs) have been widely used in our daily lives, for example, in the areas of sunscreens, cosmetics, toothpastes, food products, and nanomedical reagents. Recently, increasing concern has been raised about their neurotoxicity, but the mechanisms underlying such toxic effects are still unknown. In this work, we employed a human neuroblastoma cell line (SH-SY5Y) to study the effects of TiO2 NPs on neurological systems. Our results showed that TiO2 NPs did not affect cell viability but induced noticeable morphological changes until 100 μg ml-1. Immunofluorescence detection showed disorder, disruption, retraction, and decreased intensity of the microtubules after TiO2 NPs treatment. Both α and β tubule expressions did not change in the TiO2 NP-treated group, but the percentage of soluble tubules was increased. A microtubule dynamic study in living cells indicated that TiO2 NPs caused a lower growth rate and a higher shortening rate of microtubules as well as shortened lifetimes of de novo microtubules. TiO2 NPs did not cause changes in the expression and phosphorylation state of tau proteins, but a tau-TiO2 NP interaction was observed. TiO2 NPs could interact with tubule heterodimers, microtubules and tau proteins, which led to the instability of microtubules, thus contributing to the neurotoxicity of TiO2 NPs.Titanium dioxide (TiO2) nanoparticles (NPs) have been widely used in our daily lives, for example, in the areas of sunscreens, cosmetics, toothpastes, food products, and nanomedical reagents. Recently, increasing concern has been raised about their neurotoxicity, but the mechanisms underlying such toxic effects are still unknown. In this work, we employed a human neuroblastoma cell line (SH-SY5Y) to study the effects of TiO2 NPs on neurological systems. Our results showed that TiO2 NPs did not affect cell viability but induced noticeable morphological changes until 100 μg ml-1. Immunofluorescence detection showed disorder
Dissecting the molecular mechanism underlying the intimate relationship between cellulose microfibrils and cortical microtubules.

PubMed

Lei, Lei; Li, Shundai; Bashline, Logan; Gu, Ying

2014-01-01

A central question in plant cell development is how the cell wall determines directional cell expansion and therefore the final shape of the cell. As the major load-bearing component of the cell wall, cellulose microfibrils are laid down transversely to the axis of elongation, thus forming a spring-like structure that reinforces the cell laterally and while favoring longitudinal expansion in most growing cells. Mounting evidence suggests that cortical microtubules organize the deposition of cellulose microfibrils, but the precise molecular mechanisms linking microtubules to cellulose organization have remained unclear until the recent discovery of cellulose synthase interactive protein 1 , a linker protein between the cortical microtubules and the cellulose biosynthesizing machinery. In this review, we will focus on the intimate relationship between cellulose microfibrils and cortical microtubules, in particular, we will discuss microtubule arrangement and cell wall architecture, the linkage between cellulose synthase complexes and microtubules, and the feedback mechanisms between cell wall and microtubules.
Inhibition of beta-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules.

PubMed

Cheng, Guangmao; Qiao, Fei; Gallien, Thomas N; Kuppuswamy, Dhandapani; Cooper, George

2005-03-01

Decreased beta-adrenergic receptor (beta-AR) number occurs both in animal models of cardiac hypertrophy and failure and in patients. beta-AR recycling is an important mechanism for the beta-AR resensitization that maintains a normal complement of cell surface beta-ARs. We have shown that 1) in severe pressure overload cardiac hypertrophy, there is extensive microtubule-associated protein 4 (MAP4) decoration of a dense microtubule network; and 2) MAP4 microtubule decoration inhibits muscarinic acetylcholine receptor recycling in neuroblastoma cells. We asked here whether MAP4 microtubule decoration inhibits beta-AR recycling in adult cardiocytes. [(3)H]CGP-12177 was used as a beta-AR ligand, and feline cardiocytes were isolated and infected with adenovirus containing MAP4 (AdMAP4) or beta-galactosidase (Adbeta-gal) cDNA. MAP4 decorated the microtubules extensively only in AdMAP4 cardiocytes. beta-AR agonist exposure reduced cell surface beta-AR number comparably in AdMAP4 and Adbeta-gal cardiocytes; however, after agonist withdrawal, the cell surface beta-AR number recovered to 78.4 +/- 2.9% of the pretreatment value in Adbeta-gal cardiocytes but only to 56.8 +/- 1.4% in AdMAP4 cardiocytes (P < 0.01). This result was confirmed in cardiocytes isolated from transgenic mice having cardiac-restricted MAP4 overexpression. In functional terms of cAMP generation, beta-AR agonist responsiveness of AdMAP4 cells was 47% less than that of Adbeta-gal cells. We conclude that MAP4 microtubule decoration interferes with beta-AR recycling and that this may be one mechanism for beta-AR downregulation in heart failure.
Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling.

PubMed

Sacristan, Carlos; Kops, Geert J P L

2015-01-01

Error-free chromosome segregation relies on stable connections between kinetochores and spindle microtubules. The spindle assembly checkpoint (SAC) monitors such connections and relays their absence to the cell cycle machinery to delay cell division. The molecular network at kinetochores that is responsible for microtubule binding is integrated with the core components of the SAC signaling system. Molecular-mechanistic understanding of how the SAC is coupled to the kinetochore-microtubule interface has advanced significantly in recent years. The latest insights not only provide a striking view of the dynamics and regulation of SAC signaling events at the outer kinetochore but also create a framework for understanding how that signaling may be terminated when kinetochores and microtubules connect. Copyright © 2014 Elsevier Ltd. All rights reserved.
Regulators of spindle microtubules and their mechanisms: Living together matters.

PubMed

Lakshmi, R Bhagya; Nair, Vishnu M; Manna, Tapas K

2018-02-01

Development and survival of all eukaryotic organisms depend on equal partitioning of their chromosomes between the two newly formed daughter cells during mitosis. The mitotic spindle performs the task of physically segregating the chromosomes through multiple stages of mitosis. During this process, kinetochore-microtubule attachment requires to be selectively stabilized to hold the chromosomes, but at the same time, it has to be flexible enough to allow kinetochore microtubule dynamicity and chromosome movements. Research during the last decade or so has identified a number of proteins associated with the spindle microtubule plus ends that regulate these processes and orchestrate forces to spatially organize and separate the chromosomes. In this review, we describe the molecular details of those regulators and their mechanisms of action at the kinetochore-microtubule interface. © 2018 IUBMB Life, 70(2):101-111, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

EB1 contributes to microtubule bundling and organization, along with root growth, in Arabidopsis thaliana.

PubMed

Molines, Arthur T; Marion, Jessica; Chabout, Salem; Besse, Laetitia; Dompierre, Jim P; Mouille, Grégory; Coquelle, Frédéric M

2018-06-26

Microtubules are involved in plant development and adaptation to their environment, but the sustaining molecular mechanisms remain elusive. Microtubule-End-Binding 1 (EB1) proteins participate in directional root growth in Arabidopsis thaliana. However, a connection to the underlying microtubule array has not been established yet. We show here that EB1 proteins contribute to the organization of cortical microtubules in growing epidermal plant cells, without significant modulation of microtubule dynamics. Using super-resolution STED microscopy and an original quantification approach, we also demonstrate a significant reduction of apparent microtubule bundling in cytoplasmic-EB1-deficient plants, suggesting a function for EB1 in the interaction between adjacent microtubules. Furthermore, we observed root growth defects in EB1-deficient plants, which are not related to cell division impairment. Altogether, our results support a role for EB1 proteins in root development, in part by maintaining the organization of cortical microtubules. © 2018. Published by The Company of Biologists Ltd.
NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

PubMed Central

Harkcom, William T.; Ghosh, Ananda K.; Sung, Matthew S.; Matov, Alexandre; Brown, Kevin D.; Giannakakou, Paraskevi; Jaffrey, Samie R.

2014-01-01

Nicotinamide adenine dinucleotide (NAD+) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD+. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD+ levels. We find that these effects of NAD+ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD+ on microtubule polymers. Taken together, these data demonstrate that NAD+ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606
Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation.

PubMed

Casenghi, Martina; Meraldi, Patrick; Weinhart, Ulrike; Duncan, Peter I; Körner, Roman; Nigg, Erich A

2003-07-01

In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.
Kinesin-8 Motors Improve Nuclear Centering by Promoting Microtubule Catastrophe

NASA Astrophysics Data System (ADS)

Glunčić, Matko; Maghelli, Nicola; Krull, Alexander; Krstić, Vladimir; Ramunno-Johnson, Damien; Pavin, Nenad; Tolić, Iva M.

2015-02-01

In fission yeast, microtubules push against the cell edge, thereby positioning the nucleus in the cell center. Kinesin-8 motors regulate microtubule catastrophe; however, their role in nuclear positioning is not known. Here we develop a physical model that describes how kinesin-8 motors affect nuclear centering by promoting a microtubule catastrophe. Our model predicts the improved centering of the nucleus in the presence of motors, which we confirmed experimentally in living cells. The model also predicts a characteristic time for the recentering of a displaced nucleus, which is supported by our experiments where we displaced the nucleus using optical tweezers.
Structural Basis for Induction of Peripheral Neuropathy by Microtubule-Targeting Cancer Drugs.

PubMed

Smith, Jennifer A; Slusher, Barbara S; Wozniak, Krystyna M; Farah, Mohamed H; Smiyun, Gregoriy; Wilson, Leslie; Feinstein, Stuart; Jordan, Mary Ann

2016-09-01

Peripheral neuropathy is a serious, dose-limiting side effect of cancer treatment with microtubule-targeting drugs. Symptoms present in a "stocking-glove" distribution, with longest nerves affected most acutely, suggesting a length-dependent component to the toxicity. Axonal transport of ATP-producing mitochondria along neuronal microtubules from cell body to synapse is crucial to neuronal function. We compared the effects of the drugs paclitaxel and ixabepilone that bind along the lengths of microtubules and the drugs eribulin and vincristine that bind at microtubule ends, on mitochondrial trafficking in cultured human neuronal SK-N-SH cells and on axonal transport in mouse sciatic nerves. Antiproliferative concentrations of paclitaxel and ixabepilone significantly inhibited the anterograde transport velocity of mitochondria in neuronal cells, whereas eribulin and vincristine inhibited transport only at significantly higher concentrations. Confirming these observations, anterogradely transported amyloid precursor protein accumulated in ligated sciatic nerves of control and eribulin-treated mice, but not in paclitaxel-treated mice, indicating that paclitaxel inhibited anterograde axonal transport, whereas eribulin did not. Electron microscopy of sciatic nerves of paclitaxel-treated mice showed reduced organelle accumulation proximal to the ligation consistent with inhibition of anterograde (kinesin based) transport by paclitaxel. In contrast, none of the drugs significantly affected retrograde (dynein based) transport in neuronal cells or mouse nerves. Collectively, these results suggest that paclitaxel and ixabepilone, which bind along the lengths and stabilize microtubules, inhibit kinesin-based axonal transport, but not dynein-based transport, whereas the microtubule-destabilizing drugs, eribulin and vincristine, which bind preferentially to microtubule ends, have significantly less effect on all microtubule-based axonal transport. Cancer Res; 76(17); 5115-23. �
Mechanochemical Modeling of Dynamic Microtubule Growth Involving Sheet-to-Tube Transition

PubMed Central

Ji, Xiang-Ying; Feng, Xi-Qiao

2011-01-01

Microtubule dynamics is largely influenced by nucleotide hydrolysis and the resultant tubulin configuration changes. The GTP cap model has been proposed to interpret the stabilizing mechanisms of microtubule growth from the view of hydrolysis effects. Besides, the growth of a microtubule involves the closure of a curved sheet at its growing end. The curvature conversion from the longitudinal direction to the circumferential direction also helps to stabilize the successive growth, and the curved sheet is referred to as the conformational cap. However, there still lacks theoretical investigation on the mechanical–chemical coupling growth process of microtubules. In this paper, we study the growth mechanisms of microtubules by using a coarse-grained molecular method. First, the closure process involving a sheet-to-tube transition is simulated. The results verify the stabilizing effect of the sheet structure and predict that the minimum conformational cap length that can stabilize the growth is two dimers. Then, we show that the conformational cap and the GTP cap can function independently and harmoniously, signifying the pivotal role of mechanical factors. Furthermore, based on our theoretical results, we describe a Tetris-like growth style of microtubules: the stochastic tubulin assembly is regulated by energy and harmonized with the seam zipping such that the sheet keeps a practically constant length during growth. PMID:22205994
Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons

PubMed Central

Janning, Dennis; Igaev, Maxim; Sündermann, Frederik; Brühmann, Jörg; Beutel, Oliver; Heinisch, Jürgen J.; Bakota, Lidia; Piehler, Jacob; Junge, Wolfgang; Brandt, Roland

2014-01-01

The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP–tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin–microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport. PMID:25165145
Stockpile Dismantlement Database Training Materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1993-11-01

This document, the Stockpile Dismantlement Database (SDDB) training materials is designed to familiarize the user with the SDDB windowing system and the data entry steps for Component Characterization for Disposition. The foundation of information required for every part is depicted by using numbered graphic and text steps. The individual entering data is lead step by step through generic and specific examples. These training materials are intended to be supplements to individual on-the-job training.
ICPS Turnover GSDO Employee Event

NASA Image and Video Library

2017-11-07

In the Space Station Processing Facility at NASA's Kennedy Space Center in Florida, a ceremony is underway marking the agency's Spacecraft/Payload Integration and Evolution (SPIE) organization formally turning over processing of the Space Launch System (SLS) rocket's Interim Cryogenic Propulsion Stage (ICPS), to the center's Ground Systems Development and Operations (GSDO) Directorate. The ICPS is seen on the left in its shipping container and is the first integrated piece of flight hardware to arrive in preparation for the uncrewed Exploration Mission-1. With the Orion attached, the ICPS sits atop the SLS rocket and will provide the spacecraft with the additional thrust needed to travel tens of thousands of miles beyond the Moon.
Computational Study of Pseudo-phosphorylation of the Microtubule associated Protein Tau

NASA Astrophysics Data System (ADS)

Prokopovich, Dmitriy; Larini, Luca

This computational study focuses on the effect of pseudo-phosphorylation on the aggregation of the microtubule associated protein tau. In the axon of the neuron, tau regulates the assembly of microtubules in the cytoskeleton. This is important for both stabilization of and transport across the microtubules. One of the hallmarks of the Alzheimer's disease is that tau is hyper-phosphorylated and aggregates into neurofibrillary tangles that lay waste to the neurons. It is not known if hyper-phosphorylation directly causes the aggregation of tau into tangles. Experimentally, pseudo-phosphorylation mimics the effects of phosphorylation by mutating certain residues of the protein chain into charged residues. In this study, we will consider the fragment called PHF43 that belongs to the microtubule binding region and has been shown to readily aggregate.
A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation

PubMed Central

Mogessie, Binyam; Roth, Daniel; Rahil, Zainab; Straube, Anne

2015-01-01

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell–cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule–microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. DOI: http://dx.doi.org/10.7554/eLife.05697.001 PMID:25898002
Polychlorinated biphenyls in the atmosphere of Taizhou, a major e-waste dismantling area in China.

PubMed

Han, Wenliang; Feng, Jialiang; Gu, Zeping; Wu, Minghong; Sheng, Guoying; Fu, Jiamo

2010-01-01

PM2.5, total suspended particles (TSP) and gas phase samples were collected at two sites of Taizhou, a major e-waste dismantling area in China. Concentrations, seasonal variations, congener profiles, gas-particle partitioning and size distribution of the atmospheric polychlorinated biphenyls (PCBs) were studied to assess the current state of atmospheric PCBs after the phase out of massive historical dismantling of PCBs containing e-wastes. The average sigma38PCBs concentration in the ambient air (TSP plus gas phase) near the e-waste dismantling area was (12,407 +/- 9592) pg/m3 in winter, which was substantially lower than that found one decade ago. However, the atmospheric PCBs level near the e-waste dismantling area was 54 times of the reference urban site, indicating that the impact of the historical dismantling of PCBs containing e-wastes was still significant. Tri-Penta-CBs were dominant homologues, consisting with their dominant global production. Size distribution of particle-bound PCBs showed that higher chlorinated CBs tended to partition more to the fine particles, facilitating its long range air transportation.
Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos

NASA Technical Reports Server (NTRS)

Danilchik, Michael V.; Bedrick, Steven D.; Brown, Elizabeth E.; Ray, Kimberly

2003-01-01

In dividing Xenopus eggs, furrowing is accompanied by expansion of a new domain of plasma membrane in the cleavage plane. The source of the new membrane is known to include a store of oogenetically produced exocytotic vesicles, but the site where their exocytosis occurs has not been described. Previous work revealed a V-shaped array of microtubule bundles at the base of advancing furrows. Cold shock or exposure to nocodazole halted expansion of the new membrane domain, which suggests that these microtubules are involved in the localized exocytosis. In the present report, scanning electron microscopy revealed collections of pits or craters, up to approximately 1.5 micro m in diameter. These pits are evidently fusion pores at sites of recent exocytosis, clustered in the immediate vicinity of the deepening furrow base and therefore near the furrow microtubules. Confocal microscopy near the furrow base of live embryos labeled with the membrane dye FM1-43 captured time-lapse sequences of individual exocytotic events in which irregular patches of approximately 20 micro m(2) of unlabeled membrane abruptly displaced pre-existing FM1-43-labeled surface. In some cases, stable fusion pores, approximately 2 micro m in diameter, were seen at the surface for up to several minutes before suddenly delivering patches of unlabeled membrane. To test whether the presence of furrow microtubule bundles near the surface plays a role in directing or concentrating this localized exocytosis, membrane expansion was examined in embryos exposed to D(2)O to induce formation of microtubule monasters randomly under the surface. D(2)O treatment resulted in a rapid, uniform expansion of the egg surface via random, ectopic exocytosis of vesicles. This D(2)O-induced membrane expansion was completely blocked with nocodazole, indicating that the ectopic exocytosis was microtubule-dependent. Results indicate that exocytotic vesicles are present throughout the egg subcortex, and that the presence of
The natural naphthoquinone plumbagin exhibits antiproliferative activity and disrupts the microtubule network through tubulin binding.

PubMed

Acharya, Bipul R; Bhattacharyya, Bhabatarak; Chakrabarti, Gopal

2008-07-29

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.
Cold Trap Dismantling and Sodium Removal at a Fast Breeder Reactor - 12327

DOE Office of Scientific and Technical Information (OSTI.GOV)

Graf, A.; Petrick, H.; Stutz, U.

2012-07-01

The first German prototype Fast Breeder Nuclear Reactor (KNK) is currently being dismantled after being the only operating Fast Breeder-type reactor in Germany. As this reactor type used sodium as a coolant in its primary and secondary circuit, seven cold traps containing various amounts of partially activated sodium needed to be disposed of as part of the dismantling. The resulting combined difficulties of radioactive contamination and high chemical reactivity were handled by treating the cold traps differently depending on their size and the amount of sodium contained inside. Six small cold traps were processed onsite by cutting them up intomore » small parts using a band saw under a protective atmosphere. The sodium was then converted to sodium hydroxide by using water. The remaining large cold trap could not be handled in the same way due to its dimensions (2.9 m x 1.1 m) and the declared amount of sodium inside (1,700 kg). It was therefore manually dismantled inside a large box filled with a protective atmosphere, while the resulting pieces were packaged for later burning in a special facility. The experiences gained by KNK during this process may be advantageous for future dismantling projects in similar sodium-cooled reactors worldwide. The dismantling of a prototype fast breeder reactor provides the challenge not only to dismantle radioactive materials but also to handle sodium-contaminated or sodium-containing components. The treatment of sodium requires additional equipment and installations to ensure a safe handling. Since it is not permitted to bring sodium into a repository, all sodium has to be neutralized either through a controlled reaction with water or by incinerating. The resulting components can be disposed of as normal radioactive waste with no further conditions. The handling of sodium needs skilled and experienced workers to minimize the inherent risks. And the example of the disposal of the large KNK cold trap shows the interaction with
Simultaneous 3D tracking of passive tracers and microtubule bundles in an active gel

NASA Astrophysics Data System (ADS)

Fan, Yi; Breuer, Kenneth S.; Fluids Team

Kinesin-driven microtubule bundles generate a spontaneous flow in unconfined geometries. They exhibit properties of active matter, including the emergence of collective motion, reduction of apparent viscosity and consumption of local energy. Here we present results from 3D tracking of passive tracers (using Airy rings and 3D scanning) synchronized with 3D measurement of the microtubule bundles motion. This technique is applied to measure viscosity variation and collective flow in a confined geometry with particular attention paid to the self-pumping system recently reported by Wu et al. (2016). Results show that the viscosity in an equilibrium microtubule network is around half that of the isotropic unbundled microtubule solution. Cross-correlations of the active microtubule network and passive tracers define a neighborhood around microtubule bundles in which passive tracers are effectively transported. MRSEC NSF.
Functional organization of mitotic microtubules. Physical chemistry of the in vivo equilibrium system.

PubMed Central

Inoué, S; Fuseler, J; Salmon, E D; Ellis, G W

1975-01-01

Equilibrium between mitotic microtubules and tubulin is analyzed, using birefringence of mitotic spindle to measure microtubule concentration in vivo. A newly designed temperature-controlled slide and miniature, thermostated hydrostatic pressure chamber permit rapid alteration of temperature and of pressure. Stress birefringence of the windows is minimized, and a system for rapid recording of compensation is incorporated, so that birefringence can be measured to 0.1 nm retardation every few seconds. Both temperature and pressure data yield thermodynamic values (delta H similar to 35 kcal/mol, delta S similar to 120 entropy units [eu], delta V similar to 400 ml/mol of subunit polymerized) consistent with the explanation that polymerization of tubulin is entropy driven and mediated by hydrophobic interactions. Kinetic data suggest pseudo-zero-order polymerization and depolymerization following rapid temperature shifts, and a pseudo-first-order depolymerization during anaphase at constant temperature. The equilibrium properties of the in vivo mitotic microtubules are compared with properties of isolated brain tubules. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 12 FIGURE 13 FIGURE 14 FIGURE 19 PMID:1139037
Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

PubMed

Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

2010-07-13

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.
Potential mechanisms of resistance to microtubule inhibitors.

PubMed

Kavallaris, Maria; Annereau, Jean-Philippe; Barret, Jean-Marc

2008-06-01

Antimitotic drugs targeting the microtubules, such as the taxanes and vinca alkaloids, are widely used in the treatment of neoplastic diseases. Development of drug resistance over time, however, limits the efficacy of these agents and poses a clinical challenge to long-term improvement of patient outcomes. Understanding the mechanism(s) of drug resistance becomes paramount to allowing for alternative, if not improved, therapeutic options that might circumvent this challenge. Vinflunine, a novel microtubule inhibitor, has shown superior preclinical antitumor activity, and displays a different pattern of resistance, compared with other agents in the vinca alkaloid class.
Vitamin K3 disrupts the microtubule networks by binding to tubulin: a novel mechanism of its antiproliferative activity.

PubMed

Acharya, Bipul R; Choudhury, Diptiman; Das, Amlan; Chakrabarti, Gopal

2009-07-28

Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.

Controlling self-assembly of microtubule spools via kinesin motor density

PubMed Central

Lam, A.T.; Curschellas, C.; Krovvidi, D.; Hess, H.

2014-01-01

Active self-assembly, in which non-thermal energy is consumed by the system to put together building blocks, allows the creation of non-equilibrium structures and active materials. Microtubule spools assembled in gliding assays are one example of such non-equilibrium structures, capable of storing bending energies on the order of 105 kT. Although these structures arise spontaneously in experiments, the origin of microtubule spooling has long been debated. Here, using a stepwise kinesin gradient, we demonstrate that spool assembly can be controlled by the surface density of kinesin motors, showing that pinning of microtubules due to dead motors plays a dominant role in spool initiation. PMID:25269076
Controlling self-assembly of microtubule spools via kinesin motor density.

PubMed

Lam, A T; Curschellas, C; Krovvidi, D; Hess, H

2014-11-21

Active self-assembly, in which non-thermal energy is consumed by the system to put together building blocks, allows the creation of non-equilibrium structures and active materials. Microtubule spools assembled in gliding assays are one example of such non-equilibrium structures, capable of storing bending energies on the order of 10(5) kT. Although these structures arise spontaneously in experiments, the origin of microtubule spooling has long been debated. Here, using a stepwise kinesin gradient, we demonstrate that spool assembly can be controlled by the surface density of kinesin motors, showing that pinning of microtubules due to dead motors plays a dominant role in spool initiation.
[Study on the determination of 28 inorganic elements in sunflower seeds by ICP-OES/ICP-MS].

PubMed

Liu, Hong-Wei; Qin, Zong-Hui; Xie, Hua-Lin; Cao, Shu

2013-01-01

The present paper describes a simple method for the determination of trace elements in sunflower seeds by using inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma spectrometry (ICP-MS). HNO3 + H2O2 were used to achieve the complete decomposition of the organic matrix in a closed-vessel microwave oven. The contents of 10 trace elements (Al, B, Ca, Fe, K, Mg, Na, Si, P and S) in sunflower seeds were determined by ICP-OES while 18 trace elements (As, Ba, Cd, Co, Cr, Cu, Li, Mn, Mo, Ni, Pb, Rb, Sr, Sn, Sb, Ti, V and Zn) were determined by ICP-MS. The rice reference material (GBW10045) was used as standard reference materials. The results showed a good agreement between measured and certified values for all analytes. The concentrations of necessary micro elements Ca, K, Mg, P and S were higher. This method was simple, sensitive and precise and can perform simultaneous multi-elements determination of sunflower seeds.
Hypothesis: NDL proteins function in stress responses by regulating microtubule organization

PubMed Central

Khatri, Nisha; Mudgil, Yashwanti

2015-01-01

N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants. PMID:26583023
Hypothesis: NDL proteins function in stress responses by regulating microtubule organization.

PubMed

Khatri, Nisha; Mudgil, Yashwanti

2015-01-01

N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants.
Dear microtubule, I see you

DOE PAGES

Nogales, Eva

2016-11-01

This essay summarizes my personal journey toward the atomic visualization of microtubules and a mechanistic understanding of how these amazing polymers work. During this journey, I have been witness and partaker in the blooming of a technique I love—cryo-electron microscopy.
Dear microtubule, I see you

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nogales, Eva

This essay summarizes my personal journey toward the atomic visualization of microtubules and a mechanistic understanding of how these amazing polymers work. During this journey, I have been witness and partaker in the blooming of a technique I love—cryo-electron microscopy.
The performance of single and multi-collector ICP-MS instruments for fast and reliable 34S/32S isotope ratio measurements†

PubMed Central

Pröfrock, Daniel; Irrgeher, Johanna; Prohaska, Thomas

2016-01-01

The performance and validation characteristics of different single collector inductively coupled plasma mass spectrometers based on different technical principles (ICP-SFMS, ICP-QMS in reaction and collision modes, and ICP-MS/MS) were evaluated in comparison to the performance of MC ICP-MS for fast and reliable S isotope ratio measurements. The validation included the determination of LOD, BEC, measurement repeatability, within-lab reproducibility and deviation from certified values as well as a study on instrumental isotopic fractionation (IIF) and the calculation of the combined standard measurement uncertainty. Different approaches of correction for IIF applying external intra-elemental IIF correction (aka standard-sample bracketing) using certified S reference materials and internal inter-elemental IIF (aka internal standardization) correction using Si isotope ratios in MC ICP-MS are explained and compared. The resulting combined standard uncertainties of examined ICP-QMS systems were not better than 0.3–0.5% (uc,rel), which is in general insufficient to differentiate natural S isotope variations. Although the performance of the single collector ICP-SFMS is better (single measurement uc,rel = 0.08%), the measurement reproducibility (>0.2%) is the major limit of this system and leaves room for improvement. MC ICP-MS operated in the edge mass resolution mode, applying bracketing for correction of IIF, provided isotope ratio values with the highest quality (relative combined measurement uncertainty: 0.02%; deviation from the certified value: <0.002%). PMID:27812369
The performance of single and multi-collector ICP-MS instruments for fast and reliable 34S/32S isotope ratio measurements.

PubMed

Hanousek, Ondrej; Brunner, Marion; Pröfrock, Daniel; Irrgeher, Johanna; Prohaska, Thomas

2016-11-14

The performance and validation characteristics of different single collector inductively coupled plasma mass spectrometers based on different technical principles (ICP-SFMS, ICP-QMS in reaction and collision modes, and ICP-MS/MS) were evaluated in comparison to the performance of MC ICP-MS for fast and reliable S isotope ratio measurements. The validation included the determination of LOD, BEC, measurement repeatability, within-lab reproducibility and deviation from certified values as well as a study on instrumental isotopic fractionation (IIF) and the calculation of the combined standard measurement uncertainty. Different approaches of correction for IIF applying external intra-elemental IIF correction (aka standard-sample bracketing) using certified S reference materials and internal inter-elemental IIF (aka internal standardization) correction using Si isotope ratios in MC ICP-MS are explained and compared. The resulting combined standard uncertainties of examined ICP-QMS systems were not better than 0.3-0.5% ( u c,rel ), which is in general insufficient to differentiate natural S isotope variations. Although the performance of the single collector ICP-SFMS is better (single measurement u c,rel = 0.08%), the measurement reproducibility (>0.2%) is the major limit of this system and leaves room for improvement. MC ICP-MS operated in the edge mass resolution mode, applying bracketing for correction of IIF, provided isotope ratio values with the highest quality (relative combined measurement uncertainty: 0.02%; deviation from the certified value: <0.002%).
Verification of Dismantlement of Nuclear Warheads and Controls on Nuclear Materials

DTIC Science & Technology

1993-01-12

754001- 40-SS00 Staclaed Form 296 (Rov. 2.-9) Pwww-esu o s IS ŖS-wa Contents EXECUTIVE SUMMARY 1 1 INTRODUCTION 7 1.1 Study Charge ...for monitoring both non-proliferation and dismantlement. 6 1 INTRODUCTION 1.1 Study Charge This study addresses the question of monitorin6 future...Co59 + n 19 barn 5 yr 7 Ni59 NijM + n 4.4 barn 10s yr e- capture Ni63 Ni62 + n 15 barn 92 yr p 32 p 3l + n 0.19 barn 14 day f- S 3 5 SM + n 0.27 barn
Differential interactions of the formins INF2, mDia1, and mDia2 with microtubules

PubMed Central

Gaillard, Jeremie; Ramabhadran, Vinay; Neumanne, Emmanuelle; Gurel, Pinar; Blanchoin, Laurent; Vantard, Marylin; Higgs, Henry N.

2011-01-01

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (Kd < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements. PMID:21998204
The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner

PubMed Central

Bell, Kayla M.; Cha, Hyo Keun; Sindelar, Charles V.; Cochran, Jared C.

2017-01-01

Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in Saccharomyces cerevisiae, can move bidirectionally along microtubules, switching directionality according to biochemical conditions, a behavior that remains largely unexplained. To this end, we used biochemical rate and equilibrium constant measurements as well as cryo-electron microscopy methodologies to investigate the microtubule interactions of the Cin8 motor domain. These experiments unexpectedly revealed that, whereas Cin8 ATPase kinetics fell within measured ranges for kinesins (especially kinesin-5 proteins), approximately four motors can bind each αβ-tubulin dimer within the microtubule lattice. This result contrasted with those observations on other known kinesins, which can bind only a single “canonical” site per tubulin dimer. Competition assays with human kinesin-5 (Eg5) only partially abrogated this behavior, indicating that Cin8 binds microtubules not only at the canonical site, but also one or more separate (“noncanonical”) sites. Moreover, we found that deleting the large, class-specific insert in the microtubule-binding loop 8 reverts Cin8 to one motor per αβ-tubulin in the microtubule. The novel microtubule-binding mode of Cin8 identified here provides a potential explanation for Cin8 clustering along microtubules and potentially may contribute to the mechanism for direction reversal. PMID:28701465
Using SAFRAN Software to Assess Radiological Hazards from Dismantling of Tammuz-2 Reactor Core at Al-tuwaitha Nuclear Site

NASA Astrophysics Data System (ADS)

Abed Gatea, Mezher; Ahmed, Anwar A.; jundee kadhum, Saad; Ali, Hasan Mohammed; Hussein Muheisn, Abbas

2018-05-01

The Safety Assessment Framework (SAFRAN) software has implemented here for radiological safety analysis; to verify that the dose acceptance criteria and safety goals are met with a high degree of confidence for dismantling of Tammuz-2 reactor core at Al-tuwaitha nuclear site. The activities characterizing, dismantling and packaging were practiced to manage the generated radioactive waste. Dose to the worker was considered an endpoint-scenario while dose to the public has neglected due to that Tammuz-2 facility is located in a restricted zone and 30m berm surrounded Al-tuwaitha site. Safety assessment for dismantling worker endpoint-scenario based on maximum external dose at component position level in the reactor pool and internal dose via airborne activity while, for characterizing and packaging worker endpoints scenarios have been done via external dose only because no evidence for airborne radioactivity hazards outside the reactor pool. The in-situ measurements approved that reactor core components are radiologically activated by Co-60 radioisotope. SAFRAN results showed that the maximum received dose for workers are (1.85, 0.64 and 1.3mSv/y) for activities dismantling, characterizing and packaging of reactor core components respectively. Hence, the radiological hazards remain below the low level hazard and within the acceptable annual dose for workers in radiation field
A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast.

