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Sample records for cortical microtubules stomata

  1. Microtubule-associated protein AtMPB2C plays a role in organization of cortical microtubules, stomata patterning, and tobamovirus infectivity.

    PubMed

    Ruggenthaler, Pia; Fichtenbauer, Daniela; Krasensky, Julia; Jonak, Claudia; Waigmann, Elisabeth

    2009-03-01

    AtMPB2C is the Arabidopsis (Arabidopsis thaliana) homolog of MPB2C, a microtubule-associated host factor of tobacco mosaic virus movement protein that was been previously identified in Nicotiana tabacum. To analyze the endogenous function of AtMPB2C and its role in viral infections, transgenic Arabidopsis plant lines stably overexpressing green fluorescent protein (GFP)-AtMPB2C were established. The GFP-AtMPB2C fusion protein was detectable in various cell types and organs and localized at microtubules in a punctuate pattern or in filaments. To determine whether overexpression impacted on the cortical microtubular cytoskeleton, GFP-AtMPB2C-overexpressing plants were compared to known microtubular marker lines. In rapidly elongated cell types such as vein cells and root cells, GFP-AtMPB2C overexpression caused highly unordered assemblies of cortical microtubules, a disturbed, snake-like microtubular shape, and star-like crossing points of microtubules. Phenotypically, GFP-AtMPB2C transgenic plants showed retarded growth but were viable and fertile. Seedlings of GFP-AtMPB2C transgenic plants were characterized by clockwise twisted leaves, clustered stomata, and enhanced drought tolerance. GFP-AtMPB2C-overexpressing plants showed increased resistance against oilseed rape mosaic virus, a close relative of tobacco mosaic virus, but not against cucumber mosaic virus when compared to Arabidopsis wild-type plants. These results suggest that AtMPB2C is involved in the alignment of cortical microtubules, the patterning of stomata, and restricting tobamoviral infections.

  2. Microtubule-Associated Protein AtMPB2C Plays a Role in Organization of Cortical Microtubules, Stomata Patterning, and Tobamovirus Infectivity1

    PubMed Central

    Ruggenthaler, Pia; Fichtenbauer, Daniela; Krasensky, Julia; Jonak, Claudia; Waigmann, Elisabeth

    2009-01-01

    AtMPB2C is the Arabidopsis (Arabidopsis thaliana) homolog of MPB2C, a microtubule-associated host factor of tobacco mosaic virus movement protein that was been previously identified in Nicotiana tabacum. To analyze the endogenous function of AtMPB2C and its role in viral infections, transgenic Arabidopsis plant lines stably overexpressing green fluorescent protein (GFP)-AtMPB2C were established. The GFP-AtMPB2C fusion protein was detectable in various cell types and organs and localized at microtubules in a punctuate pattern or in filaments. To determine whether overexpression impacted on the cortical microtubular cytoskeleton, GFP-AtMPB2C-overexpressing plants were compared to known microtubular marker lines. In rapidly elongated cell types such as vein cells and root cells, GFP-AtMPB2C overexpression caused highly unordered assemblies of cortical microtubules, a disturbed, snake-like microtubular shape, and star-like crossing points of microtubules. Phenotypically, GFP-AtMPB2C transgenic plants showed retarded growth but were viable and fertile. Seedlings of GFP-AtMPB2C transgenic plants were characterized by clockwise twisted leaves, clustered stomata, and enhanced drought tolerance. GFP-AtMPB2C-overexpressing plants showed increased resistance against oilseed rape mosaic virus, a close relative of tobacco mosaic virus, but not against cucumber mosaic virus when compared to Arabidopsis wild-type plants. These results suggest that AtMPB2C is involved in the alignment of cortical microtubules, the patterning of stomata, and restricting tobamoviral infections. PMID:19074626

  3. Cortical microtubule rearrangements and cell wall patterning

    PubMed Central

    Oda, Yoshihisa

    2015-01-01

    Plant cortical microtubules, which form a highly ordered array beneath the plasma membrane, play essential roles in determining cell shape and function by directing the arrangement of cellulosic and non-cellulosic compounds on the cell surface. Interphase transverse arrays of cortical microtubules self-organize through their dynamic instability and inter-microtubule interactions, and by branch-form microtubule nucleation and severing. Recent studies revealed that distinct spatial signals including ROP GTPase, cellular geometry, and mechanical stress regulate the behavior of cortical microtubules at the subcellular and supercellular levels, giving rise to dramatic rearrangements in the cortical microtubule array in response to internal and external cues. Increasing evidence indicates that negative regulators of microtubules also contribute to the rearrangement of the cortical microtubule array. In this review, I summarize recent insights into how the rearrangement of the cortical microtubule array leads to proper, flexible cell wall patterning. PMID:25904930

  4. Arabidopsis MICROTUBULE-ASSOCIATED PROTEIN18 functions in directional cell growth by destabilizing cortical microtubules.

    PubMed

    Wang, Xia; Zhu, Lei; Liu, Baoquan; Wang, Che; Jin, Lifeng; Zhao, Qian; Yuan, Ming

    2007-03-01

    Microtubule-associated proteins (MAPs) play important roles in the regulation of microtubule function in cells. We describe Arabidopsis thaliana MAP18, which binds to microtubules and inhibits tubulin polymerization in vitro and colocalizes along cortical microtubules as patches of dot-like structures. MAP18 is expressed mostly in the expanding cells. Cells overexpressing MAP18 in Arabidopsis exhibit various growth phenotypes with loss of polarity. Cortical microtubule arrays were significantly altered in cells either overexpressing MAP18 or where it had been downregulated by RNA interference (RNAi). The cortical microtubules were more sensitive to treatment with microtubule-disrupting drugs when MAP18 was overexpressed, but more resistant when MAP18 was eliminated in cells expressing MAP18 RNAi. Our study demonstrated that MAP18 may play a role in regulating directional cell growth and cortical microtubule organization by destabilizing microtubules.

  5. Cortical microtubule contacts position the spindle in C. elegans embryos.

    PubMed

    Kozlowski, Cleopatra; Srayko, Martin; Nedelec, Francois

    2007-05-04

    Interactions between microtubules and the cell cortex play a critical role in positioning organelles in a variety of biological contexts. Here we used Caenorhabditis elegans as a model system to study how cortex-microtubule interactions position the mitotic spindle in response to polarity cues. Imaging EBP-2::GFP and YFP::alpha-tubulin revealed that microtubules shrink soon after cortical contact, from which we propose that cortical adaptors mediate microtubule depolymerization energy into pulling forces. We also observe association of dynamic microtubules to form astral fibers that persist, despite the catastrophe events of individual microtubules. Computer simulations show that these effects, which are crucially determined by microtubule dynamics, can explain anaphase spindle oscillations and posterior displacement in 3D.

  6. Cortical microtubules in sweet clover columella cells developed in microgravity

    NASA Technical Reports Server (NTRS)

    Hilaire, E.; Paulsen, A. Q.; Brown, C. S.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Electron micrographs of columella cells from sweet clover seedlings grown and fixed in microgravity revealed longitudinal and cross sectioned cortical microtubules. This is the first report demonstrating the presence and stability of this network in plants in microgravity.

  7. 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.

  8. An improved quantitative analysis method for plant cortical microtubules.

    PubMed

    Lu, Yi; Huang, Chenyang; Wang, Jia; Shang, Peng

    2014-01-01

    The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD) algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1) image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM) algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies.

  9. A mechanism for reorientation of cortical microtubule arrays driven by microtubule severing.

    PubMed

    Lindeboom, Jelmer J; Nakamura, Masayoshi; Hibbel, Anneke; Shundyak, Kostya; Gutierrez, Ryan; Ketelaar, Tijs; Emons, Anne Mie C; Mulder, Bela M; Kirik, Viktor; Ehrhardt, David W

    2013-12-06

    Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array.

  10. Cortical microtubule arrays are initiated from a nonrandom prepattern driven by atypical microtubule initiation.

    PubMed

    Lindeboom, Jelmer J; Lioutas, Antonios; Deinum, Eva E; Tindemans, Simon H; Ehrhardt, David W; Emons, Anne Mie C; Vos, Jan W; Mulder, Bela M

    2013-03-01

    The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.

  11. Anillin promotes astral microtubule-directed cortical myosin polarization

    PubMed Central

    Tse, Yu Chung; Piekny, Alisa; Glotzer, Michael

    2011-01-01

    Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle–directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex. PMID:21737681

  12. Structure of cortical microtubule arrays in plant cells

    PubMed Central

    1978-01-01

    Serial sectioning was used to track the position and measure the lengths of cortical microtubules in glutaraldehyde-osmium tetroxide- fixed root tip cells. Microtubules lying against the longitudinal walls during interphase, those overlying developing xylem thickenings, and those in pre-prophase bands are oriented circumferentially but on average are only about one-eighth of the cell circumference in length, i.e., 2-4 micrometer. The arrays consist of overlapping component microtubules, interconnected by cross bridges where they are grouped and also connected to the plasma membrane. Microtubule lengths vary greatly in any given array, but the probability that any pass right around the cell is extremely low. The majority of the microtubule terminations lie in statistically random positions in the arrays, but nonrandomness in the form of groups of terminations and terminations in short lines parallel to the axis of cell elongation has been observed. Low temperature induces microtubule shortening and increases the frequency of C-shaped terminations over the 1.7% found under normal conditions; colchicine and high pressures produce abnormally large proportions of very short microtubules amongst those that survive the treatments. Deuterium oxide (D2O) treatment probably induces the formation of additional microtubules as distinct from increasing the length of those already present. The distribution of C-shaped terminations provides evidence for at least local polarity in the arrays. The validity of the findings is discussed, along with implications for the development, maintenance, and orientation of the arrays and their possible relationship to the orientation of cellulose deposition. PMID:350889

  13. Microtubule Initiation from the Nuclear Surface Controls Cortical Microtubule Growth Polarity and Orientation in Arabidopsis thaliana

    PubMed Central

    Ambrose, Chris; Wasteneys, Geoffrey O.

    2014-01-01

    The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth. PMID:25008974

  14. Microtubules are essential for guard-cell function in Vicia and Arabidopsis.

    PubMed

    Eisinger, William; Ehrhardt, David; Briggs, Winslow

    2012-05-01

    Radially arranged cortical microtubules are a prominent feature of guard cells. Guard cells expressing GFP-tubulin showed consistent changes in the appearance of microtubules when stomata opened or closed. Guard cells showed fewer microtubule structures as stomata closed, whether induced by transfer to darkness, ABA, hydrogen peroxide, or sodium hydrogen carbonate. Guard cells kept in the dark (closed stomata) showed increases in microtubule structures and stomatal aperture on light treatment. GFP-EB1, marking microtubule growing plus ends, showed no change in number of plus ends or velocity of assembly on stomatal closure. Since the number of growing plus ends and the rate of plus-end growth did not change when microtubule structure numbers declined, microtubule instability and/or rearrangement must be responsible for the apparent loss of microtubules. Guard cells with closed stomata showed more cytosolic GFP-fluorescence than those with open stomata as cortical microtubules became disassembled, although with a large net loss in total fluorescence. Microtubule-targeted drugs blocked guard-cell function in Vicia and Arabidopsis. Oryzalin disrupted guard-cell microtubules and prevented stomatal opening and taxol stabilized guard-cell microtubules and delayed stomatal closure. Gas exchange measurements indicated that the transgenes for fluorescent-labeled proteins did not disrupt normal stomatal function. These dynamic changes in guard-cell microtubules combined with our inhibitor studies provide evidence for an active role of microtubules in guard-cell function.

  15. Drosophila Katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

    PubMed Central

    Zhang, Dong; Grode, Kyle D.; Stewman, Shannon; Diaz, Daniel; Liebling, Emily; Rath, Uttama; Riera, Tania; Currie, Joshua; Buster, Daniel W.; Asenjo, Ana B.; Sosa, Hernando J.; Ross, Jennifer; Ma, Ao; Rogers, Stephen L.; Sharp, David J.

    2011-01-01

    Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila Katanin, Dm-Kat60, functions to generate a dynamic cortical-microtubule interface in interphase cells. In S2 cells, Dm-Kat60 concentrates at the interphase cell cortex where it suppresses the polymerization of microtubule plus-ends thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes to the leading edge migratory D17 cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes MTs from their ends. Based on these data, we propose that Dm-Kat60 removes tubulin from microtubule ends or lattice that contact specific cortical sites to preventing stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in MT behaviors involved in cell migration. PMID:21378981

  16. Organization of cortical microtubules in graviresponding maize roots

    NASA Technical Reports Server (NTRS)

    Blancaflor, E. B.; Hasenstein, K. H.

    1993-01-01

    Immunofluorescence labeling of cortical microtubules (MTs) was used to investigate the relationship between MT arrangement and changes in growth rate of the upper and lower sides of horizontally placed roots of maize (Zea mays L. cv. Merit). Cap cells and cells of the elongation zone of roots grown vertically in light or darkness showed MT arrangements that were transverse (perpendicular) to the growth direction. Microtubules of cells basal to the elongation zone typically showed oblique orientation. Two hours after horizontal reorientation, cap cells of gravicompetent, light-grown and curving roots contained MTs parallel to the gravity vector. The MT arrangement on the upper side of the elongation zone remained transverse but the MTs of the outer four to five layers of cortical cells along the lower side of the elongation zone showed reorientation parallel to the axis of the root. The MTs of the lower epidermis retained their transverse orientation. Dark-grown roots did not curve and did not show reorientation of MTs in cells of the root cap or elongation zone. The data indicate that MT depolymerization and reorientation is correlated with reduction in growth rate, and that MT reorientation is one of the steps of growth control of graviresponding roots.

  17. Calmodulin immunolocalization to cortical microtubules is calcium independent

    SciTech Connect

    Fisher, D.D.; Cyr, R.J.

    1992-01-01

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 [mu]M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  18. Calmodulin immunolocalization to cortical microtubules is calcium independent

    SciTech Connect

    Fisher, D.D.; Cyr, R.J.

    1992-12-31

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 {mu}M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 {mu}M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  19. Cortical microtubule reorganization in protoplasts isolated from Brassica napus hypocotyl is affected by gravity.

    PubMed

    Skagen, E B

    1998-07-01

    Researchers studied the effect of simulated weightlessness on the reorganization of cortical microtubules in Brassica napus hypocotyl protoplasts. The reorganization of cortical microtubules in protoplasts exposed to simulated weightlessness was examined using a fast rotating one-axis clinostat for 1-4 days and with a three dimensional clinostat and a free fall machine for 48 hours.

  20. Interplay between kinesin-1 and cortical dynein during axonal outgrowth and microtubule organization in Drosophila neurons

    PubMed Central

    del Castillo, Urko; Winding, Michael; Lu, Wen; Gelfand, Vladimir I

    2015-01-01

    In this study, we investigated how microtubule motors organize microtubules in Drosophila neurons. We showed that, during the initial stages of axon outgrowth, microtubules display mixed polarity and minus-end-out microtubules push the tip of the axon, consistent with kinesin-1 driving outgrowth by sliding antiparallel microtubules. At later stages, the microtubule orientation in the axon switches from mixed to uniform polarity with plus-end-out. Dynein knockdown prevents this rearrangement and results in microtubules of mixed orientation in axons and accumulation of microtubule minus-ends at axon tips. Microtubule reorganization requires recruitment of dynein to the actin cortex, as actin depolymerization phenocopies dynein depletion, and direct recruitment of dynein to the membrane bypasses the actin requirement. Our results show that cortical dynein slides ‘minus-end-out’ microtubules from the axon, generating uniform microtubule arrays. We speculate that differences in microtubule orientation between axons and dendrites could be dictated by differential activity of cortical dynein. DOI: http://dx.doi.org/10.7554/eLife.10140.001 PMID:26615019

  1. Survival of the Aligned: Ordering of the Plant Cortical Microtubule Array

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

    The cortical array is a structure consisting of highly aligned microtubules which plays a crucial role in the characteristic uniaxial expansion of all growing plant cells. Recent experiments have shown polymerization-driven collisions between the membrane-bound cortical microtubules, suggesting a possible mechanism for their alignment. We present both a coarse-grained theoretical model and stochastic particle-based simulations of this mechanism, and we compare the results from these complementary approaches. Our results indicate that collisions that induce depolymerization are sufficient to generate the alignment of microtubules in the cortical array.