PubMed

Yukawa, Masashi; Kawakami, Tomoki; Okazaki, Masaki; Kume, Kazunori; Tang, Ngang Heok; Toda, Takashi

2017-12-01

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end-directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end-directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly. © 2017 Yukawa et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells

PubMed Central

Mukhtar, Eiman; Adhami, Vaqar Mustafa; Sechi, Mario; Mukhtar, Hasan

2015-01-01

Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies suggested that fisetin binds to β-tubulin with superior affinity compared to paclitaxel. Fisetin treatment of human prostate cancer cells resulted in robust up-regulation of microtubule associated proteins (MAP)-2 and -4. In addition, fisetin treated cells were enriched in α-tubulin acetylation, an indication of stabilization of microtubules. Fisetin significantly inhibited PCa cell proliferation, migration, and invasion. Nudc, a protein associated with microtubule motor dynein/dynactin complex that regulates microtubule dynamics, was inhibited with fisetin treatment. Further, fisetin treatment of a P-glycoprotein overexpressing multidrug-resistant cancer cell line NCI/ADR-RES inhibited the viability and colony formation. Our results offer in vitro proof-of-concept for fisetin as a microtubule targeting agent. We suggest that fisetin could be developed as an adjuvant for treatment of prostate and other cancer types. PMID:26235140
Determination of 238u/235u, 236u/238u and uranium concentration in urine using sf-icp-ms and mc-icp-ms: an interlaboratory comparison.

PubMed

Parrish, Randall R; Thirlwall, Matthew F; Pickford, Chris; Horstwood, Matthew; Gerdes, Axel; Anderson, James; Coggon, David

2006-02-01

Accidental exposure to depleted or enriched uranium may occur in a variety of circumstances. There is a need to quantify such exposure, with the possibility that the testing may post-date exposure by months or years. Therefore, it is important to develop a very sensitive test to measure precisely the isotopic composition of uranium in urine at low levels of concentration. The results of an interlaboratory comparison using sector field (SF)-inductively coupled plasma-mass spectrometry (ICP-MS) and multiple collector (MC)-ICP-MS for the measurement of uranium concentration and U/U and U/U isotopic ratios of human urine samples are presented. Three urine samples were verified to contain uranium at 1-5 ng L and shown to have natural uranium isotopic composition. Portions of these urine batches were doped with depleted uranium (DU) containing small quantities of U, and the solutions were split into 100 mL and 400 mL aliquots that were subsequently measured blind by three laboratories. All methods investigated were able to measure accurately U/U with precisions of approximately 0.5% to approximately 4%, but only selected MC-ICP-MS methods were capable of consistently analyzing U/U to reasonable precision at the approximately 20 fg L level of U abundance. Isotope dilution using a U tracer demonstrates the ability to measure concentrations to better than +/-4% with the MC-ICP-MS method, though sample heterogeneity in urine samples was shown to be problematic in some cases. MC-ICP-MS outperformed SF-ICP-MS methods, as was expected. The MC-ICP-MS methodology described is capable of measuring to approximately 1% precision the U/U of any sample of human urine over the entire range of uranium abundance down to <1 ng L, and detecting very small amounts of DU contained therein.
Evidence against impaired brain microtubule protein polymerization at high glucose concentrations or during diabetes mellitus.

PubMed

Eaker, E Y; Angelastro, J M; Purich, D L; Sninsky, C A

1991-06-01

Previous studies suggest that brain microtubule protein exposed to high glucose levels or isolated from diabetic rats can become glucosylated and that this impairs GTP-induced microtubule polymerization. We set out to extend that investigation to define the mechanistic basis for inhibition of microtubule assembly during diabetes or on incubation at high glucose levels. Rat and bovine brain microtubule protein was purified by cycles of polymerization/depolymerization. When microtubules were incubated for 1 h in either buffer or buffer containing glucose (up to 165 mM), there was no difference in polymerization, a finding contrary to the earlier study. Other rats were injected with vehicle or streptozotocin (90 mg/kg) to induce diabetes as evidenced by serum glucose in excess of 300 mg%, and at 4 weeks, brain microtubule protein was isolated by the polymerization cycling method. Again, there was no difference in the amount or purity of isolated microtubule protein between control or diabetic rats. We also observed no increase in microtubule glucosylation, and GTP-induced polymerization in vitro was indistinguishable for protein derived from brains of normal rats and rats with diabetes as measured by turbidity or electron microscopy. Our results suggest that in vitro incubation with glucose or in vivo elevation of glucose during diabetes fails to impair microtubule polymerization, pointing to other mechanisms for the neuropathy associated with diabetes.
Systems and methods for dismantling a nuclear reactor

DOEpatents

Heim, Robert R; Adams, Scott Ryan; Cole, Matthew Denver; Kirby, William E; Linnebur, Paul Damon

2014-10-28

Systems and methods for dismantling a nuclear reactor are described. In one aspect the system includes a remotely controlled heavy manipulator ("manipulator") operatively coupled to a support structure, and a control station in a non-contaminated portion of a workspace. The support structure provides the manipulator with top down access into a bioshield of a nuclear reactor. At least one computing device in the control station provides remote control to perform operations including: (a) dismantling, using the manipulator, a graphite moderator, concrete walls, and a ceiling of the bioshield, the manipulator being provided with automated access to all internal portions of the bioshield; (b) loading, using the manipulator, contaminated graphite blocks from the graphite core and other components from the bioshield into one or more waste containers; and (c) dispersing, using the manipulator, dust suppression and contamination fixing spray to contaminated matter.
Multivalency of NDC80 in the outer kinetochore is essential to track shortening microtubules and generate forces

PubMed Central

2018-01-01

Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved feature of kinetochores, points of attachment of chromosomes to spindle microtubules. This may enable multivalent attachments to microtubules, with implications that remain unexplored. Using recombinant human kinetochore components, we show that while single NDC80 complexes do not track depolymerizing microtubules, reconstituted particles containing the NDC80 receptor CENP-T bound to three or more NDC80 complexes do so effectively, as expected for a kinetochore force coupler. To study multivalency systematically, we engineered modules allowing incremental addition of NDC80 complexes. The modules’ residence time on microtubules increased exponentially with the number of NDC80 complexes. Modules with two or more complexes tracked depolymerizing microtubules with increasing efficiencies, and stalled and rescued microtubule depolymerization in a force-dependent manner when conjugated to cargo. Our observations indicate that NDC80, rather than through biased diffusion, tracks depolymerizing microtubules by harnessing force generated during microtubule disassembly. PMID:29629870
A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iimori, Makoto; Ozaki, Kanako; Chikashige, Yuji

2012-02-01

Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, themore » mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.« less

Study of Opto-electronic Properties of a Single Microtubule in the Microwave Regime

DTIC Science & Technology

2013-01-09

apparently microtubule grows continuously from tubulins, in that growth randomly triggered rapid growth and long silence modes are superimposed by...synchrony and de-synchrony glues. When such synchrony is introduced in an artificial cell like environment, microtubule does not grow freely, similar to...NOTES 14. ABSTRACT The research establishes the supremacy of synchrony by proving that though apparently microtubule grows continuously from
Push or Pull? -- Cryo-Electron Microscopy of Microtubule's Dynamic Instability and Its Roles in the Kinetochore

NASA Astrophysics Data System (ADS)

Wang, Hong-Wei

2009-03-01

Microtubule is a biopolymer made up of alpha-beta-tubulin heterodimers. The tubulin dimers assemble head-to-tail as protofilaments and about 13 protofilaments interact laterally to form a hollow cylindrical structure which is the microtubule. As the major cytoskeleton in all eukaryotic cells, microtubules have the intrinsic property to switch stochastically between growth and shrinkage phases, a phenomenon termed as their dynamic instability. Microtubule's dynamic instability is closely related to the types of nucleotide (GTP or GDP) that binds to the beta-tubulin. We have biochemically trapped two types of assembly states of tubulin with GTP or GDP bound representing the polymerizing and depolymerizing ends of microtubules respectively. Using cryo-electron microscopy, we have elucidated the structures of these intermediate assemblies, showing that tubulin protofilaments demonstrate various curvatures and form different types of lateral interactions depending on the nucleotide states of tubulin and the temperature. Our work indicates that during the microtubule's dynamic cycle, tubulin undergoes various assembly states. These states, different from the straight microtubule, lend the highly dynamic and complicated behavior of microtubules. Our study of microtubule's interaction with certain kinetochore complexes suggests that the intermediate assemblies are responsible for specific mechanical forces that are required during the mitosis or meiosis. Our discoveries strongly suggest that a microtubule is a molecular machine rather than a simple cellular scaffold.
CENTROSOMES AND MICROTUBULES DURING MEIOSIS IN THE MUSHROOM BOLETUS RUBINELLUS

PubMed Central

McLaughlin, David J.

1971-01-01

The double centrosome in the basidium of Boletus rubinellus has been observed in three planes with the electron microscope at interphase preceding nuclear fusion, at prophase I, and at interphase I. It is composed of two components connected by a band-shaped middle part. At anaphase I a single, enlarged centrosome is found at the spindle pole, which is attached to the cell membrane. Microtubules mainly oriented parallel to the longitudinal axis of the basidium are present at prefusion, prophase I and interphase I. Cytoplasmic microtubules are absent when the spindle is present. The relationship of the centrosome in B. rubinellus to that in other organisms and the role of the cytoplasmic microtubules are discussed. PMID:4329156
Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

PubMed Central

Borek, Weronika E.; Groocock, Lynda M.; Samejima, Itaru; Zou, Juan; de Lima Alves, Flavia; Rappsilber, Juri; Sawin, Kenneth E.

2015-01-01

Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation. PMID:26243668
EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

PubMed

Wang, Yi-Long; Chen, Hui; Zhan, Yi-Qun; Yin, Rong-Hua; Li, Chang-Yan; Ge, Chang-Hui; Yu, Miao; Yang, Xiao-Ming

2016-08-17

EWSR1, participating in transcription and splicing, has been identified as a translocation partner for various transcription factors, resulting in translocation, which in turn plays crucial roles in tumorigenesis. Recent studies have investigated the role of EWSR1 in mitosis. However, the effect of EWSR1 on mitosis is poorly understood. Here, we observed that depletion of EWSR1 resulted in cell cycle arrest in the mitotic phase, mainly due to an increase in the time from nuclear envelope breakdown to metaphase, resulting in a high percentage of unaligned chromosomes and multipolar spindles. We also demonstrated that EWSR1 is a spindle-associated protein that interacts with α-tubulin during mitosis. EWSR1 depletion increased the cold-sensitivity of spindle microtubules, and decreased the rate of spindle assembly. EWSR1 regulated the level of microtubule acetylation in the mitotic spindle; microtubule acetylation was rescued in EWSR1-depleted mitotic cells following suppression of HDAC6 activity by its specific inhibitor or siRNA treatment. In summary, these results suggest that EWSR1 regulates the acetylation of microtubules in a cell cycle-dependent manner through its dynamic location on spindle MTs, and may be a novel regulator for mitosis progress independent of its translocation.
Motor-mediated microtubule self-organization in dilute and semi-dilute filament solutions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swaminathan, S.; Ziebert, F.; Aranson, I. S.

We study molecular motor-induced microtubule self-organization in dilute and semi-dilute filament solutions. In the dilute case, we use a probabilistic model of microtubule interaction via molecular motors to investigate microtubule bundle dynamics. Microtubules are modeled as polar rods interacting through fully inelastic, binary collisions. Our model indicates that initially disordered systems of interacting rods exhibit an orientational instability resulting in spontaneous ordering. We study the existence and dynamic interaction of microtubule bundles analytically and numerically. Our results reveal a long term attraction and coalescing of bundles indicating a clear coarsening in the system; microtubule bundles concentrate into fewer orientations onmore » a slow logarithmic time scale. In semi-dilute filament solutions, multiple motors can bind a filament to several others and, for a critical motor density, induce a transition to an ordered phase with a nonzero mean orientation. Motors attach to a pair of filaments and walk along the pair bringing them into closer alignment. We develop a spatially homogenous, mean-field theory that explicitly accounts for a force-dependent detachment rate of motors, which in turn affects the mean and the fluctuations of the net force acting on a filament. We show that the transition to the oriented state can be both continuous and discontinuous when the force-dependent detachment of motors is important.« less
Measurement of technetium-99 in Marshall Islands soil samples by ICP-MS

PubMed

Tagami; Uchida; Hamilton; Robison

2000-07-01

Extraction techniques for recovery of technetium-99 (99Tc) for Inductively Coupled Plasma Mass Spectrometry (ICP-MS) measurements were evaluated using soil samples collected from the Marshall Islands. The results of three different extraction techniques were compared: (MI) acid leaching of Tc from ashed soil; (M2) acid leaching of Tc from raw dry soil; and (M3) Tc volatilization from ashed soil using a combustion apparatus. Total Tc recoveries varied considerably between the extraction techniques but each method yielded similar analytical results for 99Tc. Applications of these extraction techniques to a series of environmental samples and ICP-MS measurements have yielded first data on the 99Tc content of Marshall Islands soil samples contaminated with close-in radioactive fallout from nuclear weapons testing. The 99Tc activity concentration in the soil samples ranged between 0.1 and 1.1 mBq g(-1) dry weight (dw). The limit of detection for 99Tc by ICP-MS was 0.17 mBq per sample or 0.014 mBq g(-1) dw under standard operating conditions.
Single-cell analysis by ICP-MS/MS as a fast tool for cellular bioavailability studies of arsenite.

PubMed

Meyer, S; López-Serrano, A; Mitze, H; Jakubowski, N; Schwerdtle, T

2018-01-24

Single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) has become a powerful and fast tool to evaluate the elemental composition at a single-cell level. In this study, the cellular bioavailability of arsenite (incubation of 25 and 50 μM for 0-48 h) has been successfully assessed by SC-ICP-MS/MS for the first time directly after re-suspending the cells in water. This procedure avoids the normally arising cell membrane permeabilization caused by cell fixation methods (e.g. methanol fixation). The reliability and feasibility of this SC-ICP-MS/MS approach with a limit of detection of 0.35 fg per cell was validated by conventional bulk ICP-MS/MS analysis after cell digestion and parallel measurement of sulfur and phosphorus.
[Application of ICP-MS and ICP-AES for Studying on Source Apportionment of PM2.5 during Haze Weather in Urban Beijing].

PubMed

Chen, Xi; Du, Peng; Guan, Qing; Feng, Xu; Xu, Dong-qun; Lin, Shao-bin

2015-06-01

To investigate the characteristics of chemical constitute and pollution sources of aerosol fine particulate matter during haze-fog day in Beijing in winter 2013. The samples of PM2.5 were collected in Beijing from January to February, 2013. The technique of ICP-MS and ICP-AES coupled with procedure of bathing-ultrasonic extraction was applied to determine the concentration of 40 elements in the aerosol samples to analyze the characteristics of elements distribution statistically. The absolute principal factor method was used to apportion the pollution sources of PM2.5 during the haze weather in Beijing city in winter 2013. The results showed that during the period of sampling, the volume concentration of Li, Mn, Pb, S etc. obeyed normal distribution approximately, and according to National Ambient Air Quality Standard issued by Ministry of Environmental Protection of the People's Republic of China, the geometric mean concentration of As was twice the annual limit of standard reference, while Pb of some aerosol samples beyond the annual limit of standard reference respectively. The mass fraction of Fe, Zn, Pb, Ti accounted for over 0.1%, while that of Mn, Cu, As, Se etc. 0.01%. These elements were primary inorganic pollutants, and especially the hazards and sources of As and Pb should be concerned. There were 6 main pollution sources were chosen by the factor analysis method, including industrial dust and human beings activities, biomass combustion and building dust, soil and sand dusts, fossil fuel, electronic waste and metal smelting, with the variance contribution rate of 40.3%, 27.0%, 9.1%, 4.9%, 4.8% and 4.6% respectively. ICP-MS and ICP-AES can be applied to analyzing multi-elements in PM2.5 accurately and quickly to facilitate source apportionment, and it indicated that the relevant pollution sources should be considered and the effect of regional transferring of haze pollution sources should be taken into account, and specific measures should be taken for
Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells.

PubMed

Mukhtar, Eiman; Adhami, Vaqar Mustafa; Sechi, Mario; Mukhtar, Hasan

2015-10-28

Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies suggested that fisetin binds to β-tubulin with superior affinity compared to paclitaxel. Fisetin treatment of human prostate cancer cells resulted in robust up-regulation of microtubule associated proteins (MAP)-2 and -4. In addition, fisetin treated cells were enriched in α-tubulin acetylation, an indication of stabilization of microtubules. Fisetin significantly inhibited PCa cell proliferation, migration, and invasion. Nudc, a protein associated with microtubule motor dynein/dynactin complex that regulates microtubule dynamics, was inhibited with fisetin treatment. Further, fisetin treatment of a P-glycoprotein overexpressing multidrug-resistant cancer cell line NCI/ADR-RES inhibited the viability and colony formation. Our results offer in vitro proof-of-concept for fisetin as a microtubule targeting agent. We suggest that fisetin could be developed as an adjuvant for treatment of prostate and other cancer types. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
GDP-to-GTP exchange on the microtubule end can contribute to the frequency of catastrophe

PubMed Central

Piedra, Felipe-Andrés; Kim, Tae; Garza, Emily S.; Geyer, Elisabeth A.; Burns, Alexander; Ye, Xuecheng; Rice, Luke M.

2016-01-01

Microtubules are dynamic polymers of αβ-tubulin that have essential roles in chromosome segregation and organization of the cytoplasm. Catastrophe—the switch from growing to shrinking—occurs when a microtubule loses its stabilizing GTP cap. Recent evidence indicates that the nucleotide on the microtubule end controls how tightly an incoming subunit will be bound (trans-acting GTP), but most current models do not incorporate this information. We implemented trans-acting GTP into a computational model for microtubule dynamics. In simulations, growing microtubules often exposed terminal GDP-bound subunits without undergoing catastrophe. Transient GDP exposure on the growing plus end slowed elongation by reducing the number of favorable binding sites on the microtubule end. Slower elongation led to erosion of the GTP cap and an increase in the frequency of catastrophe. Allowing GDP-to-GTP exchange on terminal subunits in simulations mitigated these effects. Using mutant αβ-tubulin or modified GTP, we showed experimentally that a more readily exchangeable nucleotide led to less frequent catastrophe. Current models for microtubule dynamics do not account for GDP-to-GTP exchange on the growing microtubule end, so our findings provide a new way of thinking about the molecular events that initiate catastrophe. PMID:27146111
Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma.

PubMed

Landen, Jaren W; Lang, Roland; McMahon, Steve J; Rusan, Nasser M; Yvon, Anne-Marie; Adams, Ashley W; Sorcinelli, Mia D; Campbell, Ross; Bonaccorsi, Paola; Ansel, John C; Archer, David R; Wadsworth, Patricia; Armstrong, Cheryl A; Joshi, Harish C

2002-07-15

Cellular microtubules, polymers of tubulin, alternate relentlessly between phases of growth and shortening. We now show that noscapine, a tubulin-binding agent, increases the time that cellular microtubules spend idle in a paused state. As a result, most mammalian cell types observed arrest in mitosis in the presence of noscapine. We demonstrate that noscapine-treated murine melanoma B16LS9 cells do not arrest in mitosis but rather become polyploid followed by cell death, whereas primary melanocytes reversibly arrest in mitosis and resume a normal cell cycle after noscapine removal. Furthermore, in a syngeneic murine model of established s.c. melanoma, noscapine treatment resulted in an 85% inhibition of tumor volume on day 17 when delivered by gavage compared with untreated animals (P 0.01), without evidence of toxicity to the spleen, liver, duodenum, bone marrow, or peripheral blood. This inhibition was greater than that seen in vivo by paclitaxel (Taxol) alone and similar to the inhibition of tumor volume observed when noscapine was combined with paclitaxel. Importantly, noscapine also demonstrated the ability to significantly inhibit melanoma progression by 83% on day 18 when delivered in drinking water (P 0.01) and conferred a significant survival advantage (P 0.01). Our results demonstrate that p.o.-administered noscapine significantly inhibits the progression of melanoma cells through alterations in microtubule dynamics, with no detected toxicity to the host. Consequently, noscapine could be a valuable chemotherapeutic agent, alone or in combination, for the treatment of advanced melanoma.
The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

PubMed

Fujita, Miki; Lechner, Bettina; Barton, Deborah A; Overall, Robyn L; Wasteneys, Geoffrey O

2012-02-01

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule-cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.
Elemental profiling and geographical differentiation of Ethiopian coffee samples through inductively coupled plasma-optical emission spectroscopy (ICP-OES), ICP-mass spectrometry (ICP-MS) and direct mercury analyzer (DMA).

PubMed

Habte, Girum; Hwang, In Min; Kim, Jae Sung; Hong, Joon Ho; Hong, Young Sin; Choi, Ji Yeon; Nho, Eun Yeong; Jamila, Nargis; Khan, Naeem; Kim, Kyong Su

2016-12-01

This study was aimed to establish the elemental profiling and provenance of coffee samples collected from eleven major coffee producing regions of Ethiopia. A total of 129 samples were analyzed for forty-five elements using inductively coupled plasma (ICP)-optical emission spectroscopy (OES), ICP-mass spectrometry (MS) and direct mercury analyzer (DMA). Among the macro elements, K showed the highest levels whereas Fe was found to have the lowest concentration values. In all the samples, Ca, K, Mg, P and S contents were statistically significant (p<0.05). Micro elements showed the concentrations order of: Mn>Cu>Sr>Zn>Rb>Ni>B. Contents of the trace elements were lower than the permissible standard values. Inter-regions differentiation by cluster analysis (CA), linear discriminant analysis (LDA) and principal component analysis (PCA) showed that micro and trace elements are the best chemical descriptors of the analyzed coffee samples. Copyright © 2016 Elsevier Ltd. All rights reserved.
Spectraplakins promote microtubule-mediated axonal growth by functioning as structural MAPs and EB1-dependent +TIPs

PubMed Central

Alves-Silva, J.; Sánchez-Soriano, N.; Beaven, R.; Klein, M.; Parkin, J.; Millard, T.H.; Bellen, H. J; Venken, K. J.T.; Ballestrem, C.; Kammerer, R.A.; Prokop, A.

2013-01-01

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homologue Short stop/Shot similarly cause severe axon shortening and microtubule disorganisation. How spectraplakins perform these functions is not known. Here we show that axonal growth promoting roles of Shot require interaction with EB1 (End binding protein) at polymerising plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot’s carboxyterminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organisation. In addition, we report that Shot localises along microtubule shafts and stabilises them against pharmacologically induced depolymerisation. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (plus end regulators) and structural MAPs (microtubule associated proteins). From our data we deduce a model that relates the different features of the spectraplakin carboxy-terminus to the two functions of Shot during axonal growth. PMID:22764224
In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation

PubMed Central

Matsuyama, Akihisa; Shimazu, Tadahiro; Sumida, Yuko; Saito, Akiko; Yoshimatsu, Yasuhiro; Seigneurin-Berny, Daphné; Osada, Hiroyuki; Komatsu, Yasuhiko; Nishino, Norikazu; Khochbin, Saadi; Horinouchi, Sueharu; Yoshida, Minoru

2002-01-01

Trichostatin A (TSA) inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin (TPX) cannot inhibit HDAC6, a cytoplasmic member of class II HDACs. We took advantage of this differential sensitivity of HDAC6 to TSA and TPX to identify its substrates. Using this approach, α-tubulin was identified as an HDAC6 substrate. HDAC6 deacetylated α-tubulin both in vivo and in vitro. Our investigations suggest that HDAC6 controls the stability of a dynamic pool of microtubules. Indeed, we found that highly acetylated microtubules observed after TSA treatment exhibited delayed drug-induced depolymerization and that HDAC6 overexpression prompted their induced depolymerization. Depolymerized tubulin was rapidly deacetylated in vivo, whereas tubulin acetylation occurred only after polymerization. We therefore suggest that acetylation and deacetylation are coupled to the microtubule turnover and that HDAC6 plays a key regulatory role in the stability of the dynamic microtubules. PMID:12486003
Developments in ICP-MS: electrochemically modulated liquid chromatography for the clean-up of ICP-MS blanks and reduction of matrix effects by flow injection ICP-MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gross, Cory Thomas

2008-01-01

The focus of this dissertation is the development of techniques with which to enhance the existing abilities of inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS is a powerful technique for trace metal analysis in samples of many types, but like any technique it has certain strengths and weaknesses. Attempts are made to improve upon those strengths and to overcome certain weaknesses.
The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

PubMed Central

Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

2013-01-01

Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258
Microtubule Abnormalities Underlying Gulf War Illness in Neurons from Human-Induced Pluripotent Cells

DTIC Science & Technology

2016-09-01

AWARD NUMBER: W81XWH-15-1-0433 TITLE: Microtubule Abnormalities Underlying Gulf War Illness in Neurons from Human -Induced Pluripotent Cells...2015 - 31 Aug 2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Microtubule Abnormalities Underlying Gulf War Illness in Neurons from Human -Induced...functions to normal in neurons derived from human pluripotent cells exposed to Gulf War toxins. 15. SUBJECT TERMS microtubule, neuron, Gulf War Illness
Optical properties of template synthesized nanowalled ZnS microtubules

NASA Astrophysics Data System (ADS)

Kumar, Rajesh; Chakarvarti, S. K.

2007-12-01

Electrodeposition is a versatile technique combining low processing cost with ambient conditions that can be used to prepare metallic, polymeric and semiconducting nano/micro structures. In the present work, track-etch membranes (TEMs) of makrofol (KG) have been used as templates for synthesis of ZnS nanowalled microtubules using electrodeposition technique. The morphology of the microtubules was characterized by scanning electron microscopy. Size effects on the band gap of tubules have also been studied by UV-visible spectrophotometer.

Buckling analysis of orthotropic protein microtubules under axial and radial compression based on couple stress theory.

PubMed

Beni, Yaghoub Tadi; Zeverdejani, M Karimi; Mehralian, Fahimeh

2017-10-01

Protein microtubules (MTs) are one of the important intercellular components and have a vital role in the stability and strength of the cells. Due to applied external loads, protein microtubules may be involved buckling phenomenon. Due to impact of protein microtubules in cell reactions, it is important to determine their critical buckling load. Considering nature of protein microtubules, various parameters are effective on microtubules buckling. The small size of microtubules and also lack of uniformity of MTs properties in different directions caused the necessity of accuracy in the analysis of these bio-structure. In fact, microtubules must be considered as a size dependent cylinder, which behave as an orthotropic material. Hence, in the present work using first-order shear deformation model (FSDT), the buckling equations of anisotropic MTs are derived based on new modified couple stress theory (NMCST). After solving the stability equations, the influences of various parameters are measured on the MTs critical buckling load. Copyright © 2017 Elsevier Inc. All rights reserved.
Axonal Transport: How High Microtubule Density Can Compensate for Boundary Effects in Small-Caliber Axons

PubMed Central

Wortman, Juliana C.; Shrestha, Uttam M.; Barry, Devin M.; Garcia, Michael L.; Gross, Steven P.; Yu, Clare C.

2014-01-01

Long-distance intracellular axonal transport is predominantly microtubule-based, and its impairment is linked to neurodegeneration. In this study, we present theoretical arguments that suggest that near the axon boundaries (walls), the effective viscosity can become large enough to impede cargo transport in small (but not large) caliber axons. Our theoretical analysis suggests that this opposition to motion increases rapidly as the cargo approaches the wall. We find that having parallel microtubules close enough together to enable a cargo to simultaneously engage motors on more than one microtubule dramatically enhances motor activity, and thus minimizes the effects of any opposition to transport. Even if microtubules are randomly placed in axons, we find that the higher density of microtubules found in small-caliber axons increases the probability of having parallel microtubules close enough that they can be used simultaneously by motors on a cargo. The boundary effect is not a factor in transport in large-caliber axons where the microtubule density is lower. PMID:24559984
Dinitroaniline herbicide resistance and the microtubule cytoskeleton.

PubMed

Anthony; Hussey

1999-03-01

Dinitroaniline herbicides have been used for pre-emergence weed control for the past 25 years in cotton, soybean, wheat and oilseed crops. Considering their long persistence and extensive use, resistance to dinitroanilines is fairly rare. However, the most widespread dinitroaniline-resistant weeds, the highly resistant (R) and the intermediate (I) biotypes of the invasive goosegrass Eleusine indica, are now infesting more than 1000 cotton fields in the southern states of the USA. The molecular basis of this resistance has been identified, and found to be a point mutation in a major microtubule cytoskeletal protein, alpha-tubulin. These studies have served both to explain the establishment of resistance and to reveal fundamental properties of tubulin gene expression and microtubule structure.
A candidate reference method using ICP-MS for sweat chloride quantification.

PubMed

Collie, Jake T; Massie, R John; Jones, Oliver A H; Morrison, Paul D; Greaves, Ronda F

2016-04-01

The aim of the study was to develop a method for sweat chloride (Cl) quantification using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to present to the Joint Committee for Traceability in Laboratory Medicine (JCTLM) as a candidate reference method for the diagnosis of cystic fibrosis (CF). Calibration standards were prepared from sodium chloride (NaCl) to cover the expected range of sweat Cl values. Germanium (Ge) and scandium (Sc) were selected as on-line (instrument based) internal standards (IS) and gallium (Ga) as the off-line (sample based) IS. The method was validated through linearity, accuracy and imprecision studies as well as enrolment into the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) for sweat electrolyte testing. Two variations of the ICP-MS method were developed, an on-line and off-line IS, and compared. Linearity was determined up to 225 mmol/L with a limit of quantitation of 7.4 mmol/L. The off-line IS demonstrated increased accuracy through the RCPAQAP performance assessment (CV of 1.9%, bias of 1.5 mmol/L) in comparison to the on-line IS (CV of 8.0%, bias of 3.8 mmol/L). Paired t-tests confirmed no significant differences between sample means of the two IS methods (p=0.53) or from each method against the RCPAQAP target values (p=0.08 and p=0.29). Both on and off-line IS methods generated highly reproducible results and excellent linear comparison to the RCPAQAP target results. ICP-MS is a highly accurate method with a low limit of quantitation for sweat Cl analysis and should be recognised as a candidate reference method for the monitoring and diagnosis of CF. Laboratories that currently practice sweat Cl analysis using ICP-MS should include an off-line IS to help negate any pre-analytical errors.
STUDIES ON THE MICROTUBULES IN HELIOZOA

PubMed Central

Tilney, Lewis G.; Hiramoto, Yukio; Marsland, Douglas

1966-01-01

Electron microscope preparations were made of specimens of Actinosphaerium nucleofilum fixed in glutaraldehyde before, during, and after exposure to high pressures (4,000 to 8,000 psi). A study of this material showed that, although other organelles were relatively stable, the microtubular elements of the axopodia and cytosome became unstable under pressure. Their rapid disintegration under pressure was correlated with beading and retraction of the axopodia. Moreover, after the release of pressure, microtubules reappeared as soon as, or sooner than the reextension of the axopodia. The rate of disintegration increased as the pressure was raised. At 4,000 psi, few if any tubules remained after 10 min, whereas at 6,000 and 8,000 psi the disintegration was much more rapid. Some adaptational reorganization of the microtubules and axopodia occurred while relatively low pressures were maintained. This was accompanied by an actual elongation of the axopodia in specimens maintained for 20 min at 4,000 psi, but was confined to knoblike axopodial remnants in animals kept at 6,000 psi. No regeneration of tubules or axopodia occurred at 8,000 psi. The presence of fibers and a finely fibrillar material in pressurized animals suggests that these may be derivatives of microtubular disintegration. This evidence, though purely morphological, is consistent with the hypothesis that microtubules play an important role not only in maintaining the formstability of the axopodia, but also in the active process by which the axopodia reextend themselves after retraction. PMID:5920198
The role of microtubules in contractile ring function.

PubMed

Conrad, A H; Paulsen, A Q; Conrad, G W

1992-05-01

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.
The role of microtubules in contractile ring function

NASA Technical Reports Server (NTRS)

Conrad, A. H.; Paulsen, A. Q.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1992-01-01

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.
Dictyoceratidan poisons: Defined mark on microtubule-tubulin dynamics.

PubMed

Gnanambal K, Mary Elizabeth; Lakshmipathy, Shailaja Vommi

2016-03-01

Tubulin/microtubule assembly and disassembly is characterized as one of the chief processes during cell growth and division. Hence drugs those perturb these process are considered to be effective in killing fast multiplying cancer cells. There is a collection of natural compounds which disturb microtubule/tubulin dis/assemblage and there have been a lot of efforts concerted in the marine realm too, to surveying such killer molecules. Close to half the natural compounds shooting out from marine invertebrates are generally with no traceable definite mechanisms of action though may be tough anti-cancerous hits at nanogram levels, hence fatefully those discoveries conclude therein without a capacity of translation from laboratory to pharmacy. Astoundingly at least 50% of natural compounds which have definite mechanisms of action causing disorders in tubulin/microtubule kinetics have an isolation history from sponges belonging to the Phylum: Porifera. Poriferans have always been a wonder worker to treat cancers with a choice of, yet precise targets on cancerous tissues. There is a specific order: Dictyoceratida within this Phylum which has contributed to yielding at least 50% of effective compounds possessing this unique mechanism of action mentioned above. However, not much notice is driven to Dictyoceratidans alongside the order: Demospongiae thus dictating the need to know its select microtubule/tubulin irritants since the unearthing of avarol in the year 1974 till date. Hence this review selectively pinpoints all the compounds, noteworthy derivatives and analogs stemming from order: Dictyoceratida focusing on the past, present and future. Copyright © 2016 Elsevier Inc. All rights reserved.
Comparison of ship dismantling processes in India and the U.S.