  2. Cortical Microtubule Arrays Are Initiated from a Nonrandom Prepattern Driven by Atypical Microtubule Initiation1[W][OA

    PubMed Central

    Lindeboom, Jelmer J.; Lioutas, Antonios; Deinum, Eva E.; Tindemans, Simon H.; Ehrhardt, David W.; Emons, Anne Mie C.; Vos, Jan W.; Mulder, Bela M.

    2013-01-01

    The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation. PMID:23300168

  3. An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii

    PubMed Central

    Liu, Jun; He, Yudou; Benmerzouga, Imaan; Sullivan, William J.; Morrissette, Naomi S.; Murray, John M.; Hu, Ke

    2016-01-01

    Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy. PMID:26680740

  4. Astral Microtubule Pivoting Promotes Their Search for Cortical Anchor Sites during Mitosis in Budding Yeast

    PubMed Central

    Baumgärtner, Stephan; Tolić, Iva M.

    2014-01-01

    Positioning of the mitotic spindle is crucial for proper cell division. In the budding yeast Saccharomyces cerevisiae, two mechanisms contribute to spindle positioning. In the Kar9 pathway, astral microtubules emanating from the daughter-bound spindle pole body interact via the linker protein Kar9 with the myosin Myo2, which moves the microtubule along the actin cables towards the neck. In the dynein pathway, astral microtubules off-load dynein onto the cortical anchor protein Num1, which is followed by dynein pulling on the spindle. Yet, the mechanism by which microtubules target cortical anchor sites is unknown. Here we quantify the pivoting motion of astral microtubules around the spindle pole bodies, which occurs during spindle translocation towards the neck and through the neck. We show that this pivoting is largely driven by the Kar9 pathway. The microtubules emanating from the daughter-bound spindle pole body pivot faster than those at the mother-bound spindle pole body. The Kar9 pathway reduces the time needed for an astral microtubule inside the daughter cell to start pulling on the spindle. Thus, we propose a new role for microtubule pivoting: By pivoting around the spindle pole body, microtubules explore the space laterally, which helps them search for cortical anchor sites in the context of spindle positioning in budding yeast. PMID:24721997

  5. Dual Role for Microtubules in Regulating Cortical Contractility during Cytokinesis

    PubMed Central

    Murthy, Kausalya; Wadsworth, Patricia

    2008-01-01

    Microtubules stimulate contractile ring formation in the equatorial cortex and simultaneously suppress contractility in the polar cortex; how they accomplish these differing activities is incompletely understood. We measured the behavior of GFP-actin in mammalian cells treated with nocodazole under conditions that either completely eliminate microtubules or selectively disassemble astral microtubules. Selective disassembly of astral microtubules resulted functional contractile rings that were wider than controls and had altered dynamic activity, as measured by FRAP. Complete microtubule disassembly or selective loss of astral microtubules resulted in wave-like contractile behavior of actin in the non-equatorial cortex and mislocalization of myosin II and Rho. FRAP experiments showed that both contractility and actin polymerization contributed to the wave-like behavior of actin. Wave-like, contractile behavior in anaphase cells was Rho-dependent. We conclude that dynamic astral microtubules function to suppress Rho activation in the nonequatorial cortex, limiting the contractile activity of the polar cortex. PMID:18559890

  6. Dynamic regulation of cortical microtubule organization through prefoldin-DELLA interaction.

    PubMed

    Locascio, Antonella; Blázquez, Miguel A; Alabadí, David

    2013-05-06

    Plant morphogenesis relies on specific patterns of cell division and expansion. It is well established that cortical microtubules influence the direction of cell expansion, but less is known about the molecular mechanisms that regulate microtubule arrangement. Here we show that the phytohormones gibberellins (GAs) regulate microtubule orientation through physical interaction between the nuclear-localized DELLA proteins and the prefoldin complex, a cochaperone required for tubulin folding. In the presence of GA, DELLA proteins are degraded, and the prefoldin complex stays in the cytoplasm and is functional. In the absence of GA, the prefoldin complex is localized to the nucleus, which severely compromises α/β-tubulin heterodimer availability, affecting microtubule organization. The physiological relevance of this molecular mechanism was confirmed by the observation that the daily rhythm of plant growth was accompanied by coordinated oscillation of DELLA accumulation, prefoldin subcellular localization, and cortical microtubule reorientation.

  7. 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

  8. Strain rate does not affect cortical microtubule orientation in the isolated epidermis of sunflower hypocotyls.

    PubMed

    Burian, A; Hejnowicz, Z

    2010-05-01

    A hypothesis exists that external and internal factors affect the orientation of cortical microtubules in as much as these lead to changes in cell elongation rate. Factors that stimulate elongation are proposed to lead to transverse microtubule orientation, whereas factors that inhibit elongation lead to longitudinal orientation. The elongation rate is equal to the rate of longitudinal irreversible strain in cell walls. Incubated epidermis peeled from sunflower hypocotyls does not extend unless it is stretched by loading and the pH of the incubation medium is appropriately low. Thus, peels provide a convenient model to investigate the relationship between longitudinal strain rate and cortical microtubule orientation. In the present study, it was found that peeling affects microtubule orientation. Peels were incubated for several hours in Murashige & Skoog medium (both unbuffered and buffered) to attain a steady state of microtubule orientation before loading. The effects of loading and pH on strain rate and orientation of microtubules under the outer epidermal walls were examined in three portions of peels positioned with respect to the cotyledonary node. Appropriate loading caused longitudinal strain of peels at pH 4.5 but not at pH 6.5. However, no clear effect of strain rate on microtubule orientation in the peels was observed. Independent of applied load and pH of the incubation medium, the microtubule orientation remained unchanged, i.e. orientation was mainly oblique. Our results show that strain rate does not affect cortical microtubule orientation in isolated epidermis of the sunflower hypocotyl model system, although orientation could be changed by white light.

  9. Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

    PubMed Central

    Bouchet, Benjamin P; Gough, Rosemarie E; Ammon, York-Christoph; van de Willige, Dieudonnée; Post, Harm; Jacquemet, Guillaume; Altelaar, AF Maarten; Heck, Albert JR; Goult, Benjamin T; Akhmanova, Anna

    2016-01-01

    The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules. DOI: http://dx.doi.org/10.7554/eLife.18124.001 PMID:27410476

  10. Microtubule-targeting drugs rescue axonal swellings in cortical neurons from spastin knockout mice

    PubMed Central

    Fassier, Coralie; Tarrade, Anne; Peris, Leticia; Courageot, Sabrina; Mailly, Philippe; Dalard, Cécile; Delga, Stéphanie; Roblot, Natacha; Lefèvre, Julien; Job, Didier; Hazan, Jamilé; Curmi, Patrick A.; Melki, Judith

    2013-01-01

    SUMMARY Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP), a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease. PMID:22773755

  11. 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.

  12. 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

  13. Patterns of cortical microtubules formed in epidermis of Beta vulgaris L. roots under clinorotation

    NASA Astrophysics Data System (ADS)

    Shevchenko, G. V.

    1999-01-01

    Changes of cortical microtubules (MTs) from the normal transverse arrangement were observed in epidermal cells of Beta vulgaris roots under clinorotation. We hypothesize that the epidermis is sensitive to clinorotation and that the microtubular cytoskeleton plays a key role in the ensuing growth response.

  14. The dynamics of plus end polarization and microtubule assembly during Xenopus cortical rotation.

    PubMed

    Olson, David J; Oh, Denise; Houston, Douglas W

    2015-05-15

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. 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

  16. Cobtorin target analysis reveals that pectin functions in the deposition of cellulose microfibrils in parallel with cortical microtubules.

    PubMed

    Yoneda, Arata; Ito, Takuya; Higaki, Takumi; Kutsuna, Natsumaro; Saito, Tamio; Ishimizu, Takeshi; Osada, Hiroyuki; Hasezawa, Seiichiro; Matsui, Minami; Demura, Taku

    2010-11-01

    Cellulose and pectin are major components of primary cell walls in plants, and it is believed that their mechanical properties are important for cell morphogenesis. It has been hypothesized that cortical microtubules guide the movement of cellulose microfibril synthase in a direction parallel with the microtubules, but the mechanism by which this alignment occurs remains unclear. We have previously identified cobtorin as an inhibitor that perturbs the parallel relationship between cortical microtubules and nascent cellulose microfibrils. In this study, we searched for the protein target of cobtorin, and we found that overexpression of pectin methylesterase and polygalacturonase suppressed the cobtorin-induced cell-swelling phenotype. Furthermore, treatment with polygalacturonase restored the deposition of cellulose microfibrils in the direction parallel with cortical microtubules, and cobtorin perturbed the distribution of methylated pectin. These results suggest that control over the properties of pectin is important for the deposition of cellulose microfibrils and/or the maintenance of their orientation parallel with the cortical microtubules.

  17. [The role of cortical microtubules in moss protonemal cells during dehydration/rehydration cycle].

    PubMed

    Chen, Zhi-Ling; Ouyang, Hao-Miao; Liu, Xiang-Lin; Xia, Gui-Xian

    2003-05-01

    Plant cells response to water deficit through a variety of physiological processes. In this work, we studied the function of microtubule cytoskeleton during dehydration/rehydration cycle in moss (Atrichum undulatum) protonemal cells as a model system. The morphological and cytological change of protonemal cells during dehydration and rehydration cycle were first investigated. Under normal conditions, protonemal cells showed bright green colour and appeared wet and fresh. Numerous chloroplasts distributed regularly throughout the cytoplasm in each cell. After dehydration treatment, protonemal cells lost most of their chlorophylls and turned to look yellow and dry. In addition, dehydration caused plasmolysis in these cells. Upon rehydration, the cells could recover completely from the dehydrated state. These results indicated that moss had a remarkable intrinsic ability to survive from the extreme drought stress. Microtubule, an important component of cytoskeleton, is considered to play crucial roles in the responses to some environmental stresses such as cold and light. To see if it is also involved in the drought tolerance, dynamic organization of microtubules in protonemal cells of Atrichum undulatum subjected to drought and rehydration were examined by indirect immunofluorescence combined with confocal lasersharp scanning microscopy. The cortical microtubules were arranged into a fine structure with a predominant orientation parallel to the long axis of the cells in the control cells. After dehydration, the microtubule organization was remarkablly altered and the fine microtubule structure disappeared whereas some thicker cables formed. When the cells were grown under rehydration conditions, the fine microtubule arrays reappeared. These results provided a piece of evidence that microtubules play a role in the cellular responses to drought stress in moss. Furthermore, we analyzed the effects of the microtubule-disrupting agent colchicine on the morphology recovery

  18. Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs

    PubMed Central

    1991-01-01

    During the first cell cycle, the vegetal cortex of the fertilized frog egg is translocated over the cytoplasm. This process of cortical rotation creates regional cytoplasmic differences important in later development, and appears to involve an array of aligned microtubules that forms transiently beneath the vegetal cortex. We have investigated how these microtubules might be involved in generating movement by analyzing isolated cortices and sections of Xenopus laevis and Rana pipiens eggs. First, the polarity of the cortical microtubules was determined using the "hook" assay. Almost all microtubules had their plus ends pointing in the direction of cortical rotation. Secondly, the association of microtubules with other cytoplasmic elements was examined. Immunofluorescence revealed that cytokeratin filaments coalign with the microtubules. The timing of their appearance and their position on the cytoplasmic side of the microtubules suggested that they are not involved directly in generating movement. ER was visualized with the dye DiIC16(3) and by immunofluorescence with anti- BiP (Bole, D. G., L. M. Hendershot, and J. F. Kearney, 1986. J. Cell Biol. 102:1558-1566). One layer of ER was found closely underlying the plasma membrane at all times. An additional, deeper layer formed in association with the microtubules of the array. Antibodies to sea urchin kinesin (Ingold, A. L., S. A. Cohn, and J. M. Scholey. 1988. J. Cell Biol. 107:2657-2667) detected antigens associated with both the ER and microtubules. On immunoblots they recognized microtubule associated polypeptide(s) of approximately 115 kD from Xenopus eggs. These observations are consistent with a role for kinesin in creating movement between the microtubules and ER, which leads in turn to the cortical rotation. PMID:1714912

  19. Maternal Dead-End1 is required for vegetal cortical microtubule assembly during Xenopus axis specification

    PubMed Central

    Mei, Wenyan; Jin, Zhigang; Lai, Fangfang; Schwend, Tyler; Houston, Douglas W.; King, Mary Lou; Yang, Jing

    2013-01-01

    Vertebrate axis specification is an evolutionarily conserved developmental process that relies on asymmetric activation of Wnt signaling and subsequent organizer formation on the future dorsal side of the embryo. Although roles of Wnt signaling during organizer formation have been studied extensively, it is unclear how the Wnt pathway is asymmetrically activated. In Xenopus and zebrafish, the Wnt pathway is triggered by dorsal determinants, which are translocated from the vegetal pole to the future dorsal side of the embryo shortly after fertilization. The transport of dorsal determinants requires a unique microtubule network formed in the vegetal cortex shortly after fertilization. However, molecular mechanisms governing the formation of vegetal cortical microtubule arrays are not fully understood. Here we report that Dead-End 1 (Dnd1), an RNA-binding protein required for primordial germ cell development during later stages of embryogenesis, is essential for Xenopus axis specification. We show that knockdown of maternal Dnd1 specifically interferes with the formation of vegetal cortical microtubules. This, in turn, impairs translocation of dorsal determinants, the initiation of Wnt signaling, organizer formation, and ultimately results in ventralized embryos. Furthermore, we found that Dnd1 binds to a uridine-rich sequence in the 3′-UTR of trim36, a vegetally localized maternal RNA essential for vegetal cortical microtubule assembly. Dnd1 anchors trim36 to the vegetal cortex in the egg, promoting high concentrations of Trim36 protein there. Our work thus demonstrates a novel and surprising function for Dnd1 during early development and provides an important link between Dnd1, mRNA localization, the microtubule cytoskeleton and axis specification. PMID:23615278

  20. MDP25, a novel calcium regulatory protein, mediates hypocotyl cell elongation by destabilizing cortical microtubules in Arabidopsis.

    PubMed

    Li, Jiejie; Wang, Xianling; Qin, Tao; Zhang, Yan; Liu, Xiaomin; Sun, Jingbo; Zhou, Yuan; Zhu, Lei; Zhang, Ziding; Yuan, Ming; Mao, Tonglin

    2011-12-01

    The regulation of hypocotyl elongation is important for plant growth. Microtubules play a crucial role during hypocotyl cell elongation. However, the molecular mechanism underlying this process is not well understood. In this study, we describe a novel Arabidopsis thaliana microtubule-destabilizing protein 25 (MDP25) as a negative regulator of hypocotyl cell elongation. We found that MDP25 directly bound to and destabilized microtubules to enhance microtubule depolymerization in vitro. The seedlings of mdp25 mutant Arabidopsis lines had longer etiolated hypocotyls. In addition, MDP25 overexpression resulted in significant overall shortening of hypocotyl cells, which exhibited destabilized cortical microtubules and abnormal cortical microtubule orientation, suggesting that MDP25 plays a crucial role in the negative regulation of hypocotyl cell elongation. Although MDP25 localized to the plasma membrane under normal conditions, increased calcium levels in cells caused MDP25 to partially dissociate from the plasma membrane and move into the cytosol. Cellular MDP25 bound to and destabilized cortical microtubules, resulting in their reorientation, and subsequently inhibited hypocotyl cell elongation. Our results suggest that MDP25 exerts its function on cortical microtubules by responding to cytoplasmic calcium levels to mediate hypocotyl cell elongation.

  1. Intercourse between cell wall and cytoplasm exemplified by arabinogalactan proteins and cortical microtubules.

    PubMed

    Driouich, Azeddine; Baskin, Tobias I

    2008-12-01

    How does a plant cell sense and respond to the status of its cell wall? Intercourse between cell wall and cytoplasm has long been supposed to involve arabinogalactan proteins, in part because many of them are anchored to the plasma membrane. Disrupting arabinogalactan proteins has recently been shown to disrupt the array of cortical microtubules present just inside the plasma membrane, implying that microtubules and arabinogalactan proteins interact. In this article, we assess possibilities for how this interaction might be mediated. First, we consider microdomains in the plasma membrane (lipid rafts), which have been alleged to link internal and external regions of the plasma membrane; however, the characteristics and even the existence of these domains remains controversial. Next, we point out that disrupting the synthesis of cellulose also can disrupt microtubules and consider whether arabinogalactan proteins are part of a network linking microtubules and nascent microfibrils. Finally, we outline several signaling cascades that could transmit information from arabinogalactan proteins to microtubules through channels of cellular communication. These diverse possibilities highlight the work that remains to be done before we can understand how plant cells communicate across their membranes.

  2. Cortical microtubule as a sensor and target of nitric oxide signal during the defence responses to Verticillium dahliae toxins in Arabidopsis.

    PubMed

    Shi, Fu-Mei; Yao, Lin-Lin; Pei, Bao-Lei; Zhou, Qun; Li, Xiu-Li; Li, Yun; Li, Ying-Zhang

    2009-04-01

    The molecular mechanisms of signal transduction of plants in response to Verticillium dahliae (VD) are not known. Here, we show that Arabidopsis reacts to VD-toxins with a rapid burst of nitric oxide (NO) and cortical microtubule destabilization. VD-toxins treatment triggered a disruption of cortical microtubules network. This disruption can be influenced by NO production. However, cortical microtubule disruptions were not involved in regulating the NO production. The results indicated that NO may act as an upstream signalling molecule to trigger the depolymerization of cortical microtubule. Cortical microtubules may act as a target of NO signal and as a sensor to mediate the activation of PR-1 gene expression. These results suggested that NO production and cortical microtubule dynamics appeared to be parts of the important signalling system and are involved in the defence mechanisms to VD-toxins in Arabidopsis.

  3. Microtubule and cortical forces determine platelet size during vascular platelet production.

    PubMed

    Thon, Jonathan N; Macleod, Hannah; Begonja, Antonija Jurak; Zhu, Jie; Lee, Kun-Chun; Mogilner, Alex; Hartwig, John H; Italiano, Joseph E

    2012-05-22

    Megakaryocytes release large preplatelet intermediates into the sinusoidal blood vessels. Preplatelets convert into barbell-shaped proplatelets in vitro to undergo repeated abscissions that yield circulating platelets. These observations predict the presence of circular-preplatelets and barbell-proplatelets in blood, and two fundamental questions in platelet biology are what are the forces that determine barbell-proplatelet formation, and how is the final platelet size established. Here we provide insights into the terminal mechanisms of platelet production. We quantify circular-preplatelets and barbell-proplatelets in human blood in high-resolution fluorescence images, using a laser scanning cytometry assay. We demonstrate that force constraints resulting from cortical microtubule band diameter and thickness determine barbell-proplatelet formation. Finally, we provide a mathematical model for the preplatelet to barbell conversion. We conclude that platelet size is limited by microtubule bundling, elastic bending, and actin-myosin-spectrin cortex forces.

  4. Cortical microtubule-associated ER sites: organization centers of cell polarity and communication.

    PubMed

    Peña, Eduardo José; Heinlein, Manfred

    2013-12-01

    Anisotropic cell growth and the ability of plant cells to communicate within and across the borders of cellular and supracellular domains depends on the ability of the cells to dynamically establish polarized networks able to deliver structural and informational macromolecules to distinct cellular sites. Studies of organelle movements and transport of endogenous and viral proteins suggest that organelle and macromolecular trafficking pathways involve transient or stable interactions with cortical microtubule-associated endoplasmic reticulum sites (C-MERs). The observations suggest that C-MERs may function as cortical hubs that organize cargo exchange between organelles and allow the recruitment, assembly, and subsequently site-specific delivery of macromolecular complexes. We propose that viruses interact with such hubs for replication and intercellular spread. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Mechanism of dynamic reorientation of cortical microtubules due to mechanical stress.

    PubMed

    Muratov, Alexander; Baulin, Vladimir A

    2015-12-01

    Directional growth caused by gravitropism and corresponding bending of plant cells has been explored since 19th century, however, many aspects of mechanisms underlying the perception of gravity at the molecular level are still not well known. Perception of gravity in root and shoot gravitropisms is usually attributed to gravisensitive cells, called statocytes, which exploit sedimentation of macroscopic and heavy organelles, amyloplasts, to sense the direction of gravity. Gravity stimulus is then transduced into distal elongation zone, which is several mm far from statocytes, where it causes stretching. It is suggested that gravity stimulus is conveyed by gradients in auxin flux. We propose a theoretical model that may explain how concentration gradients and/or stretching may indirectly affect the global orientation of cortical microtubules, attached to the cell membrane and induce their dynamic reorientation perpendicular to the gradients. In turn, oriented microtubule arrays direct the growth and orientation of cellulose microfibrils, forming part of the cell external skeleton and determine the shape of the cell. Reorientation of microtubules is also observed in reaction to light in phototropism and mechanical bending, thus suggesting universality of the proposed mechanism.

  6. Role of membrane sterols and cortical microtubules in gravity resistance in plants

    NASA Astrophysics Data System (ADS)

    Hoson, T.; Koizumi, T.; Matsumoto, S.; Kumasaki, S.; Soga, K.; Wakabayashi, K.; Sakaki, T.

    Resistance to the gravitational force is a principal graviresponse in plants comparable to gravitropism Nevertheless only limited information has been obtained for this graviresponse We have examined mechanisms of signal perception transformation and transduction of the perceived signal and response to the transduced signal in gravity resistance using hypergravity conditions produced by centrifugation In Arabidopsis hypocotyls hypergravity treatment greatly increased the expression level of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase HMGR which catalyzes a reaction producing mevalonic acid a key precursor of terpenoids such as membrane sterols Geranyl diphosphate synthase gene was also up-regulated by hypergravity whereas the expression of other genes involved in membrane lipid metabolism was not influenced Hypergravity caused an increase in sterol content in azuki bean epicotyls but not in phospholipid glycolipid or fatty acid content Also hypergravity did not influence fatty acid composition in any lipid class Thus the effect of hypergravity on membrane lipid metabolism was specific for sterol synthesis On the other hand alpha- and beta-tubulin genes were up-regulated by hypergravity treatment in Arabidopsis hypocotyls Hypergravity also induced reorientation of cortical microtubules in azuki epicotyls the percentage of epidermal cells with transverse microtubles was decreased whereas that with longitudinal microtubules was increased Inhibitors of HMGR action and microtubule-disrupting agents completely prevented the gravity resistance

  7. Abscisic acid induces ectopic outgrowth in epidermal cells through cortical microtubule reorganization in Arabidopsis thaliana

    PubMed Central

    Takatani, Shogo; Hirayama, Takashi; Hashimoto, Takashi; Takahashi, Taku; Motose, Hiroyasu

    2015-01-01

    Abscisic acid (ABA) regulates seed maturation, germination and various stress responses in plants. The roles of ABA in cellular growth and morphogenesis, however, remain to be explored. Here, we report that ABA induces the ectopic outgrowth of epidermal cells in Arabidopsis thaliana. Seedlings of A. thaliana germinated and grown in the presence of ABA developed ectopic protrusions in the epidermal cells of hypocotyls, petioles and cotyledons. One protrusion was formed in the middle of each epidermal cell. In the hypocotyl epidermis, two types of cell files are arranged alternately into non-stoma cell files and stoma cell files, ectopic protrusions being restricted to the non-stoma cell files. This suggests the presence of a difference in the degree of sensitivity to ABA or in the capacity of cells to form protrusions between the two cell files. The ectopic outgrowth was suppressed in ABA insensitive mutants, whereas it was enhanced in ABA hypersensitive mutants. Interestingly, ABA-induced ectopic outgrowth was also suppressed in mutants in which microtubule organization was compromised. Furthermore, cortical microtubules were disorganized and depolymerized by the ABA treatment. These results suggest that ABA signaling induces ectopic outgrowth in epidermal cells through microtubule reorganization. PMID:26068445

  8. Rac-GTPases Regulate Microtubule Stability and Axon Growth of Cortical GABAergic Interneurons.

    PubMed

    Tivodar, Simona; Kalemaki, Katerina; Kounoupa, Zouzana; Vidaki, Marina; Theodorakis, Kostas; Denaxa, Myrto; Kessaris, Nicoletta; de Curtis, Ivan; Pachnis, Vassilis; Karagogeos, Domna

    2015-09-01

    Cortical interneurons are characterized by extraordinary functional and morphological diversity. Although tremendous progress has been made in uncovering molecular and cellular mechanisms implicated in interneuron generation and function, several questions still remain open. Rho-GTPases have been implicated as intracellular mediators of numerous developmental processes such as cytoskeleton organization, vesicle trafficking, transcription, cell cycle progression, and apoptosis. Specifically in cortical interneurons, we have recently shown a cell-autonomous and stage-specific requirement for Rac1 activity within proliferating interneuron precursors. Conditional ablation of Rac1 in the medial ganglionic eminence leads to a 50% reduction of GABAergic interneurons in the postnatal cortex. Here we examine the additional role of Rac3 by analyzing Rac1/Rac3 double-mutant mice. We show that in the absence of both Rac proteins, the embryonic migration of medial ganglionic eminence-derived interneurons is further impaired. Postnatally, double-mutant mice display a dramatic loss of cortical interneurons. In addition, Rac1/Rac3-deficient interneurons show gross cytoskeletal defects in vitro, with the length of their leading processes significantly reduced and a clear multipolar morphology. We propose that in the absence of Rac1/Rac3, cortical interneurons fail to migrate tangentially towards the pallium due to defects in actin and microtubule cytoskeletal dynamics.

  9. Cortical microtubule nucleation can organise the cytoskeleton of Drosophila oocytes to define the anteroposterior axis

    PubMed Central

    Khuc Trong, Philipp; Doerflinger, Hélène; Dunkel, Jörn; St Johnston, Daniel; Goldstein, Raymond E

    2015-01-01

    Many cells contain non-centrosomal arrays of microtubules (MTs), but the assembly, organisation and function of these arrays are poorly understood. We present the first theoretical model for the non-centrosomal MT cytoskeleton in Drosophila oocytes, in which bicoid and oskar mRNAs become localised to establish the anterior-posterior body axis. Constrained by experimental measurements, the model shows that a simple gradient of cortical MT nucleation is sufficient to reproduce the observed MT distribution, cytoplasmic flow patterns and localisation of oskar and naive bicoid mRNAs. Our simulations exclude a major role for cytoplasmic flows in localisation and reveal an organisation of the MT cytoskeleton that is more ordered than previously thought. Furthermore, modulating cortical MT nucleation induces a bifurcation in cytoskeletal organisation that accounts for the phenotypes of polarity mutants. Thus, our three-dimensional model explains many features of the MT network and highlights the importance of differential cortical MT nucleation for axis formation. DOI: http://dx.doi.org/10.7554/eLife.06088.001 PMID:26406117

  10. The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization

    PubMed Central

    Miller, Rita K.; Matheos, Dina; Rose, Mark D.

    1999-01-01

    In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)– Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Δ, pea2Δ, bud6Δ, and bni1Δ, required for normal polarization and actin cytoskeleton functions and, of these, bni1Δ affected Kar9p localization most severely. Like kar9Δ, bni1Δ mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Δ, the bni1Δ mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Δ bni1Δ double mutants suggested that Kar9p retained some function in bni1Δ mitotic cells. Unlike the polarization mutants, kar9Δ shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Δ. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks. PMID:10085294

  11. Modelling the role of catastrophe, crossover and katanin-mediated severing in the self-organisation of plant cortical microtubules.

    PubMed

    Mace, Alex; Wang, Wenjia

    2015-12-01

    Plant cortical microtubules can form ordered arrays through interactions among themselves. When an incident microtubule collides with a barrier microtubule it may entrain if below a certain angle. Else it undergoes collision induced catastrophe (CIC) or crosses over the barrier microtubule. It has been proposed that katanin is necessary to create order by severing these crossover sites. The authors present a three-state computational model using Arabidopsis thaliana data to show how spontaneous catastrophe, the probability of CIC versus crossover, and katanin-mediated severing at the crossover sites affect microtubule ordering. The results of the systematic simulations show that (1), the microtubule order is more sensitive to the catastrophe rate than the rescue rate; (2), at 21°C, peak order is observed at 0.3 CIC and order decreases as CIC increases; and (3) at 0.2 CIC, katanin severing acting uniformly at all crossover sites is able to create order within a biologically reasonable time frame, but at lower CICs this becomes unrealistically fast. This would imply that at lower CIC levels preferential crossover site targeting and severing activity regulators would be required for katanin to bring about order.

  12. MDP25, A Novel Calcium Regulatory Protein, Mediates Hypocotyl Cell Elongation by Destabilizing Cortical Microtubules in Arabidopsis[C][W][OA

    PubMed Central

    Li, Jiejie; Wang, Xianling; Qin, Tao; Zhang, Yan; Liu, Xiaomin; Sun, Jingbo; Zhou, Yuan; Zhu, Lei; Zhang, Ziding; Yuan, Ming; Mao, Tonglin

    2011-01-01

    The regulation of hypocotyl elongation is important for plant growth. Microtubules play a crucial role during hypocotyl cell elongation. However, the molecular mechanism underlying this process is not well understood. In this study, we describe a novel Arabidopsis thaliana microtubule-destabilizing protein 25 (MDP25) as a negative regulator of hypocotyl cell elongation. We found that MDP25 directly bound to and destabilized microtubules to enhance microtubule depolymerization in vitro. The seedlings of mdp25 mutant Arabidopsis lines had longer etiolated hypocotyls. In addition, MDP25 overexpression resulted in significant overall shortening of hypocotyl cells, which exhibited destabilized cortical microtubules and abnormal cortical microtubule orientation, suggesting that MDP25 plays a crucial role in the negative regulation of hypocotyl cell elongation. Although MDP25 localized to the plasma membrane under normal conditions, increased calcium levels in cells caused MDP25 to partially dissociate from the plasma membrane and move into the cytosol. Cellular MDP25 bound to and destabilized cortical microtubules, resulting in their reorientation, and subsequently inhibited hypocotyl cell elongation. Our results suggest that MDP25 exerts its function on cortical microtubules by responding to cytoplasmic calcium levels to mediate hypocotyl cell elongation. PMID:22209764

  13. Kinesin-4 Functions in Vesicular Transport on Cortical Microtubules and Regulates Cell Wall Mechanics during Cell Elongation in Plants.

    PubMed

    Kong, Zhaosheng; Ioki, Motohide; Braybrook, Siobhan; Li, Shundai; Ye, Zheng-Hua; Julie Lee, Yuh-Ru; Hotta, Takashi; Chang, Anny; Tian, Juan; Wang, Guangda; Liu, Bo

    2015-07-01

    In plants, anisotropic cell expansion depends on cortical microtubules that serve as tracks along which macromolecules and vesicles are transported by the motor kinesins of unknown identities. We used cotton (Gossypium hirsutum) fibers that underwent robust elongation to discover kinesins that are involved in cell elongation and found Gh KINESIN-4A expressed abundantly. The motor was detected by immunofluorescence on vesicle-like structures that were associated with cortical microtubules. In Arabidopsis thaliana, the orthologous motor At KINESIN-4A/FRA1, previously implicated in cellulose deposition during secondary growth in fiber cells, was examined by live-cell imaging in cells expressing the fluorescently tagged functional protein. The motor decorated vesicle-like particles that exhibit a linear movement along cortical microtubules with an average velocity of 0.89 μm/min, which was significantly different from those linked to cellulose biosynthesis. We also discovered that At KINESIN-4A/FRA1 and the related At KINESIN-4C play redundant roles in cell wall mechanics, cell elongation, and the axial growth of various vegetative and reproductive organs, as the loss of At KINESIN-4C greatly enhanced the defects caused by a null mutation at the KINESIN-4A/FRA1 locus. The double mutant displayed a lack of cell wall softening at normal stages of rapid cell elongation. Furthermore, enhanced deposition of arabinose-containing carbohydrate was detected in the kinesin-4 mutants. Our findings established a connection between the Kinesin-4-based transport of cargoes containing non-cellulosic components along cortical microtubules and cell wall mechanics and cell elongation in flowering plants.