NASA Astrophysics Data System (ADS)

Ahluwalia, Rashpal S.; Sibal, Pooja; Govindarajulu, Sriram

2004-03-01

This paper compares ship-dismantling processes in India and the U.S. The information for India was collected during an informal visit to the ship dismantling sites in Alang, India. The information for the U.S. was obtained from the MARAD report. For a 10,000-ton passenger ship, the Indian contractor makes a profit of about 24% compared to a loss of about 15% in the U.S. The loss in the US is primarily due to high labor costs, compliance to safety and health regulations and lack of market for used components and scrap metal.
Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

PubMed

Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

2015-08-10

Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. Copyright © 2015 Elsevier Inc. All rights reserved.
Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds

PubMed Central

Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

2015-01-01

SUMMARY Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin “clouds” are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10’s role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. PMID:26235048
The engine of microtubule dynamics comes into focus.

PubMed

Mitchison, T J

2014-05-22

In this issue, Alushin et al. report high-resolution structures of three states of the microtubule lattice: GTP-bound, which is stable to depolymerization; unstable GDP-bound; and stable Taxol and GDP-bound. By comparing these structures at near-atomic resolution, they are able to propose a detailed model for how GTP hydrolysis destabilizes the microtubule and thus powers dynamic instability and chromosome movement. Destabilization of cytoskeleton filaments by nucleotide hydrolysis is an important general principle in cell dynamics, and this work represents a major step forward on a problem with a long history. Copyright © 2014 Elsevier Inc. All rights reserved.
Potassium Stable Isotopic Compositions Measured by High-Resolution MC-ICP-MS

NASA Technical Reports Server (NTRS)

Morgan, Leah E.; Lloyd, Nicholas S.; Ellam, Robert M.; Simon, Justin I.

2012-01-01

Potassium isotopic (K-41/K-39) compositions are notoriously difficult to measure. TIMS measurements are hindered by variable fractionation patterns throughout individual runs and too few isotopes to apply an internal spike method for instrumental mass fractionation corrections. Internal fractionation corrections via the K-40/K-39 ratio can provide precise values but assume identical K-40/K-39 ratios (e.g. 0.05% (1sigma) in [1]); this is appropriate in some cases (e.g. identifying excess K-41) but not others (e.g., determining mass fractionation effects and metrologically traceable isotopic abundances). SIMS analyses have yielded measurements with 0.25% precisions (1sigma) [2]. ICP-MS analyses are significantly affected by interferences from molecular species such as Ar-38H(+) and Ar-40H(+) and instrument mass bias. Single collector ICP-MS instruments in "cold plasma" mode have yielded uncertainties as low as 2% (1sigma, e.g. [3]). Although these precisions may be acceptable for some concentration determinations, they do not resolve isotopic variation in terrestrial materials. Here we present data from a series of measurements made on the Thermo Scientific NEPTUNE Plus multi-collector ICP-MS that demonstrate the ability to make K-41/K-39 ratio measurements with 0.07% precisions (1sigma). These data, collected on NIST K standards, indicate the potential for MC-ICP-MS measurements to look for K isotopic variations at the sub-permil level. The NEPTUNE Plus can sufficiently resolve 39K and 41K from the interfering 38ArH+ and 40ArH+ peaks in wet cold plasma and high-resolution mode. Measurements were made on small but flat, interference-free, plateaus (ca. 50 ppm by mass width for K-41). Although ICP-MS does not yield accurate K-41/K-39 values due to significant instrumental mass fractionation (ca. 6%), this bias can be sufficiently stable over the time required for several measurements so that relative K-41/K-39 values can be precisely determined via sample
Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.

PubMed

Leung, C L; Sun, D; Zheng, M; Knowles, D R; Liem, R K

1999-12-13

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends-PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH(2) terminus. However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest-specific protein, Gas2. In this paper, we demonstrate that the NH(2)-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.
Cold stability of microtubules in wood-forming tissues of conifers during seasons of active and dormant cambium.

PubMed

Begum, Shahanara; Shibagaki, Masaki; Furusawa, Osamu; Nakaba, Satoshi; Yamagishi, Yusuke; Yoshimoto, Joto; Jin, Hyun-O; Sano, Yuzou; Funada, Ryo

2012-01-01

The cold stability of microtubules during seasons of active and dormant cambium was analyzed in the conifers Abies firma, Abies sachalinensis and Larix leptolepis by immunofluorescence microscopy. Samples were fixed at room temperature and at a low temperature of 2-3°C to examine the effects of low temperature on the stability of microtubules. Microtubules were visible in cambium, xylem cells and phloem cells after fixation at room temperature during seasons of active and dormant cambium. By contrast, fixation at low temperature depolymerized microtubules in cambial cells, differentiating tracheids, differentiating xylem ray parenchyma and phloem ray parenchyma cells during the active season. However, similar fixation did not depolymerize microtubules during cambial dormancy in winter. Our results indicate that the stability of microtubules in cambial cells and cambial derivatives at low temperature differs between seasons of active and dormant cambium. Moreover, the change in the stability of microtubules that we observed at low temperature might be closely related to seasonal changes in the cold tolerance of conifers. In addition, low-temperature fixation depolymerized microtubules in cambial cells and differentiating cells that had thin primary cell walls, while such low-temperature fixation did not depolymerize microtubules in differentiating secondary xylem ray parenchyma cells and tracheids that had thick secondary cell walls. The stability of microtubules at low temperature appears to depend on the structure of the cell wall, namely, primary or secondary. Therefore, we propose that the secondary cell wall might be responsible for the cold stability of microtubules in differentiating secondary xylem cells of conifers.
Human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore–microtubule attachments

PubMed Central

Zelter, Alex; Riffle, Michael; MacCoss, Michael J.; Asbury, Charles L.; Davis, Trisha N.

2018-01-01

Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore–microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex–microtubule interaction is sufficiently strong to make a significant contribution to kinetochore–microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Cross-linking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together, our results indicate that the Ska complex can directly strengthen the kinetochore–microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule. PMID:29487209
Sustainable design for automotive products: dismantling and recycling of end-of-life vehicles.

PubMed

Tian, Jin; Chen, Ming

2014-02-01

The growth in automotive production has increased the number of end-of-life vehicles (ELVs) annually. The traditional approach ELV processing involves dismantling, shredding, and landfill disposal. The "3R" (i.e., reduce, reuse, and recycle) principle has been increasingly employed in processing ELVs, particularly ELV parts, to promote sustainable development. The first step in processing ELVs is dismantling. However, certain parts of the vehicle are difficult to disassemble and use in practice. The extended producer responsibility policy requires carmakers to contribute in the processing of scrap cars either for their own developmental needs or for social responsibility. The design for dismantling approach can be an effective solution to the existing difficulties in dismantling ELVs. This approach can also provide guidelines in the design of automotive products. This paper illustrates the difficulty of handling polymers in dashboards. The physical properties of polymers prevent easy separation and recycling by using mechanical methods. Thus, dealers have to rely on chemical methods such as pyrolysis. Therefore, car designers should use a single material to benefit dealers. The use of materials for effective end-of-life processing without sacrificing the original performance requirements of the vehicle should be explored. Copyright © 2013 Elsevier Ltd. All rights reserved.
Chooz A, First Pressurized Water Reactor to be Dismantled in France - 13445

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boucau, Joseph; Mirabella, C.; Nilsson, Lennart

2013-07-01

Nine commercial nuclear power plants have been permanently shut down in France to date, of which the Chooz A plant underwent an extensive decommissioning and dismantling program. Chooz Nuclear Power Station is located in the municipality of Chooz, Ardennes region, in the northeast part of France. Chooz B1 and B2 are 1,500 megawatt electric (MWe) pressurized water reactors (PWRs) currently in operation. Chooz A, a 305 MWe PWR implanted in two caves within a hill, began operations in 1967 and closed in 1991, and will now become the first PWR in France to be fully dismantled. EDF CIDEN (Engineering Centermore » for Dismantling and Environment) has awarded Westinghouse a contract for the dismantling of its Chooz A reactor vessel (RV). The project began in January 2010. Westinghouse is leading the project in a consortium with Nuvia France. The project scope includes overall project management, conditioning of the reactor vessel (RV) head, RV and RV internals segmentation, reactor nozzle cutting for lifting the RV out of the pit and seal it afterwards, dismantling of the RV thermal insulation, ALARA (As Low As Reasonably Achievable) forecast to ensure acceptable doses for the personnel, complementary vacuum cleaner to catch the chips during the segmentation work, needs and facilities, waste characterization and packaging, civil work modifications, licensing documentation. The RV and RV internals will be segmented based on the mechanical cutting technology that Westinghouse applied successfully for more than 13 years. The segmentation activities cover the cutting and packaging plan, tooling design and qualification, personnel training and site implementation. Since Chooz A is located inside two caves, the project will involve waste transportation from the reactor cave through long galleries to the waste buffer area. The project will end after the entire dismantling work is completed, and the waste storage is outside the caves and ready to be shipped either to the ANDRA
Evaluation of prognostic and predictive value of microtubule associated protein tau in two independent cohorts.

PubMed

Baquero, Maria T; Lostritto, Karen; Gustavson, Mark D; Bassi, Kimberly A; Appia, Franck; Camp, Robert L; Molinaro, Annette M; Harris, Lyndsay N; Rimm, David L

2011-11-02

Microtubule associated proteins (MAPs) endogenously regulate microtubule stabilization and have been reported as prognostic and predictive markers for taxane response. The microtubule stabilizer, MAP-tau, has shown conflicting results. We quantitatively assessed MAP-tau expression in two independent breast cancer cohorts to determine prognostic and predictive value of this biomarker. MAP-tau expression was evaluated in the retrospective Yale University breast cancer cohort (n = 651) using tissue microarrays and also in the TAX 307 cohort, a clinical trial randomized for TAC versus FAC chemotherapy (n = 140), using conventional whole tissue sections. Expression was measured using the AQUA method for quantitative immunofluorescence. Scores were correlated with clinicopathologic variables, survival, and response to therapy. Assessment of the Yale cohort using Cox univariate analysis indicated an improved overall survival (OS) in tumors with a positive correlation between high MAP-tau expression and overall survival (OS) (HR = 0.691, 95% CI = 0.489-0.974; P = 0.004). Kaplan Meier analysis showed 10-year survival for 65% of patients with high MAP-tau expression compared to 52% with low expression (P = .006). In TAX 307, high expression was associated with significantly longer median time to tumor progression (TTP) regardless of treatment arm (33.0 versus 23.4 months, P = 0.010) with mean TTP of 31.2 months. Response rates did not differ by MAP-tau expression (P = 0.518) or by treatment arm (P = 0.584). Quantitative measurement of MAP-tau expression has prognostic value in both cohorts, with high expression associated with longer TTP and OS. Differences by treatment arm or response rate in low versus high MAP-tau groups were not observed, indicating that MAP-tau is not associated with response to taxanes and is not a useful predictive marker for taxane-based chemotherapy.
Evaluation of prognostic and predictive value of microtubule associated protein tau in two independent cohorts

PubMed Central

2011-01-01

Introduction Microtubule associated proteins (MAPs) endogenously regulate microtubule stabilization and have been reported as prognostic and predictive markers for taxane response. The microtubule stabilizer, MAP-tau, has shown conflicting results. We quantitatively assessed MAP-tau expression in two independent breast cancer cohorts to determine prognostic and predictive value of this biomarker. Methods MAP-tau expression was evaluated in the retrospective Yale University breast cancer cohort (n = 651) using tissue microarrays and also in the TAX 307 cohort, a clinical trial randomized for TAC versus FAC chemotherapy (n = 140), using conventional whole tissue sections. Expression was measured using the AQUA method for quantitative immunofluorescence. Scores were correlated with clinicopathologic variables, survival, and response to therapy. Results Assessment of the Yale cohort using Cox univariate analysis indicated an improved overall survival (OS) in tumors with a positive correlation between high MAP-tau expression and overall survival (OS) (HR = 0.691, 95% CI = 0.489-0.974; P = 0.004). Kaplan Meier analysis showed 10-year survival for 65% of patients with high MAP-tau expression compared to 52% with low expression (P = .006). In TAX 307, high expression was associated with significantly longer median time to tumor progression (TTP) regardless of treatment arm (33.0 versus 23.4 months, P = 0.010) with mean TTP of 31.2 months. Response rates did not differ by MAP-tau expression (P = 0.518) or by treatment arm (P = 0.584). Conclusions Quantitative measurement of MAP-tau expression has prognostic value in both cohorts, with high expression associated with longer TTP and OS. Differences by treatment arm or response rate in low versus high MAP-tau groups were not observed, indicating that MAP-tau is not associated with response to taxanes and is not a useful predictive marker for taxane-based chemotherapy. PMID:21888627

Msd1/SSX2IP-dependent microtubule anchorage ensures spindle orientation and primary cilia formation

PubMed Central

Hori, Akiko; Ikebe, Chiho; Tada, Masazumi; Toda, Takashi

2014-01-01

Anchoring microtubules to the centrosome is critical for cell geometry and polarity, yet the molecular mechanism remains unknown. Here we show that the conserved human Msd1/SSX2IP is required for microtubule anchoring. hMsd1/SSX2IP is delivered to the centrosome in a centriolar satellite-dependent manner and binds the microtubule-nucleator γ-tubulin complex. hMsd1/SSX2IP depletion leads to disorganised interphase microtubules and misoriented mitotic spindles with reduced length and intensity. Furthermore, hMsd1/SSX2IP is essential for ciliogenesis, and during zebrafish embryogenesis, knockdown of its orthologue results in ciliary defects and disturbs left-right asymmetry. We propose that the Msd1 family comprises conserved microtubule-anchoring proteins. PMID:24397932
Inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS for isotope analysis of long-lived radionuclides

NASA Astrophysics Data System (ADS)

Becker, J. Sabine

2005-04-01

For a few years now inductively coupled plasma mass spectrometry has been increasingly used for precise and accurate determination of isotope ratios of long-lived radionuclides at the trace and ultratrace level due to its excellent sensitivity, good precision and accuracy. At present, ICP-MS and also laser ablation ICP-MS are applied as powerful analytical techniques in different fields such as the characterization of nuclear materials, recycled and by-products (e.g., spent nuclear fuel or depleted uranium ammunitions), radioactive waste control, in environmental monitoring and in bioassay measurements, in health control, in geochemistry and geochronology. Especially double-focusing sector field ICP mass spectrometers with single ion detector or with multiple ion collector device have been used for the precise determination of long-lived radionuclides isotope ratios at very low concentration levels. Progress has been achieved by the combination of ultrasensitive mass spectrometric techniques with effective separation and enrichment procedures in order to improve detection limits or by the introduction of the collision cell in ICP-MS for reducing disturbing interfering ions (e.g., of 129Xe+ for the determination of 129I). This review describes the state of the art and the progress of ICP-MS and laser ablation ICP-MS for isotope ratio measurements of long-lived radionuclides in different sample types, especially in the main application fields of characterization of nuclear and radioactive waste material, environmental research and health controls.
Purification and characterization of sheep brain cold-stable microtubules.

PubMed Central

Pirollet, F; Job, D; Fischer, E H; Margolis, R L

1983-01-01

The isolation of cold-stable microtubules in high yields, described previously only from rodents, was extended to the brain of higher animals. Under optimal conditions, yields of 30 mg of cold-stable microtubles per 100 g of sheep brain could be obtained routinely. Material purified by two polymerization cycles displayed the same stability to cold temperature or to millimolar concentrations of calcium and the same lability to calmodulin and to ATP as did the purified material obtained from the rat [Job, D., Rauch, C.T., Fischer, E.H. & Margolis, R.L. (1982) Biochemistry 21, 509]. Furthermore, DE-52 chromatography of this material yielded a fraction that restored cold stability when added to cold-labile microtubules. Known to bind to calmodulin and to enhance microtubule assembly, tau proteins had no cold-stabilizing activity. Protein profiles of the cold-stabilizing fraction from sheep and rat brain were similar to one another but showed no protein bands corresponding to the tau proteins. Images PMID:6572919
Environmental applications of single collector high resolution ICP-MS.

PubMed

Krachler, Michael

2007-08-01

The number of environmental applications of single collector high resolution ICP-MS (HR-ICP-MS) has increased rapidly in recent years. There are many factors that contribute to make HR-ICP-MS a very powerful tool in environmental analysis. They include the extremely low detection limits achievable, tremendously high sensitivity, the ability to separate ICP-MS signals of the analyte from spectral interferences, enabling the reliable determination of many trace elements, and the reasonable precision of isotope ratio measurements. These assets are improved even further using high efficiency sample introduction systems. Therefore, external factors such as the stability of laboratory blanks are frequently the limiting factor in HR-ICP-MS analysis rather than the detection power. This review aims to highlight the most recent applications of HR-ICP-MS in this sector, focusing on matrices and applications where the superior capabilities of the instrumental technique are most useful and often ultimately required.
Analysis and comparison of glass fragments by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and ICP-MS.

PubMed

Trejos, Tatiana; Montero, Shirly; Almirall, José R

2003-08-01

The discrimination potential of Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) is compared with previously reported solution ICP-MS methods using external calibration (EC) with internal standardization and a newly reported solution isotope dilution (ID) method for the analysis of two different glass populations. A total of 91 different glass samples were used for the comparison study; refractive index and elemental composition were measured by the techniques mentioned above. One set consisted of 45 headlamps taken from a variety of automobiles that represents a range of 20 years of manufacturing dates. A second set consisted of 46 automotive glasses (side windows, rear windows, and windshields) representing casework glass from different vehicle manufacturers over several years. The element menu for the LA-ICP-MS and EC-ICP-MS methods include Mg, Al, Ca, Mn, Ce, Ti, Zr, Sb, Ga, Ba, Rb, Sm, Sr, Hf, La, and Pb. The ID method was limited to the analysis of two isotopes each of Mg, Sr, Zr, Sb, Ba, Sm, Hf, and Pb. Laser ablation analyses were performed with a Q switched Nd:YAG, 266 nm, 6 mJ output energy laser. The laser was used in depth profile mode while sampling using a 50 microm spot size for 50 sec at 10 Hz (500 shots). The typical bias for the analysis of NIST 612 by LA-ICP-MS was less than 5% in all cases and typically better than 5% for most isotopes. The precision for the vast majority of the element menu was determined generally less than 10% for all the methods when NIST 612 was measured (40 microg x g(-1)). Method detection limits (MDL) for the EC and LA-ICP-MS methods were similar and generally reported as less than 1 microg x g(-1) for the analysis of NIST 612. While the solution sample introduction methods using EC and ID presented excellent sensitivity and precision, these methods have the disadvantages of destroying the sample, and also involve complex sample preparation. The laser ablation method was simpler, faster, and
Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells.

PubMed

Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka; Stearns, Tim

2017-09-14

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.
Stabilization, not polymerization, of microtubules inhibits the nuclear translocation of STATs in adipocytes.

PubMed

Gleason, Evanna L; Hogan, Jessica C; Stephens, Jacqueline M

2004-12-17

Signal transducers and activators of transcriptions (STATs) are a family of latent transcription factors which are activated by a variety of growth factors and cytokines in many cell types. However, the mechanism by which these transcription factors translocate to the nucleus is poorly understood. The goal of this study was to determine the requirement of microfilaments and microtubules for cytokine induced STAT activation in cultured adipocytes. We used seven different actin-specific and microtubule-specific agents that are well-established effectors of these cytoskeletal networks. Our results clearly demonstrate that inhibition of microfilaments or the prevention of microtubule polymerization has no effect on the ability of STATs to be tyrosine phosphorylated or to translocate to the nucleus. However, we observed that paclitaxel, a microtubule stabilizer, resulted in a significant decrease in the nuclear translocation of STATs without affecting the cytosolic tyrosine phosphorylation of these transcription factors. In summary, our results demonstrate that the dynamic instability, but not the polymerization, of microtubules contributes to nuclear translocation of STAT proteins in adipocytes.
Inter-dependent apical microtubule and actin dynamics orchestrate centrosome retention and neuronal delamination

PubMed Central

Kasioulis, Ioannis

2017-01-01

Detachment of newborn neurons from the neuroepithelium is required for correct neuronal architecture and functional circuitry. This process, also known as delamination, involves adherens-junction disassembly and acto-myosin-mediated abscission, during which the centrosome is retained while apical/ciliary membranes are shed. Cell-biological mechanisms mediating delamination are, however, poorly understood. Using live-tissue and super-resolution imaging, we uncover a centrosome-nucleated wheel-like microtubule configuration, aligned with the apical actin cable and adherens-junctions within chick and mouse neuroepithelial cells. These microtubules maintain adherens-junctions while actin maintains microtubules, adherens-junctions and apical end-foot dimensions. During neuronal delamination, acto-myosin constriction generates a tunnel-like actin-microtubule configuration through which the centrosome translocates. This movement requires inter-dependent actin and microtubule activity, and we identify drebrin as a potential coordinator of these cytoskeletal dynamics. Furthermore, centrosome compromise revealed that this organelle is required for delamination. These findings identify new cytoskeletal configurations and regulatory relationships that orchestrate neuronal delamination and may inform mechanisms underlying pathological epithelial cell detachment. PMID:29058679
Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino.

PubMed

Rosales-Nieves, Alicia E; Johndrow, James E; Keller, Lani C; Magie, Craig R; Pinto-Santini, Delia M; Parkhurst, Susan M

2006-04-01

The actin-nucleation factors Spire and Cappuccino (Capu) regulate the onset of ooplasmic streaming in Drosophila melanogaster. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. Here, we demonstrate that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms has not been addressed. We find that SpireD does not have crosslinking activity, whereas SpireC is a potent crosslinker. We show that SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. We propose that Rho1, cappuccino and spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments.
The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules

PubMed Central

Green, Rebecca A.; Mayers, Jonathan R.; Wang, Shaohe; Lewellyn, Lindsay; Desai, Arshad; Audhya, Anjon

2013-01-01

Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring–derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution. PMID:24217623
Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration.

PubMed

Daou, Pascale; Hasan, Salma; Breitsprecher, Dennis; Baudelet, Emilie; Camoin, Luc; Audebert, Stéphane; Goode, Bruce L; Badache, Ali

2014-03-01

Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge.
Construction of artificial cilia from microtubules and kinesins through a well-designed bottom-up approach.

PubMed

Sasaki, Ren; Kabir, Arif Md Rashedul; Inoue, Daisuke; Anan, Shizuka; Kimura, Atsushi P; Konagaya, Akihiko; Sada, Kazuki; Kakugo, Akira

2018-04-05

Self-organized structures of biomolecular motor systems, such as cilia and flagella, play key roles in the dynamic processes of living organisms, like locomotion or the transportation of materials. Although fabrication of such self-organized structures from reconstructed biomolecular motor systems has attracted much attention in recent years, a systematic construction methodology is still lacking. In this work, through a bottom-up approach, we fabricated artificial cilia from a reconstructed biomolecular motor system, microtubule/kinesin. The artificial cilia exhibited a beating motion upon the consumption, by the kinesins, of the chemical energy obtained from the hydrolysis of adenosine triphosphate (ATP). Several design parameters, such as the length of the microtubules, the density of the kinesins along the microtubules, the depletion force among the microtubules, etc., have been identified, which permit tuning of the beating frequency of the artificial cilia. The beating frequency of the artificial cilia increases upon increasing the length of the microtubules, but declines for the much longer microtubules. A high density of the kinesins along the microtubules is favorable for the beating motion of the cilia. The depletion force induced bundling of the microtubules accelerated the beating motion of the artificial cilia and increased the beating frequency. This work helps understand the role of self-assembled structures of the biomolecular motor systems in the dynamics of living organisms and is expected to expedite the development of artificial nanomachines, in which the biomolecular motors may serve as actuators.
Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration

PubMed Central

Daou, Pascale; Hasan, Salma; Breitsprecher, Dennis; Baudelet, Emilie; Camoin, Luc; Audebert, Stéphane; Goode, Bruce L.; Badache, Ali

2014-01-01

Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge. PMID:24403606
Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis

PubMed Central

Wandke, Cornelia; Barisic, Marin; Sigl, Reinhard; Rauch, Veronika; Wolf, Frank; Amaro, Ana C.; Tan, Chia H.; Pereira, Antonio J.; Kutay, Ulrike; Maiato, Helder; Meraldi, Patrick

2012-01-01

Chromokinesins are microtubule plus end–directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar spindle formation but the functions of the human homologues, hKID (KIF22) and KIF4A, are poorly understood. By using RNAi-mediated protein knockdown in human cells, we find that only co-depletion delayed progression through mitosis in a Mad2-dependent manner. Depletion of hKID caused abnormal chromosome arm orientation, delayed chromosome congression, and sensitized cells to nocodazole. Knockdown of KIF4A increased the number and length of microtubules, altered kinetochore oscillations, and decreased kinetochore microtubule flux. These changes were associated with failures in establishing a tight metaphase plate and an increase in anaphase lagging chromosomes. Co-depletion of both chromokinesins aggravated chromosome attachment failures, which led to mitotic arrest. Thus, hKID and KIF4A contribute independently to the rapid and correct attachment of chromosomes by controlling the positioning of chromosome arms and the dynamics of microtubules, respectively. PMID:22945934
Spatial confinement of active microtubule networks induces large-scale rotational cytoplasmic flow

PubMed Central

Suzuki, Kazuya; Miyazaki, Makito; Takagi, Jun; Itabashi, Takeshi; Ishiwata, Shin’ichi

2017-01-01

Collective behaviors of motile units through hydrodynamic interactions induce directed fluid flow on a larger length scale than individual units. In cells, active cytoskeletal systems composed of polar filaments and molecular motors drive fluid flow, a process known as cytoplasmic streaming. The motor-driven elongation of microtubule bundles generates turbulent-like flow in purified systems; however, it remains unclear whether and how microtubule bundles induce large-scale directed flow like the cytoplasmic streaming observed in cells. Here, we adopted Xenopus egg extracts as a model system of the cytoplasm and found that microtubule bundle elongation induces directed flow for which the length scale and timescale depend on the existence of geometrical constraints. At the lower activity of dynein, kinesins bundle and slide microtubules, organizing extensile microtubule bundles. In bulk extracts, the extensile bundles connected with each other and formed a random network, and vortex flows with a length scale comparable to the bundle length continually emerged and persisted for 1 min at multiple places. When the extracts were encapsulated in droplets, the extensile bundles pushed the droplet boundary. This pushing force initiated symmetry breaking of the randomly oriented bundle network, leading to bundles aligning into a rotating vortex structure. This vortex induced rotational cytoplasmic flows on the length scale and timescale that were 10- to 100-fold longer than the vortex flows emerging in bulk extracts. Our results suggest that microtubule systems use not only hydrodynamic interactions but also mechanical interactions to induce large-scale temporally stable cytoplasmic flow. PMID:28265076
Examining the technology acceptance for dismantling of waste printed circuit boards in light of recycling and environmental concerns.

PubMed

Duan, Huabo; Hou, Kun; Li, Jinhui; Zhu, Xiaodong

2011-03-01

The dismantling of printed circuit board assemblies (PCBAs) and the recovery of their useful materials can lead to serious environmental impacts mainly due to their complicated physical structure and the variety of toxic elements contained in their material composition. So far, less attention has been paid to their responsible recycling compared to that of bare printed circuit boards. Combined with other materials recovery process, proper dismantling of PCBAs is beneficial to conserve scarce resources, reuse the components, and eliminate or safely dispose of hazardous materials. In analyzing the generation, resources potential and hazardous risk of scrap PCBAs, technologies used for the dismantling of waste PCBAs have been widely investigated and reviewed from the aspects of both industrial application and laboratory-scale studies. In addition, the feasibility of PCBA dismantling has been discussed, the determinants of which, including the heating conditions and mechanical properties have been identified. Moreover, this paper evaluates the environmental consequences caused by the dismantling of PCBAs. Copyright © 2010 Elsevier Ltd. All rights reserved.
Moonlighting microtubule-associated proteins: regulatory functions by day and pathological functions at night.

PubMed

Oláh, J; Tőkési, N; Lehotzky, A; Orosz, F; Ovádi, J

2013-11-01

The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeletal network. Cytoskeleton comprises fibrous protein networks of microtubules, actin, and intermediate filaments. These filamentous polymer structures are highly dynamic and undergo constant and rapid reorganization during cellular processes. The microtubular system plays a crucial role in the brain, as it is involved in an enormous number of cellular events including cell differentiation and pathological inclusion formation. These multifarious functions of microtubules can be achieved by their decoration with proteins/enzymes that exert specific effects on the dynamics and organization of the cytoskeleton and mediate distinct functions due to their moonlighting features. This mini-review focuses on two aspects of the microtubule cytoskeleton. On the one hand, we describe the heteroassociation of tubulin/microtubules with metabolic enzymes, which in addition to their catalytic activities stabilize microtubule structures via their cross-linking functions. On the other hand, we focus on the recently identified moonlighting tubulin polymerization promoting protein, TPPP/p25. TPPP/p25 is a microtubule-associated protein and it displays distinct physiological or pathological (aberrant) functions; thus it is a prototype of Neomorphic Moonlighting Proteins. The expression of TPPP/p25 is finely controlled in the human brain; this protein is indispensable for the development of projections of oligodendrocytes that are responsible for the ensheathment of axons. The nonphysiological, higher or lower TPPP/p25 level leads to distinct CNS diseases. Mechanisms contributing to the control of microtubule stability and dynamics by metabolic enzymes and TPPP/p25 will be discussed. Copyright © 2013 Wiley Periodicals, Inc.
TONNEAU2/FASS Regulates the Geometry of Microtubule Nucleation and Cortical Array Organization in Interphase Arabidopsis Cells[C][W

PubMed Central

Kirik, Angela; Ehrhardt, David W.; Kirik, Viktor

2012-01-01

Organization of microtubules into ordered arrays involves spatial and temporal regulation of microtubule nucleation. Here, we show that acentrosomal microtubule nucleation in plant cells involves a previously unknown regulatory step that determines the geometry of microtubule nucleation. Dynamic imaging of interphase cortical microtubules revealed that the ratio of branching to in-bundle microtubule nucleation on cortical microtubules is regulated by the Arabidopsis thaliana B′′ subunit of protein phosphatase 2A, which is encoded by the TONNEAU2/FASS (TON2) gene. The probability of nucleation from γ-tubulin complexes localized at the cell cortex was not affected by a loss of TON2 function, suggesting a specific role of TON2 in regulating the nucleation geometry. Both loss of TON2 function and ectopic targeting of TON2 to the plasma membrane resulted in defects in cell shape, suggesting the importance of TON2-mediated regulation of the microtubule cytoskeleton in cell morphogenesis. Loss of TON2 function also resulted in an inability for cortical arrays to reorient in response to light stimulus, suggesting an essential role for TON2 and microtubule branching nucleation in reorganization of microtubule arrays. Our data establish TON2 as a regulator of interphase microtubule nucleation and provide experimental evidence for a novel regulatory step in the process of microtubule-dependent nucleation. PMID:22395485
A novel dismantling process of waste printed circuit boards using water-soluble ionic liquid.

PubMed

Zeng, Xianlai; Li, Jinhui; Xie, Henghua; Liu, Lili

2013-10-01

Recycling processes for waste printed circuit boards (WPCBs) have been well established in terms of scientific research and field pilots. However, current dismantling procedures for WPCBs have restricted the recycling process, due to their low efficiency and negative impacts on environmental and human health. This work aimed to seek an environmental-friendly dismantling process through heating with water-soluble ionic liquid to separate electronic components and tin solder from two main types of WPCBs-cathode ray tubes and computer mainframes. The work systematically investigates the influence factors, heating mechanism, and optimal parameters for opening solder connections on WPCBs during the dismantling process, and addresses its environmental performance and economic assessment. The results obtained demonstrate that the optimal temperature, retention time, and turbulence resulting from impeller rotation during the dismantling process, were 250 °C, 12 min, and 45 rpm, respectively. Nearly 90% of the electronic components were separated from the WPCBs under the optimal experimental conditions. This novel process offers the possibility of large industrial-scale operations for separating electronic components and recovering tin solder, and for a more efficient and environmentally sound process for WPCBs recycling. Copyright © 2013 Elsevier Ltd. All rights reserved.
Dismantling the nuclear research reactor Thetis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michiels, P.

The research reactor Thetis, in service since 1967 and stopped in 2003, is part of the laboratories of the institution of nuclear science of the University of Ghent. The reactor, of the pool-type, was used as a neutron-source for the production of radio-isotopes and for activation analyses. The reactor is situated in a water pool with inner diameter of 3 m. and a depth of 7.5 m. The reactor core is situated 5.3 m under water level. Besides the reactor, the pool contains pneumatic loops, handling tools, graphite blocks for neutron moderation and other experimental equipment. The building houses storagemore » rooms for fissile material and sources, a pneumatic circuit for transportation of samples, primary and secondary cooling circuits, water cleaning resin circuits, a ventilation system and other necessary devices. Because of the experimental character of the reactor, laboratories with glove boxes and other tools were needed and are included in the dismantling program. The building is in 3 levels with a crawl-space. The ground-floor contains the ventilation installation, the purification circuits with tanks, cooling circuits and pneumatic transport system. On the first floor, around the reactor hall, the control-room, visiting area, end-station for pneumatic transport, waste-storage room, fuel storage room and the labs are located. The second floor contains a few laboratories and end stations of the two high speed transfer tubes. The lowest level of the pool is situated under ground level. The reactor has been operated at a power of 150 kW and had a max operating power of 250 kW. Belgoprocess has been selected to decommission the reactor, the labs, storage halls and associated circuits to free release the building for conventional reuse and for the removal of all its internals as legal defined. Besides the dose-rate risk and contamination risk, there is also an asbestos risk of contamination. During construction of the installation, asbestos-containing materials

Analysis of high-purity germanium dioxide by ETV-ICP-AES with preliminary concentration of trace elements.

PubMed

Medvedev, Nickolay S; Shaverina, Anastasiya V; Tsygankova, Alphiya R; Saprykin, Anatoly I

2016-08-01

The paper presents a combined technique of germanium dioxide analysis by inductively coupled plasma atomic emission spectrometry (ICP-AES) with preconcentration of trace elements by distilling off matrix and electrothermal (ETV) introduction of the trace elements concentrate into the ICP. Evaluation of metrological characteristics of the developed technique of high-purity germanium dioxide analysis was performed. The limits of detection (LODs) for 25 trace elements ranged from 0.05 to 20ng/g. The accuracy of proposed technique is confirmed by "added-found" («or spiking») experiment and comparing the results of ETV-ICP-AES and ICP-AES analysis of high purity germanium dioxide samples. Copyright © 2016 Elsevier B.V. All rights reserved.
Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat)

NASA Technical Reports Server (NTRS)

Lewis, M. L.; Reynolds, J. L.; Cubano, L. A.; Hatton, J. P.; Lawless, B. D.; Piepmeier, E. H.

1998-01-01

Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.
Interim Cryogenic Propulsion Stage (ICPS) Handover Signing

NASA Image and Video Library

2017-10-26

Meeting in the Launch Control Center of NASA's Kennedy Space Center in Florida, officials of the agency's Spacecraft/Payload Integration and Evolution (SPIE) organization formally turn over processing of the Space Launch System (SLS) rocket's Interim Cryogenic Propulsion Stage (ICPS) to the center's Ground Systems Development and Operations (GSDO) directorate. The ICPS is the first integrated piece of flight hardware to arrive in preparation for the uncrewed Exploration Mission-1. With the Orion attached, the ICPS sits atop the SLS rocket and will provide the spacecraft with the additional thrust needed to travel tens of thousands of miles beyond the Moon.
Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells

PubMed Central

Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka

2017-01-01

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next. PMID:28906251
Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation

PubMed Central

Rickman, Jamie; Duellberg, Christian; Cade, Nicholas I.; Griffin, Lewis D.; Surrey, Thomas

2017-01-01

Growing microtubules are protected from depolymerization by the presence of a GTP or GDP/Pi cap. End-binding proteins of the EB1 family bind to the stabilizing cap, allowing monitoring of its size in real time. The cap size has been shown to correlate with instantaneous microtubule stability. Here we have quantitatively characterized the properties of cap size fluctuations during steady-state growth and have developed a theory predicting their timescale and amplitude from the kinetics of microtubule growth and cap maturation. In contrast to growth speed fluctuations, cap size fluctuations show a characteristic timescale, which is defined by the lifetime of the cap sites. Growth fluctuations affect the amplitude of cap size fluctuations; however, cap size does not affect growth speed, indicating that microtubules are far from instability during most of their time of growth. Our theory provides the basis for a quantitative understanding of microtubule stability fluctuations during steady-state growth. PMID:28280102
Microtubule-organizing centers and nucleating sites in land plants.

PubMed

Vaughn, K C; Harper, J D

1998-01-01

Microtubule-organizing centers (MTOCs) are morphologically diverse cellular sites involved in the nucleation and organization of microtubules (MTs). These structures are synonymous with the centrosome in mammalian cells. In most land plant cells, however, no such structures are observed and some have argued that plant cells may not have MTOCs. This review summarizes a number of experimental approaches toward the elucidation of those subcellular sites involved in microtubule nucleation and organization. In lower land plants, structurally well-defined MTOCs are present, such as the blepharoplast, multilayered structure, and polar organizer. In higher plants, much of the nucleation and organization of MTs occurs on the nuclear envelope or other endomembranes, such as the plasmalemma and smooth (tubular) endoplasmic reticulum. In some instances, one endomembrane may serve as a site of nucleation whereas others serve as the site of organization. Structural and motor microtubule-associated proteins also appear to be involved in MT nucleation and organization. Immunochemical evidence indicates that at least several of the proteins found in mammalian centrosomes, gamma-tubulin, centrin, pericentrin, and polypeptides recognized by the monoclonal antibodies MPM-2, 6C6, and C9 also recognize putative lower land plant MTOCs, indicating shared mechanisms of nucleation/organization in plants and animals. The most recent data from tubulin incorporation in vivo, mutants with altered MT organization, and molecular studies indicate the potential of these research tools in investigation of MTOCs in plants.
ICPS Turnover GSDO Employee Event

NASA Image and Video Library

2017-11-07

Mike Bolger, Ground Systems Development and Operations Program manager at NASA's Kennedy Space Center, speaks to guests during a ceremony in the high bay of the Space Station Processing Facility. The event marked the milestone of the Space Launch System rocket's Interim Cryogenic Propulsion Stage (ICPS) being turned over from NASA's Spacecraft/Payload Integration and Evolution organization to the spaceport's Ground Systems Development and Operations directorate. The ICPS is the first integrated piece of flight hardware to arrive in preparation for the uncrewed Exploration Mission-1.
Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1

PubMed Central

Tikhonenko, Irina; Irizarry, Karen; Khodjakov, Alexey; Koonce, Michael P.

2015-01-01

It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm. PMID:26298292
Low level detection of Cs-135 and Cs-137 in environmental samples by ICP-MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liezers, Martin; Farmer, Orville T.; Thomas, Linda MP

2009-10-01

The measurement of the fission product cesium isotopes 135Cs and 137Cs at low femtogram (fg) 10-15 levels in ground water by Inductively Coupled Plasma-Mass Spectrometry ICP-MS is reported. To eliminate the potential natural barium isobaric interference on the cesium isotopes, in-line chromatographic separation of the cesium from barium was performed followed by high sensitivity ICP-MS analysis. A high efficiency desolvating nebulizer system was employed to maximize ICP-MS sensitivity ~10cps/femtogram. The three sigma detection limit measured for 135Cs was 2fg/ml (0.1uBq/ml) and for 137Cs 0.9fg/ml (0.0027Bq/ml) with analysis time of less than 30 minutes/sample. Cesium detection and 135/137 isotope ratio measurementmore » at very low femtogram levels using this method in a ground water matrix is also demonstrated.« less
Pick-N-Pull Auto Dismantlers, Kansas City, LLC Inc.

EPA Pesticide Factsheets

The EPA is providing notice of a proposed Administrative Penalty Assessment against Pick-N-Pull Auto Dismantlers, Kansas City, LLC, a subsidiary of Schnitzer Steel Industries, Inc., for alleged violations at its facilities at 8012 East Truman Rd., Kansas C
Taking directions: the role of microtubule-bound nucleation in the self-organization of the plant cortical array

NASA Astrophysics Data System (ADS)

Deinum, Eva E.; Tindemans, Simon H.; Mulder, Bela M.

2011-10-01

The highly aligned cortical microtubule array of interphase plant cells is a key regulator of anisotropic cell expansion. Recent computational and analytical work has shown that the non-equilibrium self-organization of this structure can be understood on the basis of experimentally observed collisional interactions between dynamic microtubules attached to the plasma membrane. Most of these approaches assumed that new microtubules are homogeneously and isotropically nucleated on the cortical surface. Experimental evidence, however, shows that nucleation mostly occurs from other microtubules and under specific relative angles. Here, we investigate the impact of directed microtubule-bound nucleations on the alignment process using computer simulations. The results show that microtubule-bound nucleations can increase the degree of alignment achieved, decrease the timescale of the ordering process and widen the regime of dynamic parameters for which the system can self-organize. We establish that the major determinant of this effect is the degree of co-alignment of the nucleations with the parent microtubule. The specific role of sideways branching nucleations appears to allow stronger alignment while maintaining a measure of overall spatial homogeneity. Finally, we investigate the suggestion that observed persistent rotation of microtubule domains can be explained through a handedness bias in microtubule-bound nucleations, showing that this is possible only for an extreme bias and over a limited range of parameters.
Blocking ESCRT-Mediated Envelopment Inhibits Microtubule-Dependent Trafficking of Alphaherpesviruses In Vitro

PubMed Central

Kharkwal, Himanshu; Smith, Caitlin G.

2014-01-01

ABSTRACT Herpes simplex virus (HSV) and, as reported here, pseudorabies virus (PRV) utilize the ESCRT apparatus to drive cytoplasmic envelopment of their capsids. Here, we demonstrate that blocking ESCRT-mediated envelopment using the dominant-negative inhibitor Vps4A-EQ (Vps4A in which glutamate [E] at position 228 in the ATPase active site is replaced by a glutamine [Q]) reduced the ability of HSV and PRV particles to subsequently traffic along microtubules in vitro. HSV and PRV capsid-associated particles with bound green fluorescent protein (GFP)-labeled Vps4A-EQ were readily detected by fluorescence microscopy in cytoplasmic extracts of infected cells. These Vps4A-EQ-associated capsid-containing particles bound to microtubules in vitro but were unable to traffic along them. Using a PRV strain expressing a fluorescent capsid and a fluorescently tagged form of the envelope protein gD, we found that similar numbers of gD-positive and gD-negative capsid-associated particles accumulated in cytoplasmic extracts under our conditions. Both classes of PRV particle bound to microtubules in vitro with comparable efficiency, and similar results were obtained for HSV using anti-gD immunostaining. The gD-positive and gD-negative PRV capsids were both capable of trafficking along microtubules in vitro; however, motile gD-positive particles were less numerous and their trafficking was more sensitive to the inhibitory effects of Vps4A-EQ. We discuss our data in the context of microtubule-mediated trafficking of naked and enveloped alphaherpesvirus capsids. IMPORTANCE The alphaherpesviruses include several important human pathogens. These viruses utilize microtubule-mediated transport to travel through the cell cytoplasm; however, the molecular mechanisms of trafficking are not well understood. In this study, we have used a cell-free system to examine the requirements for microtubule trafficking and have attempted to distinguish between the movement of so-called “naked” and
Maintenance of dendritic spine morphology by partitioning-defective 1b through regulation of microtubule growth.

PubMed

Hayashi, Kenji; Suzuki, Atsushi; Hirai, Syu-ichi; Kurihara, Yasuyuki; Hoogenraad, Casper C; Ohno, Shigeo

2011-08-24

Dendritic spines are postsynaptic structures that receive excitatory synaptic input from presynaptic terminals. Actin and its regulatory proteins play a central role in morphogenesis of dendritic spines. In addition, recent studies have revealed that microtubules are indispensable for the maintenance of mature dendritic spine morphology by stochastically invading dendritic spines and regulating dendritic localization of p140Cap, which is required for actin reorganization. However, the regulatory mechanisms of microtubule dynamics remain poorly understood. Partitioning-defective 1b (PAR1b), a cell polarity-regulating serine/threonine protein kinase, is thought to regulate microtubule dynamics by inhibiting microtubule binding of microtubule-associated proteins. Results from the present study demonstrated that PAR1b participates in the maintenance of mature dendritic spine morphology in mouse hippocampal neurons. Immunofluorescent analysis revealed PAR1b localization in the dendrites, which was concentrated in dendritic spines of mature neurons. PAR1b knock-down cells exhibited decreased mushroom-like dendritic spines, as well as increased filopodia-like dendritic protrusions, with no effect on the number of protrusions. Live imaging of microtubule plus-end tracking proteins directly revealed decreases in distance and duration of microtubule growth following PAR1b knockdown in a neuroblastoma cell line and in dendrites of hippocampal neurons. In addition, reduced accumulation of GFP-p140Cap in dendritic protrusions was confirmed in PAR1b knock-down neurons. In conclusion, the present results suggested a novel function for PAR1b in the maintenance of mature dendritic spine morphology by regulating microtubule growth and the accumulation of p140Cap in dendritic spines.
Chlamydia Hijacks ARF GTPases To Coordinate Microtubule Posttranslational Modifications and Golgi Complex Positioning.

PubMed

Wesolowski, Jordan; Weber, Mary M; Nawrotek, Agata; Dooley, Cheryl A; Calderon, Mike; St Croix, Claudette M; Hackstadt, Ted; Cherfils, Jacqueline; Paumet, Fabienne

2017-05-02

The intracellular bacterium Chlamydia trachomatis develops in a parasitic compartment called the inclusion. Posttranslationally modified microtubules encase the inclusion, controlling the positioning of Golgi complex fragments around the inclusion. The molecular mechanisms by which Chlamydia coopts the host cytoskeleton and the Golgi complex to sustain its infectious compartment are unknown. Here, using a genetically modified Chlamydia strain, we discovered that both posttranslationally modified microtubules and Golgi complex positioning around the inclusion are controlled by the chlamydial inclusion protein CT813/CTL0184/InaC and host ARF GTPases. CT813 recruits ARF1 and ARF4 to the inclusion membrane, where they induce posttranslationally modified microtubules. Similarly, both ARF isoforms are required for the repositioning of Golgi complex fragments around the inclusion. We demonstrate that CT813 directly recruits ARF GTPases on the inclusion membrane and plays a pivotal role in their activation. Together, these results reveal that Chlamydia uses CT813 to hijack ARF GTPases to couple posttranslationally modified microtubules and Golgi complex repositioning at the inclusion. IMPORTANCE Chlamydia trachomatis is an important cause of morbidity and a significant economic burden in the world. However, how Chlamydia develops its intracellular compartment, the so-called inclusion, is poorly understood. Using genetically engineered Chlamydia mutants, we discovered that the effector protein CT813 recruits and activates host ADP-ribosylation factor 1 (ARF1) and ARF4 to regulate microtubules. In this context, CT813 acts as a molecular platform that induces the posttranslational modification of microtubules around the inclusion. These cages are then used to reposition the Golgi complex during infection and promote the development of the inclusion. This study provides the first evidence that ARF1 and ARF4 play critical roles in controlling posttranslationally modified
Drosophila Spastin Regulates Synaptic Microtubule Networks and Is Required for Normal Motor Function

PubMed Central

Sherwood, Nina Tang; Sun, Qi; Xue, Mingshan; Zhang, Bing

2004-01-01

The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord. PMID:15562320
α-Synuclein Fibrils Exhibit Gain of Toxic Function, Promoting Tau Aggregation and Inhibiting Microtubule Assembly*

PubMed Central

Oikawa, Takayuki; Nonaka, Takashi; Terada, Makoto; Tamaoka, Akira; Hisanaga, Shin-ichi; Hasegawa, Masato

2016-01-01

α-Synuclein is the major component of Lewy bodies and Lewy neurites in Parkinson disease and dementia with Lewy bodies and of glial cytoplasmic inclusions in multiple system atrophy. It has been suggested that α-synuclein fibrils or intermediate protofibrils in the process of fibril formation may have a toxic effect on neuronal cells. In this study, we investigated the ability of soluble monomeric α-synuclein to promote microtubule assembly and the effects of conformational changes of α-synuclein on Tau-promoted microtubule assembly. In marked contrast to previous findings, monomeric α-synuclein had no effect on microtubule polymerization. However, both α-synuclein fibrils and protofibrils inhibited Tau-promoted microtubule assembly. The inhibitory effect of α-synuclein fibrils was greater than that of the protofibrils. Dot blot overlay assay and spin-down techniques revealed that α-synuclein fibrils bind to Tau and inhibit microtubule assembly by depleting the Tau available for microtubule polymerization. Using various deletion mutants of α-synuclein and Tau, the acidic C-terminal region of α-synuclein and the basic central region of Tau were identified as regions involved in the binding. Furthermore, introduction of α-synuclein fibrils into cultured cells overexpressing Tau protein induced Tau aggregation. These results raise the possibility that α-synuclein fibrils interact with Tau, inhibit its function to stabilize microtubules, and also promote Tau aggregation, leading to dysfunction of neuronal cells. PMID:27226637
Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization.

PubMed

Shen, Jia; Ma, Hailin; Zhang, Tiancheng; Liu, Hui; Yu, Linghua; Li, Guosheng; Li, Huishuang; Hu, Meichun

2017-01-01

The tubulin/microtubule system, which is an integral component of the cytoskeleton, plays an essential role in mitosis. Targeting mitotic progression by disturbing microtubule dynamics is a rational strategy for cancer treatment. Microtubule polymerization assay was performed to examine the effect of Magnolol (a novel natural phenolic compound isolated from Magnolia obovata) on cellular microtubule polymerization in human non-small cell lung cancer (NSCLC) cells. Cell cycle analysis, mitotic index assay, cell proliferation assay, colony formation assay, western blotting analysis of cell cycle regulators, Annexin V-FITC/PI staining, and live/dead viability staining were carried out to investigate the Magnolol's inhibitory effect on proliferation and viability of NSCLS cells in vitro. Xenograft model of human A549 NSCLC tumor was used to determine the Magnolol's efficacy in vivo. Magnolol treatment effectively inhibited cell proliferation and colony formation of NSCLC cells. Further study proved that Magnolol induced the mitotic phase arrest and inhibited G2/M progression in a dose-dependent manner, which were mechanistically associated with expression alteration of a series of cell cycle regulators. Furthermore, Magnolol treatment disrupted the cellular microtubule organization via inhibiting the polymerization of microtubule. We also found treatment with NSCLC cells with Magnolol resulted in apoptosis activation through a p53-independent pathway, and autophgy induction via down-regulation of the Akt/mTOR pathway. Finally, Magnolol treatment significantly suppressed the NSCLC tumor growth in mouse xenograft model in vivo. These findings identify Magnolol as a promising candidate with anti-microtubule polymerization activity for NSCLC treatment. © 2017 The Author(s). Published by S. Karger AG, Basel.
Mitochondria drive autophagy pathology via microtubule disassembly

PubMed Central

Arduíno, Daniela M.; Esteves, A. Raquel; Cardoso, Sandra Morais

2013-01-01

Neurons are exquisitely dependent on quality control systems to maintain a healthy intracellular environment. A permanent assessment of protein and organelle “quality” allows a coordinated action between repair and clearance of damage proteins and dysfunctional organelles. Impairments in the intracellular clearance mechanisms in long-lived postmitotic cells, like neurons, result in the progressive accumulation of damaged organelles and aggregates of aberrant proteins. Using cells bearing Parkinson disease (PD) patients’ mitochondria, we demonstrated that aberrant accumulation of autophagosomes in PD, commonly interpreted as an abnormal induction of autophagy, is instead due to defective autophagic clearance. This defect is a consequence of alterations in the microtubule network driven by mitochondrial dysfunction that hinder mitochondria and autophagosome trafficking. We uncover mitochondria and microtubule-directed traffic as main players in the regulation of autophagy in PD. PMID:23075854
Prefoldin 6 is required for normal microtubule dynamics and organization in Arabidopsis

PubMed Central

Gu, Ying; Deng, Zhiping; Paredez, Alexander R.; DeBolt, Seth; Wang, Zhi-Yong; Somerville, Chris

2008-01-01

Newly translated tubulin molecules undergo a series of complex interactions with nascent chain-binding chaperones, including prefoldin (PFD) and chaperonin-containing TCP-1 (CCT). By screening for oryzalin hypersensitivity, we identified several mutants of Arabidopsis that have lesions in PFD subunits. The pfd6–1 mutant exhibits a range of microtubule defects, including hypersensitivity to oryzalin, defects in cell division, cortical array organization, and microtubule dynamicity. Consistent with phenotypic analysis, proteomic analysis indicates several isoforms of tubulins were reduced in pfd6–1. These results support the concept that the function of microtubules is critically dependent on the absolute amount of tubulins. PMID:19004800
Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets.

PubMed

Quick, Quincy A

2018-01-26

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents.

Lessons from bacterial homolog of tubulin, FtsZ for microtubule dynamics.

PubMed

Battaje, Rachana Rao; Panda, Dulal

2017-09-01

FtsZ, a homolog of tubulin, is found in almost all bacteria and archaea where it has a primary role in cytokinesis. Evidence for structural homology between FtsZ and tubulin came from their crystal structures and identification of the GTP box. Tubulin and FtsZ constitute a distinct family of GTPases and show striking similarities in many of their polymerization properties. The differences between them, more so, the complexities of microtubule dynamic behavior in comparison to that of FtsZ, indicate that the evolution to tubulin is attributable to the incorporation of the complex functionalities in higher organisms. FtsZ and microtubules function as polymers in cell division but their roles differ in the division process. The structural and partial functional homology has made the study of their dynamic properties more interesting. In this review, we focus on the application of the information derived from studies on FtsZ dynamics to study microtubule dynamics and vice versa. The structural and functional aspects that led to the establishment of the homology between the two proteins are explained to emphasize the network of FtsZ and microtubule studies and how they are connected. © 2017 Society for Endocrinology.
Emerging roles of apoptotic microtubules during the execution phase of apoptosis.

PubMed

Oropesa Ávila, Manuel; Fernández Vega, Alejandro; Garrido Maraver, Juan; Villanueva Paz, Marina; De Lavera, Isabel; De La Mata, Mario; Cordero, Mario D; Alcocer Gómez, Elizabet; Delgado Pavón, Ana; Álvarez Córdoba, Mónica; Cotán, David; Sánchez-Alcázar, José Antonio

2015-09-01

Apoptosis is a genetically programmed energy-dependent process of cell demise, characterized by specific morphological and biochemical events in which the activation of caspases has an essential role. During apoptosis the cytoskeleton participates actively in characteristic morphological rearrangements of the dying cell. This reorganisation has been assigned mainly to actinomyosin ring contraction, while microtubule and intermediate filaments are depolymerized at early stages of apoptosis. However, recent reports have showed that microtubules are reformed during the execution phase of apoptosis organizing an apoptotic microtubule network (AMN). AMN is organized behind plasma membrane, forming a cortical structure. Apoptotic microtubules repolymerization takes place in many cell types and under different apoptotic inducers. It has been hypothesized that AMN is critical for maintaining plasma membrane integrity and cell morphology during the execution phase of apoptosis. AMN disorganization leads apoptotic cells to secondary necrosis and the release of potential toxic molecules which can damage neighbor cells and promotes inflammation. Therefore, AMN formation during physiological apoptosis or in pathological apoptosis induced by anti-cancer treatments is essential for tissue homeostasis and the prevention of additional cell damage and inflammation. © 2015 Wiley Periodicals, Inc.
[Determination of diborane in the air of workplace by ICP-AES].

PubMed

Ding, Chun-Guang; Zhang, Jing; Yan, Hui-Fang

2011-06-01

A sampling method was established to collect diborane in the air of workplace and an ICP-AES method was developed to determine the Boron in desorbed solution. Diborane in the air of workplace was collected by solid sorbent tube filled with oxidant impregnated activated carbon. The adsorbed diborane was desorbed into 3% H2O2 aqueous, and then the desorbed Boron was determined by ICP-AES. The sampling efficiency of this method was 99.6% with the desorption efficiency of diborane with 5.660 microg and 56.6 microg spiked were 90.9% and 99.5%, respectively. Both the intra-and inter-precision RSD were less than 8%. The standard curve of this method ranged from 0.1 to 10.0 microg/ml (Boron), and the LOD and LOQ were 0.011 mg/m3 and 0.035 mg/m3 (15L samples) respectively. The method established was suitable for diborane sampling and determination in the air of workplace.
Rapid and simple determination of selenium in blood serum by inductively coupled plasma-mass spectrometry (ICP-MS).

PubMed

Labat, L; Dehon, B; Lhermitte, M

2003-05-01

An inductively coupled plasma mass spectrometer (ICP-MS) with a rapid sample-preparative procedure was used for the determination of selenium in blood serum. Blood serum was prepared by dilution in an acidic solution consisting of nitric acid (1%), X-triton (0.1%) and 1-butanol (0.8%). A calibration curve was established for 1-40 microg mL(-1) (r(2)>0.99). The limit of detection was 0.5 microg mL(-1). Repeatability and intermediate precision were satisfactory with relative standard deviations (RSD) of 2.0% and 3.2%, respectively. This method was easily applied to reference materials with satisfactory accuracy. Good correlation (r(2)=0.96) was observed between ICP-MS and atomic absorption spectrometry (AAS) for the determination of (82)Se in blood serum from 23 patients. These results suggest that the sample preparative procedure coupled with ICP-MS can be used for the routine determination of (82)Se in human blood serum.
LIM kinase inhibitors disrupt mitotic microtubule organization and impair tumor cell proliferation

PubMed Central

Mardilovich, Katerina; Baugh, Mark; Crighton, Diane; Kowalczyk, Dominika; Gabrielsen, Mads; Munro, June; Croft, Daniel R.; Lourenco, Filipe; James, Daniel; Kalna, Gabriella; McGarry, Lynn; Rath, Oliver; Shanks, Emma; Garnett, Mathew J.; McDermott, Ultan; Brookfield, Joanna; Charles, Mark; Hammonds, Tim; Olson, Michael F.

2015-01-01

The actin and microtubule cytoskeletons are critically important for cancer cell proliferation, and drugs that target microtubules are widely-used cancer therapies. However, their utility is compromised by toxicities due to dose and exposure. To overcome these issues, we characterized how inhibition of the actin and microtubule cytoskeleton regulatory LIM kinases could be used in drug combinations to increase efficacy. A previously-described LIMK inhibitor (LIMKi) induced dose-dependent microtubule alterations that resulted in significant mitotic defects, and increased the cytotoxic potency of microtubule polymerization inhibitors. By combining LIMKi with 366 compounds from the GSK Published Kinase Inhibitor Set, effective combinations were identified with kinase inhibitors including EGFR, p38 and Raf. These findings encouraged a drug discovery effort that led to development of CRT0105446 and CRT0105950, which potently block LIMK1 and LIMK2 activity in vitro, and inhibit cofilin phosphorylation and increase αTubulin acetylation in cells. CRT0105446 and CRT0105950 were screened against 656 cancer cell lines, and rhabdomyosarcoma, neuroblastoma and kidney cancer cells were identified as significantly sensitive to both LIMK inhibitors. These large-scale screens have identified effective LIMK inhibitor drug combinations and sensitive cancer types. In addition, the LIMK inhibitory compounds CRT0105446 and CRT0105950 will enable further development of LIMK-targeted cancer therapy. PMID:26540348
RSK2 signals through stathmin to promote microtubule dynamics and tumor metastasis

PubMed Central

Alesi, GN; Jin, L; Li, D; Magliocca, KR; Kang, Y; Chen, ZG; Shin, DM; Khuri, FR; Kang, S

2017-01-01

Metastasis is responsible for >90% of cancer-related deaths. Complex signaling in cancer cells orchestrates the progression from a primary to a metastatic cancer. However, the mechanisms of these cellular changes remain elusive. We previously demonstrated that p90 ribosomal S6 kinase 2 (RSK2) promotes tumor metastasis. Here we investigated the role of RSK2 in the regulation of microtubule dynamics and its potential implication in cancer cell invasion and tumor metastasis. Stable knockdown of RSK2 disrupted microtubule stability and decreased phosphorylation of stathmin, a microtubule-destabilizing protein, at serine 16 in metastatic human cancer cells. We found that RSK2 directly binds and phosphorylates stathmin at the leading edge of cancer cells. Phosphorylation of stathmin by RSK2 reduced stathmin-mediated microtubule depolymerization. Moreover, overexpression of phospho-mimetic mutant stathmin S16D significantly rescued the decreased invasive and metastatic potential mediated by RSK2 knockdown in vitro and in vivo. Furthermore, stathmin phosphorylation positively correlated with RSK2 expression and metastatic cancer progression in primary patient tumor samples. Our finding demonstrates that RSK2 directly phosphorylates stathmin and regulates microtubule polymerization to provide a pro-invasive and pro-metastatic advantage to cancer cells. Therefore, the RSK2–stathmin pathway represents a promising therapeutic target and a prognostic marker for metastatic human cancers. PMID:27041561
Kinetochore-independent chromosome segregation driven by lateral microtubule bundles

PubMed Central

Muscat, Christina C; Torre-Santiago, Keila M; Tran, Michael V; Powers, James A; Wignall, Sarah M

2015-01-01

During cell division, chromosomes attach to spindle microtubules at sites called kinetochores, and force generated at the kinetochore-microtubule interface is the main driver of chromosome movement. Surprisingly, kinetochores are not required for chromosome segregation on acentrosomal spindles in Caenorhabditis elegans oocytes, but the mechanism driving chromosomes apart in their absence is not understood. In this study, we show that lateral microtubule–chromosome associations established during prometaphase remain intact during anaphase to facilitate separation, defining a novel form of kinetochore-independent segregation. Chromosome dynamics during congression and segregation are controlled by opposing forces; plus-end directed forces are mediated by a protein complex that forms a ring around the chromosome center and dynein on chromosome arms provides a minus-end force. At anaphase onset, ring removal shifts the balance between these forces, triggering poleward movement along lateral microtubule bundles. This represents an elegant strategy for controlling chromosomal movements during cell division distinct from the canonical kinetochore-driven mechanism. DOI: http://dx.doi.org/10.7554/eLife.06462.001 PMID:26026148
Cellulose synthase interactive protein 1 (CSI1) mediates the intimate relationship between cellulose microfibrils and cortical microtubules.

PubMed

Lei, Lei; Li, Shundai; Gu, Ying

2012-07-01

Cellulose is synthesized at the plasma membrane by protein complexes known as cellulose synthase complexes (CSCs). The cellulose-microtubule alignment hypothesis states that there is a causal link between the orientation of cortical microtubules and orientation of nascent cellulose microfibrils. The mechanism behind the alignment hypothesis is largely unknown. CESA interactive protein 1 (CSI1) interacts with CSCs and potentially links CSCs to the cytoskeleton. CSI1 not only co-localizes with CSCs but also travels bi-directionally in a speed indistinguishable from CSCs. The linear trajectories of CSI1-RFP coincide with the underlying microtubules labeled by YFP-TUA5. In the absence of CSI1, both the distribution and the motility of CSCs are defective and the alignment of CSCs and microtubules is disrupted. These observations led to the hypothesis that CSI1 directly mediates the interaction between CSCs and microtubules. In support of this hypothesis, CSI1 binds to microtubules directly by an in vitro microtubule-binding assay. In addition to a role in serving as a messenger from microtubule to CSCs, CSI1 labels SmaCCs/MASCs, a compartment that has been proposed to be involved in CESA trafficking and/or delivery to the plasma membrane.
Cellulose synthase interactive protein 1 (CSI1) mediates the intimate relationship between cellulose microfibrils and cortical microtubules

PubMed Central

Lei, Lei; Li, Shundai; Gu, Ying

2012-01-01

Cellulose is synthesized at the plasma membrane by protein complexes known as cellulose synthase complexes (CSCs). The cellulose-microtubule alignment hypothesis states that there is a causal link between the orientation of cortical microtubules and orientation of nascent cellulose microfibrils. The mechanism behind the alignment hypothesis is largely unknown. CESA interactive protein 1 (CSI1) interacts with CSCs and potentially links CSCs to the cytoskeleton. CSI1 not only co-localizes with CSCs but also travels bi-directionally in a speed indistinguishable from CSCs. The linear trajectories of CSI1-RFP coincide with the underlying microtubules labeled by YFP-TUA5. In the absence of CSI1, both the distribution and the motility of CSCs are defective and the alignment of CSCs and microtubules is disrupted. These observations led to the hypothesis that CSI1 directly mediates the interaction between CSCs and microtubules. In support of this hypothesis, CSI1 binds to microtubules directly by an in vitro microtubule-binding assay. In addition to a role in serving as a messenger from microtubule to CSCs, CSI1 labels SmaCCs/MASCs, a compartment that has been proposed to be involved in CESA trafficking and/or delivery to the plasma membrane. PMID:22751327
Emission of mitochondrial biophotons and their effect on electrical activity of membrane via microtubules.