  14. Modification of growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls grown under microgravity conditions in space.

    PubMed

    Soga, Kouichi; Yamazaki, Chiaki; Kamada, Motoshi; Tanigawa, Naoki; Kasahara, Haruo; Yano, Sachiko; Kojo, Kei H; Kutsuna, Natsumaro; Kato, Takehide; Hashimoto, Takashi; Kotake, Toshihisa; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2017-09-01

    We carried out a space experiment, denoted as Aniso Tubule, to examine the effects of microgravity on the growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls, using lines in which microtubules are visualized by labelling tubulin or microtubule-associated proteins (MAPs) with green fluorescent protein (GFP). In all lines, GFP-tubulin6 (TUB6)-, basic proline-rich protein1 (BPP1)-GFP- and spira1-like3 (SP1L3)-GFP-expressing using a constitutive promoter, and spiral2 (SPR2)-GFP- and GFP-65 kDa MAP-1 (MAP65-1)-expressing using a native promoter, the length of hypocotyls grown under microgravity conditions in space was longer than that grown at 1 g conditions on the ground. In contrast, the diameter of hypocotyls grown under microgravity conditions was smaller than that of the hypocotyls grown at 1 g. The percentage of cells with transverse microtubules was increased under microgravity conditions, irrespective of the lines. Also, the average angle of the microtubules with respect to the transverse cell axis was decreased in hypocotyls grown under microgravity conditions. When GFP fluorescence was quantified in hypocotyls of GFP-MAP65-1 and SPR2-GFP lines, microgravity increased the levels of MAP65-1, which appears to be involved in the maintenance of transverse microtubule orientation. However, the levels of SPR2 under microgravity conditions were comparable to those at 1 g. These results suggest that the microgravity-induced increase in the levels of MAP65-1 is involved in increase in the transverse microtubules, which may lead to modification of growth anisotropy, thereby developing longer and thinner hypocotyls under microgravity conditions in space. This article is protected by copyright. All rights reserved.

  15. Time course and auxin sensitivity of cortical microtubule reorientation in maize roots

    NASA Technical Reports Server (NTRS)

    Blancaflor, E. B.; Hasenstein, K. H.

    1995-01-01

    The kinetics of MT [microtubule] reorientation in primary roots of Zea mays cv. Merit, were examined 15, 30, 45, and 60 min after horizontal positioning. Confocal microscopy of longitudinal tissue sections showed no change in MT orientation 15 and 30 min after horizontal placement. However, after 45 and 60 min, MTs of the outer 4-5 cortical cell layers along the lower side were reoriented. In order to test whether MT reorientation during graviresponse is caused by an auxin gradient, we examined the organization of MTs in roots that were incubated for 1 h in solutions containing 10(-9) to 10(-6) M IAA. IAA treatment at 10(-8) M or less showed no major or consistent changes but 10(-7) M IAA resulted in MT reorientation in the cortex. The auxin effect does not appear to be acid-induced since benzoic acid (10(-5) M) did not cause MT reorientation. The region closest to the maturation zone was most sensitive to IAA. The data indicate that early stages of gravity induced curvature occur in the absence of MT reorientation but sustained curvature leads to reoriented MTs in the outer cortex. Growth inhibition along the lower side of graviresponding roots appears to result from asymmetric distribution of auxin following gravistimulation.

  16. Time course and auxin sensitivity of cortical microtubule reorientation in maize roots

    NASA Technical Reports Server (NTRS)

    Blancaflor, E. B.; Hasenstein, K. H.

    1995-01-01

    The kinetics of MT [microtubule] reorientation in primary roots of Zea mays cv. Merit, were examined 15, 30, 45, and 60 min after horizontal positioning. Confocal microscopy of longitudinal tissue sections showed no change in MT orientation 15 and 30 min after horizontal placement. However, after 45 and 60 min, MTs of the outer 4-5 cortical cell layers along the lower side were reoriented. In order to test whether MT reorientation during graviresponse is caused by an auxin gradient, we examined the organization of MTs in roots that were incubated for 1 h in solutions containing 10(-9) to 10(-6) M IAA. IAA treatment at 10(-8) M or less showed no major or consistent changes but 10(-7) M IAA resulted in MT reorientation in the cortex. The auxin effect does not appear to be acid-induced since benzoic acid (10(-5) M) did not cause MT reorientation. The region closest to the maturation zone was most sensitive to IAA. The data indicate that early stages of gravity induced curvature occur in the absence of MT reorientation but sustained curvature leads to reoriented MTs in the outer cortex. Growth inhibition along the lower side of graviresponding roots appears to result from asymmetric distribution of auxin following gravistimulation.

  17. Speedy small stomata?

    PubMed

    Raven, John A

    2014-04-01

    Recent work has made progress in relating the size of stomata to stomatal functioning and, in particular, the speed of opening and closing and its implications. Calculations of the influence of stomatal size on the potential rate of osmolarity increase, assuming size-independent ion influx rate per unit area of guard cell plasmalemma set at the value found in large (60 μm long) stomata, show that 10 μm long stomata could have at least a 6-fold higher rate of osmolarity increase. There could be a corresponding decrease in the time taken in going from the closed to the fully open state from about 1h to about 10 min; this is approximately the range found for stomata.. However, there are no data on the rate of stomatal movement over a sufficient size range to test this suggestion. Faster opening requires, assuming optimal allocation, a higher activity of the required enzymes per unit volume of guard cells. This is explored for cytosolic carbonic anhydrase which is needed in guard cells, at least in the light, for malic acid synthesis which is involved in stomatal opening in most stomata. Faster opening and closing of smaller than of larger stomata could allow closer tracking of environmental (mainly light) variations, although the available data are not adequate to determine if such a greater tracking occurs. The range of speeds of stomatal movement is similar to that for photoinhibition-related phenomena, despite the very different mechanisms involved.

  18. Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants

    PubMed Central

    Sotiriou, P.; Giannoutsou, E.; Panteris, E.; Apostolakos, P.; Galatis, B.

    2016-01-01

    Background and aims This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. Methods Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. Results In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. Conclusions The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: 1067–1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination

  19. Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells

    PubMed Central

    Zhang, Hui-ming; Talbot, Mark J.; McCurdy, David W.; Patrick, John W.; Offler, Christina E.

    2015-01-01

    Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca2+ levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane–microtubule inter-relationship is discussed. PMID:26136268

  20. A RhoGEF and Rho family GTPase-activating protein complex links the contractile ring to cortical microtubules at the onset of cytokinesis.

    PubMed

    Somers, W Gregory; Saint, Robert

    2003-01-01

    The mechanism that positions the cytokinetic contractile ring is unknown, but derives from the spindle midzone. We show that an interaction between the Rho GTP exchange factor, Pebble, and the Rho family GTPase-activating protein, RacGAP50C, connects the contractile ring to cortical microtubules at the site of furrowing in D. melanogaster cells. Pebble regulates actomyosin organization, while RacGAP50C and its binding partner, the Pavarotti kinesin-like protein, regulate microtubule bundling. All three factors are required for cytokinesis. As furrowing begins, these proteins colocalize to a cortical equatorial ring. We propose that RacGAP50C-Pavarotti complexes travel on cortical microtubules to the cell equator, where they associate with the Pebble RhoGEF to position contractile ring formation and coordinate F-actin and microtubule remodeling during cytokinesis.

  1. Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells

    PubMed Central

    Carreno, Sébastien; Kouranti, Ilektra; Glusman, Edith Szafer; Fuller, Margaret T.; Echard, Arnaud; Payre, François

    2008-01-01

    Cell division requires cell shape changes involving the localized reorganization of cortical actin, which must be tightly linked with chromosome segregation operated by the mitotic spindle. How this multistep process is coordinated remains poorly understood. In this study, we show that the actin/membrane linker moesin, the single ERM (ezrin, radixin, and moesin) protein in Drosophila melanogaster, is required to maintain cortical stability during mitosis. Mitosis onset is characterized by a burst of moesin activation mediated by a Slik kinase–dependent phosphorylation. Activated moesin homogenously localizes at the cortex in prometaphase and is progressively restricted at the equator in later stages. Lack of moesin or inhibition of its activation destabilized the cortex throughout mitosis, resulting in severe cortical deformations and abnormal distribution of actomyosin regulators. Inhibiting moesin activation also impaired microtubule organization and precluded stable positioning of the mitotic spindle. We propose that the spatiotemporal control of moesin activation at the mitotic cortex provides localized cues to coordinate cortical contractility and microtubule interactions during cell division. PMID:18283112

  2. GSK-3 Activity Is Critical for the Orientation of the Cortical Microtubules and the Dorsoventral Axis Determination in Zebrafish Embryos

    PubMed Central

    Shao, Ming; Lin, Yushuang; Liu, Zhongzhen; Zhang, Ying; Wang, Lifeng; Liu, Changbin; Zhang, Hongwei

    2012-01-01

    The formation of dorsal-ventral (D–V) axis is the earliest event that breaks the radial symmetry and determines the bilateral body plan of a vertebrate embryo, however, the maternal control of this process is not fully understood. Here, we discovered a new dorsalizing window of acute lithium treatment, which covers only less than 10 minutes after fertilization. Lithium treatment in this window was not able to reverse the ventralized phenotype in tokkeabi (tkk) mutant embryos, and its dorsalizing activity on wild-type embryos was inhibited by nocodazole co-treatment. These evidences indicate that the underlying mechanism is independent of a direct activation of Wnt/β-catenin signaling, but depends on the upstream level of the microtubule mediated dorsal determinant transport. In order to identify the target of lithium in this newly discovered sensitive window, GSK-3 inhibitor IX as well as the IMPase inhibitor L690, 330 treatments were performed. We found that only GSK-3 inhibitor IX treatment mimicked the lithium treatment in the dorsalizing activity. Further study showed that the parallel pattern of cortical microtubules in the vegetal pole region and the directed migration of the Wnt8a mRNA were randomized by either lithium or GSK-3 inhibitor IX treatment. These results thus revealed an early and critical role of GSK-3 activity that regulates the orientation of the cortical microtubules and the directed transport of the dorsal determinants in zebrafish embryos. PMID:22574208

  3. Regulation and subcellular localization of the microtubule-destabilizing stathmin family phosphoproteins in cortical neurons.

    PubMed

    Gavet, Olivier; El Messari, Saïd; Ozon, Sylvie; Sobel, André

    2002-06-01

    Stathmin is a ubiquitous cytosolic phosphoprotein, preferentially expressed in the nervous system, and the generic element of a protein family that includes the neural-specific proteins SCG10, SCLIP, and RB3 and its splice variants, RB3' and RB3". All phosphoproteins of the family share with stathmin its tubulin binding and microtubule (MT)-destabilizing activities. To understand better the specific roles of these proteins in neuronal cells, we performed a comparative study of their expression, regulation, and intracellular distribution in embryonic cortical neurons in culture. We found that stathmin is highly expressed ( approximately 0.25% of total proteins) and uniformly present in the various neuronal compartments (cell body, dendrites, axon, growth cones). It appeared mainly unphosphorylated or weakly phosphorylated on one site, and antisera to specific phosphorylated sites (serines 16, 25, or 38) did not reveal a differential regulation of its phosphorylation among neuronal cell compartments. However, they revealed a subpopulation of cells in which stathmin was highly phosphorylated on serine 16, possibly by CaM kinase II also active in a similar subpopulation. The other proteins of the stathmin family are expressed about 100-fold less than stathmin in partially distinct neuronal populations, RB3 being detected in only about 20% of neurons in culture. In contrast to stathmin, they are each mostly concentrated at the Golgi apparatus and are also present along dendrites and axons, including growth cones. Altogether, our results suggest that the different members of the stathmin family have complementary, at least partially distinct functions in neuronal cell regulation, in particular in relation to MT dynamics. Copyright 2002 Wiley-Liss, Inc.

  4. Tensile Tissue Stress Affects the Orientation of Cortical Microtubules in the Epidermis of Sunflower Hypocotyl.

    PubMed

    Hejnowicz; Rusin; Rusin

    2000-03-01

    In turgid multicellular organs, it is convenient to differentiate between the two kinds of tensile forces acting in cell walls as a result of turgor pressure. The primary forces occur both in situ and in cells isolated from the organ, whereas the secondary forces occur only in situ. The latter are an unavoidable physical consequence of the variation in mechanical parameters of tissues forming layers or strands. The most rigid tissue is under maximal tensile force, whereas the least rigid is under maximal compressive force. These forces cause tissue stresses (that is, certain tissues are under tensile stress, whereas others are under compressive stress in the organ). The primary and secondary forces result in primary and secondary stress in cell walls, respectively. The anisotropy of the primary stress is a function of cell shape. For instance, in cylindric cells the anisotropy expressed as the ratio of longitudinal to transverse stresses is 0.5. The anisotropy of the secondary stress is a function of the compound structure of the organ. For example, in the epidermis of sunflower hypocotyl, the longitudinal secondary stress is much higher than the transverse stress. The primary and secondary stresses are superimposed, and, as a consequence, the stress anisotropy in the outer thick walls of epidermal cells is greater than 1. These outer epidermal walls transmit most of the tissue stress. When the epidermis is peeled but remains turgid, only primary stress remains, but loading of the peel can reestablish the original stress anisotropy. We studied the effect of stress anisotropy changes on the orientation of cortical microtubules (CMTs) in the sunflower hypocotyl epidermis. We showed that changes in stress anisotropy cause the CMT orientation to change in the direction of maximal wall stress. In situ, the relatively high tensile tissue stress in the epidermis causes maximal stress in the longitudinal direction and relatively steep CMT orientation. When the tissue

  5. Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants.

    PubMed

    Sotiriou, P; Giannoutsou, E; Panteris, E; Apostolakos, P; Galatis, B

    2016-03-01

    This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: : 1067-1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril

  6. Cellular basis for the automorphic curvature of rice coleoptiles on a three-dimensional clinostat: possible involvement of reorientation of cortical microtubules.

    PubMed

    Saiki, Mizue; Fujita, Hiroshi; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Yamashita, Masamichi; Hoson, Takayuki

    2005-06-01

    Coleoptiles of rice (Oryza sativa L.) show a spontaneous (automorphic) curvature toward the caryopsis under microgravity conditions. The possible involvement of the reorientation of cortical microtubules in automorphic curvature was studied in rice coleoptiles grown on a three-dimensional clinostat. When rice seedlings that had been grown in the normal gravitational field were transferred to the clinostat in the dark, cortical microtubules of epidermal cells in the dorsal side of the coleoptiles oriented more transversely than the ventral side within 0.5 h. The rotation on the clinostat also increased the cell wall extensibility in the dorsal side and decreased the extensibility in the ventral side, and induced automorphic curvature. The reorientation of cortical microtubules preceded the changes in the cell wall extensibility and the curvature. The irradiation of rice seedlings with white light from above inhibited microtubule reorientation and changes in the cell wall extensibility, as well as curvature of coleoptiles. Also, colchicine, applied to the bending region of coleoptiles, partially inhibited the automorphic curvature. These results suggest that reorientation of cortical microtubules is involved in causing automorphic curvature in rice coleoptiles on the clinostat.