PubMed

Rahnama, Majid; Tuszynski, Jack A; Bókkon, István; Cifra, Michal; Sardar, Peyman; Salari, Vahid

2011-03-01

In this paper we argue that, in addition to electrical and chemical signals propagating in the neurons of the brain, signal propagation takes place in the form of biophoton production. This statement is supported by recent experimental confirmation of photon guiding properties of a single neuron. We have investigated the interaction of mitochondrial biophotons with microtubules from a quantum mechanical point of view. Our theoretical analysis indicates that the interaction of biophotons and microtubules causes transitions/fluctuations of microtubules between coherent and incoherent states. A significant relationship between the fluctuation function of microtubules and alpha-EEG diagrams is elaborated on in this paper. We argue that the role of biophotons in the brain merits special attention. © Imperial College Press
Development of evaluation models of manpower needs for dismantling the dry conversion process-related equipment in uranium refining and conversion plant (URCP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sari Izumo; Hideo Usui; Mitsuo Tachibana

Evaluation models for determining the manpower needs for dismantling various types of equipment in uranium refining and conversion plant (URCP) have been developed. The models are widely applicable to other uranium handling facilities. Additionally, a simplified model was developed for easily and accurately calculating the manpower needs for dismantling dry conversion process-related equipment (DP equipment). It is important to evaluate beforehand project management data such as manpower needs to prepare an optimized decommissioning plan and implement effective dismantling activity. The Japan Atomic Energy Agency (JAEA) has developed the project management data evaluation system for dismantling activities (PRODIA code), which canmore » generate project management data using evaluation models. For preparing an optimized decommissioning plan, these evaluation models should be established based on the type of nuclear facility and actual dismantling data. In URCP, the dry conversion process of reprocessed uranium and others was operated until 1999, and the equipment related to the main process was dismantled from 2008 to 2011. Actual data such as manpower for dismantling were collected during the dismantling activities, and evaluation models were developed using the collected actual data on the basis of equipment classification considering the characteristics of uranium handling facility. (authors)« less
Microtubule-regulating proteins and cAMP-dependent signaling in neuroblastoma differentiation.

PubMed

Muñoz-Llancao, Pablo; de Gregorio, Cristian; Las Heras, Macarena; Meinohl, Christopher; Noorman, Kevin; Boddeke, Erik; Cheng, Xiaodong; Lezoualc'h, Frank; Schmidt, Martina; Gonzalez-Billault, Christian

2017-03-01

Neurons are highly differentiated cells responsible for the conduction and transmission of information in the nervous system. The proper function of a neuron relies on the compartmentalization of their intracellular domains. Differentiated neuroblastoma cells have been extensively used to study and understand the physiology and cell biology of neuronal cells. Here, we show that differentiation of N1E-115 neuroblastoma cells is more pronounced upon exposure of a chemical analog of cyclic AMP (cAMP), db-cAMP. We next analysed the expression of key microtubule-regulating proteins in differentiated cells and the expression and activation of key cAMP players such as EPAC, PKA and AKAP79/150. Most of the microtubule-promoting factors were up regulated during differentiation of N1E-115 cells, while microtubule-destabilizing proteins were down regulated. We observed an increase in tubulin post-translational modifications related to microtubule stability. As expected, db-cAMP increased PKA- and EPAC-dependent signalling. Consistently, pharmacological modulation of EPAC activity instructed cell differentiation, number of neurites, and neurite length in N1E-115 cells. Moreover, disruption of the PKA-AKAP interaction reduced these morphometric parameters. Interestingly, PKA and EPAC act synergistically to induce neuronal differentiation in N1E-115. Altogether these results show that the changes observed in the differentiation of N1E-115 cells proceed by regulating several microtubule-stabilizing factors, and the acquisition of a neuronal phenotype is a process involving concerted although independent functions of EPAC and PKA. © 2017 Wiley Periodicals, Inc.
[Determination of 24 metal elements and their compounds in air of workplace by ICP-AES].

PubMed

Wang, Xiang; Qiu, Jianguo; Zhao, Zhonglin; Guo, Ying

2014-06-01

To establish a method for determination of the levels of 24 metal elements and their compounds in the air of workplace by inductively coupled plasma-atomic emission spectroscopy (ICP- AES). Sampling filters were digested by microwave, and diluted to 25 ml. Twenty-four elements (Mg, Ni, K, Mo, Zn, Ca, Ba, Pb, Mn, Cd, Cr, Co, Cu, Sr, Bi, Tl, Sn, Li, Sb, Zr, In, V, Y, and Be) were simultaneously measured by ICP-AES. The detection limits for 24 elements were 0.001∼0.029 mg/L; liner correlation coefficient r values were all equal to or above 0.9994; the relative standard derivations were less than 5%; the recovery rates were 91.2%∼103.9%; the degradation rates in 7 days were less than 9.7%. ICP-AES technique is a simple, rapid, accurate, and reliable method, which can be used to measure 24 metal elements and their compounds in the air of workplace.
[Development of ICP-OES, ICP-MS and GF-AAS Methods for Simultaneous Quantification of Lead, Total Arsenic and Cadmium in Soft Drinks].

PubMed

Kataoka, Yohei; Watanabe, Takahiro; Hayashi, Tomoko; Teshima, Reiko; Matsuda, Rieko

2015-01-01

In this study, we developed methods to quantify lead, total arsenic and cadmium contained in various kinds of soft drinks, and we evaluated their performance. The samples were digested by common methods to prepare solutions for measurement by ICP-OES, ICP-MS and graphite furnace atomic absorption spectrometry (GF-AAS). After digestion, internal standard was added to the digestion solutions for measurements by ICP-OES and ICP-MS. For measurement by GF-AAS, additional purification of the digestion solution was conducted by back-extraction of the three metals into nitric acid solution after extraction into an organic solvent with ammonium pyrrolidine dithiocarbamate. Performance of the developed methods were evaluated for eight kinds of soft drinks.
Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

PubMed Central

Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

2016-01-01

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110
Insights into EB1 structure and the role of its C-terminal domain for discriminating microtubule tips from the lattice

PubMed Central

Buey, Rubén M.; Mohan, Renu; Leslie, Kris; Walzthoeni, Thomas; Missimer, John H.; Menzel, Andreas; Bjelić, Saša; Bargsten, Katja; Grigoriev, Ilya; Smal, Ihor; Meijering, Erik; Aebersold, Ruedi; Akhmanova, Anna; Steinmetz, Michel O.

2011-01-01

End-binding proteins (EBs) comprise a conserved family of microtubule plus end–tracking proteins. The concerted action of calponin homology (CH), linker, and C-terminal domains of EBs is important for their autonomous microtubule tip tracking, regulation of microtubule dynamics, and recruitment of numerous partners to microtubule ends. Here we report the detailed structural and biochemical analysis of mammalian EBs. Small-angle X-ray scattering, electron microscopy, and chemical cross-linking in combination with mass spectrometry indicate that EBs are elongated molecules with two interacting CH domains, an arrangement reminiscent of that seen in other microtubule- and actin-binding proteins. Removal of the negatively charged C-terminal tail did not affect the overall conformation of EBs; however, it increased the dwell times of EBs on the microtubule lattice in microtubule tip–tracking reconstitution experiments. An even more stable association with the microtubule lattice was observed when the entire negatively charged C-terminal domain of EBs was replaced by a neutral coiled-coil motif. In contrast, the interaction of EBs with growing microtubule tips was not significantly affected by these C-terminal domain mutations. Our data indicate that long-range electrostatic repulsive interactions between the C-terminus and the microtubule lattice drive the specificity of EBs for growing microtubule ends. PMID:21737692
Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy.

PubMed

Yan, Si; Guo, Changmiao; Hou, Guangjin; Zhang, Huilan; Lu, Xingyu; Williams, John Charles; Polenova, Tatyana

2015-11-24

Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.
Actin and microtubule networks contribute differently to cell response for small and large strains

NASA Astrophysics Data System (ADS)

Kubitschke, H.; Schnauss, J.; Nnetu, K. D.; Warmt, E.; Stange, R.; Kaes, J.

2017-09-01

Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.
Teloplasm formation in a leech, Helobdella triserialis, is a microtubule-dependent process.

PubMed

Astrow, S H; Holton, B; Weisblat, D A

1989-10-01

Fertilized eggs of the leech Helobdella triserialis undergo a cytoplasmic reorganization which generates domains of nonyolky cytoplasm, called teloplasm, at the animal and vegetal poles. The segregation of teloplasm to one cell of the eight-cell embryo is responsible for a unique developmental fate of that cell, i.e., to give rise to segmental ectoderm and mesoderm. We have studied the cytoplasmic movements that generate teloplasm using time-lapse video microscopy; the formation and migration of rings of nonyolky cytoplasm were visualized using transmitted light, while the movements of mitochondria into these rings were monitored with epifluorescence after labeling embryos with rhodamine 123, a fluorescent mitochondrial dye. To examine the likelihood that cytoskeletal elements play a role in the mechanism of teloplasm formation in Helobdella, we examined the distribution of microtubules and microfilaments during the first cell cycle by indirect immunofluorescence and rhodamine-phalloidin labeling, respectively. The cortex of the early embryo contained a network of microtubules many of which were oriented parallel to the cell surface. As teloplasm formation ensued, microtubule networks became concentrated in the animal and the vegetal cortex relative to the equatorial cortex. More extensive microtubule arrays were found within the rings of teloplasm. Actin filaments appeared in the form of narrow rings in the cortex, but these varied apparently randomly from embryo to embryo in terms of number, size, and position. The role of microtubules and microfilaments in teloplasm formation was tested using depolymerizing agents. Teloplasm formation was blocked by microtubule inhibitors, but not by microfilament inhibitors. These results differ significantly from those obtained in embryos of the oligochaete Tubifex hattai, suggesting that the presumably homologous cytoplasmic reorganizations seen in these two annelids have different cytoskeletal dependencies.
Reassessing the roles of PIN proteins and anticlinal microtubules during pavement cell morphogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belteton, Samuel; Sawchuk, Megan G.; Donohoe, Bryon S.

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxinmore » gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here we used Arabidopsis reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells, and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls.« less

Intrahepatic Cholestasis of Pregnancy (ICP) in U.S. Latinas and Chileans: Clinical features, Ancestry Analysis, and Admixture Mapping.

PubMed

Bull, Laura N; Hu, Donglei; Shah, Sohela; Temple, Luisa; Silva, Karla; Huntsman, Scott; Melgar, Jennifer; Geiser, Mary T; Sanford, Ukina; Ortiz, Juan A; Lee, Richard H; Kusanovic, Juan P; Ziv, Elad; Vargas, Juan E

2015-01-01

In the Americas, women with Indigenous American ancestry are at increased risk of intrahepatic cholestasis of pregnancy (ICP), relative to women of other ethnicities. We hypothesized that ancestry-related genetic factors contribute to this increased risk. We collected clinical and laboratory data, and performed biochemical assays on samples from U.S. Latinas and Chilean women, with and without ICP. The study sample included 198 women with ICP (90 from California, U.S., and 108 from Chile) and 174 pregnant control women (69 from California, U.S., and 105 from Chile). SNP genotyping was performed using Affymetrix arrays. We compared overall genetic ancestry between cases and controls, and used a genome-wide admixture mapping approach to screen for ICP susceptibility loci. We identified commonalities and differences in features of ICP between the 2 countries and determined that cases had a greater proportion of Indigenous American ancestry than did controls (p = 0.034). We performed admixture mapping, taking country of origin into account, and identified one locus for which Native American ancestry was associated with increased risk of ICP at a genome-wide level of significance (P = 3.1 x 10(-5), Pcorrected = 0.035). This locus has an odds ratio of 4.48 (95% CI: 2.21-9.06) for 2 versus zero Indigenous American chromosomes. This locus lies on chromosome 2, with a 10 Mb 95% confidence interval which does not contain any previously identified hereditary 'cholestasis genes.' Our results indicate that genetic factors contribute to the risk of developing ICP in the Americas, and support the utility of clinical and genetic studies of ethnically mixed populations for increasing our understanding of ICP.
Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

PubMed Central

Colombié, Nathalie; Głuszek, A. Agata; Meireles, Ana M.; Ohkura, Hiroyuki

2013-01-01

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which “amplifies” spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle. PMID:23785300
Artificial neural networks can be effectively used to model changes of intracranial pressure (ICP) during spinal surgery using different non invasive ICP surrogate estimators.

PubMed

Watad, Abdulla; Bragazzi, Nicola L; Bacigaluppi, Susanna; Amital, Howard; Watad, Samaa; Sharif, Kassem; Bisharat, Bishara; Siri, Anna; Mahamid, Ala; Abu Ras, Hakim; Nasr, Ahmed; Bilotta, Federico; Robba, Chiara; Adawi, Mohammad

2018-02-23

Artificial Intelligence (AI) techniques play a major role in anesthesiology, even though their importance is often overlooked. In the extant literature, AI approaches, such as Artificial Neural Networks (ANNs), have been underutilized, mainly being used to model patient's consciousness state, to predict the precise amount of anesthetic gases, the level of analgesia, or the need of anesthesiological blocks, among others. In the field of neurosurgery, ANNs have been effectively applied to the diagnosis and prognosis of cerebral tumors, seizures, low back pain, and also to the monitoring of intracranial pressure (ICP). A MultiLayer Perceptron (MLP), which is a feedforward ANN, with hyperbolic tangent as activation function in the input/hidden layers, softmax as activation function in the output layer, and cross-entropy as error function, was used to model the impact of prone versus supine position and the use of positive end expiratory pressure (PEEP) on ICP in a sample of 30 patients undergoing spinal surgery. Different non invasive surrogate estimations of ICP have been used and compared: namely, mean optic nerve sheath diameter (ONSD), non invasive estimated cerebral perfusion pressure (NCPP), pulsatility index (PI), ICP derived from PI (ICP-PI), and flow velocity diastolic formula (FVDICP). ONSD proved to be a more robust surrogate estimation of ICP, with a predictive power of 75%, whilst the power of NCPP, ICP-PI, PI, and FVDICP were 60.5%, 54.8%, 53.1%, and 47.7%, respectively. Our MLP analysis confirmed our findings previously obtained with regression, correlation, multivariate Receiving Operator Curve (multi-ROC) analyses. ANNs can be successfully used to predict the effects of prone versus supine position and PEEP on ICP in patients undergoing spinal surgery using different non invasive surrogate estimators of ICP.
NudEL targets dynein to microtubule ends through LIS1.

PubMed

Li, Jun; Lee, Wei-Lih; Cooper, John A

2005-07-01

Dynein is a minus-end-directed microtubule motor with critical roles in mitosis, membrane transport and intracellular transport. Several proteins regulate dynein activity, including dynactin, LIS1 (refs 2, 3) and NudEL (NudE-like). Here, we identify a NUDEL homologue in budding yeast and name it Ndl1. The ndl1delta null mutant shows decreased targeting of dynein to microtubule plus ends, an essential element of the model for dynein function. We find that Ndl1 regulates dynein targeting through LIS1, with which it interacts biochemically, but not through CLIP170, another plus-end protein involved in dynein targeting. Ndl1 is found at far fewer microtubule ends than are LIS1 and dynein. However, when Ndl1 is present at a plus end, the molar amount of Ndl1 approaches that of LIS1 and dynein. We propose a model in which Ndl1 binds transiently to the plus end to promote targeting of LIS1, which cooperatively recruits dynein.
Evaluation of the accuracy of the determination of lead isotope ratios in wine by ICP MS using quadrupole, multicollector magnetic sector and time-of-flight analyzers.

PubMed

Barbaste, M; Halicz, L; Galy, A; Medina, B; Emteborg, H; C Adams, F; Lobinski, R

2001-04-12

Different mass analysers [(quadrupole (Q), time-of-flight (TOF) and multicollector (MC) sector-field (SF)] of ions produced in an inductively coupled plasma were evaluated for the determination of lead isotope ratios in wine samples. A population of 20 wines of different origin including two reference wines from the EC Standards, Measurement and Testing Programme with concentrations varying between 7-140 mug Pb l(-1) was investigated. Wines were analyzed directly by Q ICP MS and MC ICP MS. The poor sensitivity of the TOF instrument, further aggravated by matrix signal suppression, did not allow the acquisition of data for wine samples that contained less than 50 mug l(-1) in the direct sample introduction mode. The separation and preconcentration of lead were therefore required. The precision obtained for the (206)Pb/(207)Pb and (208)Pb/(206)Pb were similar and equal to 0.14-2.7% for Q ICP MS, 0.04-0.17% for TOF ICP MS and 0.01-0.12% for MC ICP MS. The precision for (206)Pb/(204)Pb was 0.44-5.29, 0.15-1.7, 0.08-1.6%, respectively. On the level of accuracy, the data from TOF ICP MS and MC ICP MS were in good agreement. The accuracy of Q ICP MS data was judged satisfactory in comparison with the other techniques but their poor precision was a significant obstacle on the way of using these data for the determination of the geographic origin of wine.
The hepatitis E virus open reading frame 3 product interacts with microtubules and interferes with their dynamics.

PubMed

Kannan, Harilakshmi; Fan, Sumin; Patel, Deendayal; Bossis, Ioannis; Zhang, Yan-Jin

2009-07-01

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated alpha-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.
The conceptual solutions concerning decommissioning and dismantling of Russian civil nuclear powered ships

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kulikov, Konstantin N.; Nizamutdinov, Rinat A.; Abramov, Andrey N.

From 1959 up to 1991 nine civil nuclear powered ships were built in Russia: eight ice-breakers and one lash lighter carrier (cargo ship). At the present time three of them were taking out of service: ice-breaker 'Lenin' is decommissioned as a museum and is set for storage in the port of Murmansk, nuclear ice-breakers 'Arktika' and 'Sibir' are berthing. The ice-breakers carrying rad-wastes appear to be a possible source of radiation contamination of Murmansk region and Kola Bay because the ship long-term storage afloat has the negative effect on hull's structures. As the result of this under the auspices ofmore » the Federal Targeted Program 'Nuclear and Radiation Safety of Russia for 2008 and the period until 2015' the conception and projects of decommissioning of nuclear-powered ships are developed by the State corporation Rosatom with the involvement of companies of United Shipbuilding Corporation. In developing the principal provisions of conception of decommissioning and dismantling of icebreakers the technical and economic assessment of dismantling options in ship-repairing enterprises of North-West of Russia was performed. The paper contains description of options, research procedure, analysis of options of decommissioning and dismantling of nuclear ice-breakers, taking into account the principle of optimization of potential radioactive effect to personnel, human population and environment. The report's conclusions contain the recommendations for selection of option for development of nuclear icebreaker decommissioning and dismantling projects. (authors)« less
Colchicine Depolymerizes Microtubules, Increases Junctophilin-2, and Improves Right Ventricular Function in Experimental Pulmonary Arterial Hypertension.

PubMed

Prins, Kurt W; Tian, Lian; Wu, Danchen; Thenappan, Thenappan; Metzger, Joseph M; Archer, Stephen L

2017-05-31

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by obstructive pulmonary vascular remodeling and right ventricular (RV) dysfunction. Although RV function predicts outcomes in PAH, mechanisms of RV dysfunction are poorly understood, and RV-targeted therapies are lacking. We hypothesized that in PAH, abnormal microtubular structure in RV cardiomyocytes impairs RV function by reducing junctophilin-2 (JPH2) expression, resulting in t-tubule derangements. Conversely, we assessed whether colchicine, a microtubule-depolymerizing agent, could increase JPH2 expression and enhance RV function in monocrotaline-induced PAH. Immunoblots, confocal microscopy, echocardiography, cardiac catheterization, and treadmill testing were used to examine colchicine's (0.5 mg/kg 3 times/week) effects on pulmonary hemodynamics, RV function, and functional capacity. Rats were treated with saline (n=28) or colchicine (n=24) for 3 weeks, beginning 1 week after monocrotaline (60 mg/kg, subcutaneous). In the monocrotaline RV, but not the left ventricle, microtubule density is increased, and JPH2 expression is reduced, with loss of t-tubule localization and t-tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves t-tubule morphology in RV cardiomyocytes. Colchicine therapy diminishes RV hypertrophy, improves RV function, and enhances RV-pulmonary artery coupling. Colchicine reduces small pulmonary arteriolar thickness and improves pulmonary hemodynamics. Finally, colchicine increases exercise capacity. Monocrotaline-induced PAH causes RV-specific derangement of microtubules marked by reduction in JPH2 and t-tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves both t-tubule architecture and RV function. Colchicine also reduces adverse pulmonary vascular remodeling. These results provide biological plausibility for a clinical trial to repurpose colchicine as a RV-directed therapy for PAH
Stable kinetochore-microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells.

PubMed

Tauchman, Eric C; Boehm, Frederick J; DeLuca, Jennifer G

2015-12-01

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore-microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore-microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal.
The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition

PubMed Central

Applewhite, Derek A.; Grode, Kyle D.; Duncan, Mara C.; Rogers, Stephen L.

2013-01-01

Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis—much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a “closed” conformation through interactions between its NH2-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH2-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex. PMID:23885120
Direct observation shows superposition and large scale flexibility within cytoplasmic dynein motors moving along microtubules

NASA Astrophysics Data System (ADS)

Imai, Hiroshi; Shima, Tomohiro; Sutoh, Kazuo; Walker, Matthew L.; Knight, Peter J.; Kon, Takahide; Burgess, Stan A.

2015-09-01

Cytoplasmic dynein is a dimeric AAA+ motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA+ rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dynein's directionality and unusual stepping behaviour.
Stable kinetochore–microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells

PubMed Central

Tauchman, Eric C.; Boehm, Frederick J.; DeLuca, Jennifer G.

2015-01-01

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore–microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal. PMID:26620470
A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics

PubMed Central

Geyer, Elisabeth A; Burns, Alexander; Lalonde, Beth A; Ye, Xuecheng; Piedra, Felipe-Andres; Huffaker, Tim C; Rice, Luke M

2015-01-01

Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking. DOI: http://dx.doi.org/10.7554/eLife.10113.001 PMID:26439009
The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity

PubMed Central

Wieczorek, Michal; Tcherkezian, Joseph; Bernier, Cynthia; Prota, Andrea E.; Chaaban, Sami; Rolland, Yannève; Godbout, Claude; Hancock, Mark A.; Arezzo, Joseph C.; Ocal, Ozhan; Rocha, Cecilia; Olieric, Natacha; Hall, Anita; Ding, Hui; Bramoullé, Alexandre; Annis, Matthew G.; Zogopoulos, George; Harran, Patrick G.; Wilkie, Thomas M.; Brekken, Rolf A.; Siegel, Peter M.; Steinmetz, Michel O.; Shore, Gordon C.; Brouhard, Gary J.; Roulston, Anne

2017-01-01

Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety. PMID:27856798
Accurate and precise determination of isotopic ratios by MC-ICP-MS: a review.

PubMed

Yang, Lu

2009-01-01

For many decades the accurate and precise determination of isotope ratios has remained a very strong interest to many researchers due to its important applications in earth, environmental, biological, archeological, and medical sciences. Traditionally, thermal ionization mass spectrometry (TIMS) has been the technique of choice for achieving the highest accuracy and precision. However, recent developments in multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) have brought a new dimension to this field. In addition to its simple and robust sample introduction, high sample throughput, and high mass resolution, the flat-topped peaks generated by this technique provide for accurate and precise determination of isotope ratios with precision reaching 0.001%, comparable to that achieved with TIMS. These features, in combination with the ability of the ICP source to ionize nearly all elements in the periodic table, have resulted in an increased use of MC-ICP-MS for such measurements in various sample matrices. To determine accurate and precise isotope ratios with MC-ICP-MS, utmost care must be exercised during sample preparation, optimization of the instrument, and mass bias corrections. Unfortunately, there are inconsistencies and errors evident in many MC-ICP-MS publications, including errors in mass bias correction models. This review examines "state-of-the-art" methodologies presented in the literature for achievement of precise and accurate determinations of isotope ratios by MC-ICP-MS. Some general rules for such accurate and precise measurements are suggested, and calculations of combined uncertainty of the data using a few common mass bias correction models are outlined.
In vitro polymerization of microtubules with a fullerene derivative.

PubMed

Ratnikova, Tatsiana A; Govindan, Praveen Nedumpully; Salonen, Emppu; Ke, Pu Chun

2011-08-23

Fullerene derivative C(60)(OH)(20) inhibited microtubule polymerization at low micromolar concentrations. The inhibition was mainly attributed to the formation of hydrogen bonding between the nanoparticle and the tubulin heterodimer, the building block of the microtubule, as evidenced by docking and molecular dynamics simulations. Our circular dichroism spectroscopy measurement indicated changes in the tubulin secondary structures, while our guanosine-5'-triphosphate hydrolysis assay showed hindered release of inorganic phosphate by the nanoparticle. Isothermal titration calorimetry revealed that C(60)(OH)(20) binds to tubulin at a molar ratio of 9:1 and with a binding constant of 1.3 ± 0.16 × 10(6) M(-1), which was substantiated by the binding site and binding energy analysis using docking and molecular dynamics simulations. Our simulations further suggested that occupancy by the nanoparticles at the longitudinal contacts between tubulin dimers within a protofilament or at the lateral contacts of the M-loop and H5 and H12 helices of neighboring tubulins could also influence the polymerization process. This study offered a new molecular-level insight on how nanoparticles may reshape the assembly of cytoskeletal proteins, a topic of essential importance for illuminating cell response to engineered nanoparticles and for the advancement of nanomedicine. © 2011 American Chemical Society
Direct determination of halogens in powdered geological and environmental samples using isotope dilution laser ablation ICP-MS

NASA Astrophysics Data System (ADS)

Boulyga, Sergei F.; Heumann, Klaus G.

2005-04-01

Laser ablation inductively coupled plasma isotope dilution mass spectrometry (LA-ICP-IDMS) with a special laser ablation system for bulk analyses (LINA-Spark(TM)-Atomiser) was applied for direct determinations of chlorine, bromine, and iodine in rock and sediment samples. Special attention was focused on possible inter-halogen fractionations and analyte/spike isotope fractionations by using LA-ICP-MS and LA-ICP-IDMS, respectively. A variation of Br/Cl and I/Cl element intensity ratios by a factor of 1.3-3 was observed when changing the nebulizer gas flow rate in the range of 0.84-1.0 L min-1 and the laser power density in the range of 2-10 GW cm-2, respectively. When using an internal standard for halogen quantification in LA-ICP-MS, this inter-element fractionation can cause systematic errors, which can be avoided by applying the isotope dilution technique. However, at high laser power densities (>5.7 GW cm-2 for iodine and >4.0 GW cm-2 for bromine and chlorine) the corresponding measured isotope ratio of the isotope-diluted sample deviates significantly from the target value. Under optimised conditions concentrations in the range of 30 [mu]g g-1-16 × 103 [mu]g g-1 for chlorine, <2-140 [mu]g g-1 for bromine, and <0.1-31 [mu]g g-1 for iodine were determined by LA-ICP-IDMS in two sediment reference materials (SRM 1646, SRM 2704) and three rock reference samples (GS-N, Granite; BX-N, Bauxite; DT-N, Disthene), which have not been certified for these halogens. The sediment results agree well within the given uncertainties with indicative values by different methods and the results of the rock samples with those obtained by negative thermal ionisation isotope dilution mass spectrometry. The detection limits of LA-ICP-IDMS are 8 [mu]g g-1 for chlorine, 1.7 [mu]g g-1 for bromine, and 0.1 [mu]g g-1 for iodine.
Alzheimer Aβ disrupts the mitotic spindle and directly inhibits mitotic microtubule motors

PubMed Central

Borysov, Sergiy I; Granic, Antoneta; Padmanabhan, Jaya; Walczak, Claire E

2011-01-01

Chromosome mis-segregation and aneuploidy are greatly induced in Alzheimer disease and models thereof by mutant forms of the APP and PS proteins and by their product, the Aβ peptide. Here we employ human somatic cells and Xenopus egg extracts to show that Aβ impairs the assembly and maintenance of the mitotic spindle. Mechanistically, these defects result from Aβ's inhibition of mitotic motor kinesins, including Eg5, KIF4A and MCAK. In vitro studies show that oligomeric Aβ directly inhibits recombinant MCAK by a noncompetitive mechanism. In contrast, inhibition of Eg5 and KIF4A is competitive with respect to both ATP and microtubules, indicating that Aβ interferes with their interactions with the microtubules of the mitotic spindle. Consistently, increased levels of polymerized microtubules or of the microtubule stabilizing protein Tau significantly decrease the inhibitory effect of Aβ on Eg5 and KIF4A. Together, these results indicate that by disrupting the interaction between specific kinesins and microtubules and by exerting a direct inhibitory effect on the motor activity, excess Aβ deregulates the mechanical forces that govern the spindle and thereby leads to the generation of defective mitotic structures. The resulting defect in neurogenesis can account for the over 30% aneuploid/hyperploid, degeneration-prone neurons observed in Alzheimer disease brain. The finding of mitotic motors including Eg5 in mature post-mitotic neurons implies that their inhibition by Aβ may also disrupt neuronal function and plasticity. PMID:21566458
Provenance establishment of coffee using solution ICP-MS and ICP-AES.

PubMed

Valentin, Jenna L; Watling, R John

2013-11-01

Statistical interpretation of the concentrations of 59 elements, determined using solution based inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma emission spectroscopy (ICP-AES), was used to establish the provenance of coffee samples from 15 countries across five continents. Data confirmed that the harvest year, degree of ripeness and whether the coffees were green or roasted had little effect on the elemental composition of the coffees. The application of linear discriminant analysis and principal component analysis of the elemental concentrations permitted up to 96.9% correct classification of the coffee samples according to their continent of origin. When samples from each continent were considered separately, up to 100% correct classification of coffee samples into their countries, and plantations of origin was achieved. This research demonstrates the potential of using elemental composition, in combination with statistical classification methods, for accurate provenance establishment of coffee. Copyright © 2013 Elsevier Ltd. All rights reserved.
Microtubule Dynamics Scale with Cell Size to Set Spindle Length and Assembly Timing.

PubMed

Lacroix, Benjamin; Letort, Gaëlle; Pitayu, Laras; Sallé, Jérémy; Stefanutti, Marine; Maton, Gilliane; Ladouceur, Anne-Marie; Canman, Julie C; Maddox, Paul S; Maddox, Amy S; Minc, Nicolas; Nédélec, François; Dumont, Julien

2018-05-21

Successive cell divisions during embryonic cleavage create increasingly smaller cells, so intracellular structures must adapt accordingly. Mitotic spindle size correlates with cell size, but the mechanisms for this scaling remain unclear. Using live cell imaging, we analyzed spindle scaling during embryo cleavage in the nematode Caenorhabditis elegans and sea urchin Paracentrotus lividus. We reveal a common scaling mechanism, where the growth rate of spindle microtubules scales with cell volume, which explains spindle shortening. Spindle assembly timing is, however, constant throughout successive divisions. Analyses in silico suggest that controlling the microtubule growth rate is sufficient to scale spindle length and maintain a constant assembly timing. We tested our in silico predictions to demonstrate that modulating cell volume or microtubule growth rate in vivo induces a proportional spindle size change. Our results suggest that scalability of the microtubule growth rate when cell size varies adapts spindle length to cell volume. Copyright © 2018 Elsevier Inc. All rights reserved.

CEP295 interacts with microtubules and is required for centriole elongation.

PubMed

Chang, Ching-Wen; Hsu, Wen-Bin; Tsai, Jhih-Jie; Tang, Chieh-Ju C; Tang, Tang K

2016-07-01

Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules. © 2016. Published by The Company of Biologists Ltd.
CEP295 interacts with microtubules and is required for centriole elongation

PubMed Central

Chang, Ching-Wen; Hsu, Wen-Bin; Tsai, Jhih-Jie; Tang, Chieh-Ju C.

2016-01-01

ABSTRACT Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules. PMID:27185865
Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets

PubMed Central

Quick, Quincy A.

2018-01-01

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents. PMID:29373494
Stability and function of a putative microtubule-organizing center in the human parasite Toxoplasma gondii

PubMed Central

Leung, Jacqueline M.; He, Yudou; Zhang, Fangliang; Hwang, Yu-Chen; Nagayasu, Eiji; Liu, Jun; Murray, John M.; Hu, Ke

2017-01-01

The organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and show that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 (ΔkinesinAΔapr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live ΔkinesinAΔapr1 parasites and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and invade host cells, as well as decreased secretion of effectors important for these processes. Together the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis. PMID:28331073
Challenges and opportunities in the high-resolution cryo-EM visualization of microtubules and their binding partners.

PubMed

Nogales, Eva; Kellogg, Elizabeth H

2017-10-01

As non-crystallizable polymers, microtubules have been the target of cryo-electron microscopy (cryo-EM) studies since the technique was first established. Over the years, image processing strategies have been developed that take care of the unique, pseudo-helical symmetry of the microtubule. With recent progress in data quality and data processing, cryo-EM reconstructions are now reaching resolutions that allow the generation of atomic models of microtubules and the factors that bind them. These include cellular partners that contribute to microtubule cellular functions, or small ligands that interfere with those functions in the treatment of cancer. The stage is set to generate a family portrait for all identified microtubule interacting proteins and to use cryo-EM as a drug development tool in the targeting of tubulin. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.
Chromokinesin Kid and kinetochore kinesin CENP-E differentially support chromosome congression without end-on attachment to microtubules.

PubMed

Iemura, Kenji; Tanaka, Kozo

2015-03-06

Chromosome congression is the alignment of chromosomes at the spindle equator, and is a prerequisite for faithful chromosome segregation. Recent data suggest that before kinetochores attach to the end of microtubules (end-on attachment), chromosomes can move along microtubules towards the spindle equator through attachment of kinetochores to the lateral surface of microtubules (lateral attachment). Here we address this mechanism, focusing on the contribution of two mitotic motors, Kid and CENP-E. In cells depleted of Hec1, which is essential for end-on attachment, chromosomes show partial and transient congression. This transient congression is further perturbed by co-depletion of Kid, suggesting its role in chromosome congression. In comparison, CENP-E suppresses chromosome congression, probably by tethering kinetochores to short, unstable microtubules, and works in congression only when microtubules are stabilized. Our results may reflect the differential contributions of Kid and CENP-E in chromosome congression in physiological conditions where stabilized microtubules are becoming increased.
[Determination of high concentrations of rubidium chloride by ICP-OES].