  7. Simulation of the effects of microtubules in the cortical rotation of amphibian embryos in normal and zero gravity.

    PubMed

    Nouri, Comron; Tuszynski, Jack A; Wiebe, Mark W; Gordon, Richard

    2012-09-01

    This paper reports the results of computer modeling of microtubules that end up in the cortical region of a one-cell amphibian embryo, prior to the first cell division. Microtubules are modeled as initially randomly oriented semi-flexible rods, represented by several lines of point-masses interacting with one another like masses on springs with longitudinal and transverse stiffness. They are also considered to be space-filling rods floating in a viscous fluid (cytoplasm) experiencing drag forces and buoyancy from the fluid under a variable gravity field to test gravitational effects. Their randomly distributed interactions with the surrounding spherical container (the cell membrane) have a statistical nonzero average that creates a torque causing a rotational displacement between the cytoplasm and the rigid cortex. The simulation has been done for zero and normal gravity and it validates the observation that cortical rotation occurs in microgravity as well as on Earth. The speed of rotation depends on gravity, but is still substantial in microgravity.

  8. Stomata and pathogens

    PubMed Central

    Gudesblat, Gustavo E; Torres, Pablo S

    2009-01-01

    Bacteria and fungi are capable of triggering stomatal closure through pathogen-associated molecular patterns (PAMPs), which prevents penetration through these pores. Therefore, the stomata can be considered part of the plant innate immune response. Some pathogens have evolved mechanisms to evade stomatal defense. The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc), which infects plants of the Brassicaceae family mainly through hydathodes, has also been reported to infect plants through stomata. A recent report shows that penetration of Xcc in Arabidopsis leaves through stomata depends on a secreted small molecule whose synthesis is under control of the rpf/diffusible signal factor (DSF) cell-to-cell signaling system, which also controls genes involved in biofilm formation and pathogenesis. The same reports shows that Arabidopsis ROS- and PAMP-activated MAP kinase 3 (MPK3) is essential for stomatal innate response. Other recent and past findings about modulation of stomatal behaviour by pathogens are also discussed. In all, these findings support the idea that PAMP-triggered stomatal closure might be a more effective and widespread barrier against phytopathogens than previously thought, which has in turn led to the evolution in pathogens of several mechanisms to evade stomatal defense. PMID:20514224

  9. A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting

    PubMed Central

    Byron, Adam; Askari, Janet A.; Humphries, Jonathan D.; Jacquemet, Guillaume; Koper, Ewa J.; Warwood, Stacey; Choi, Colin K.; Stroud, Matthew J.; Chen, Christopher S.; Knight, David; Humphries, Martin J.

    2015-01-01

    Integrin activation, which is regulated by allosteric changes in receptor conformation, enables cellular responses to the chemical, mechanical and topological features of the extracellular microenvironment. A global view of how activation state converts the molecular composition of the region proximal to integrins into functional readouts is, however, lacking. Here, using conformation-specific monoclonal antibodies, we report the isolation of integrin activation state-dependent complexes and their characterization by mass spectrometry. Quantitative comparisons, integrating network, clustering, pathway and image analyses, define multiple functional protein modules enriched in a conformation-specific manner. Notably, active integrin complexes are specifically enriched for proteins associated with microtubule-based functions. Visualization of microtubules on micropatterned surfaces and live cell imaging demonstrate that active integrins establish an environment that stabilizes microtubules at the cell periphery. These data provide a resource for the interrogation of the global molecular connections that link integrin activation to adhesion signalling. PMID:25609142

  10. Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

    PubMed

    Zhang, Hui-ming; Talbot, Mark J; McCurdy, David W; Patrick, John W; Offler, Christina E

    2015-09-01

    Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  11. Efficient event-driven simulations shed new light on microtubule organization in the plant cortical array

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

    The dynamics of the plant microtubule cytoskeleton is a paradigmatic example of the complex spatiotemporal processes characterising life at the cellular scale. This system is composed of large numbers of spatially extended particles, each endowed with its own intrinsic stochastic dynamics, and is capable of non-equilibrium self-organisation through collisional interactions of these particles. To elucidate the behaviour of such a complex system requires not only conceptual advances, but also the development of appropriate computational tools to simulate it. As the number of parameters involved is large and the behaviour is stochastic, it is essential that these simulations be fast enough to allow for an exploration of the phase space and the gathering of sufficient statistics to accurately pin down the average behaviour as well as the magnitude of fluctuations around it. Here we describe a simulation approach that meets this requirement by adopting an event-driven methodology that encompasses both the spontaneous stochastic changes in microtubule state as well as the deterministic collisions. In contrast with finite time step simulations this technique is intrinsically exact, as well as several orders of magnitude faster, which enables ordinary PC hardware to simulate systems of ˜ 10^3 microtubules on a time scale ˜ 10^{3} faster than real time. In addition we present new tools for the analysis of microtubule trajectories on curved surfaces. We illustrate the use of these methods by addressing a number of outstanding issues regarding the importance of various parameters on the transition from an isotropic to an aligned and oriented state.

  12. 3D reconstruction of cortical microtubules using multi-angle total internal reflection fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Jin, Luhong; Xiu, Peng; Zhou, Xiaoxu; Fan, Jiannan; Kuang, Cuifang; Liu, Xu; Xu, Yingke

    2017-01-01

    Total internal reflection fluorescence microscopy (TIRFM) has been widely used in biomedical research to visualize cellular processes near the cell surface. In this study, a novel multi-angle ring-illuminated TIRFM system, equipped with two galvo mirrors that are on conjugate plan of a 4f optical system was developed. Multi-angle TIRFM generates images with different penetration depths through the controlled variation of the incident angle of illuminating laser. We presented a method to perform three-dimensional (3-D) reconstruction of microtubules from multi-angle TIRFM images. The performance of our method was validated in simulated microtubules with variable signal-to-noise ratios (SNR) and the axial resolution and accuracy of reconstruction were evaluated in selecting different numbers of illumination angles or in different SNR conditions. In U373 cells, we reconstructed the 3-D localization of microtubules near the cell surface with high resolution using over a hundred different illumination angles. Theoretically, the presented TIRFM setup and 3-D reconstruction method can achieve 40 nm axial resolution in experimental conditions where SNR is as low as 2, with 35 different illumination angles. Moreover, our system and reconstruction method have the potential to be used in live cells to track membrane dynamics in 3-D.

  13. Auxin deprivation induces a developmental switch in maize somatic embryogenesis involving redistribution of microtubules and actin filaments from endoplasmic to cortical cytoskeletal arrays.

    PubMed

    Samaj, J; Baluska, F; Pretová, A; Volkmann, D

    2003-06-01

    A developmental switch from non-polar pre-embryogenic units to polarized transition units in maize embryogenic callus is caused by auxin deprivation from the culture medium. This switch is accompanied by cytoskeletal rearrangements in embryogenic cells. An immunofluorescence study revealed prominent endoplasmic microtubules and actin filament meshworks radiating from the nuclear surfaces in pre-embryogenic cells growing on medium supplemented with auxin. On the other hand, parallel-organized cortical microtubules and cortical actin filament networks are inherently associated with polarized embryogenic cells of transition units growing on medium without auxin. These results indicate that fine-tuning of the dynamic equilibrium between endoplasmic and cortical cytoskeletal arrays is important for progress in somatic embryogenesis.

  14. Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots. II. Role of Cortical Microtubules and Cellulose Microfibrils1

    PubMed Central

    Baskin, Tobias I.; Meekes, Herman T.H.M.; Liang, Benjamin M.; Sharp, Robert E.

    1999-01-01

    We tested the hypothesis that the degree of anisotropic expansion of plant tissues is controlled by the degree of alignment of cortical microtubules or cellulose microfibrils. Previously, for the primary root of maize (Zea mays L.), we quantified spatial profiles of expansion rate in length, radius, and circumference and the degree of growth anisotropy separately for the stele and cortex, as roots became thinner with time from germination or in response to low water potential (B.M. Liang, A.M. Dennings, R.E. Sharp, T.I. Baskin [1997] Plant Physiol 115:101–111). Here, for the same material, we quantified microtubule alignment with indirect immunofluorescence microscopy and microfibril alignment throughout the cell wall with polarized-light microscopy and from the innermost cell wall layer with electron microscopy. Throughout much of the growth zone, mean orientations of microtubules and microfibrils were transverse, consistent with their parallel alignment specifying the direction of maximal expansion rate (i.e. elongation). However, where microtubule alignment became helical, microfibrils often made helices of opposite handedness, showing that parallelism between these elements was not required for helical orientations. Finally, contrary to the hypothesis, the degree of growth anisotropy was not correlated with the degree of alignment of either microtubules or microfibrils. The mechanisms plants use to specify radial and tangential expansion rates remain uncharacterized. PMID:9952465

  15. Positioning of microtubule organizing centers by cortical pushing and pulling forces

    NASA Astrophysics Data System (ADS)

    Pavin, Nenad; Laan, Liedewij; Ma, Rui; Dogterom, Marileen; Jülicher, Frank

    2012-10-01

    Positioning of microtubule (MT) organizing centers with respect to the confining geometry of cells depends on pushing and/or pulling forces generated by MTs that interact with the cell cortex (Dogterom et al 2005 Curr. Opin. Cell Biol. 17 67-74). How, in living cells, these forces lead to proper positioning is still largely an open question. Recently, it was shown by in vitro experiments using artificial microchambers that in a square geometry, MT asters center more reliably by a combination of pulling and pushing forces than by pushing forces alone (Laan et al 2012a Cell 148 502-14). These findings were explained by a physical description of aster mechanics that includes slipping of pushing MT ends along chamber boundaries. In this paper, we extend that theoretical work by studying the influence of the shape of the confining geometry on the positioning process. We find that pushing and pulling forces can have centering or off-centering behavior in different geometries. Pushing forces center in a one-dimensional and a square geometry, but lead to off-centering in a circle if slipping is sufficiently pronounced. Pulling forces, however, do not center in a one-dimensional geometry, but improve centering in a circle and a square. In an elongated stadium geometry, positioning along the short axis depends mainly on pulling forces, while positioning along the long axis depends mainly on pushing forces. Our theoretical results suggest that different positioning strategies could be used by different cell types.

  16. Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation.

    PubMed

    Yabuuchi, Takatoshi; Nakai, Tomonori; Sonobe, Seiji; Yamauchi, Daisuke; Mineyuki, Yoshinobu

    2015-01-01

    Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.

  17. Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation

    PubMed Central

    Yabuuchi, Takatoshi; Nakai, Tomonori; Sonobe, Seiji; Yamauchi, Daisuke; Mineyuki, Yoshinobu

    2015-01-01

    Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation. PMID:26237087

  18. Arabidopsis MAP65-4 plays a role in phragmoplast microtubule organization and marks the cortical cell division site.

    PubMed

    Li, Haoge; Sun, Baojuan; Sasabe, Michiko; Deng, Xingguang; Machida, Yasunori; Lin, Honghui; Julie Lee, Y-R; Liu, Bo

    2017-07-01

    The evolutionarily conserved MAP65 family proteins bundle anti-parallel microtubules (MTs). In Arabidopsis thaliana, mutations in the MAP65-3 gene lead to serious defects in MT organization in the phragmoplast and cause failures in cytokinesis. However, the functions of other ArabidopsisMAP65 isoforms are largely unknown. MAP65 functions were analyzed based on genetic interactions among different map65 mutations. Live-cell imaging and immunolocalization experiments revealed dynamic activities of two closely related MAP65 proteins in dividing cells. The map65-4 mutation caused synthetic lethality with map65-3 although map65-4 alone did not cause a noticeable phenotype. Furthermore, the introduction of an extra copy of the MAP65-4 gene significantly suppressed defects in cytokinesis and seedling growth caused by map65-3 because of restoring MT engagement in the spindle midzone. During mitosis, MAP65-4 first appeared at the preprophase band and persisted at the cortical division site afterwards. It was also concentrated on MTs in the spindle midzone and the phragmoplast. In the absence of MAP65-3, MAP65-4 exhibited greatly enhanced localization in the midzone of developing phragmoplast. Therefore, we have uncovered redundant but differential contributions of MAP65-3 and MAP65-4 to engaging and bundling anti-parallel MTs in the phragmoplast and disclosed a novel action of MAP65-4 at the cortical cell division site. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  19. Molecular Evolution of Grass Stomata.

    PubMed

    Chen, Zhong-Hua; Chen, Guang; Dai, Fei; Wang, Yizhou; Hills, Adrian; Ruan, Yong-Ling; Zhang, Guoping; Franks, Peter J; Nevo, Eviatar; Blatt, Michael R

    2017-02-01

    Grasses began to diversify in the late Cretaceous Period and now dominate more than one third of global land area, including three-quarters of agricultural land. We hypothesize that their success is likely attributed to the evolution of highly responsive stomata capable of maximizing productivity in rapidly changing environments. Grass stomata harness the active turgor control mechanisms present in stomata of more ancient plant lineages, maximizing several morphological and developmental features to ensure rapid responses to environmental inputs. The evolutionary development of grass stomata appears to have been a gradual progression. Therefore, understanding the complex structures, developmental events, regulatory networks, and combinations of ion transporters necessary to drive rapid stomatal movement may inform future efforts towards breeding new crop varieties.

  20. Early asymmetries in maternal transcript distribution associated with a cortical microtubule network and a polar body in the beetle Tribolium castaneum.

    PubMed

    Peel, Andrew D; Averof, Michalis

    2010-11-01

    The localization of maternal mRNAs during oogenesis plays a central role in axial specification in some insects. Here we describe a polar body-associated asymmetry in maternal transcript distribution in pre-blastoderm eggs of the beetle Tribolium castaneum. Since the position of the polar body marks the future dorsal side of the embryo, we have investigated whether this asymmetry in mRNA distribution plays a role in dorsal-ventral axis specification. Whilst our results suggest polar body-associated transcripts do not play a significant role in specifying the DV axis, at least during early embryogenesis, we do find that the polar body is closely associated with a cortical microtubule network (CMN), which may play a role in the localization of transcripts during oogenesis. Transcripts of the gene T.c.pangolin co-localize with the CMN at the time of their anterior localization during oogenesis and their anterior localization is disrupted by the microtubule-depolymerizing agent colcemid.

  1. Hexavalent chromium-induced differential disruption of cortical microtubules in some Fabaceae species is correlated with acetylation of α-tubulin.

    PubMed

    Eleftheriou, Eleftherios P; Adamakis, Ioannis-Dimosthenis S; Michalopoulou, Vasiliki A

    2016-03-01

    The effects of hexavalent chromium [Cr(VI)] on the cortical microtubules (MTs) of five species of the Fabaceae family (Vicia faba, Pisum sativum, Vigna sinensis, Vigna angularis, and Medicago sativa) were investigated by confocal laser scanning microscopy after immunolocalization of total tubulin with conventional immunofluorescence techniques and of acetylated α-tubulin with the specific 6-11B-1 monoclonal antibody. Moreover, total α-tubulin and acetylated α-tubulin were quantified by Western immunoblotting and scanning densitometry. Results showed the universality of Cr(VI) detrimental effects to cortical MTs, which proved to be a sensitive and reliable subcellular marker for monitoring Cr(VI) toxicity in plant cells. However, a species-specific response was recorded, and a correlation of MT disturbance with the acetylation status of α-tubulin was demonstrated. In V. faba, MTs were depolymerized at the gain of cytoplasmic tubulin background and displayed low α-tubulin acetylation, while in P. sativum, V. sinensis, V. angularis, and M. sativa, MTs became bundled and changed orientation from perpendicular to oblique or longitudinal. Bundled MTs were highly acetylated as determined by both immunofluorescence and Western immunoblotting. Tubulin acetylation in P. sativum and M. sativa preceded MT bundling; in V. sinensis it followed MT derangement, while in V. angularis the two phenomena coincided. Total α-tubulin remained constant in all treatments. Should acetylation be an indicator of MT stabilization, it is deduced that bundled MTs became stabilized, lost their dynamic properties, and were rendered inactive. Results of this report allow the conclusion that Cr(VI) toxicity disrupts MTs and deranges the MT-mediated functions either by depolymerizing or stabilizing them.

  2. A Three-Dimensional Computer Simulation Model Reveals the Mechanisms for Self-Organization of Plant Cortical Microtubules into Oblique Arrays

    PubMed Central

    Eren, Ezgi Can; Gautam, Natarajan

    2010-01-01

    The noncentrosomal cortical microtubules (CMTs) of plant cells self-organize into a parallel three-dimensional (3D) array that is oriented transverse to the cell elongation axis in wild-type plants and is oblique in some of the mutants that show twisted growth. To study the mechanisms of CMT array organization, we developed a 3D computer simulation model based on experimentally observed properties of CMTs. Our computer model accurately mimics transverse array organization and other fundamental properties of CMTs observed in rapidly elongating wild-type cells as well as the defective CMT phenotypes observed in the Arabidopsis mor1-1 and fra2 mutants. We found that CMT interactions, boundary conditions, and the bundling cutoff angle impact the rate and extent of CMT organization, whereas branch-form CMT nucleation did not significantly impact the rate of CMT organization but was necessary to generate polarity during CMT organization. We also found that the dynamic instability parameters from twisted growth mutants were not sufficient to generate oblique CMT arrays. Instead, we found that parameters regulating branch-form CMT nucleation and boundary conditions at the end walls are important for forming oblique CMT arrays. Together, our computer model provides new mechanistic insights into how plant CMTs self-organize into specific 3D arrangements. PMID:20519434

  3. Leaf Stomata as Bioindicators: Stimulating Student Research

    ERIC Educational Resources Information Center

    Case, Steven B.

    2006-01-01

    Stomata are the pores on leaves through which carbon dioxide, oxygen, and water vapor are exchanged with the atmosphere. Researchers have found that leaf stomatal densities change in response to several environmental variables, including humidity, light intensity, and atmospheric levels of carbon dioxide, a greenhouse gas (Van Der Burgh, Dilcher,…

  4. Leaf Stomata as Bioindicators: Stimulating Student Research

    ERIC Educational Resources Information Center

    Case, Steven B.

    2006-01-01

    Stomata are the pores on leaves through which carbon dioxide, oxygen, and water vapor are exchanged with the atmosphere. Researchers have found that leaf stomatal densities change in response to several environmental variables, including humidity, light intensity, and atmospheric levels of carbon dioxide, a greenhouse gas (Van Der Burgh, Dilcher,…

  5. Microtubule-microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes.

    PubMed

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill; Gelfand, Vladimir I

    2016-08-23

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

  6. Analysis of Cortical Flow Models In Vivo

    PubMed Central

    Benink, Hélène A.; Mandato, Craig A.; Bement, William M.

    2000-01-01

    Cortical flow, the directed movement of cortical F-actin and cortical organelles, is a basic cellular motility process. Microtubules are thought to somehow direct cortical flow, but whether they do so by stimulating or inhibiting contraction of the cortical actin cytoskeleton is the subject of debate. Treatment of Xenopus oocytes with phorbol 12-myristate 13-acetate (PMA) triggers cortical flow toward the animal pole of the oocyte; this flow is suppressed by microtubules. To determine how this suppression occurs and whether it can control the direction of cortical flow, oocytes were subjected to localized manipulation of either the contractile stimulus (PMA) or microtubules. Localized PMA application resulted in redirection of cortical flow toward the site of application, as judged by movement of cortical pigment granules, cortical F-actin, and cortical myosin-2A. Such redirected flow was accelerated by microtubule depolymerization, showing that the suppression of cortical flow by microtubules is independent of the direction of flow. Direct observation of cortical F-actin by time-lapse confocal analysis in combination with photobleaching showed that cortical flow is driven by contraction of the cortical F-actin network and that microtubules suppress this contraction. The oocyte germinal vesicle serves as a microtubule organizing center in Xenopus oocytes; experimental displacement of the germinal vesicle toward the animal pole resulted in localized flow away from the animal pole. The results show that 1) cortical flow is directed toward areas of localized contraction of the cortical F-actin cytoskeleton; 2) microtubules suppress cortical flow by inhibiting contraction of the cortical F-actin cytoskeleton; and 3) localized, microtubule-dependent suppression of actomyosin-based contraction can control the direction of cortical flow. We discuss these findings in light of current models of cortical flow. PMID:10930453

  7. Characterization of the role of calcium in regulating the microtubule-destabilizing activity of MDP25.

    PubMed

    Qin, Tao; Li, Jiejie; Yuan, Ming; Mao, Tonglin

    2012-07-01

    Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins. Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.

  8. Arabidopsis AUGMIN Subunit8 Is a Microtubule Plus-End Binding Protein That Promotes Microtubule Reorientation in Hypocotyls[C][W

    PubMed Central

    Cao, Lingyan; Wang, Linhai; Zheng, Min; Cao, Hong; Ding, Lian; Zhang, Xiaolan; Fu, Ying

    2013-01-01

    In plant cells, cortical microtubules provide tracks for cellulose-synthesizing enzymes and regulate cell division, growth, and morphogenesis. The role of microtubules in these essential cellular processes depends on the spatial arrangement of the microtubules. Cortical microtubules are reoriented in response to changes in cell growth status and cell shape. Therefore, an understanding of the mechanism that underlies the change in microtubule orientation will provide insight into plant cell growth and morphogenesis. This study demonstrated that AUGMIN subunit8 (AUG8) in Arabidopsis thaliana is a novel microtubule plus-end binding protein that participates in the reorientation of microtubules in hypocotyls when cell elongation slows down. AUG8 bound to the plus ends of microtubules and promoted tubulin polymerization in vitro. In vivo, AUG8 was recruited to the microtubule branch site immediately before nascent microtubules branched out. It specifically associated with the plus ends of growing cortical microtubules and regulated microtubule dynamics, which facilitated microtubule reorientation when microtubules changed their growth trajectory or encountered obstacle microtubules during microtubule reorientation. This study thus reveals a novel mechanism underlying microtubule reorientation that is critical for modulating cell elongation in Arabidopsis. PMID:23735294

  9. CYTOPLASMIC MICROTUBULES

    PubMed Central

    Slautterback, David B.

    1963-01-01

    Small cytoplasmic tubules are present in the interstitial cells and cnidoblasts of hydra. They are referred to here as "microtubules." These tubular elements have an outside diameter of 180 A and an inside diameter of 80 A. By difference, the membranous wall is estimated to be 50 A thick. The maximum length of the microtubules cannot be determined from thin sections but is known to exceed 1.5 µ. In the interstitial cells the microtubules are found in the intercellular bridges, free in the cytoplasm and in association with the centrioles. In the cnidoblast they form a framework around the developing nematocyst and in late stages are related to the cnidocil forming a tight skein in the basal part of the cell. Especially in this cell, confluence of microtubules with small spherical vesicles of the Golgi complex has been observed. It is proposed that these tubules function in the transport of water, ions, or small molecules. PMID:14079495

  10. Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose Synthesis in Arabidopsis.

    PubMed

    Liu, Zengyu; Schneider, Rene; Kesten, Christopher; Zhang, Yi; Somssich, Marc; Zhang, Youjun; Fernie, Alisdair R; Persson, Staffan

    2016-08-08

    Cellulose is the most abundant biopolymer on Earth and is the major contributor to plant morphogenesis. Cellulose is synthesized by plasma membrane-localized cellulose synthase complexes (CSCs). Nascent cellulose microfibrils become entangled in the cell wall, and further catalysis therefore drives the CSC forward through the membrane: a process guided by cortical microtubules via the protein CSI1/POM2. Still, it is unclear how the microtubules can withstand the forces generated by the motile CSCs to effectively direct CSC movement. Here, we identified a family of microtubule-associated proteins, the cellulose synthase-microtubule uncouplings (CMUs), that located as static puncta along cortical microtubules. Functional disruption of the CMUs caused lateral microtubule displacement and compromised microtubule-based guidance of CSC movement. CSCs that traversed the microtubules interacted with the microtubules via CSI1/POM2, which prompted the lateral microtubule displacement. Hence, we have revealed how microtubules can withstand the propulsion of the CSCs during cellulose biosynthesis and thus sustain anisotropic plant cell growth. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Evolution and development of monocot stomata.

    PubMed

    Rudall, Paula J; Chen, Elisabeth D; Cullen, Erin

    2017-08-09

    Leaves of monocots are typically linear with parallel venation, though a few taxa have broad leaves. Studies of stomatal patterning and development in monocots required updating in the context of rapidly improving knowledge of both the phylogenetic and development-genetic context of monocots that facilitate studies of character evolution. We used an existing microscope-slide collection to obtain data on stomatal structure across all the major monocot clades, including some species with relatively broad leaves. In addition, we used both light and electron microscopy to study stomatal development in 16 selected species. We evaluated these data in a phylogenetic context to assess stomatal character evolution. Mature stomatal patterning in monocots can be broadly categorized as anomocytic, paracytic-nonoblique, and paracytic/tetracytic oblique, depending on the presence, development, and arrangement of lateral subsidiary cells. Stomatal meristemoids invariably result from an asymmetric mitosis in monocots. In species where lateral subsidiary cells are present, they are perigene cells. Among monocots with relatively broad leaves, stomatal orientation is linear-axial in most taxa, but transverse in Lapageria and Stemona, and random in Dioscorea and some Araceae. Amplifying divisions are apparently absent in monocots. Anomocytic stomata represent the likely ancestral (plesiomorphic) condition in monocots, though multiple evolutionary transitions and reversals have occurred. Paracytic-nonoblique stomata with highly modified perigene lateral neighbor cells characterize grasses and other Poales. The presence of anomocytic stomata in Japonolirion and Tofieldia reinforces the concept that these two genera have retained many ancestral monocot features and are critical in understanding character evolution in monocots. © 2017 Botanical Society of America.

  12. The ANGUSTIFOLIA gene of Arabidopsis, a plant CtBP gene, regulates leaf-cell expansion, the arrangement of cortical microtubules in leaf cells and expression of a gene involved in cell-wall formation

    PubMed Central

    Kim, Gyung-Tae; Shoda, Keiko; Tsuge, Tomohiko; Cho, Kiu-Hyung; Uchimiya, Hirofumi; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Tsukaya, Hirokazu

    2002-01-01

    We previously showed that the ANGUSTIFOLIA (AN) gene regulates the width of leaves of Arabidopsis thaliana, by controlling the polar elongation of leaf cells. In the present study, we found that the abnormal arrangement of cortical microtubules (MTs) in an leaf cells appeared to account entirely for the abnormal shape of the cells. It suggested that the AN gene might regulate the polarity of cell growth by controlling the arrangement of cortical MTs. We cloned the AN gene using a map-based strategy and identified it as the first member of the CtBP family to be found in plants. Wild-type AN cDNA reversed the narrow-leaved phenotype and the abnormal arrangement of cortical MTs of the an-1 mutation. In the animal kingdom, CtBPs self-associate and act as co-repressors of transcription. The AN protein can also self-associate in the yeast two-hybrid system. Furthermore, microarray analysis suggested that the AN gene might regulate the expression of certain genes, e.g. the gene involved in formation of cell walls, MERI5. A discussion of the molecular mechanisms involved in the leaf shape regulation is presented based on our observations. PMID:11889033

  13. Stronger net posterior cortical forces and asymmetric microtubule arrays produce simultaneous centration and rotation of the pronuclear complex in the early Caenorhabditis elegans embryo

    PubMed Central

    Coffman, Valerie C.; McDermott, Matthew B. A.; Shtylla, Blerta; Dawes, Adriana T.

    2016-01-01

    Positioning of microtubule-organizing centers (MTOCs) incorporates biochemical and mechanical cues for proper alignment of the mitotic spindle and cell division site. Current experimental and theoretical studies in the early Caenorhabditis elegans embryo assume remarkable changes in the origin and polarity of forces acting on the MTOCs. These changes must occur over a few minutes, between initial centration and rotation of the pronuclear complex and entry into mitosis, and the models do not replicate in vivo timing of centration and rotation. Here we propose a model that incorporates asymmetry in the microtubule arrays generated by each MTOC, which we demonstrate with in vivo measurements, and a similar asymmetric force profile to that required for posterior-directed spindle displacement during mitosis. We find that these asymmetries are capable of and important for recapitulating the simultaneous centration and rotation of the pronuclear complex observed in vivo. The combination of theoretical and experimental evidence provided here offers a unified framework for the spatial organization and forces needed for pronuclear centration, rotation, and spindle displacement in the early C. elegans embryo. PMID:27733624

  14. Recent advances in the research of lymphatic stomata.

    PubMed

    Wang, Zi-Bin; Li, Meng; Li, Ji-Cheng

    2010-05-01

    Lymphatic stomata are small openings of lymphatic capillaries on the free surface of the mesothelium. The peritoneal cavity, pleural cavity, and pericardial cavity are connected with lymphatic system via these small openings, which have the function of active absorption. The ultrastructure of the lymphatic stomata and their absorption from the body cavities are important clinically, such as ascites elimination, neoplasm metastasis, and inflammatory reaction. The lymphatic stomata play an important role in the physiological and pathological conditions. Our previous study indicated for the first time that nitric oxide (NO) could regulate the opening and absorption of the lymphatic stomata. It could decrease the level of free intracellular calcium [Ca(2+)] through increasing the cyclic guanosine monophosphate (cGMP) level in the rat peritoneal mesothelial cells, thus regulating the lymphatic stomata. This process is related with the NO-cGMP-[Ca(2+)] signal pathway. In this review, we summarize the recent advances in understanding the development and the function of the lymphatic stomata. The ultrastructure and regulations of the lymphatic stomata are also discussed in this review.

  15. Effects of stomata clustering on leaf gas exchange.

    PubMed

    Lehmann, Peter; Or, Dani

    2015-09-01

    A general theoretical framework for quantifying the stomatal clustering effects on leaf gaseous diffusive conductance was developed and tested. The theory accounts for stomatal spacing and interactions among 'gaseous concentration shells'. The theory was tested using the unique measurements of Dow et al. (2014) that have shown lower leaf diffusive conductance for a genotype of Arabidopsis thaliana with clustered stomata relative to uniformly distributed stomata of similar size and density. The model accounts for gaseous diffusion: through stomatal pores; via concentration shells forming at pore apertures that vary with stomata spacing and are thus altered by clustering; and across the adjacent air boundary layer. Analytical approximations were derived and validated using a numerical model for 3D diffusion equation. Stomata clustering increases the interactions among concentration shells resulting in larger diffusive resistance that may reduce fluxes by 5-15%. A similar reduction in conductance was found for clusters formed by networks of veins. The study resolves ambiguities found in the literature concerning stomata end-corrections and stomatal shape, and provides a new stomata density threshold for diffusive interactions of overlapping vapor shells. The predicted reduction in gaseous exchange due to clustering, suggests that guard cell function is impaired, limiting stomatal aperture opening.

  16. EXTRACELLULAR MICROTUBULES

    PubMed Central

    Bouck, G. Benjamin

    1969-01-01

    Mastigonemes (Flimmer) from the sperm of Ascophyllum and Fucus were found to consist of a tripartite structure—a ca. 2000-A tapered basal region, a closed microtubular shaft, and a group of terminal filaments. Each of these regions appears to be constructed of globular subunits with a center-to-center distance of about 45 A. The mastigoneme microtubule is of smaller diameter (170–190 A) than cytoplasmic microtubules in these or other plant cells. During the initial stages of flagellar ontogeny, structures similar to mastigonemes (presumptive mastigonemes) are found within membrane-limited sacs in the cytoplasm or within the perinuclear space. Mastigonemes at this time are generally not found on the flagellar surface. Later, when the anterior flagellum acquires mastigonemes, the presumptive mastigonemes are absent from the cytoplasm. The regularity of attachment of mastigonemes to the flagellar surface suggests that specific attachment sites are constructed on the plasma membrane during flagellar ontogeny. No evidence for penetration of the mastigoneme through the plasma membrane was obtained. The origin and structure of mastigonemes are discussed in relation to reports of the origin and structure of other microtubular systems. PMID:5812471

  17. 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.