PubMed

Zhong, Yuan; Sun, Bai; Li, Hai-jun; Wang, Tao; Li, Wu; Song, Peng-sheng

2015-01-01

The method of ICP-OES for the direct determination of high content of rubidium in rubidium chloride solutions was studied through mass dilution method and optimizing parameters of the instrument in the present paper. It can reduce the times of dilution and the error introduced by the dilution, and improve the accuracy of determination results of rubidium. Through analyzing the sensitivity of the three detection spectral lines for rubidium ion, linearly dependent coefficient and the relative errors of the determination results, the spectral line of Rb 780. 023 nm was chosen as the most suitable wavelength to measure the high content of rubidium in the rubidium chloride solutions. It was found that the instrument parameters of ICP-OES such as the atomizer flow, the pump speed and the high-frequency power are the major factors for the determination of rubidium ion in the rubidium chloride solutions. As we know instrument parameters of ICP-OES have an important influence on the atomization efficiency as well as the emissive power of the spectral lines of rubidium, they are considered as the significant factors for the determination of rubidium. The optimization parameters of the instrument were obtained by orthogonal experiments and further single factor experiment, which are 0. 60 L . min-1 of atomizer flow, 60 r . min-1 of pump speed, and 1 150 W of high-frequency power. The same experiments were repeated a week later with the optimization parameters of the instrument, and the relative errors of the determination results are less than 0. 5% when the concentration of rubidium chloride ranged from 0. 09% to 0. 18%. As the concentration of rubidium chloride is 0. 06%, the relative errors of the determination results are -1. 7%. The determination of lithium chloride and potassium chloride in the high concentration of the aqueous solutions was studied under the condition of similar instrument parameters. It was found by comparison that the determination results of lithium
Dismantling of Radium-226 Coal Level Gauges: Encountered Problems and How to Solve

DOE Office of Scientific and Technical Information (OSTI.GOV)

Punnachaiya, M.; Nuanjan, P.; Moombansao, K.

2006-07-01

This paper describes the techniques for dismantling of disused-sealed Radium-226 (Ra-226) coal level gauges which the source specifications and documents were not available, including problems occurred during dismantling stage and the decision making in solving all those obstacles. The 2 mCi (20 pieces), 6 mCi (20 pieces) and 6.6 mCi (30 pieces) of Ra-226 hemi-spherically-shaped with lead-filled coal level gauges were used in industrial applications for electric power generation. All sources needed to be dismantled for further conditioning as requested by the International Atomic Energy Agency (IAEA). One of the 2 mCi Ra-226 source was dismantled under the supervision ofmore » IAEA expert. Before conditioning period, each of the 6 mCi and 6.6 mCi sources were dismantled and inspected. It was found that coal level gauges had two different source types: the sealed cylindrical source (diameter 2 cm x 2 cm length) locked with spring in lead housing for 2 mCi and 6.6 mCi; while the 6 mCi was an embedded capsule inside source holder stud assembly in lead-filled housing. Dismantling Ra-226 coal level gauges comprised of 6 operational steps: confirmation of the surface dose rate on each source activity, calculation of working time within the effective occupational dose limit, cutting the weld of lead container by electrical blade, confirmation of the Ra-226 embedded capsule size using radiation scanning technique and gamma radiography, automatic sawing of the source holder stud assembly, and transferring the source to store in lead safe box. The embedded length of 6 mCi Ra-226 capsule in its diameter 2 cm x 14.7 cm length stud assembly was identified, the results from scanning technique and radiographic film revealed the embedded source length of about 2 cm, therefore all the 6 mCi sources were safely cut at 3 cm using the automatic saw. Another occurring problem was one of the 6.6 mCi spring type source stuck inside its housing because the spring was deformed and there
Metabolite profiling with HPLC-ICP-MS as a tool for in vivo characterization of imaging probes.

PubMed

Boros, Eszter; Pinkhasov, Omar R; Caravan, Peter

2018-01-01

Current analytical methods for characterizing pharmacokinetic and metabolic properties of positron emission tomography (PET) and single photon emission computed tomography (SPECT) probes are limited. Alternative methods to study tracer metabolism are needed. The study objective was to assess the potential of high performance liquid chromatography - inductively coupled plasma - mass spectrometry (HPLC-ICP-MS) for quantification of molecular probe metabolism and pharmacokinetics using stable isotopes. Two known peptide-DOTA conjugates were chelated with nat Ga and nat In. Limit of detection of HPLC-ICP-MS for 69 Ga and 115 In was determined. Rats were administered 50-150 nmol of Ga- and/or In-labeled probes, blood was serially sampled, and plasma analyzed by HPLC-ICP-MS using both reverse phase and size exclusion chromatography. The limits of detection were 0.16 pmol for 115 In and 0.53 pmol for 69 Ga. Metabolites as low as 0.001 %ID/g could be detected and transchelation products identified. Simultaneous administration of Ga- and In-labeled probes allowed the determination of pharmacokinetics and metabolism of both probes in a single animal. HPLC-ICP-MS is a robust, sensitive and radiation-free technique to characterize the pharmacokinetics and metabolism of imaging probes.
A centrosomal protein FOR20 regulates microtubule assembly dynamics and plays a role in cell migration.

PubMed

Srivastava, Shalini; Panda, Dulal

2017-08-10

Here, we report that a centrosomal protein FOR20 [FOP (FGFR1 (fibroblast growth factor receptor 1) oncogene protein)-like protein of molecular mass of 20 kDa; also named as C16orf63, FLJ31153 or PHSECRG2] can regulate the assembly and stability of microtubules. Both FOR20 IgG antibody and GST (glutathione S -transferase)-tagged FOR20 could precipitate tubulin from the HeLa cell extract, indicating a possible interaction between FOR20 and tubulin. FOR20 was also detected in goat brain tissue extract and it cycled with microtubule-associated proteins. Furthermore, FOR20 bound to purified tubulin and inhibited the assembly of tubulin in vitro. The overexpression of FOR20 depolymerized interphase microtubules and the depletion of FOR20 prevented nocodazole-induced depolymerization of microtubules in HeLa cells. In addition, the depletion of FOR20 suppressed the dynamics of individual microtubules in live HeLa cells. FOR20-depleted MDA-MB-231 cells displayed zigzag motion and migrated at a slower rate than the control cells, indicating that FOR20 plays a role in directed cell migration. The results suggested that the centrosomal protein FOR20 is a new member of the microtubule-associated protein family and that it regulates the assembly and dynamics of microtubules. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
Dissociation of the Tubulin-sequestering and Microtubule Catastrophe-promoting Activities of Oncoprotein 18/Stathmin

PubMed Central

Howell, Bonnie; Larsson, Niklas; Gullberg, Martin; Cassimeris, Lynne

1999-01-01

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100–147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5–25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminal–truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not
On the alignment of cellulose microfibrils by cortical microtubules: a review and a model.

PubMed

Baskin, T I

2001-01-01

The hypothesis that microtubules align microfibrils, termed the alignment hypothesis, states that there is a causal link between the orientation of cortical microtubules and the orientation of nascent microfibrils. I have assessed the generality of this hypothesis by reviewing what is known about the relation between microtubules and microfibrils in a wide group of examples: in algae of the family Characeae, Closterium acerosum, Oocystis solitaria, and certain genera of green coenocytes and in land plant tip-growing cells, xylem, diffusely growing cells, and protoplasts. The salient features about microfibril alignment to emerge are as follows. Cellulose microfibrils can be aligned by cortical microtubules, thus supporting the alignment hypothesis. Alignment of microfibrils can occur independently of microtubules, showing that an alternative to the alignment hypothesis must exist. Microfibril organization is often random, suggesting that self-assembly is insufficient. Microfibril organization differs on different faces of the same cell, suggesting that microfibrils are aligned locally, not with respect to the entire cell. Nascent microfibrils appear to associate tightly with the plasma membrane. To account for these observations, I present a model that posits alignment to be mediated through binding the nascent microfibril. The model, termed templated incorporation, postulates that the nascent microfibril is incorporated into the cell wall by binding to a scaffold that is oriented; further, the scaffold is built and oriented around either already incorporated microfibrils or plasma membrane proteins, or both. The role of cortical microtubules is to bind and orient components of the scaffold at the plasma membrane. In this way, spatial information to align the microfibrils may come from either the cell wall or the cell interior, and microfibril alignment with and without microtubules are subsets of a single mechanism.
Studies of LA-ICP-MS on quartz glasses at different wavelengths of a Nd:YAG laser.

PubMed

Becker, J S; Tenzler, D

2001-07-01

The capability of LA-ICP-MS for determination of trace impurities in transparent quartz glasses was investigated. Due to low or completely lacking absorption of laser radiation, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) proves difficult on transparent solids, and in particular the quantification of measurement results is problematic in these circumstances. Quartz glass reference materials of various compositions were studied by using a Nd:YAG laser system with focused laser radiation of wavelengths of 1064 nm, 532 nm and 266 nm, and an ICP-QMS (Elan 6000, Perkin Elmer). The influence of ICP and laser ablation conditions in the analysis of quartz glasses of different compositions was investigated, with the laser power density in the region of interaction between laser radiation and solid surface determining the ablation process. The trace element concentration was determined via calibration curves recorded with the aid of quartz glass reference materials. Under optimized measuring conditions the correlation coefficients of the calibration curves are in the range of 0.9-1. The relative sensitivity factors of the trace elements determined in the quartz glass matrix are 0.1-10 for most of the trace elements studied by LA-ICP-MS. The detection limits of the trace elements in quartz glass are in the low ng/g to pg/g range.
Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy.

PubMed

Flex, Elisabetta; Niceta, Marcello; Cecchetti, Serena; Thiffault, Isabelle; Au, Margaret G; Capuano, Alessandro; Piermarini, Emanuela; Ivanova, Anna A; Francis, Joshua W; Chillemi, Giovanni; Chandramouli, Balasubramanian; Carpentieri, Giovanna; Haaxma, Charlotte A; Ciolfi, Andrea; Pizzi, Simone; Douglas, Ganka V; Levine, Kara; Sferra, Antonella; Dentici, Maria Lisa; Pfundt, Rolph R; Le Pichon, Jean-Baptiste; Farrow, Emily; Baas, Frank; Piemonte, Fiorella; Dallapiccola, Bruno; Graham, John M; Saunders, Carol J; Bertini, Enrico; Kahn, Richard A; Koolen, David A; Tartaglia, Marco

2016-10-06

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αβ-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective β-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/β-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with
Prickle isoforms control the direction of tissue polarity by microtubule independent and dependent mechanisms.

PubMed

Sharp, Katherine A; Axelrod, Jeffrey D

2016-02-10

Planar cell polarity signaling directs the polarization of cells within the plane of many epithelia. While these tissues exhibit asymmetric localization of a set of core module proteins, in Drosophila, more than one mechanism links the direction of core module polarization to the tissue axes. One signaling system establishes a polarity bias in the parallel, apical microtubules upon which vesicles containing core proteins traffic. Swapping expression of the differentially expressed Prickle isoforms, Prickle and Spiny-legs, reverses the direction of core module polarization. Studies in the proximal wing and the anterior abdomen indicated that this results from their differential control of microtubule polarity. Prickle and Spiny-legs also control the direction of polarization in the distal wing (D-wing) and the posterior abdomen (P-abd). We report here that this occurs without affecting microtubule polarity in these tissues. The direction of polarity in the D-wing is therefore likely determined by a novel mechanism independent of microtubule polarity. In the P-abd, Prickle and Spiny-legs interpret at least two directional cues through a microtubule-polarity-independent mechanism. © 2016. Published by The Company of Biologists Ltd.
Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis.

PubMed

Belteton, Samuel A; Sawchuk, Megan G; Donohoe, Bryon S; Scarpella, Enrico; Szymanski, Daniel B

2018-01-01

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis ( Arabidopsis thaliana ) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls. © 2018 American Society of Plant Biologists. All Rights Reserved.
Characterization Of Nuclear Materials Using Time-Of-Flight ICP-MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buerger, Stefan; Riciputi, Lee R; Bostick, Debra A

2006-01-01

The investigation of illicit trafficking of nuclear materials, nuclear safeguards analysis, and non-proliferation control requires sensitive and isotope-selective detection methods to gain crucial nuclear forensic information like isotope 'fingerprints' and multi-element signatures. The advantage of time-of-flight (TOF) mass spectrometry - quasi-simultaneous multi-mass analysis - combined with an inductively coupled plasma (ICP) ion source provides an analytical instrument with multi-element and multi-isotope capability and good detection limits. A TOF-ICP-MS system thus appears to be an advantageous choice for the investigation and characterization of nuclear materials. We present here results using a GBC OptiMass 8000 time-of-flight ICP-MS for the isotope screening ofmore » solid samples by laser ablation and the multi-element determination of impurities in uranium ore concentrates using matrix matched standards. A laser ablation system (New Wave Research, UP 213) coupled to the TOF-ICP-MS instrument has been used to optimize the system for analysis of non-radioactive metal samples of natural isotopic composition for a variety of elements including Cu, Sr, Zr, Mo, Cd, In, Ba, Ta, W, Re, Pt, and Pb in pure metals, alloys, and glasses to explore precision, accuracy, and detection limits. Similar methods were then applied to measure uranium. When the laser system is optimized, no mass bias correction is required. Precision and accuracy for the determination of the isotopic composition is typically 1 - 3% for elemental concentrations of as little as 50 ppm in the matrix, with no requirement for sample preparation. The laser ablation precision and accuracy are within ~10x of the instrumental limits for liquid analysis (0.1%). We have investigated the capabilities of the TOF-ICP-MS for the analysis of impurities in uranium matrices. Matrix matching has been used to develop calibration curves for a range of impurities (alkaline, earth-alkaline, transition metals, and
Determination of alkyllead compounds by HPLC/ICP using a glass-frit nebulizer ICP interface

NASA Astrophysics Data System (ADS)

Ibrahim, Mona; Nisamaneepong, Wipawan; Haas, David L.; Caruso, Joseph A.

The glass-frit nebulizer, by forming a very fine mist, has improved the ability of the ICP to accept the introduction of organic solvents with high evaporation rates. The reversed-phase chromatographic separation of TML and TEL, and their determination with glass frit nebulization ICP was accomplished with various mobile phases and columns. The separation of several trialkyllead salts also was studied on a strong cation exchange column, but these compounds were not determined with the glass frit nebulizer interface. Detection limits as low as 33 pg s -1 for TML and 100 pg s -1 for TEL and precision of 3.4% for TML and 6.9% relative standard deviation for TEL were obtained.
The Problem Child: Provocations toward Dismantling the Carceral State

ERIC Educational Resources Information Center

Meiners, Erica R.

2017-01-01

In this essay Erica R. Meiners argues that those committed to dismantling our nation's deep and racialized investments in policing and imprisoning must analyze how the flexible category of "the child," and its figurative powers, operate in complex ways to punish communities and naturalize and expand criminalization and surveillance.…
Aβ-mediated spine changes in the hippocampus are microtubule-dependent and can be reversed by a subnanomolar concentration of the microtubule-stabilizing agent epothilone D

PubMed Central

Penazzi, Lorène; Tackenberg, Christian; Ghori, Adnan; Golovyashkina, Nataliya; Niewidok, Benedikt; Selle, Karolin; Ballatore, Carlo; Smith, Amos B.; Bakota, Lidia; Brandt, Roland

2016-01-01

Dendritic spines represent the major postsynaptic input of excitatory synapses. Loss of spines and changes in their morphology correlate with cognitive impairment in Alzheimer’s disease (AD) and are thought to occur early during pathology. Therapeutic intervention at a preclinical stage of AD to modify spine changes might thus be warranted. To follow the development and to potentially interfere with spine changes over time, we established a long term ex vivo model from organotypic cultures of the hippocampus from APP transgenic and control mice. The cultures exhibit spine loss in principal hippocampal neurons, which closely resembles the changes occurring in vivo, and spine morphology progressively changes from mushroom-shaped to stubby. We demonstrate that spine changes are completely reversed within few days after blocking amyloid-β (Aβ) production with the gamma-secretase inhibitor DAPT. We show that the microtubule disrupting drug nocodazole leads to spine loss similar to Aβ expressing cultures and suppresses DAPT-mediated spine recovery in slices from APP transgenic mice. Finally, we report that epothilone D (EpoD) at a subnanomolar concentration, which slightly stabilizes microtubules in model neurons, completely reverses Aβ-induced spine loss and increases thin spine density. Taken together the data indicate that Aβ causes spine changes by microtubule destabilization and that spine recovery requires microtubule polymerization. Moreover, our results suggest that a low, subtoxic concentration of EpoD is sufficient to reduce spine loss during the preclinical stage of AD. PMID:26772969

Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte.

PubMed

Dahlgaard, Katja; Raposo, Alexandre A S F; Niccoli, Teresa; St Johnston, Daniel

2007-10-01

Mutants in the actin nucleators Cappuccino and Spire disrupt the polarized microtubule network in the Drosophila oocyte that defines the anterior-posterior axis, suggesting that microtubule organization depends on actin. Here, we show that Cappuccino and Spire organize an isotropic mesh of actin filaments in the oocyte cytoplasm. capu and spire mutants lack this mesh, whereas overexpressed truncated Cappuccino stabilizes the mesh in the presence of Latrunculin A and partially rescues spire mutants. Spire overexpression cannot rescue capu mutants, but prevents actin mesh disassembly at stage 10B and blocks late cytoplasmic streaming. We also show that the actin mesh regulates microtubules indirectly, by inhibiting kinesin-dependent cytoplasmic flows. Thus, the Capu pathway controls alternative states of the oocyte cytoplasm: when active, it assembles an actin mesh that suppresses kinesin motility to maintain a polarized microtubule cytoskeleton. When inactive, unrestrained kinesin movement generates flows that wash microtubules to the cortex.
Proteolysis of microtubule associated protein 2 and sensitivity of pancreatic tumours to docetaxel

PubMed Central

Veitia, R; David, S; Barbier, P; Vantard, M; Gounon, P; Bissery, M C; Fellous, A

2000-01-01

We have studied the state of microtubule associated protein 2 (MAP2) in the pancreatic ductal adenocarcinomas P03 and P02 (sensitive and refractory to docetaxel respectively) since they express the corresponding mRNA and MAP2-related peptides. Immunohistochemical localization showed that in tumour P03 the MAP2-related peptides are highly expressed and confined to the epithelial malignant cells while in P02 the intensity of the immunostaining is lower. However, anti α-tubulin staining followed a similar pattern suggesting that the net amount of macromolecular structures in the sensitive tumour is higher than in the refractory one. This may explain its higher sensitivity to docetaxel, because tubulin assembled into microtubules is the target of the drug. We found that protein extracts from both tumours differed in their proteolytic activity on rat brain MAP2. Since the proteolysis pattern obtained was similar to the one produced by Cathepsin D, we studied the effect of MAP2 proteolysed by this enzyme on microtubule formation in vitro. Proteolysis was found to increase the tendency of tubulin to assemble into macromolecular structures (microtubules and aggregates) in the presence of docetaxel. This suggests that in vivo proteolysis of MAP2 might increase microtubule alterations and potentiate the antitumour effect of docetaxel. © 2000 Cancer Research Campaign PMID:10945505
An integrated analysis for determining the geographical origin of medicinal herbs using ICP-AES/ICP-MS and (1)H NMR analysis.

PubMed

Kwon, Yong-Kook; Bong, Yeon-Sik; Lee, Kwang-Sik; Hwang, Geum-Sook

2014-10-15

ICP-MS and (1)H NMR are commonly used to determine the geographical origin of food and crops. In this study, data from multielemental analysis performed by ICP-AES/ICP-MS and metabolomic data obtained from (1)H NMR were integrated to improve the reliability of determining the geographical origin of medicinal herbs. Astragalus membranaceus and Paeonia albiflora with different origins in Korea and China were analysed by (1)H NMR and ICP-AES/ICP-MS, and an integrated multivariate analysis was performed to characterise the differences between their origins. Four classification methods were applied: linear discriminant analysis (LDA), k-nearest neighbour classification (KNN), support vector machines (SVM), and partial least squares-discriminant analysis (PLS-DA). Results were compared using leave-one-out cross-validation and external validation. The integration of multielemental and metabolomic data was more suitable for determining geographical origin than the use of each individual data set alone. The integration of the two analytical techniques allowed diverse environmental factors such as climate and geology, to be considered. Our study suggests that an appropriate integration of different types of analytical data is useful for determining the geographical origin of food and crops with a high degree of reliability. Copyright © 2014 Elsevier Ltd. All rights reserved.
The spectraplakin Short stop is an essential microtubule regulator involved in epithelial closure in Drosophila

PubMed Central

Takács, Zsanett; Vilmos, Péter; Lénárt, Péter; Röper, Katja; Erdélyi, Miklós

2017-01-01

ABSTRACT Dorsal closure of the Drosophila embryonic epithelium provides an excellent model system for the in vivo analysis of molecular mechanisms regulating cytoskeletal rearrangements. In this study, we investigated the function of the Drosophila spectraplakin Short stop (Shot), a conserved cytoskeletal structural protein, during closure of the dorsal embryonic epithelium. We show that Shot is essential for the efficient final zippering of the opposing epithelial margins. By using isoform-specific mutant alleles and genetic rescue experiments with truncated Shot variants, we demonstrate that Shot functions as an actin–microtubule cross-linker in mediating zippering. At the leading edge of epithelial cells, Shot regulates protrusion dynamics by promoting filopodia formation. Fluorescence recovery after photobleaching (FRAP) analysis and in vivo imaging of microtubule growth revealed that Shot stabilizes dynamic microtubules. The actin- and microtubule-binding activities of Shot are simultaneously required in the same molecule, indicating that Shot is engaged as a physical crosslinker in this process. We propose that Shot-mediated interactions between microtubules and actin filaments facilitate filopodia formation, which promotes zippering by initiating contact between opposing epithelial cells. PMID:28062848
Birefringence of single and bundled microtubules.

PubMed

Oldenbourg, R; Salmon, E D; Tran, P T

1998-01-01

We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07 nm. The peak retardance of a small bundle increased linearly with the number of MTs in the bundle. Axonemes (prepared from sea urchin sperm) had a peak retardance 20 times higher than that of single MTs, in accordance with the nine doublets and two singlets arrangement of parallel MTs in the axoneme. Measured filament retardance decreased when the filament was defocused or the numerical aperture of the imaging system was decreased. However, the retardance "area," which we defined as the image retardance integrated along a line perpendicular to the filament axis, proved to be independent of focus and of numerical aperture. These results are in good agreement with a theory that we developed for measuring retardances with imaging optics. Our theoretical concept is based on Wiener's theory of mixed dielectrics, which is well established for nonimaging applications. We extend its use to imaging systems by considering the coherence region defined by the optical set-up. Light scattered from within that region interferes coherently in the image point. The presence of a filament in the coherence region leads to a polarization dependent scattering cross section and to a finite retardance measured in the image point. Similar to resolution measurements, the linear dimension of the coherence region for retardance measurements is on the order lambda/(2 NA), where lambda is the wavelength of light and NA is the numerical aperture of the illumination and imaging lenses.
Birefringence of single and bundled microtubules.

PubMed Central

Oldenbourg, R; Salmon, E D; Tran, P T

1998-01-01

We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07 nm. The peak retardance of a small bundle increased linearly with the number of MTs in the bundle. Axonemes (prepared from sea urchin sperm) had a peak retardance 20 times higher than that of single MTs, in accordance with the nine doublets and two singlets arrangement of parallel MTs in the axoneme. Measured filament retardance decreased when the filament was defocused or the numerical aperture of the imaging system was decreased. However, the retardance "area," which we defined as the image retardance integrated along a line perpendicular to the filament axis, proved to be independent of focus and of numerical aperture. These results are in good agreement with a theory that we developed for measuring retardances with imaging optics. Our theoretical concept is based on Wiener's theory of mixed dielectrics, which is well established for nonimaging applications. We extend its use to imaging systems by considering the coherence region defined by the optical set-up. Light scattered from within that region interferes coherently in the image point. The presence of a filament in the coherence region leads to a polarization dependent scattering cross section and to a finite retardance measured in the image point. Similar to resolution measurements, the linear dimension of the coherence region for retardance measurements is on the order lambda/(2 NA), where lambda is the wavelength of light and NA is the numerical aperture of the illumination and imaging lenses. PMID:9449366
Predicting the stochastic guiding of kinesin-driven microtubules in microfabricated tracks: a statistical-mechanics-based modeling approach.

PubMed

Lin, Chih-Tin; Meyhofer, Edgar; Kurabayashi, Katsuo

2010-01-01

Directional control of microtubule shuttles via microfabricated tracks is key to the development of controlled nanoscale mass transport by kinesin motor molecules. Here we develop and test a model to quantitatively predict the stochastic behavior of microtubule guiding when they mechanically collide with the sidewalls of lithographically patterned tracks. By taking into account appropriate probability distributions of microscopic states of the microtubule system, the model allows us to theoretically analyze the roles of collision conditions and kinesin surface densities in determining how the motion of microtubule shuttles is controlled. In addition, we experimentally observe the statistics of microtubule collision events and compare our theoretical prediction with experimental data to validate our model. The model will direct the design of future hybrid nanotechnology devices that integrate nanoscale transport systems powered by kinesin-driven molecular shuttles.
Microtubule-Targeting Agents Enter the Central Nervous System (CNS): Double-edged Swords for Treating CNS Injury and Disease.

PubMed

Hur, Eun-Mi; Lee, Byoung Dae

2014-12-01

Microtubules have been among the most successful targets in anticancer therapy and a large number of microtubule-targeting agents (MTAs) are in various stages of clinical development for the treatment of several malignancies. Given that injury and diseases in the central nervous system (CNS) are accompanied by acute or chronic disruption of the structural integrity of neurons and that microtubules provide structural support for the nervous system at cellular and intracellular levels, microtubules are emerging as potential therapeutic targets for treating CNS disorders. It has been postulated that exogenous application of MTAs might prevent the breakdown or degradation of microtubules after injury or during neurodegeneration, which will thereby aid in preserving the structural integrity and function of the nervous system. Here we review recent evidence that supports this notion and also discuss potential risks of targeting microtubules as a therapy for treating nerve injury and neurodegenerative diseases.
Effects of Microtubule and Actin Inhibitors on Cryptococcus neoformans Examined by Scanning and Transmission Electron Microscopy.

PubMed

Kopecká, Marie

2014-01-01

Cryptococcus neoformans is one of the most important human fungal pathogens. Its cells contain rich microtubules required for nuclear division and rich F-actin cytoskeletons for cell division. Disruption of microtubules by a microtubule inhibitor should block nuclear division, and disruption of F-actin by an actin inhibitor should block cell division. We investigated the effects of microtubule and actin inhibitors to find out whether the cytoskeletons of C. neoformans can become a new anti-fungal target for the inhibition of cell division, when examined at the ultrastructural level. Cells treated with the microtubule inhibitors vincristine (VIN) and methyl benzimidazole-2-ylcarbamate (BCM) and the actin inhibitor latrunculin A (LA), in yeast extract peptone dextrose medium, were examined by scanning (SEM) and transmission electron microscopy (TEM), and the cell number was counted using a Bürker chamber. After 2 days of inhibition with VIN, BCM or LA, the cells did not divide, but later, resistant, proliferating cells appeared in all samples. With combined microtubule and actin inhibitors (VIN + LA or BCM + LA), cells did not divide during 6 or even 14 days, and no resistant cells originated. TEM showed that the inhibited cells were without cytoplasm and were dead; only empty cell walls persisted with reduced capsules, shown on SEM. Combined microtubule and actin inhibitors (VIN + LA or BCM + LA), have lethal effects on C. neoformans cells and no resistant cells originate. © 2015 S. Karger AG, Basel
Lateral motion and bending of microtubules studied with a new single-filament tracking routine in living cells.

PubMed

Pallavicini, Carla; Levi, Valeria; Wetzler, Diana E; Angiolini, Juan F; Benseñor, Lorena; Despósito, Marcelo A; Bruno, Luciana

2014-06-17

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells
Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

PubMed Central

Pallavicini, Carla; Levi, Valeria; Wetzler, Diana E.; Angiolini, Juan F.; Benseñor, Lorena; Despósito, Marcelo A.; Bruno, Luciana

2014-01-01

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in
Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity

PubMed Central

Matthies, Heinrich J.G.; Baskin, Ronald J.; Hawley, R. Scott

2001-01-01

NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NODDTW and NOD“DR2”) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport. PMID:11739796
NudEL targets dynein to microtubule ends through LIS1

PubMed Central

Li, Jun; Lee, Wei-Lih; Cooper, John A.

2006-01-01

Dynein is a minus-end-directed microtubule motor with critical roles in mitosis, membrane transport and intracellular transport. Several proteins regulate dynein activity, including dynactin1, LIS1 (refs 2, 3) and NudEL (NudE-like)2,4–8. Here, we identify a NUDEL homologue in budding yeast and name it Ndl1. The ndl1Δ null mutant shows decreased targeting of dynein to microtubule plus ends, an essential element of the model for dynein function. We find that Ndl1 regulates dynein targeting through LIS1, with which it interacts biochemically, but not through CLIP170, another plus-end protein involved in dynein targeting9. Ndl1 is found at far fewer microtubule ends than are LIS1 and dynein. However, when Ndl1 is present at a plus end, the molar amount of Ndl1 approaches that of LIS1 and dynein. We propose a model in which Ndl1 binds transiently to the plus end to promote targeting of LIS1, which cooperatively recruits dynein. PMID:15965467
Steps toward dismantling poverty for working, poor women.

PubMed

Froehlich, Jeanette

2005-01-01

The majority of the world's poor people are women and many of them spend long hours doing paid and unpaid work. Pay inequities between men and women persist and income inequalities between the rich and poor are deepening. Working poor females, especially working poor mothers, struggle against considerable odds. This situation, and some steps that healthcare professionals can take toward dismantling poverty are addressed.
High-precision Ru isotopic measurements by multi-collector ICP-MS.

PubMed

Becker, Harry; Dalpe, Claude; Walker, Richard J

2002-06-01

Ruthenium isotopic data for a pure Aldrich ruthenium nitrate solution obtained using a Nu Plasma multi collector inductively coupled plasma-mass spectrometer (MC-ICP-MS) shows excellent agreement (better than 1 epsilon unit = 1 part in 10(4)) with data obtained by other techniques for the mass range between 96 and 101 amu. External precisions are at the 0.5-1.7 epsilon level (2sigma). Higher sensitivity for MC ICP-MS compared to negative thermal ionization mass spectrometry (N-TIMS) is offset by the uncertainties introduced by relatively large mass discrimination and instabilities in the plasma source-ion extraction region that affect the long-term reproducibility. Large mass bias correction in ICP mass spectrometry demands particular attention to be paid to the choice of normalizing isotopes. Because of its position in the mass spectrum and the large mass bias correction, obtaining precise and accurate abundance data for 104Ru by MC-ICP-MS remains difficult. Internal and external mass bias correction schemes in this mass range may show similar shortcomings if the isotope of interest does not lie within the mass range covered by the masses used for normalization. Analyses of meteorite samples show that if isobaric interferences from Mo are sufficiently large (Ru/Mo < 10(4)), uncertainties on the Mo interference correction propagate through the mass bias correction and yield inaccurate results for Ru isotopic compositions. Second-order linear corrections may be used to correct for these inaccuracies, but such results are generally less precise than N-TIMS data.
An agent-based model contrasts opposite effects of dynamic and stable microtubules on cleavage furrow positioning

PubMed Central

Odell, Garrett M.; Foe, Victoria E.

2008-01-01

From experiments by Foe and von Dassow (Foe, V.E., and G. von Dassow. 2008. J. Cell Biol. 183:457–470) and others, we infer a molecular mechanism for positioning the cleavage furrow during cytokinesis. Computer simulations reveal how this mechanism depends on quantitative motor-behavior details and explore how robustly this mechanism succeeds across a range of cell sizes. The mechanism involves the MKLP1 (kinesin-6) component of centralspindlin binding to and walking along microtubules to stimulate cortical contractility where the centralspindlin complex concentrates. The majority of astral microtubules are dynamically unstable. They bind most MKLP1 and suppress cortical Rho/myosin II activation because the tips of unstable microtubules usually depolymerize before MKLP1s reach the cortex. A subset of astral microtubules stabilizes during anaphase, becoming effective rails along which MKLP1 can actually reach the cortex. Because stabilized microtubules aim statistically at the equatorial spindle midplane, that is where centralspindlin accumulates to stimulate furrow formation. PMID:18955556
An agent-based model contrasts opposite effects of dynamic and stable microtubules on cleavage furrow positioning.

PubMed

Odell, Garrett M; Foe, Victoria E

2008-11-03

From experiments by Foe and von Dassow (Foe, V.E., and G. von Dassow. 2008. J. Cell Biol. 183:457-470) and others, we infer a molecular mechanism for positioning the cleavage furrow during cytokinesis. Computer simulations reveal how this mechanism depends on quantitative motor-behavior details and explore how robustly this mechanism succeeds across a range of cell sizes. The mechanism involves the MKLP1 (kinesin-6) component of centralspindlin binding to and walking along microtubules to stimulate cortical contractility where the centralspindlin complex concentrates. The majority of astral microtubules are dynamically unstable. They bind most MKLP1 and suppress cortical Rho/myosin II activation because the tips of unstable microtubules usually depolymerize before MKLP1s reach the cortex. A subset of astral microtubules stabilizes during anaphase, becoming effective rails along which MKLP1 can actually reach the cortex. Because stabilized microtubules aim statistically at the equatorial spindle midplane, that is where centralspindlin accumulates to stimulate furrow formation.
New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis.