  18. Microtubule Catastrophe and Rescue

    PubMed Central

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

    2012-01-01

    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. PMID:23092753

  19. Microtubules in viral replication and transport.

    PubMed

    Niehl, Annette; Peña, Eduardo J; Amari, Khalid; Heinlein, Manfred

    2013-07-01

    Viruses use and subvert host cell mechanisms to support their replication and spread between cells, tissues and organisms. Microtubules and associated motor proteins play important roles in these processes in animal systems, and may also play a role in plants. Although transport processes in plants are mostly actin based, studies, in particular with Tobacco mosaic virus (TMV) and its movement protein (MP), indicate direct or indirect roles of microtubules in the cell-to-cell spread of infection. Detailed observations suggest that microtubules participate in the cortical anchorage of viral replication complexes, in guiding their trafficking along the endoplasmic reticulum (ER)/actin network, and also in developing the complexes into virus factories. Microtubules also play a role in the plant-to-plant transmission of Cauliflower mosaic virus (CaMV) by assisting in the development of specific virus-induced inclusions that facilitate viral uptake by aphids. The involvement of microtubules in the formation of virus factories and of other virus-induced inclusions suggests the existence of aggresomal pathways by which plant cells recruit membranes and proteins into localized macromolecular assemblies. Although studies related to the involvement of microtubules in the interaction of viruses with plants focus on specific virus models, a number of observations with other virus species suggest that microtubules may have a widespread role in viral pathogenesis. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  20. 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.

  1. Characterization of the role of calcium in regulating the microtubule-destabilizing activity of MDP25

    PubMed Central

    Qin, Tao; Li, Jiejie; Yuan, Ming; Mao, Tonglin

    2012-01-01

    Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins.1 Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.2 PMID:22751329

  2. Lymphatic Stomata in the Adult Human Pulmonary Ligament

    PubMed Central

    Miura, Masahiro; Iobe, Hiroaki; Kudo, Tomoo; Shimazu, Yoshihito; Aoba, Takaaki; Okudela, Koji; Nagahama, Kiyotaka; Sakamaki, Kentaro; Yoshida, Maki; Nagao, Toshitaka; Nakaya, Takeo; Kurata, Atsushi; Ohtani, Osamu

    2015-01-01

    Abstract Background: Lymphatic stomata are small lymphatic openings in the serosal membrane that communicate with the serosal cavity. Although these stomata have primarily been studied in experimental mammals, little is known concerning the presence and properties of lymphatic stomata in the adult human pleura. Thus, adult human pleurae were examined for the presence or absence of lymphatic stomata. Methods and Results: A total of 26 pulmonary ligaments (13 left and 13 right) were obtained from 15 adult human autopsy cases and examined using electron and light microscopy. The microscopic studies revealed the presence of apertures fringed with D2-40-positive, CD31-positive, and cytokeratin-negative endothelial cells directly communicating with submesothelial lymphatics in all of the pulmonary ligaments. The apertures' sizes and densities varied from case to case according to the serial tissue section. The medians of these aperture sizes ranged from 2.25 to 8.75 μm in the left pulmonary ligaments and from 2.50 to 12.50 μm in the right pulmonary ligaments. The densities of the apertures ranged from 2 to 9 per mm2 in the left pulmonary ligaments and from 2 to 18 per mm2 in the right pulmonary ligaments. However, no significant differences were found regarding the aperture size (p=0.359) and density (p=0.438) between the left and the right pulmonary ligaments. Conclusions: Our study revealed that apertures exhibit structural adequacy as lymphatic stomata on the surface of the pulmonary ligament, thereby providing evidence that lymphatic stomata are present in the adult human pleura. PMID:25526320

  3. Producing Conditional Mutants for Studying Plant Microtubule Function

    SciTech Connect

    Richard Cyr

    2009-09-29

    The cytoskeleton, and in particular its microtubule component, participates in several processes that directly affect growth and development in higher plants. Normal cytoskeletal function requires the precise and orderly arrangement of microtubules into several cell cycle and developmentally specific arrays. One of these, the cortical array, is notable for its role in directing the deposition of cellulose (the most prominent polymer in the biosphere). An understanding of how these arrays form, and the molecular interactions that contribute to their function, is incomplete. To gain a better understanding of how microtubules work, we have been working to characterize mutants in critical cytoskeletal genes. This characterization is being carried out at the subcellular level using vital microtubule gene constructs. In the last year of funding colleagues have discovered that gamma-tubulin complexes form along the lengths of cortical microtubules where they act to spawn new microtubules at a characteristic 40 deg angle. This finding complements nicely the finding from our lab (which was funded by the DOE) showing that microtubule encounters are angle dependent; high angles encounters results in catastrophic collisions while low angle encounters result in favorable zippering. The finding of a 40 deg spawn of new microtubules from extant microtubule, together with aforementioned rules of encounters, insures favorable co-alignment in the array. I was invited to write a New and Views essay on this topic and a PDF is attached (News and Views policy does not permit funding acknowledgments and so I was not allowed to acknowledge support from the DOE).

  4. General theory for the mechanics of confined microtubule asters

    NASA Astrophysics Data System (ADS)

    Ma, Rui; Laan, Liedewij; Dogterom, Marileen; Pavin, Nenad; Jülicher, Frank

    2014-01-01

    In cells, dynamic microtubules organize into asters or spindles to assist positioning of organelles. Two types of forces are suggested to contribute to the positioning process: (i) microtubule-growth based pushing forces; and (ii) motor protein mediated pulling forces. In this paper, we present a general theory to account for aster positioning in a confinement of arbitrary shape. The theory takes account of microtubule nucleation, growth, catastrophe, slipping, as well as interaction with cortical force generators. We calculate microtubule distributions and forces acting on microtubule organizing centers in a sphere and in an ellipsoid. Positioning mechanisms based on both pushing forces and pulling forces can be distinguished in our theory for different parameter regimes or in different geometries. In addition, we investigate positioning of microtubule asters in the case of asymmetric distribution of motors. This analysis enables us to characterize situations relevant for Caenorrhabditis elegans embryos.

  5. Microtubule dynamics and organization

    NASA Astrophysics Data System (ADS)

    Dogterom, Marileen

    2000-03-01

    Microtubules are rigid biopolymers found in all higher order cells. They are a mayor part of the cytoskeleton, the network of protein polymers that gives the cell its shape and rigidity and allows for various forms of (intra)cellular motility. The intracellular spatial organization of the microtubule network is constantly changing as the microtubules adapt to their different functions. In part, this spatial organization depends on the assembly dynamics (including microtubule nucleation) and forces generated by the microtubules themselves. To understand these mechanisms, we study the physical aspects connected with the assembly, force generation and spatial organization of microtubules in simplified model systems, in the absence of other cellular components. We measure the forces generated by individual microtubules by making them grow against a microfabricated barrier. These experiments show that a single microtubule can generate at least several picoNewton of force, comparable to what is known for motor proteins. Theoretical modeling of force-generation by multi-protofilament polymers is used to predict force-velocity relations that can be compared to experimental data. We study the self-organization of microtubules by confining them to microfabricated chambers that mimic the geometry of living cells. The distribution of microtubule nucleation sites in these chambers is controlled to study its effect on the organization of the microtubule network. We find that so-called microtubule asters position themselves in response to forces generated by dynamic microtubules. Experiments aimed at measuring the forces acting on these asters using optical trapping techniques will be described.

  6. Cellulose microfibril alignment recovers from DCB-induced disruption despite microtubule disorganization.

    PubMed

    Himmelspach, Regina; Williamson, Richard E; Wasteneys, Geoffrey O

    2003-11-01

    Cellulose microfibril deposition patterns define the direction of plant cell expansion. To better understand how microfibril alignment is controlled, we examined microfibril orientation during cortical microtubule disruption using the temperature-sensitive mutant of Arabidopsis thaliana, mor1-1. In a previous study, it was shown that at restrictive temperature for mor1-1, cortical microtubules lose transverse orientation and cells lose growth anisotropy without any change in the parallel arrangement of cellulose microfibrils. In this study, we investigated whether a pre-existing template of well-ordered microfibrils or the presence of well-organized cortical microtubules was essential for the cell to resume deposition of parallel microfibrils. We first transiently disrupted the parallel order of microfibrils in mor1-1 using a brief treatment with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). We then analysed the alignment of recently deposited cellulose microfibrils (by field emission scanning electron microscopy) as cellulose synthesis recovered and microtubules remained disrupted at the mor1-1 mutant's non-permissive culture temperature. Despite the disordered cortical microtubules and an initially randomized wall texture, new cellulose microfibrils were deposited with parallel, transverse orientation. These results show that transverse cellulose microfibril deposition requires neither accurately transverse cortical microtubules nor a pre-existing template of well-ordered microfibrils. We also demonstrated that DCB treatments reduced the ability of cortical microtubules to form transverse arrays, supporting a role for cellulose microfibrils in influencing cortical microtubule organization.

  7. Rhizobacteria Bacillus subtilis restricts foliar pathogen entry through stomata.

    PubMed

    Kumar, Amutha Sampath; Lakshmanan, Venkatachalam; Caplan, Jeffrey L; Powell, Deborah; Czymmek, Kirk J; Levia, Delphis F; Bais, Harsh P

    2012-11-01

    Plants exist in a complex multitrophic environment, where they interact with and compete for resources with other plants, microbes and animals. Plants have a complex array of defense mechanisms, such as the cell wall being covered with a waxy cuticle serving as a potent physical barrier. Although some pathogenic fungi infect plants by penetrating through the cell wall, many bacterial pathogens invade plants primarily through stomata on the leaf surface. Entry of the foliar pathogen, Pseudomonas syringae pathovar tomato DC3000 (hereafter PstDC3000), into the plant corpus occurs through stomatal openings, and consequently a key plant innate immune response is the transient closure of stomata, which delays disease progression. Here, we present evidence that the root colonization of the rhizobacteria Bacillus subtilis FB17 (hereafter FB17) restricts the stomata-mediated pathogen entry of PstDC3000 in Arabidopsis thaliana. Root binding of FB17 invokes abscisic acid (ABA) and salicylic acid (SA) signaling pathways to close light-adapted stomata. These results emphasize the importance of rhizospheric processes and environmental conditions as an integral part of the plant innate immune system against foliar bacterial infections.

  8. JAZ2 controls stomata dynamics during bacterial invasion.

    PubMed

    Gimenez-Ibanez, Selena; Boter, Marta; Ortigosa, Andrés; García-Casado, Gloria; Chini, Andrea; Lewsey, Mathew G; Ecker, Joseph R; Ntoukakis, Vardis; Solano, Roberto

    2017-02-01

    Coronatine (COR) facilitates entry of bacteria into the plant apoplast by stimulating stomata opening. COR-induced signaling events at stomata remain unclear. We found that the COR and jasmonate isoleucine (JA-Ile) co-receptor JAZ2 is constitutively expressed in guard cells and modulates stomatal dynamics during bacterial invasion We analyzed tissue expression patterns of AtJAZ genes and measured stomata opening and pathogen resistance in loss- and gain-of-function mutants. Arabidopsis jaz2 mutants are partially impaired in pathogen-induced stomatal closing and more susceptible to Pseudomonas. Gain-of-function mutations in JAZ2 prevent stomatal reopening by COR and are highly resistant to bacterial penetration. The JAZ2 targets MYC2, MYC3 and MYC4 directly regulate the expression of ANAC19, ANAC55 and ANAC72 to modulate stomata aperture. Due to the antagonistic interactions between the salicylic acid (SA) and JA defense pathways, efforts to increase resistance to biotrophs result in enhanced susceptibility to necrotrophs, and vice versa. Remarkably, dominant jaz2Δjas mutants are resistant to Pseudomonas syringae but retain unaltered resistance against necrotrophs. Our results demonstrate the existence of a COI1-JAZ2-MYC2,3,4-ANAC19,55,72 module responsible for the regulation of stomatal aperture that is hijacked by bacterial COR to promote infection. They also provide novel strategies for crop protection against biotrophs without compromising resistance to necrotrophs.

  9. 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.

  10. The discovery of the synovial lymphatic stomata and lymphatic reabsorption in knee effusion.

    PubMed

    Ping, Zepeng; Jiang, Tingting; Wang, Chong; Chen, Zhongyi; Chen, Zhongliang; Wang, Jiaxiong; Wang, Li; Wang, Beibei; Xu, Dandan; Liu, Changming; Li, Zhongjie; Li, Ji-Cheng

    2015-06-01

    To illustrate the mechanism of lymphatic reabsorption in knee joint effusion. The current investigation employed transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques to reveal the ultrastructure of the knee synovial membrane in New Zealand rabbits and human. Ultrastructural changes of the synovial lymphatic stomata were observed by using trypan blue absorption and sodium hydroxide (NaOH) digestion methods, and the animal models of synovitis. New Zealand rabbits and human synovial membranes were composed of two types of synovial cells: type A and type B. No lymphatic stomata were found among type A synovial cells, whereas lymphatic stomata with the diameters ranging 0.74-3.26 µm were found in type B synovial cells, and some stomata were closed. After the NaOH digestion, a number of sieve pores, similar to lymphatic stomata in size and shape, were observed in the dense fibrous connective tissue underneath the type B synovial cells. After injecting trypan blue into the rabbit knee joint cavity, absorption of trypan blue through the lymphatic stomata was observed, suggesting the absorption function of the synovial lymphatic stomata. In the rabbit knee joint synovitis models, the synovial lymphatic stomata diameter enlarged. Some macrophages migrated from the lymphatic stomata, indicating that the synovial lymphatic stomata were involved in the joint effusion absorption and inflammatory response. Our study is the first to report the existence of synovial lymphatic stomata in the New Zealand rabbits and human knee joints. Lymphatic stomata may have an important role in the reabsorption of joint effusion.

  11. Dynamic Behavior of Microtubules during Dynein-dependent Nuclear Migrations of Meiotic Prophase in Fission Yeast

    PubMed Central

    Yamamoto, Ayumu; Tsutsumi, Chihiro; Kojima, Hiroaki; Oiwa, Kazuhiro; Hiraoka, Yasushi

    2001-01-01

    During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex. PMID:11739791

  12. Microtubule binding distinguishes dystrophin from utrophin

    PubMed Central

    Belanto, Joseph J.; Mader, Tara L.; Eckhoff, Michael D.; Strandjord, Dana M.; Banks, Glen B.; Gardner, Melissa K.; Lowe, Dawn A.; Ervasti, James M.