PubMed

Sugimoto, K; Williamson, R E; Wasteneys, G O

2000-12-01

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.
Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

PubMed Central

Kudalkar, Emily M.; Scarborough, Emily A.; Umbreit, Neil T.; Zelter, Alex; Gestaut, Daniel R.; Riffle, Michael; Johnson, Richard S.; MacCoss, Michael J.; Asbury, Charles L.; Davis, Trisha N.

2015-01-01

Multiple protein subcomplexes of the kinetochore cooperate as a cohesive molecular unit that forms load-bearing microtubule attachments that drive mitotic chromosome movements. There is intriguing evidence suggesting that central kinetochore components influence kinetochore–microtubule attachment, but the mechanism remains unclear. Here, we find that the conserved Mis12/MIND (Mtw1, Nsl1, Nnf1, Dsn1) and Ndc80 (Ndc80, Nuf2, Spc24, Spc25) complexes are connected by an extensive network of contacts, each essential for viability in cells, and collectively able to withstand substantial tensile load. Using a single-molecule approach, we demonstrate that an individual MIND complex enhances the microtubule-binding affinity of a single Ndc80 complex by fourfold. MIND itself does not bind microtubules. Instead, MIND binds Ndc80 complex far from the microtubule-binding domain and confers increased microtubule interaction of the complex. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a folded conformation. Together, our results suggest a previously unidentified mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore complex. PMID:26430240
Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells

NASA Astrophysics Data System (ADS)

Ory, Eleanor C.; Bhandary, Lekhana; E Boggs, Amanda; Chakrabarti, Kristi R.; Parker, Joshua; Losert, Wolfgang; Martin, Stuart S.

2017-04-01

The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems—growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

Chlamydia Hijacks ARF GTPases To Coordinate Microtubule Posttranslational Modifications and Golgi Complex Positioning

PubMed Central

Wesolowski, Jordan; Weber, Mary M.; Nawrotek, Agata; Dooley, Cheryl A.; Calderon, Mike; St. Croix, Claudette M.; Hackstadt, Ted; Cherfils, Jacqueline

2017-01-01

ABSTRACT The intracellular bacterium Chlamydia trachomatis develops in a parasitic compartment called the inclusion. Posttranslationally modified microtubules encase the inclusion, controlling the positioning of Golgi complex fragments around the inclusion. The molecular mechanisms by which Chlamydia coopts the host cytoskeleton and the Golgi complex to sustain its infectious compartment are unknown. Here, using a genetically modified Chlamydia strain, we discovered that both posttranslationally modified microtubules and Golgi complex positioning around the inclusion are controlled by the chlamydial inclusion protein CT813/CTL0184/InaC and host ARF GTPases. CT813 recruits ARF1 and ARF4 to the inclusion membrane, where they induce posttranslationally modified microtubules. Similarly, both ARF isoforms are required for the repositioning of Golgi complex fragments around the inclusion. We demonstrate that CT813 directly recruits ARF GTPases on the inclusion membrane and plays a pivotal role in their activation. Together, these results reveal that Chlamydia uses CT813 to hijack ARF GTPases to couple posttranslationally modified microtubules and Golgi complex repositioning at the inclusion. PMID:28465429
Computational Study of Pseudo-Phosphorylation and Phosphorylation of the Microtubule Associated Protein Tau

NASA Astrophysics Data System (ADS)

Prokopovich, Dmitriy; Larini, Luca

This study focuses on the effect of pseudo-phosphorylation on the aggregation of protein tau, which is very often found interacting with microtubules in the neuron. Within the axon of the neuron, tau governs the assembly of microtubules that make up the cytoskeleton. This is important for stabilization of and transport across the microtubules. One of the indications of the Alzheimer's disease is the hyper-phosphorylation and aggregation of protein tau into neurofibrillary tangles that destroy the neurons. But even experts in the field do not know if hyper-phosphorylation directly causes the aggregation of tau. In some experiments, pseudo-phosphorylation mimics the effects of phosphorylation. It does so by mutating certain residues of the protein chain into charged residues. In this computational study, we will employ a fragment of tau called PHF43. This fragment belongs to the microtubule binding region and papers published by others have indicated that it readily aggregates. Replica exchange molecular dynamics simulations were performed on the pseudo-phosphorylated, phosphorylated, and dimerized PHF43. The program used to simulate and analyze PHF43 was AMBER14.
The dynamics of plus end polarization and microtubule assembly during Xenopus cortical rotation

PubMed Central

Olson, David J.; Oh, Denise

2015-01-01

The self-organization of dorsally-directed microtubules during cortical rotation in the Xenopus egg is essential for dorsal axis formation. The mechanisms controlling this process have been problematic to analyze, owing to difficulties in visualizing microtubules in living egg. Also, the order of events occurring at the onset of cortical rotation have not been satisfactorily visualized in vivo and have been inferred from staged fixed samples. To address these issues, we have characterized the dynamics of total microtubule and plus end behavior continuously throughout cortical rotation, as well as in oocytes and unfertilized eggs. Here, we show that the nascent microtubule network forms in the cortex but associates with the deep cytoplasm at the start of rotation. Importantly, plus ends remain cortical and become increasingly more numerous and active prior to rotation, with dorsal polarization occurring rapidly after the onset of rotation. Additionally, we show that vegetally localized Trim36 is required to attenuate dynamic plus end growth, suggesting that vegetal factors are needed to locally coordinate growth in the cortex. PMID:25753733
Dismantling of Highly Contaminated Process Installations of the German Reprocessing Facility (WAK) - Status of New Remote Handling Technology - 13287

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dux, Joachim; Friedrich, Daniel; Lutz, Werner

2013-07-01

Decommissioning and dismantling of the former German Pilot Reprocessing Plant Karlsruhe (WAK) including the Vitrification Facility (VEK) is being executed in different Project steps related to the reprocessing, HLLW storage and vitrification complexes /1/. While inside the reprocessing building the total inventory of process equipment has already been dismantled and disposed of, the HLLW storage and vitrification complex has been placed out of operation since vitrification and tank rinsing procedures where finalized in year 2010. This paper describes the progress made in dismantling of the shielded boxes of the highly contaminated laboratory as a precondition to get access to themore » hot cells of the HLLW storage. The major challenges of the dismantling of this laboratory were the high dose rates up to 700 mSv/h and the locking technology for the removal of the hot cell installations. In parallel extensive prototype testing of different carrier systems and power manipulators to be applied to dismantle the HLLW-tanks and other hot cell equipment is ongoing. First experiences with the new manipulator carrier system and a new master slave manipulator with force reflection will be reported. (authors)« less
Multi-Sensor Fused Interrogation of Brain to Determine ICP Level

DTIC Science & Technology

1997-08-01

manifestations, but the decision is considerably more difficult for soldiers who are rendered immediately unconscious through blunt injury and concussion...is an example of swept sine excitation yielding low frequency resonance and attenuation data using head-down tilt to elevate ICP, and Figure 2 is an... excitation ) in an adult male excitation ) in female adult volunteer with ICP volunteer with ICP elevation induced through elevation induced through
Microtubule-dependent regulation of mitotic protein degradation

PubMed Central

Song, Ling; Craney, Allison; Rape, Michael

2014-01-01

Accurate cell division depends on tightly regulated ubiquitylation events catalyzed by the anaphase-promoting complex. Among its many substrates, the APC/C triggers the degradation of proteins that stabilize the mitotic spindle, and loss or accumulation of such spindle assembly factors can result in aneuploidy and cancer. Although critical for cell division, it has remained poorly understood how the timing of spindle assembly factor degradation is established during mitosis. Here, we report that active spindle assembly factors are protected from APC/C-dependent degradation by microtubules. In contrast, those molecules that are not bound to microtubules are highly susceptible to proteolysis and turned over immediately after APC/C-activation. The correct timing of spindle assembly factor degradation, as achieved by this regulatory circuit, is required for accurate spindle structure and function. We propose that the localized stabilization of APC/C-substrates provides a mechanism for the selective disposal of cell cycle regulators that have fulfilled their mitotic roles. PMID:24462202
Aurora A regulates the activity of HURP by controlling the accessibility of its microtubule-binding domain.

PubMed

Wong, Jim; Lerrigo, Robert; Jang, Chang-Young; Fang, Guowei

2008-05-01

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.
Transport Properties of Melanosomes along Microtubules Interpreted by a Tug-of-War Model with Loose Mechanical Coupling

PubMed Central

Bouzat, Sebastián; Levi, Valeria; Bruno, Luciana

2012-01-01

In this work, we explored theoretically the transport of organelles driven along microtubules by molecular motors of opposed polarities using a stochastic model that considers a Langevin dynamics for the cargo, independent cargo-motor linkers and stepping motion for the motors. It has been recently proposed that the stiffness of the motor plays an important role when multiple motors collectively transport a cargo. Therefore, we considered in our model the recently reported values for the stiffness of the cargo-motor linker determined in living cells (∼0.01 pN/nm, [1]) which is significantly lower than the motor stiffness obtained in in vitro assays and used in previous studies. Our model could reproduce the multimodal velocity distributions and typical trajectory characteristics including the properties of the reversions in the overall direction of motion observed during melanosome transport along microtubules in Xenopus laevis melanophores. Moreover, we explored the contribution of the different motility states of the cargo-motor system to the different modes of the velocity distributions and could identify the microscopic mechanisms of transport leading to trajectories compatible with those observed in living cells. Finally, by changing the attachment and detachment rates, the model could reproduce the different velocity distributions observed during melanosome transport along microtubules in Xenopus laevis melanophores stimulated for aggregation and dispersion. Our analysis suggests that active tug-of-war processes with loose mechanical coupling can account for several aspects of cargo transport along microtubules in living cells. PMID:22952716
A Live-Attenuated HSV-2 ICP0 − Virus Elicits 10 to 100 Times Greater Protection against Genital Herpes than a Glycoprotein D Subunit Vaccine

PubMed Central

Halford, William P.; Püschel, Ringo; Gershburg, Edward; Wilber, Andrew; Gershburg, Svetlana; Rakowski, Brandon

2011-01-01

Glycoprotein D (gD-2) is the entry receptor of herpes simplex virus 2 (HSV-2), and is the immunogen in the pharmaceutical industry's lead HSV-2 vaccine candidate. Efforts to prevent genital herpes using gD-2 subunit vaccines have been ongoing for 20 years at a cost in excess of $100 million. To date, gD-2 vaccines have yielded equivocal protection in clinical trials. Therefore, using a small animal model, we sought to determine if a live-attenuated HSV-2 ICP0 − virus would elicit better protection against genital herpes than a gD-2 subunit vaccine. Mice immunized with gD-2 and a potent adjuvant (alum+monophosphoryl lipid A) produced high titers of gD-2 antibody. While gD-2-immunized mice possessed significant resistance to HSV-2, only 3 of 45 gD-2-immunized mice survived an overwhelming challenge of the vagina or eyes with wild-type HSV-2 (MS strain). In contrast, 114 of 115 mice immunized with a live HSV-2 ICP0 − virus, 0ΔNLS, survived the same HSV-2 MS challenges. Likewise, 0ΔNLS-immunized mice shed an average 125-fold less HSV-2 MS challenge virus per vagina relative to gD-2-immunized mice. In vivo imaging demonstrated that a luciferase-expressing HSV-2 challenge virus failed to establish a detectable infection in 0ΔNLS-immunized mice, whereas the same virus readily infected naïve and gD-2-immunized mice. Collectively, these results suggest that a HSV-2 vaccine might be more likely to prevent genital herpes if it contained a live-attenuated HSV-2 virus rather than a single HSV-2 protein. PMID:21412438
F-actin and microtubule suspensions as indeterminate fluids.

PubMed

Buxbaum, R E; Dennerll, T; Weiss, S; Heidemann, S R

1987-03-20

The viscosity of F-actin and microtubule suspensions has been measured as a function of shear rate with a Weissenberg rheogoniometer. At shear rates of less than 1.0 per second the viscosity of suspensions of these two structural proteins is inversely proportional to shear rate. These results are consistent with previous in vivo measurements of the viscosity of cytoplasm. This power law implies that shear stress is independent of shear rate; that is, shear stress is a constant at all shear rates less than 1.0 per second. Thus the flow profile of these fluids is indeterminate, or nearly so. This flow property may explain several aspects of intracellular motility in living cells. Possible explanations for this flow property are based on a recent model for semidilute suspensions of rigid rods or a classical friction model for liquid crystals.
Increased microtubule assembly rates mediate chromosomal instability in colorectal cancer cells

PubMed Central

Ertych, Norman; Stolz, Ailine; Stenzinger, Albrecht; Weichert, Wilko; Kaulfuß, Silke; Burfeind, Peter; Aigner, Achim; Wordeman, Linda

2015-01-01

Chromosomal instability (CIN) is defined as the perpetual missegregation of whole chromosomes during mitosis and represents a hallmark of human cancer. However, the mechanisms causing CIN and its consequences on tumor growth are largely unknown. We identify an increase in microtubule plus end assembly rates as a fundamental trigger for CIN in CRC cells. This trigger is mediated by overexpression of the oncogene AURKA or by loss of the tumor suppressor gene CHK2, a genetic constitution found in 73% of human colorectal cancers. Increased microtubule assembly rates are associated with transient abnormalities in mitotic spindle geometry promoting the generation of lagging chromosomes and resulting in CIN. Reconstitution of proper microtubule assembly rates by chemical or genetic means suppresses CIN and thereby, unexpectedly, accelerates tumor growth in vitro and in vivo. Thus, we identify a fundamental mechanism triggering CIN in cancer cells and reveal its adverse consequence on tumor growth. PMID:24976383
PSD-95 alters microtubule dynamics via an association with EB3

PubMed Central

Sweet, Eric S.; Previtera, Michelle L.; Fernández, Jose R.; Charych, Erik I.; Tseng, Chia-Yi; Kwon, Munjin; Starovoytov, Valentin; Zheng, James Q.; Firestein, Bonnie L.

2011-01-01

Little is known about how the neuronal cytoskeleton is regulated when a dendrite decides whether to branch or not. Previously, we reported that postsynaptic density protein 95 (PSD-95) acts as a stop signal for dendrite branching. It is yet to be elucidated how PSD-95 affects the cytoskeleton and how this regulation relates to the dendritic arbor. Here, we show that the SH3 (src homology 3) domain of PSD-95 interacts with a proline-rich region within the microtubule end-binding protein EB3. Overexpression of PSD-95 or mutant EB3 results in a decreased lifetime of EB3 comets in dendrites. In line with these data, transfected rat neurons show that overexpression of PSD-95 results in less organized microtubules at dendritic branch points and decreased dendritogensis. The interaction between PSD-95 and EB3 elucidates a function for a novel region of EB3 and provides a new and important mechanism for the regulation of microtubules in determining dendritic morphology. PMID:21248129
[Convertibility of the data determined by ICP-AES and FAAS for soil available K and Na].

PubMed

Zhang, Jian-min; Wang, Meng; Ge, Xiao-ping; Wu, Jian-zhi; Ge, Ying; Li, Shi-peng; Chang, Jie

2009-05-01

In recent years, inductively coupled plasma atomic emission spectrometry (ICP-AES) have been commonly used to determine the soil available K and Na with the extraction solution of HCl-H2SO4, while previous data of soil available K and Na were measured by flame atomic absorption spectrometry (FAAS) with the extraction solution of NH4OAc. In order to utilize previous data, quest for the convertibility of the data determined by ICP-AES and FAAS, and compare the data determined by both methods, the authors chose four types of soil to determine soil available K and Na by ICP-AES and FAAS, respectively. Four types of soil represent grit soil, clay, silt from river and silt from sea, respectively. Soil samples included four types of soil and these samples represent different soil nutrition. The authors analyzed the correlations of two kinds of measured data. The paired samples t-test proves that there was significantly positively correlation between these two methods. The correlation coefficient of the data between these two methods for measuring soil available K is 0.98. The results of soil available K determined by the two methods can be conversed through the formula, y = l.14x + 6.53 (R2 = 0.91, n=24, p < 0.001). As for Na, although there is a significantly positively correlation between these two methods, the slopes of single model of clay and grit soil were different from that of general model. And so the results determined by the two methods can be conversed through different formula according to the types of soil, that is, for clay: y = l.23x + 10.03; for grit soil: y = 3.12x - 23.03; for silt: y = 0.60x. In conclusion, the authors' results showed that previous data of available K and Na measured by FAAS with the extraction solution of NH4OAc were available. And these data were comparable to the data measured by ICP-AES through definite formula The authors' results also suggested that ICP-AES was preferable when many elements were measured at the same time. Under
Single molecule FRET observation of kinesin-1’s head-tail interaction on microtubule

PubMed Central

Aoki, Takahiro; Tomishige, Michio; Ariga, Takayuki

2013-01-01

Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the micro-tubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility. PMID:27493553
In vitro systems for the study of microtubule-based cell polarity in fission yeast.

PubMed

Taberner, Núria; Lof, Andries; Roth, Sophie; Lamers, Dimitry; Zeijlemaker, Hans; Dogterom, Marileen

2015-01-01

Establishment of cell polarity is essential for processes such as growth and division. In fission yeast, as well as other species, polarity factors travel at the ends of microtubules to cortical sites where they associate with the membrane and subsequently maintain a polarized activity pattern despite their ability to diffuse in the membrane. In this chapter we present methods to establish an in vitro system that captures the essential features of this process. This bottom-up approach allows us to identify the minimal molecular requirements for microtubule-based cell polarity. We employ microfabrication techniques combined with surface functionalization to create rigid chambers with affinity for proteins, as well as microfluidic techniques to create and shape emulsion droplets with functionalized lipid boundaries. Preliminary results are shown demonstrating that a properly organized microtubule cytoskeleton can be confined to these confined spaces, and proteins traveling at the ends of growing microtubules can be delivered to their boundaries. Copyright © 2015 Elsevier Inc. All rights reserved.
Tuning microtubule dynamics to enhance cancer therapy by modulating FER-mediated CRMP2 phosphorylation.

PubMed

Zheng, Yiyan; Sethi, Ritika; Mangala, Lingegowda S; Taylor, Charlotte; Goldsmith, Juliet; Wang, Ming; Masuda, Kenta; Karaminejadranjbar, Mohammad; Mannion, David; Miranda, Fabrizio; Herrero-Gonzalez, Sandra; Hellner, Karin; Chen, Fiona; Alsaadi, Abdulkhaliq; Albukhari, Ashwag; Fotso, Donatien Chedom; Yau, Christopher; Jiang, Dahai; Pradeep, Sunila; Rodriguez-Aguayo, Cristian; Lopez-Berestein, Gabriel; Knapp, Stefan; Gray, Nathanael S; Campo, Leticia; Myers, Kevin A; Dhar, Sunanda; Ferguson, David; Bast, Robert C; Sood, Anil K; von Delft, Frank; Ahmed, Ahmed Ashour

2018-02-02

Though used widely in cancer therapy, paclitaxel only elicits a response in a fraction of patients. A strong determinant of paclitaxel tumor response is the state of microtubule dynamic instability. However, whether the manipulation of this physiological process can be controlled to enhance paclitaxel response has not been tested. Here, we show a previously unrecognized role of the microtubule-associated protein CRMP2 in inducing microtubule bundling through its carboxy terminus. This activity is significantly decreased when the FER tyrosine kinase phosphorylates CRMP2 at Y479 and Y499. The crystal structures of wild-type CRMP2 and CRMP2-Y479E reveal how mimicking phosphorylation prevents tetramerization of CRMP2. Depletion of FER or reducing its catalytic activity using sub-therapeutic doses of inhibitors increases paclitaxel-induced microtubule stability and cytotoxicity in ovarian cancer cells and in vivo. This work provides a rationale for inhibiting FER-mediated CRMP2 phosphorylation to enhance paclitaxel on-target activity for cancer therapy.
Coupling of kinesin ATP turnover to translocation and microtubule regulation: one engine, many machines.

PubMed

Friel, Claire T; Howard, Jonathon

2012-12-01

The cycle of ATP turnover is integral to the action of motor proteins. Here we discuss how variation in this cycle leads to variation of function observed amongst members of the kinesin superfamily of microtubule associated motor proteins. Variation in the ATP turnover cycle among superfamily members can tune the characteristic kinesin motor to one of the range of microtubule-based functions performed by kinesins. The speed at which ATP is hydrolysed affects the speed of translocation. The ratio of rate constants of ATP turnover in relation to association and dissociation from the microtubule influence the processivity of translocation. Variation in the rate-limiting step of the cycle can reverse the way in which the motor domain interacts with the microtubule producing non-motile kinesins. Because the ATP turnover cycle is not fully understood for the majority of kinesins, much work remains to show how the kinesin engine functions in such a wide variety of molecular machines.
Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores

PubMed Central

Kajtez, Janko; Solomatina, Anastasia; Novak, Maja; Polak, Bruno; Vukušić, Kruno; Rüdiger, Jonas; Cojoc, Gheorghe; Milas, Ana; Šumanovac Šestak, Ivana; Risteski, Patrik; Tavano, Federica; Klemm, Anna H.; Roscioli, Emanuele; Welburn, Julie; Cimini, Daniela; Glunčić, Matko; Pavin, Nenad; Tolić, Iva M.

2016-01-01

During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term ‘bridging fibre', bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape. PMID:26728792
Microtubule-Targeting Agents Eribulin and Paclitaxel Differentially Affect Neuronal Cell Bodies in Chemotherapy-Induced Peripheral Neuropathy.

PubMed

Benbow, Sarah J; Wozniak, Krystyna M; Kulesh, Bridget; Savage, April; Slusher, Barbara S; Littlefield, Bruce A; Jordan, Mary Ann; Wilson, Leslie; Feinstein, Stuart C

2017-07-01

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of anticancer treatment with microtubule-targeted agents (MTAs). The frequency of severe CIPN, which can be dose limiting and even life threatening, varies widely among different MTAs. For example, paclitaxel induces a higher frequency of severe CIPN than does eribulin. Different MTAs also possess distinct mechanisms of microtubule-targeted action. Recently, we demonstrated that paclitaxel and eribulin differentially affect sciatic nerve axons, with paclitaxel inducing more pronounced neurodegenerative effects and eribulin inducing greater microtubule stabilizing biochemical effects. Here, we complement and extend these axonal studies by assessing the effects of paclitaxel and eribulin in the cell bodies of sciatic nerve axons, housed in the dorsal root ganglia (DRG). Importantly, the microtubule network in cell bodies is known to be significantly more dynamic than in axons. Paclitaxel induced activating transcription factor 3 expression, a marker of neuronal stress/injury. Paclitaxel also increased expression levels of acetylated tubulin and end binding protein 1, markers of microtubule stability and growth, respectively. These effects are hypothesized to be detrimental to the dynamic microtubule network within the cell bodies. In contrast, eribulin had no significant effect on any of these parameters in the cell bodies. Taken together, DRG cell bodies and their axons, two distinct neuronal cell compartments, contain functionally distinct microtubule networks that exhibit unique biochemical responses to different MTA treatments. We hypothesize that these distinct mechanistic actions may underlie the variability seen in the initiation, progression, persistence, and recovery from CIPN.
Plant cytoskeleton: DELLA connects gibberellins to microtubules.

PubMed

Dixit, Ram

2013-06-03

A new study reveals that DELLA proteins directly interact with the prefoldin complex, thus regulating tubulin subunit availability in a gibberellin-dependent manner. This finding provides a mechanistic link between the growth-promoting plant hormone gibberellin and cortical microtubule organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Stability of kinetochore-microtubule attachment and the role of different KMN network components in Drosophila.

PubMed

Feijão, Tália; Afonso, Olga; Maia, André F; Sunkel, Claudio E

2013-10-01

Kinetochores bind spindle microtubules and also act as signaling centers that monitor this interaction. Defects in kinetochore assembly lead to chromosome missegregation and aneuploidy. The interaction between microtubules and chromosomes involves a conserved super-complex of proteins, known as the KNL1Mis12Ndc80 (KMN) network, composed by the KNL1 (Spc105), Mis12, and Ndc80 complexes. Previous studies indicate that all components of the network are required for kinetochore-microtubule attachment and all play relevant functions in chromosome congression, biorientation, and segregation. Here, we report a comparative study addressing the role of the different KMN components using dsRNA and in vivo fluorescence microscopy in Drosophila S2 cells allowing us to suggest that different KMN network components might perform different roles in chromosome segregation and the mitotic checkpoint signaling. Depletion of different components results in mostly lateral kinetochore-microtubule attachments that are relatively stable on depletion of Mis12 or Ndc80 but very unstable after Spc105 depletion. In vivo analysis on depletion of Mis12, Ndc80, and to some extent Spc105, shows that lateral kinetochore-microtubule interactions are still functional allowing poleward kinetochore movement. We also find that different KMN network components affect differently the localization of spindle assembly checkpoint (SAC) proteins at kinetochores. Depletion of Ndc80 and Spc105 abolishes the mitotic checkpoint, whereas depletion of Mis12 causes a delay in mitotic progression. Taken together, our results suggest that Mis12 and Ndc80 complexes help to properly orient microtubule attachment, whereas Spc105 plays a predominant role in the kinetochore-microtubule attachment as well as in the poleward movement of chromosomes, SAC response, and cell viability. Copyright © 2013 Wiley Periodicals, Inc.
Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study

PubMed Central

Zhu, Jie; Burakov, Anton; Rodionov, Vladimir

2010-01-01

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces—dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles—is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility. PMID:20980619
Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study.

PubMed

Zhu, Jie; Burakov, Anton; Rodionov, Vladimir; Mogilner, Alex

2010-12-01

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.
ICP-MS Workshop

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carman, April J.; Eiden, Gregory C.

2014-11-01

This is a short document that explains the materials that will be transmitted to LLNL and DNN HQ regarding the ICP-MS Workshop held at PNNL June 17-19th. The goal of the information is to pass on to LLNL information regarding the planning and preparations for the Workshop at PNNL in preparation of the SIMS workshop at LLNL.
Specific determination of bromate in bread by ion chromatography with ICP-MS.

PubMed

Akiyama, Takumi; Yamanaka, Michiko; Date, Yukiko; Kubota, Hiroki; Nagaoka, Megumi Hamano; Kawasaki, Yoko; Yamazaki, Takeshi; Yomota, Chikako; Maitani, Tamio

2002-12-01

A sensitive method for detecting bromate in bread by ion chromatography with inductively-coupled plasma mass spectrometry (IC/ICP-MS) was developed. Bromate was extracted from bread with water. The clean-up procedure included a 0.2 micron filter, a C18 cartridge for defatting, a silver cartridge to remove halogen anions, a centrifugal ultrafiltration unit to remove proteins, and a cation-exchange cartridge to remove silver ions. A 500 microL sample solution was applied to IC/ICP-MS. The detection limit and the quantitation limit of bromate in the solution were 0.3 ng/mL and 1.0 ng/mL, expressed as HBrO3, respectively, which corresponded to 2 ng/g and 5 ng/g, respectively, in bread. Recovery of bromate was about 90%, and the CV was about 2%. Based on the detection limit in solution and recovery from bread, the detection limit of bromate in bread was estimated to be 2 ng/g.
Dissecting EB1-microtubule interactions from every direction: using single-molecule visualization and static and dynamic binding measurements

NASA Astrophysics Data System (ADS)

Lopez, Benjamin

2015-03-01

EB1 is an important microtubule associating protein (MAP) that acts as a master coordinator of protein activity at the growing plus-end of the microtubule. We can recapitulate the plus-end binding behavior of EB1 along the entire length of a static microtubule using microtubules polymerized in the presence of the nonhydrolyzable GTP analogs GMPCPP and GTP γS instead of GTP. Through the use of single-molecule TIRF imaging we find that EB1 is highly dynamic (with a sub-second characteristic binding lifetime) and continuously diffusive while bound to the microtubule. We measure the diffusion coefficient, D, through linear fitting to mean-squared displacement of individually labeled proteins, and the binding lifetime, τ, by fitting a single exponential decay to the probability distribution of trajectory lifetimes. In agreement with measurements of other diffusive MAPs, we find that D increases and τ decreases with increasing ionic strength. We also find that D is sensitive to the choice of GTP analog: EB1 proteins bound to GTP γS polymerized microtubules have a D half of that found with GMPCPP polymerized microtubules. To compare these single-molecule measurements to the bulk binding behavior of EB1, we use TIRF imaging to measure the intensity of microtubules coated with EB1-GFP as a function of EB1 concentration. We find that EB1 binding is cooperative and both the quantity of EB1 bound and the dissociation constant are sensitive to GTP analog and ionic concentration. The correlation between binding affinity and D and the cooperative nature of EB1-microtubule binding leads to a decrease in D with increasing EB1 concentration. Interestingly, we also find an increase in τ at high EB1 concentrations, consistent with attractive EB1-microtubule interactions driving the cooperativity. To further understand the nature of the cooperativity we estimate the interaction energy by measuring the association and dissociation rates (kon and koff respectively) at different
Three-dimensional fine structure of the organization of microtubules in neurite varicosities by ultra-high voltage electron microscope tomography.

PubMed

Nishida, Tomoki; Yoshimura, Ryoichi; Endo, Yasuhisa

2017-09-01

Neurite varicosities are highly specialized compartments that are involved in neurotransmitter/ neuromodulator release and provide a physiological platform for neural functions. However, it remains unclear how microtubule organization contributes to the form of varicosity. Here, we examine the three-dimensional structure of microtubules in varicosities of a differentiated PC12 neural cell line using ultra-high voltage electron microscope tomography. Three-dimensional imaging showed that a part of the varicosities contained an accumulation of organelles that were separated from parallel microtubule arrays. Further detailed analysis using serial sections and whole-mount tomography revealed microtubules running in a spindle shape of swelling in some other types of varicosities. These electron tomographic results showed that the structural diversity and heterogeneity of microtubule organization supported the form of varicosities, suggesting that a different distribution pattern of microtubules in varicosities is crucial to the regulation of varicosities development.
Comment on "Zircon U-Th-Pb dating using LA-ICP-MS: Simultaneous U-Pb and U-Th dating on 0.1 Ma Toya Tephra, Japan" by Hisatoshi Ito

NASA Astrophysics Data System (ADS)

Guillong, M.; Schmitt, A. K.; Bachmann, O.

2015-04-01

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) of eight zircon reference materials and synthetic zircon-hafnon end-members indicate that corrections for abundance sensitivity and molecular zirconium sesquioxide ions (Zr2O3+) are critical for reliable determination of 230Th abundances in zircon. Other polyatomic interferences in the mass range 223-233 amu are insignificant. When corrected for abundance sensitivity and interferences, activity ratios of (230Th)/(238U) for the zircon reference materials we used average 1.001 ± 0.010 (1σ error; mean square of weighted deviates MSWD = 1.45; n = 8). This includes the 91500 and Plešovice zircons, which were deemed unsuitable for calibration of (230Th)/(238U) by Ito (2014). Uranium series zircon ages generated by LA-ICP-MS without mitigating (e.g., by high mass resolution) or correcting for abundance sensitivity and molecular interferences on 230Th such as those presented by Ito (2014) are potentially unreliable.
G protein betagamma subunits interact with alphabeta- and gamma-tubulin and play a role in microtubule assembly in PC12 cells.

PubMed

Montoya, Valentina; Gutierrez, Christina; Najera, Omar; Leony, Denisse; Varela-Ramirez, Armando; Popova, Juliana; Rasenick, Mark M; Das, Siddhartha; Roychowdhury, Sukla

2007-12-01

The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.
Trace analysis of high-purity graphite by LA-ICP-MS.

PubMed

Pickhardt, C; Becker, J S

2001-07-01

Laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been established as a very efficient and sensitive technique for the direct analysis of solids. In this work the capability of LA-ICP-MS was investigated for determination of trace elements in high-purity graphite. Synthetic laboratory standards with a graphite matrix were prepared for the purpose of quantifying the analytical results. Doped trace elements, concentration 0.5 microg g(-1), in a laboratory standard were determined with an accuracy of 1% to +/- 7% and a relative standard deviation (RSD) of 2-13%. Solution-based calibration was also used for quantitative analysis of high-purity graphite. It was found that such calibration led to analytical results for trace-element determination in graphite with accuracy similar to that obtained by use of synthetic laboratory standards for quantification of analytical results. Results from quantitative determination of trace impurities in a real reactor-graphite sample, using both quantification approaches, were in good agreement. Detection limits for all elements of interest were determined in the low ng g(-1) concentration range. Improvement of detection limits by a factor of 10 was achieved for analyses of high-purity graphite with LA-ICP-MS under wet plasma conditions, because the lower background signal and increased element sensitivity.
A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay.

PubMed

Liu, Bing-Hsien; Lin, Yu-Chen; Ho, Chia-Shin; Yang, Che-Chuan; Chang, Yun-Tsui; Chang, Jui-Feng; Li, Chun-Yuan; Cheng, Cheng-Shun; Huang, Jiun-Yan; Lee, Yen-Fu; Hsu, Ming-Hung; Lin, Feng-Chun; Wang, Hao-Ching; Lo, Chu-Fang; Yang, Shieh-Yueh; Wang, Han-Ching

2015-01-01

Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world's most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe3O4 magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10(-3) ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10(6) ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.
Microtubule plus-end tracking of end-binding protein 1 (EB1) is regulated by CDK5 regulatory subunit-associated protein 2

PubMed Central

Fong, Ka-Wing; Au, Franco K. C.; Jia, Yue; Yang, Shaozhong; Zhou, Liying; Qi, Robert Z.

2017-01-01

Microtubules are polar cytoskeleton filaments that extend via growth at their plus ends. Microtubule plus-end-tracking proteins (+TIPs) accumulate at these growing plus ends to control microtubule dynamics and attachment. The +TIP end-binding protein 1 (EB1) and its homologs possess an autonomous plus-end-tracking mechanism and interact with other known +TIPs, which then recruit those +TIPs to the growing plus ends. A major +TIP class contains the SXIP (Ser-X-Ile-Pro, with X denoting any amino acid residue) motif, known to interact with EB1 and its homologs for plus-end tracking, but the role of SXIP in regulating EB1 activities is unclear. We show here that an interaction of EB1 with the SXIP-containing +TIP CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) regulates several EB1 activities, including microtubule plus-end tracking, dynamics at microtubule plus ends, microtubule and α/β-tubulin binding, and microtubule polymerization. The SXIP motif fused with a dimerization domain from CDK5RAP2 significantly enhanced EB1 plus-end-tracking and microtubule-polymerizing and bundling activities, but the SXIP motif alone failed to do so. An SXIP-binding-deficient EB1 mutant displayed significantly lower microtubule plus-end tracking than the wild-type protein in transfected cells. These results suggest that EB1 cooperates with CDK5RAP2 and perhaps other SXIP-containing +TIPs in tracking growing microtubule tips. We also generated plus-end-tracking chimeras of CDK5RAP2 and the adenomatous polyposis coli protein (APC) and found that overexpression of the dimerization domains interfered with microtubule plus-end tracking of their respective SXIP-containing chimeras. Our results suggest that disruption of SXIP dimerization enables detailed investigations of microtubule plus-end-associated functions of individual SXIP-containing +TIPs. PMID:28320860
SDF1 Reduces Interneuron Leading Process Branching through Dual Regulation of Actin and Microtubules

PubMed Central

Lysko, Daniel E.; Putt, Mary

2014-01-01

Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process. PMID:24695713
Mouse ACF7 and drosophila short stop modulate filopodia formation and microtubule organisation during neuronal growth.

PubMed

Sanchez-Soriano, Natalia; Travis, Mark; Dajas-Bailador, Federico; Gonçalves-Pimentel, Catarina; Whitmarsh, Alan J; Prokop, Andreas

2009-07-15

Spectraplakins are large actin-microtubule linker molecules implicated in various processes, including gastrulation, wound healing, skin blistering and neuronal degeneration. Expression data for the mammalian spectraplakin ACF7 and genetic analyses of the Drosophila spectraplakin Short stop (Shot) suggest an important role during neurogenesis. Using three parallel neuronal culture systems we demonstrate that, like Shot, ACF7 is essential for axon extension and describe, for the first time, their subcellular functions during axonal growth. Firstly, both ACF7 and Shot regulate the organisation of neuronal microtubules, a role dependent on both the F-actin- and microtubule-binding domains. This role in microtubule organisation is probably the key mechanism underlying the roles of Shot and ACF7 in growth cone advance. Secondly, we found a novel role for ACF7 and Shot in regulating the actin cytoskeleton through their ability to control the formation of filopodia. This function in F-actin regulation requires EF-hand motifs and interaction with the translational regulator Krasavietz/eIF5C, indicating that the underlying mechanisms are completely different from those used to control microtubules. Our data provide the basis for the first mechanistic explanation for the role of Shot and ACF7 in the developing nervous system and demonstrate their ability to coordinate the organisation of both actin and microtubule networks during axonal growth.
Mouse ACF7 and Drosophila Short stop modulate filopodia formation and microtubule organisation during neuronal growth

PubMed Central

Sanchez-Soriano, Natalia; Travis, Mark; Dajas-Bailador, Federico; Gonçalves-Pimentel, Catarina; Whitmarsh, Alan J.; Prokop, Andreas

2009-01-01

Summary Spectraplakins are large actin-microtubule linker molecules implicated in various processes, including gastrulation, wound healing, skin blistering and neuronal degeneration. Expression data for the mammalian spectraplakin ACF7 and genetic analyses of the Drosophila spectraplakin Short stop (Shot) suggest an important role during neurogenesis. Using three parallel neuronal culture systems we demonstrate that, like Shot, ACF7 is essential for axon extension and describe, for the first time, their subcellular functions during axonal growth. Firstly, both ACF7 and Shot regulate the organisation of neuronal microtubules, a role dependent on both the F-actin- and microtubule-binding domains. This role in microtubule organisation is probably the key mechanism underlying the roles of Shot and ACF7 in growth cone advance. Secondly, we found a novel role for ACF7 and Shot in regulating the actin cytoskeleton through their ability to control the formation of filopodia. This function in F-actin regulation requires EF-hand motifs and interaction with the translational regulator Krasavietz/eIF5C, indicating that the underlying mechanisms are completely different from those used to control microtubules. Our data provide the basis for the first mechanistic explanation for the role of Shot and ACF7 in the developing nervous system and demonstrate their ability to coordinate the organisation of both actin and microtubule networks during axonal growth. PMID:19571116
The complications and the position of the Codman MicroSensor™ ICP device: an analysis of 549 patients and 650 Sensors.

PubMed

Koskinen, Lars-Owe D; Grayson, David; Olivecrona, Magnus

2013-11-01

Complications of and insertion depth of the Codman MicroSensor ICP monitoring device (CMS) is not well studied. To study complications and the insertion depth of the CMS in a clinical setting. We identified all patients who had their intracranial pressure (ICP) monitored using a CMS device between 2002 and 2010. The medical records and post implantation computed tomography (CT) scans were analyzed for occurrence of infection, hemorrhage and insertion depth. In all, 549 patients were monitored using 650 CMS. Mean monitoring time was 7.0 ± 4.9 days. The mean implantation depth was 21.3 ± 11.1 mm (0-88 mm). In 27 of the patients, a haematoma was identified; 26 of these were less than 1 ml, and one was 8 ml. No clinically significant bleeding was found. There was no statistically significant increase in the number of hemorrhages in presumed coagulopathic patients. The infection rate was 0.6 % and the calculated infection rate per 1,000 catheter days was 0.8. The risk for hemorrhagic and infectious complications when using the CMS for ICP monitoring is low. The depth of insertion varies considerably and should be taken into account if patients are treated with head elevation, since the pressure is measured at the tip of the sensor. To meet the need for ICP monitoring, an intraparenchymal ICP monitoring device should be preferred to the use of an external ventricular drainage (EVD).
Signatures of a macroscopic switching transition for a dynamic microtubule

NASA Astrophysics Data System (ADS)

Aparna, J. S.; Padinhateeri, Ranjith; Das, Dibyendu

2017-04-01

Characterising complex kinetics of non-equilibrium self-assembly of bio-filaments is of general interest. Dynamic instability in microtubules, consisting of successive catastrophes and rescues, is observed to occur as a result of the non-equilibrium conversion of GTP-tubulin to GDP-tubulin. We study this phenomenon using a model for microtubule kinetics with GTP/GDP state-dependent polymerisation, depolymerisation and hydrolysis of subunits. Our results reveal a sharp switch-like transition in the mean velocity of the filaments, from a growth phase to a shrinkage phase, with an associated co-existence of the two phases. This transition is reminiscent of the discontinuous phase transition across the liquid-gas boundary. We probe the extent of discontinuity in the transition quantitatively using characteristic signatures such as bimodality in velocity distribution, variance and Binder cumulant, and also hysteresis behaviour of the system. We further investigate ageing behaviour in catastrophes of the filament, and find that the multi-step nature of catastrophes is intensified in the vicinity of the switching transition. This assumes importance in the context of Microtubule Associated Proteins which have the potential of altering kinetic parameter values.
Method of low tantalum amounts determination in niobium and its compounds by ICP-OES technique.

PubMed

Smolik, Marek; Turkowska, Magdalena

2013-10-15

A method of determination of low amounts of tantalum in niobium and niobium compounds without its prior separation by means of inductively coupled plasma optical emission spectrometry (ICP-OES) has been worked out. The method involves dissolution of the analyzed samples of niobium as well as its various compounds (oxides, fluorides, chlorides, niobates(V)) in fluoride environments, precipitation of sparingly soluble niobic(tantalic) acid (Nb2O5(Ta2O5) · xH2O), converting them into soluble complex compounds by means of oxalic acid with addition of hydrogen peroxide and finally analyzing directly obtained solutions by ICP-OES. This method permits determination of Ta in niobium at the level of 10(-3)% with relatively good precision (≤ 8% RSD) and accuracy (recovery factor: 0.9-1.1). Relative differences in the results obtained by two independent methods (ICP-OES and ICP-MS) do not exceed 14%, and other elements present in niobium compounds (Ti, W, Zr, Hf, V, Mo, Fe, Cr) at the level of 10(-2)% do not affect determination. © 2013 Elsevier B.V. All rights reserved.
Decommissioning, Dismantling and Disarming: a Unique Information Showroom Inside the G2 Reactor at Marcoule Centre (France) - 12068

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volant, Emmanuelle; Garnier, Cedric

2012-07-01

The paper aims at presenting the new information showroom called 'Escom G2' (for 'Espace Communication') inaugurated by the French Atomic Energy and Alternative Energies Commission (CEA) in spring 2011. This showroom is settled directly inside the main building of the G2 nuclear reactor: a facility formerly dedicated to weapon-grade plutonium production since the late 1950's at the Marcoule nuclear centre, in south of France. After its shutdown, and reprocessing of the last spent fuels, a first dismantling step was successfully completed from 1986 to 1996. Unique in France and in Europe, Escom G2 is focused on France dismantling expertise andmore » its action for disarmament. This showroom comprises of a 300-square meters permanent exhibition, organized around four themes: France strategy for disarmament, decommissioning and dismantling technical aspects, uranium and plutonium production cycles. Each of these topics is illustrated with posters, photos, models and technical pieces from the dismantled plants. It is now used to present France's action in disarmament to highly ranked audiences such as: state representatives, diplomats, journalists... The paper explains the background story of this original project. As a matter of fact, in 1996 France was the first nuclear state to decide to shut down and dismantle its fissile material production facilities for nuclear weapons. First, the paper presents the history of the G2 reactor in the early ages of Marcoule site, its operating highlights as well as its main dismantling operations, are presented. In Marcoule, where the three industrial-scale reactors G1, G2 and G3 used to be operated for plutonium production (to be then reprocessed in the nearby UP1 plant), the initial dismantling phase has now been completed (in 1980's for G1 and in 1996 for G2 and G3). The second phase, aimed at completely dismantling these three reactors, will restart in 2020, and is directly linked to the opening of a future national storage
Accumulation of Cytoplasmic Dynein and Dynactin at Microtubule Plus Ends in Aspergillus nidulans Is Kinesin DependentV⃞

PubMed Central

Zhang, Jun; Li, Shihe; Fischer, Reinhard; Xiang, Xin

2003-01-01

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end–directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF. PMID:12686603

[Pollution Characteristics and Ecological Risk of PBDEs in Water and Sediment from an Electronic Waste Dismantling Area in Taizhou].

PubMed

Chen, Xiang-ping; Peng, Bao-qi; Lü, Su-ping; Chen, Qiang; Zhang, Yong; Huang, Chang-jiang; Dong, Qiao-xiang

2016-05-15

An e-waste dismantling industrial park of Taizhou was selected as the sampling center, within a radius of 16 km, and a total of 30 sampling sites were designed in three circles as follows: C (3 km), S (5-10 km) and R (10-16 km). Pollution characteristics and ecological risk of polybrominated diphenyl ethers (PBDEs) in water and sediments were investigated. The concentrations of PBDEs in water ranged from 9.4 to 57.2 ng · L⁻¹, with a mean value of 25.9 ng · L⁻¹; and 3.7 to 38,775 ng · g⁻¹, with an average of 2 779 ng · g⁻¹ in sediments. BDE-209 was the predominant congener. The spatial distribution patterns of PBDE levels in water and sediment were both in the following order: C > S > R. Furthermore, the concentrations of PBDEs in sediments showed significant negative correlation against the distance from the industrial park (P < 0.01). Compared with other regions around the world, the PBDEs contamination was more serious in the area, which indicated that e-waste dismantling activity was one of the significant sources for PBDEs pollution. It was estimated that a total of 30. 7 t PBDEs (including 28. 9 t BDE- 209) was discharged into surrounding environment as a result of dismantling industrial activities in last 40 years. A preliminary ecological risk assessment for PBDEs in water and sediments was conducted by hazard quotient method. The results demonstrated that the Penta-BDEs in the center of e-waste dismantling area ( a radius of 1.5 km) was at particularly high risk level and could cause serious influence on the ecological safety and human health.
Advances in the measurement of sulfur isotopes by multi-collector ICP-MS (MC-ICP- MS)

NASA Astrophysics Data System (ADS)

Ridley, W. I.; Wilson, S. A.; Anthony, M. W.

2006-12-01

The demonstrated capability to measure 34S/32S by MC-ICP-MS with a precision (2ó) of ~0.2 per mil has many potential applications in geochemistry. However, a number of obstacles limit this potential. First, to achieve the precision indicated above requires sufficient mass resolution to separate isobaric interferences of 16O2 and 17O2 on 32S and 34S, respectively. These requirements for high resolution mean overall instrument sensitivity is reduced. Second, current methods preclude analysis of samples with complex matrices, a common characteristic of sulfur-bearing geologic materials. Here, we describe and discuss a method that provides both efficient removal of matrix constituents, and provides pre-concentration of S, thus overcoming these obstacles. The method involves the separation of sulfur from matrix constituents by high pressure (1000 psi) ion chromatography (HPIC), followed by isotope measurement using MC-ICP-MS. This combination allows for analysis of liquid samples with a wide range of S concentrations. A powerful advantage of this technique is the efficient separation of many sulfur species from matrix cations and anions (for instance in a seawater or acid mine drainage matrix), as well as the separation of sulfur species, e.g., sulfate, sulfite, thiosulfate, thiocynate, from each other for isotope analysis. The automated HPIC system uses a carbonate-bicarbonate eluent with eluent suppression, and has sufficient baseline separation to collect the various sulfur species as pure fractions. The individual fractions are collected over a specific time interval based upon a pre-determined elution profile and peak retention times. The addition of a second ion exchange column into the system allows pre-concentration of sulfur species by 2-3 orders of magnitude for samples that otherwise would have sulfur concentrations too low to provide precise isotopic ratios. The S isotope ratios are measured by MC-ICP-MS using a desolvating sample introduction system, a
Apoptotic cells subjected to cold/warming exposure disorganize apoptotic microtubule network and undergo secondary necrosis.

PubMed

Oropesa-Ávila, Manuel; Fernández-Vega, Alejandro; de la Mata, Mario; Garrido-Maraver, Juan; Cotán, David; Paz, Marina Villanueva; Pavón, Ana Delgado; Cordero, Mario D; Alcocer-Gómez, Elizabet; de Lavera, Isabel; Lema, Rafael; Zaderenko, Ana Paula; Sánchez-Alcázar, José A

2014-09-01

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath the plasma membrane which plays a critical role in preserving cell morphology and plasma membrane integrity. The aim of this study was to examine the effect of cold/warming exposure on apoptotic microtubules and plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptotic H460 cells that cold/warming exposure disorganized apoptotic microtubules and allowed the access of active caspases to the cellular cortex and the cleavage of essential proteins in the preservation of plasma membrane permeability. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase and calcium ATPase pump (PMCA-4) involved in cell calcium extrusion resulted in increased plasma permeability and calcium overload leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the addition of the pan-caspase inhibitor z-VAD during cold/warming exposure that induces AMN depolymerization avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Likewise, apoptotic microtubules stabilization by taxol during cold/warming exposure also prevented cellular cortex and plasma membrane protein cleavage and secondary necrosis. Furthermore, microtubules stabilization or caspase inhibition during cold/warming exposure was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that cold/warming exposure of apoptotic cells induces secondary necrosis which can be prevented by both, microtubule stabilization or caspase inhibition.
Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis1[OPEN

PubMed Central

Sawchuk, Megan G.; Scarpella, Enrico

2018-01-01

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis (Arabidopsis thaliana) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls. PMID:29192026
Analysis of I-Br-Cl in single fluid inclusions by LA-ICP-MS

NASA Astrophysics Data System (ADS)

Giehl, C.; Fusswinkel, T.; Beermann, O.; Garbe-Schönberg, D.; Scholten, L.; Wagner, T.

2017-12-01

Halogens are excellent tracers of hydrothermal fluid sources and in-situ LA-ICP-MS analysis of Cl and Br in single fluid inclusions has provided fundamentally new insight into hydrothermal fluid flow and ore formation. There is mounting evidence that enrichment and depletion of Br relative to Cl may be caused by a number of processes beyond seawater evaporation and halite dissolution which cannot be discriminated on the basis of Br/Cl ratios alone. Expanding the analytical capabilities of fluid inclusion LA-ICP-MS analysis to include iodine would allow to discern between selective and coupled enrichment processes of Cl, Br and I, even in geologically complex samples that are inaccessible to bulk extraction techniques. We present iodine concentration data determined by LA-ICP-MS analysis of synthetic fluid inclusions, using the Sca17 scapolite reference material for external standardization (Seo et al., 2011). Iodine concentrations in Sca17 were determined using the Durango apatite standard. Four starting solutions containing I (0.3, 1.5, 27, 78 µg/g), Br (941, 1403, 2868, 4275 µg/g), Na (30.7, 94.7 mg/g), and Cl (50, 137 mg/g) (analyzed by ICP-OES and ICP-MS at CAU Kiel) were prepared by dissolving reagent grade chemical powders in ultra-pure water. Spherical inclusions (up to 40 µm) were synthesized from the starting solutions in pre-cracked, HF-treated synthetic quartz crystals which were placed in gold capsules and equilibrated at 600°C, 100/200 MPa in cold seal pressure vessels. Fluid inclusion LA-ICP-MS analysis (University of Helsinki) yielded average I concentrations in excellent agreement with the starting solutions (27.3 µg/g ± 14 %RSD for the 27 µg/g solution and 77.6 µg/g ± 8.3 %RSD for the 78 µg/g solution). Average Br and I concentrations deviate less than 10 % from solution concentration values. For the low I concentration solutions, the synthetic inclusions were too small to detect I. Thus, given suitable standard materials and sufficient
Microtubule organization in three-dimensional confined geometries: evaluating the role of elasticity through a combined in vitro and modeling approach.

PubMed

Cosentino Lagomarsino, Marco; Tanase, Catalin; Vos, Jan W; Emons, Anne Mie C; Mulder, Bela M; Dogterom, Marileen

2007-02-01

Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a confining box-like geometry, in the absence of other (active) microtubule organizing processes. This is inspired, for example, by the cortical microtubule array of elongating plant cells, where microtubules are typically organized in an aligned array transverse to the cell elongation axis. The method we adopt is a combination of analytical calculations, in which the polymers are modeled as inextensible filaments with bending elasticity confined to a two-dimensional surface that defines the limits of a three-dimensional space, and in vitro experiments, in which microtubules are polymerized from nucleation seeds in microfabricated chambers. We show that these features are sufficient to organize the polymers in aligned, coiling configurations as for example observed in plant cells. Though elasticity can account for the regularity of these arrays, it cannot account for a transverse orientation of microtubules to the cell's long axis. We therefore conclude that an additional active, force-generating process is necessary to create a coiling configuration perpendicular to the long axis of the cell.
Two-dimensional on-line detection of brominated and iodinated volatile organic compounds by ECD and ICP-MS after GC separation.

PubMed

Schwarz, A; Heumann, K G

2002-09-01

Inductively coupled plasma-mass spectrometry (ICP-MS) was coupled to a gas chromatographic (GC) system with electron capture detector (ECD), which enables relatively easy characterization and quantification of brominated and iodinated (halogenated) volatile organic compounds (HVOCs) in aquatic and air samples. The GC-ECD system is connected in series with an ICP-MS by a directly heated transfer line and an outlet port-hole for elimination of the ECD make-up gas during ignition of the plasma. The hyphenated GC-ECD/ICP-MS system provides high selectivity and sensitivity for monitoring individual HVOCs under fast chromatographic conditions. The ECD is most sensitive for the detection of chlorinated and brominated but the ICP-MS for iodinated compounds. The greatest advantage of the use of an ICP-MS is its element-specific detection, which allows clear identification of compounds in most cases. The absolute detection limits for ICP-MS are 0.5 pg for iodinated, 10 pg for brominated, and 50 pg for chlorinated HVOCs with the additional advantage that calibration is almost independent on different compounds of the same halogen. In contrast to that detection limits for ECD vary for the different halogenated compounds and lie in the range of 0.03-11 pg. The two-dimensional GC-ECD/ICP-MS instrumentation is compared with electron impact mass spectrometry (EI-MS) and microwave induced plasma atomic emission detection (MIP-AED). Even if EI-MS has additional power in identifying unknown peaks by its scan mode, the detection limits are much higher compared with GC-ECD/ICP-MS, whereas the selective ion monitoring mode (SIM) reaches similar detection limits. The MIP-AED detection limits are at the same level as EI-MS in the scan mode.
Effects of aging in catastrophe on the steady state and dynamics of a microtubule population

NASA Astrophysics Data System (ADS)

Jemseena, V.; Gopalakrishnan, Manoj

2015-05-01

Several independent observations have suggested that the catastrophe transition in microtubules is not a first-order process, as is usually assumed. Recent in vitro observations by Gardner et al. [M. K. Gardner et al., Cell 147, 1092 (2011), 10.1016/j.cell.2011.10.037] showed that microtubule catastrophe takes place via multiple steps and the frequency increases with the age of the filament. Here we investigate, via numerical simulations and mathematical calculations, some of the consequences of the age dependence of catastrophe on the dynamics of microtubules as a function of the aging rate, for two different models of aging: exponential growth, but saturating asymptotically, and purely linear growth. The boundary demarcating the steady-state and non-steady-state regimes in the dynamics is derived analytically in both cases. Numerical simulations, supported by analytical calculations in the linear model, show that aging leads to nonexponential length distributions in steady state. More importantly, oscillations ensue in microtubule length and velocity. The regularity of oscillations, as characterized by the negative dip in the autocorrelation function, is reduced by increasing the frequency of rescue events. Our study shows that the age dependence of catastrophe could function as an intrinsic mechanism to generate oscillatory dynamics in a microtubule population, distinct from hitherto identified ones.
Katanin spiral and ring structures shed light on power stroke for microtubule severing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zehr, Elena; Szyk, Agnieszka; Piszczek, Grzegorz

Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases critical for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of 3D structures, their mechanism has remained poorly understood. We report the first X-ray structure of the monomeric AAA katanin module and cryo-EM reconstructions of the hexamer in two conformations. These reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to severing enzymes and critical for their function. Our reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy ofmore » a gating protomer that tenses or relaxes inter-protomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.« less
Assessing the Contributions of Motor Enzymes and Microtubule Dynamics to Mitotic Chromosome Motions.

PubMed

McIntosh, J Richard

2017-10-06

During my graduate work with Keith Porter, I became fascinated by the mitotic spindle, an interest that has motivated much of my scientific work ever since. I began spindle studies by using electron microscopes, instruments that have made significant contributions to our understanding of spindle organization. Such instruments have helped to elucidate the distributions of spindle microtubules, the interactions among them, their molecular polarity, and their associations with both kinetochores and spindle poles. Our lab has also investigated some processes of spindle physiology: microtubule dynamics, the actions of microtubule-associated proteins (including motor enzymes), the character of forces generated by specific spindle components, and factors that control mitotic progression. Here, I give a personal perspective on some of this intellectual history and on what recent discoveries imply about the mechanisms of chromosome motion.
Content of nutritional elements in sudangrass and ryegrass determined by ICP-AES.

PubMed

Li, Wen-Xi; Lu, Jian-Wei; Seneweera, Saman P; Wu, Ji; Chen, Fang; Lu, Jun-Ming; Li, Xiao-Kun

2011-09-01

The sudangrass (Sorghum sudanense) and ryegrass (Lolium multi florum L.) rotation is a new type of cropping system, which has developed rapidly in recent years in the south of China. The contents of nutritional elements for forage grass in the sudangrass and ryegrass rotation system were determined by ICP-AES. The results showed that there were abundant and essential nutritional elements for animals in sudangrass and ryegrass. The contents of P, K, Ca, Mg, S, Fe, B, Cu, Zn and Mn for sudangrass were 0.20% -0.29%, 1.94%-2.57%, 0.62%-0.97%, 0.39%-0.69%, 0.12%-0.18%, 108.35-180.12, 3.04-5.96, 6.17-10.02, 20.37-31.36 and 46.80-101.29 mg x kg(-1), respectively. The contents of P, K, Ca, Mg, S, Fe, B, Cu, Zn, Mn for ryegrass were 0.39%-0.70%, 3.77%-5.07%, 0.61%-0.84%, 0.28% -0.47%, 0.32%-0.41%, 291.65- 632.20, 2.13-3.23, 13.29-15.19, 30.73-42.98 and 92.08-156.04 mg x kg(-1), respectively, and there were differences between various periods in nutritional elements in the two forage grasses. The application of ICP-AES could reflect fast and efficiently the content of nutritional elements for forage grass as animals feed.
Separation techniques for the clean-up of radioactive mixed waste for ICP-AES/ICP-MS analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swafford, A.M.; Keller, J.M.

1993-03-17

Two separation techniques were investigated for the clean-up of typical radioactive mixed waste samples requiring elemental analysis by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) or Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These measurements frequently involve regulatory or compliance criteria which include the determination of elements on the EPA Target Analyte List (TAL). These samples usually consist of both an aqueous phase and a solid phase which is mostly an inorganic sludge. Frequently, samples taken from the waste tanks contain high levels of uranium and thorium which can cause spectral interferences in ICP-AES or ICP-MS analysis. The removal of these interferences ismore » necessary to determine the presence of the EPA TAL elements in the sample. Two clean-up methods were studied on simulated aqueous waste samples containing the EPA TAL elements. The first method studied was a classical procedure based upon liquid-liquid extraction using tri-n- octylphosphine oxide (TOPO) dissolved in cyclohexane. The second method investigated was based on more recently developed techniques using extraction chromatography; specifically the use of a commercially available Eichrom TRU[center dot]Spec[trademark] column. Literature on these two methods indicates the efficient removal of uranium and thorium from properly prepared samples and provides considerable qualitative information on the extraction behavior of many other elements. However, there is a lack of quantitative data on the extraction behavior of elements on the EPA Target Analyte List. Experimental studies on these two methods consisted of determining whether any of the analytes were extracted by these methods and the recoveries obtained. Both methods produced similar results; the EPA target analytes were only slightly or not extracted. Advantages and disadvantages of each method were evaluated and found to be comparable.« less
Vesicle deformation by microtubules: A phase diagram

NASA Astrophysics Data System (ADS)

Emsellem, Virginie; Cardoso, Olivier; Tabeling, Patrick

1998-10-01

The experimental investigation of vesicles deformed by the growth of encapsulated microtubules shows that the axisymmetric morphologies can be classified into ovals, lemons, φ, cherries, dumbbells, and pearls. A geometrical phase diagram is established. Numerical minimization of the elastic energy of the membrane reproduces satisfactorily well the observed morphologies and the corresponding phase diagram.
Reconstitution of dynein transport to the microtubule plus end by kinesin

PubMed Central

Roberts, Anthony J; Goodman, Brian S; Reck-Peterson, Samara L

2014-01-01

Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track. DOI: http://dx.doi.org/10.7554/eLife.02641.001 PMID:24916158
Hydropathic analysis and biological evaluation of stilbene derivatives as colchicine site microtubule inhibitors with anti-leukemic activity

PubMed Central

TRIPATHI, ASHUTOSH; DURRANT, DAVID; LEE, RAY M.; BARUCHELLO, RICCARDO; ROMAGNOLI, ROMEO; SIMONI, DANIELE; KELLOGG, GLEN E.

2009-01-01

The crucial role of the microtubule in the cell division has identified tubulin as a target for the development of therapeutics for cancer; in particular tubulin is a target for antineoplastic agents that act by interfering with the dynamic stability of microtubules. A molecular modeling study was carried out to accurately represent the complex structure and the binding mode of a new class of stilbene-based tubulin inhibitors that bind at the αβ-tubulin colchicine site. Computational docking along with HINT score analysis fitted these inhibitors into the colchicine site and revealed detailed structure-activity information useful for inhibitor design. Quantitative analysis of the results was in good agreement with the in vitro antiproliferative activity of these derivatives (ranging from 3 nM to 100 μM) such that calculated and measured free energies of binding correlate with an r2 of 0.89 (standard error ± 0.85 kcal mol−1). This correlation suggests that the activity of unknown compounds may be predicted. PMID:19912057
MACF1 regulates the migration of pyramidal neurons via microtubule dynamics and GSK-3 signaling

PubMed Central

Ka, Minhan; Jung, Eui-Man; Mueller, Ulrich; Kim, Woo-Yang

2014-01-01

Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons. PMID:25224226
Shot and Patronin polarise microtubules to direct membrane traffic and biogenesis of microvilli in epithelia.

PubMed

Khanal, Ichha; Elbediwy, Ahmed; Diaz de la Loza, Maria Del Carmen; Fletcher, Georgina C; Thompson, Barry J

2016-07-01

In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. Here, we examine the hierarchy of events during cytoskeletal polarisation in Drosophila melanogaster epithelia. Core apical-basal polarity determinants polarise the spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1, CAMSAP2 and CAMSAP3 in humans) and Shortstop [Shot; MACF1 and BPAG1 (also known as DST) in humans] to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV (also known as Didum in Drosophila) actin motor to deliver the key microvillar determinant Cadherin 99C to the apical membrane to organise the biogenesis of actin microvilli. © 2016. Published by The Company of Biologists Ltd.
MACF1 regulates the migration of pyramidal neurons via microtubule dynamics and GSK-3 signaling.

PubMed

Ka, Minhan; Jung, Eui-Man; Mueller, Ulrich; Kim, Woo-Yang

2014-11-01

Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons. Copyright © 2014 Elsevier Inc. All rights reserved.
Characterization of Diamond-like Carbon (DLC) films deposited by RF ICP PECVD method

NASA Astrophysics Data System (ADS)

Oleszkiewicz, Waldemar; Kijaszek, Wojciech; Gryglewicz, Jacek; Zakrzewski, Adrian; Gajewski, Krzysztof; Kopiec, Daniel; Kamyczek, Paulina; Popko, Ewa; Tłaczała, Marek

2013-07-01

The work presents the results of a research carried out with Plasmalab Plus 100 system, manufactured by Oxford Instruments Company. The system was configured for deposition of diamond-like carbon films by ICP PECVD method. The deposition processes were carried out in CH4 or CH4/H2 atmosphere and the state of the plasma was investigated by the OES method. The RF plasma was capacitively coupled by 13.56 MHz generator with supporting ICP generator (13.56 Mhz). The deposition processes were conducted in constant value of RF generator's power and resultant value of the DC Bias. The power values of RF generator was set at 70 W and the power values of ICP generator was set at 300 W. In this work we focus on the influence of DLC film's thickness on optical, electrical and structural properties of the deposited DLC films. The quality of deposited DLC layers was examined by the Raman spectroscopy, AFM microscopy and spectroscopic ellipsometry. In the investigated DLC films the calculated sp3 content was ranging from 60 % to 70 %. The films were characterized by the refractive index ranging from 2.03 to 2.1 and extinction coefficient ranging from 0.09 to 0.12.
Using ICP-OES and SEM-EDX in biosorption studies

PubMed Central

Chojnacka, Katarzyna; Marycz, Krzysztof

2010-01-01

We have compared the analytical results obtained by inductively coupled plasma optical emission spectroscopy (ICP-OES) and by scanning electron microscopy with an energy dispersive X-ray analytical system (SEM-EDX) in order to explore the mechanism of metal ions biosorption by biomass using two independent methods. The marine macroalga Enteromorpha sp. was enriched with Cu(II), Mn(II), Zn(II), and Co(II) ions via biosorption, and the biosorption capacity of alga determined from the solution and biomass composition before and after biosorption process was compared. The first technique was used to analyze the composition of the natural and metal-loaded biomass, and additionally the composition of the solution before and after biosorption. The second technique was used to obtain a picture of the surface of natural and metal ion-loaded macroalgae, to map the elements on the cell wall of dry biomass, and to determine their concentration before and after biosorption. ICP-OES showed a better precision and lower detection limit than EDX, but SEM-EDX gave more information regarding the sample composition of Enteromorpha sp. Both techniques confirmed that biosorption is a surface phenomenon, in which alkali and alkaline earth metal ions were exchanged by metal ions from aqueous solution. Figure The advantages and disadvantages of ICP-OES and SEM-EDX techniques Electronic supplementary material The online version of this article (doi:10.1007/s00604-010-0468-0) contains supplementary material, which is available to authorized users. PMID:21423317

Some links on this page may take you to non-federal websites. Their policies may differ from this site.