    2014-01-01

    Dystrophin and utrophin are highly similar proteins that both link cortical actin filaments with a complex of sarcolemmal glycoproteins, yet localize to different subcellular domains within normal muscle cells. In mdx mice and Duchenne muscular dystrophy patients, dystrophin is lacking and utrophin is consequently up-regulated and redistributed to locations normally occupied by dystrophin. Transgenic overexpression of utrophin has been shown to significantly improve aspects of the disease phenotype in the mdx mouse; therefore, utrophin up-regulation is under intense investigation as a potential therapy for Duchenne muscular dystrophy. Here we biochemically compared the previously documented microtubule binding activity of dystrophin with utrophin and analyzed several transgenic mouse models to identify phenotypes of the mdx mouse that remain despite transgenic utrophin overexpression. Our in vitro analyses revealed that dystrophin binds microtubules with high affinity and pauses microtubule polymerization, whereas utrophin has no activity in either assay. We also found that transgenic utrophin overexpression does not correct subsarcolemmal microtubule lattice disorganization, loss of torque production after in vivo eccentric contractions, or physical inactivity after mild exercise. Finally, our data suggest that exercise-induced inactivity correlates with loss of sarcolemmal neuronal NOS localization in mdx muscle, whereas loss of in vivo torque production after eccentric contraction-induced injury is associated with microtubule lattice disorganization. PMID:24706788

  13. Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

    PubMed Central

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

    2016-01-01

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

  14. Tao-1 is a negative regulator of microtubule plus-end growth

    PubMed Central

    Liu, Tao; Rohn, Jennifer L.; Picone, Remigio; Kunda, Patricia; Baum, Buzz

    2010-01-01

    Microtubule dynamics are dominated by events at microtubule plus ends as they switch between discrete phases of growth and shrinkage. Through their ability to generate force and direct polar cell transport, microtubules help to organise global cell shape and polarity. Conversely, because plus-end binding proteins render the dynamic instability of individual microtubules sensitive to the local intracellular environment, cyto-architecture also affects the overall distribution of microtubules. Despite the importance of plus-end regulation for understanding microtubule cytoskeletal organisation and dynamics, little is known about the signalling mechanisms that trigger changes in their behaviour in space and time. Here, we identify a microtubule-associated kinase, Drosophila Tao-1, as an important regulator of microtubule stability, plus-end dynamics and cell shape. Active Tao-1 kinase leads to the destabilisation of microtubules. Conversely, when Tao-1 function is compromised, rates of cortical-induced microtubule catastrophe are reduced and microtubules contacting the actin cortex continue to elongate, leading to the formation of long microtubule-based protrusions. These data reveal a role for Tao-1 in controlling the dynamic interplay between microtubule plus ends and the actin cortex in the regulation of cell form. PMID:20647372

  15. 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)

  16. 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)

  17. Disorders of Microtubule Function in Neurons: Imaging Correlates

    PubMed Central

    Mutch, Christopher A.; Poduri, Annapurdi; Sahin, Mustafa; Barry, Brenda; Walsh, Christopher A.; Barkovich, A. James

    2015-01-01

    Background and Significance A number of recent studies have described malformations of cortical development with mutations of components of microtubules and microtubule-associated proteins. Despite examinations of large numbers of MRIs, good phenotype-genotype correlations have been elusive. Additionally, most of these studies focused exclusively on cerebral cortical findings. Materials and Methods MRIs from18 patients with confirmed tubulin mutations (8 TUBA1A, 5 TUBB2B, and 5 TUBB3) and 15 patients with known mutations of the genes encoding microtubule-associated proteins (5 LIS1, 4 DCX, and 6 DYNC1H1) were carefully visually analyzed and compared. Specific note was made of cortical gyral pattern, basal ganglia and white matter to assess internal capsular size, cortical thickness, ventricular and cisternal size, and size and contours of the brain stem, cerebellar hemispheres and vermis, and the corpus callosum of patients with tubulin and microtubule-associated protein gene mutations. Results were determined by unanimous consensus of the authors. Results All patients had abnormal MRI scans. Large proportions of patients with tubulin gene mutations were found to have multiple cortical and subcortical abnormalities including microcephaly, ventriculomegaly, abnormal gyral and sulcal patterns (termed dysgyria), small or absent corpus callosum and small pons. All patients with microtubule-associated proteins mutations also had abnormal cerebral cortices (predominantly pachygyria and agyria), but fewer subcortical abnormalities were noted. Conclusion Comparison of MRIs from patients with known mutations of tubulin genes and microtubule-associated proteins allows for the establishment of some early correlations of phenotype with genotype and may assist in identification and diagnosis of these rare disorders. PMID:26564436

  18. The role of the cytoskeleton during oriented microfibril deposition. I. Elucidation of the possible interaction between microtubules and cellulose synthetic complexes.

    PubMed

    Seagull, R W

    1983-05-01

    A detailed analysis of changes in the cytoskeletal organization during cell elongation and oriented microfibril deposition has been done in the four plant species, clover (Trifolium repens), radish (Raphanus sativus), corn (Zea mays), and sorghum (Sorghum vulgare). Microtubules of variable lengths were found in all the cells examined. Some grouping of microtubules was observed with inter microtubule distances ranging from 14 to 40 nm. Single microfilaments were often observed between parallel microtubules. During cell elongation, microtubule frequency (No./microns) was maintained, thus indicating that microtubules must be formed continuously. The parallel orientation of wall microfibrils is disrupted as they deviate around plasmodesmata and pit-fields; however the cortical microtubules, thought to be influencing microfibril orientation, exhibit no consistent deviation around pit-fields. These observations are used to argue that cortical microtubules cannot influence microfibril orientation through a direct association with cellulose synthetic complexes via microtubule cross bridges.

  19. [Advances in the research of the peritoneal lymphatic stomata in human].

    PubMed

    Li, H; Li, J

    2000-12-01

    Peritoneal lymphatic stomata are small openings of the subperitoneal lymphatic vessels on the free surface of the mesothelium. The peritoneal cavity is connected with lymphatic system via these small openings which are considered to be the main passage-way that can absorb matter from the peritoneal cavity. The lymphatic stomata are claimed to be involved in many clinic procedures, such as ascites elimination; ultrafiltration failure on the continuous ambulatory peritoneal dialysis; metastasis of tumor cells from the peritoneal cavity, and so on. It was reported that the cellular factor-NO(i.e. endothelium-derived relaxing factor, EDRF) can enhance the patency of the stomata and lymphatic absorption of the stomata by stimulating guanylate way, then increasing the concentration of the cGMP, decreasing the concentration of the [Ca2+] and as a result diastole the lymphatic stomata. Some traditional Chinese medicines, which can enhance absorption of ascites, have a regulative function on the stomata by enhancing the NO concentration.

  20. Dendrites In Vitro and In Vivo Contain Microtubules of Opposite Polarity and Axon Formation Correlates with Uniform Plus-End-Out Microtubule Orientation

    PubMed Central

    Yau, Kah Wai; Schätzle, Philipp; Tortosa, Elena; Pagès, Stéphane; Holtmaat, Anthony

    2016-01-01

    In cultured vertebrate neurons, axons have a uniform arrangement of microtubules with plus-ends distal to the cell body (plus-end-out), whereas dendrites contain mixed polarity orientations with both plus-end-out and minus-end-out oriented microtubules. Rather than non-uniform microtubules, uniparallel minus-end-out microtubules are the signature of dendrites in Drosophila and Caenorhabditis elegans neurons. To determine whether mixed microtubule organization is a conserved feature of vertebrate dendrites, we used live-cell imaging to systematically analyze microtubule plus-end orientations in primary cultures of rat hippocampal and cortical neurons, dentate granule cells in mouse organotypic slices, and layer 2/3 pyramidal neurons in the somatosensory cortex of living mice. In vitro and in vivo, all microtubules had a plus-end-out orientation in axons, whereas microtubules in dendrites had mixed orientations. When dendritic microtubules were severed by laser-based microsurgery, we detected equal numbers of plus- and minus-end-out microtubule orientations throughout the dendritic processes. In dendrites, the minus-end-out microtubules were generally more stable and comparable with plus-end-out microtubules in axons. Interestingly, at early stages of neuronal development in nonpolarized cells, newly formed neurites already contained microtubules of opposite polarity, suggesting that the establishment of uniform plus-end-out microtubules occurs during axon formation. We propose a model in which the selective formation of uniform plus-end-out microtubules in the axon is a critical process underlying neuronal polarization. SIGNIFICANCE STATEMENT Live-cell imaging was used to systematically analyze microtubule organization in primary cultures of rat hippocampal neurons, dentate granule cells in mouse organotypic slices, and layer 2/3 pyramidal neuron in somatosensory cortex of living mice. In vitro and in vivo, all microtubules have a plus-end-out orientation in axons

  1. Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process

    PubMed Central

    Hutchins, B. Ian; Wray, Susan

    2014-01-01

    Microtubules are a critical part of neuronal polarity and leading process extension, thus microtubule movement plays an important role in neuronal migration. However, the dynamics of microtubules during the forward movement of the nucleus into the leading process (nucleokinesis) is unclear and may be dependent on the cell type and mode of migration used. In particular, little is known about cytoskeletal changes during axophilic migration, commonly used in anteroposterior neuronal migration. We recently showed that leading process actin flow in migrating GnRH neurons is controlled by a signaling cascade involving IP3 receptors, CaMKK, AMPK, and RhoA. In the present study, microtubule dynamics were examined in GnRH neurons. Failure of the migration of these cells leads to the neuroendocrine disorder Kallmann Syndrome. Microtubules translocated forward along the leading process shaft during migration, but reversed direction and moved toward the nucleus when migration stalled. Blocking calcium release through IP3 receptors halted migration and induced the same reversal of microtubule translocation, while blocking cortical actin flow prevented microtubules from translocating toward the distal leading process. Super-resolution imaging revealed that microtubule plus-end tips are captured at the actin cortex through calcium-dependent mechanisms. This work shows that cortical actin flow draws the microtubule network forward through calcium-dependent capture in order to promote nucleokinesis, revealing a novel mechanism engaged by migrating neurons to facilitate movement. PMID:25505874

  2. Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process.

    PubMed

    Hutchins, B Ian; Wray, Susan

    2014-01-01

    Microtubules are a critical part of neuronal polarity and leading process extension, thus microtubule movement plays an important role in neuronal migration. However, the dynamics of microtubules during the forward movement of the nucleus into the leading process (nucleokinesis) is unclear and may be dependent on the cell type and mode of migration used. In particular, little is known about cytoskeletal changes during axophilic migration, commonly used in anteroposterior neuronal migration. We recently showed that leading process actin flow in migrating GnRH neurons is controlled by a signaling cascade involving IP3 receptors, CaMKK, AMPK, and RhoA. In the present study, microtubule dynamics were examined in GnRH neurons. Failure of the migration of these cells leads to the neuroendocrine disorder Kallmann Syndrome. Microtubules translocated forward along the leading process shaft during migration, but reversed direction and moved toward the nucleus when migration stalled. Blocking calcium release through IP3 receptors halted migration and induced the same reversal of microtubule translocation, while blocking cortical actin flow prevented microtubules from translocating toward the distal leading process. Super-resolution imaging revealed that microtubule plus-end tips are captured at the actin cortex through calcium-dependent mechanisms. This work shows that cortical actin flow draws the microtubule network forward through calcium-dependent capture in order to promote nucleokinesis, revealing a novel mechanism engaged by migrating neurons to facilitate movement.

  3. Disruption of Microtubule Integrity Initiates Mitosis during CNS Repair

    PubMed Central

    Bossing, Torsten; Barros, Claudia S.; Fischer, Bettina; Russell, Steven; Shepherd, David

    2012-01-01

    Summary Mechanisms of CNS repair have vital medical implications. We show that traumatic injury to the ventral midline of the embryonic Drosophila CNS activates cell divisions to replace lost cells. A pilot screen analyzing transcriptomes of single cells during repair pointed to downregulation of the microtubule-stabilizing GTPase mitochondrial Rho (Miro) and upregulation of the Jun transcription factor Jun-related antigen (Jra). Ectopic Miro expression can prevent midline divisions after damage, whereas Miro depletion destabilizes cortical β-tubulin and increases divisions. Disruption of cortical microtubules, either by chemical depolymerization or by overexpression of monomeric tubulin, triggers ectopic mitosis in the midline and induces Jra expression. Conversely, loss of Jra renders midline cells unable to replace damaged siblings. Our data indicate that upon injury, the integrity of the microtubule cytoskeleton controls cell division in the CNS midline, triggering extra mitosis to replace lost cells. The conservation of the identified molecules suggests that similar mechanisms may operate in vertebrates. PMID:22841498

  4. 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.

  5. The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division.

    PubMed

    Ambrose, J Christian; Shoji, Tsubasa; Kotzer, Amanda M; Pighin, Jamie A; Wasteneys, Geoffrey O

    2007-09-01

    Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.

  6. Size scaling of microtubule asters in confinement

    NASA Astrophysics Data System (ADS)

    Pelletier, James; Field, Christine; Krutkramelis, Kaspars; Fakhri, Nikta; Oakey, John; Gatlin, Jay; Mitchison, Timothy

    Microtubule asters are radial arrays of microtubules (MTs) nucleated around organizing centers (MTOCs). Across a wide range of cell types and sizes, aster positioning influences cellular organization. To investigate aster size and positioning, we reconstituted dynamic asters in Xenopus cytoplasmic extract, confined in fluorous oil microfluidic emulsions. In large droplets, we observed centering of MTOCs. In small droplets, we observed a breakdown in natural positioning, with MTOCs at the droplet edge and buckled or bundled MTs along the interface. In different systems, asters are positioned by different forces, such as pushing due to MT polymerization, or pulling due to bulk or cortical dynein. To estimate different contributions to aster positioning, we biochemically perturbed dynactin function, or MT or actin polymerization. We used carbon nanotubes to measure molecular motions and forces in asters. These experimental results inform quantitative biophysical models of aster size and positioning in confinement. JFP was supported by a Fannie and John Hertz Graduate Fellowship.

  7. 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

  8. A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes.

    PubMed

    Zou, Jianwei; Hallen, Mark A; Yankel, Christine D; Endow, Sharyn A

    2008-02-11

    The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.

  9. 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.

  10. 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.

  11. Do prokaryotes contain microtubules?

    PubMed Central

    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. Images PMID:7968920

  12. Hydrogen peroxide modulates the dynamic microtubule cytoskeleton during the defence responses to Verticillium dahliae toxins in Arabidopsis.

    PubMed

    Yao, Lin-Lin; Zhou, Qun; Pei, Bao-Lei; Li, Ying-Zhang

    2011-09-01

    The molecular mechanisms of signal transduction of plants in response to infection by Verticillium dahliae (VD) are not well understood. We previously showed that NO may act as an upstream signalling molecule to trigger the depolymerization of cortical microtubules in Arabidopsis. In the present study, we used the wild-type, and atrbohD and atrbohF mutants of Arabidopsis to explore the mechanisms of action of H(2)O(2) signals and the dynamic microtubule cytoskeleton in defence responses. We demonstrated that H(2)O(2) may also act as an upstream signalling molecule to regulate cortical microtubule depolymerization. The depolymerization of the cortical microtubules played a functional role in the signalling pathway to mediate the expression of defence genes. The results indicate that H(2)O(2) modulates the dynamic microtubule cytoskeleton to trigger the expression of defence genes against V. dahliae toxins (VD-toxins) in Arabidopsis.

  13. BRIDGES BETWEEN MICROTUBULES

    PubMed Central

    McIntosh, J. R.

    1974-01-01

    Bridges between microtubules have been studied with the electron microscope in the axostyle of Saccinobaculus and in various tubule systems of chicken testis, including the helix of tubules surrounding the elongating spermatid nucleus and the flagellum of the sperm tail. In addition to the previously described periodic bridges, evidence is presented that nonperiodic bridges exist between certain tubules. An analysis of axial spacing between adjacent nonperiodic bridges suggests that these structures are attached to periodic binding sites on the microtubule wall, but that not all the binding sites are filled. The bridges appear nonperiodic as a result of random occupancy of some fraction of the periodic sites. The distribution of these binding sites is related to the substructure of the microtubule wall as seen with negative staining and optical diffraction. PMID:4132065

  14. Microtubule-based force generation.

    PubMed

    Kent, Ian A; Lele, Tanmay P

    2017-05-01

    Microtubules are vital to many important cell processes, such as cell division, transport of cellular cargo, organelle positioning, and cell migration. Owing to their diverse functions, understanding microtubule function is an important part of cell biological research that can help in combating various diseases. For example, microtubules are an important target of chemotherapeutic drugs such as paclitaxel because of their pivotal role in cell division. Many functions of microtubules relate to the generation of mechanical forces. These forces are generally either a direct result of microtubule polymerization/depolymerization or generated by motor proteins that move processively along microtubules. In this review, we summarize recent efforts to quantify and model force generation by microtubules in the context of microtubule function. WIREs Nanomed Nanobiotechnol 2017, 9:e1428. doi: 10.1002/wnan.1428 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

  15. The dual effect of abscisic acid on stomata.

    PubMed

    Pantin, Florent; Monnet, Fabien; Jannaud, Dorothée; Costa, Joaquim Miguel; Renaud, Jeanne; Muller, Bertrand; Simonneau, Thierry; Genty, Bernard

    2013-01-01

    The classical view that the drought-related hormone ABA simply acts locally at the guard cell level to induce stomatal closure is questioned by differences between isolated epidermis and intact leaves in stomatal response to several stimuli. We tested the hypothesis that ABA mediates, in addition to a local effect, a remote effect in planta by changing hydraulic regulation in the leaf upstream of the stomata. By gravimetry, porometry to water vapour and argon, and psychrometry, we investigated the effect of exogenous ABA on transpiration, stomatal conductance and leaf hydraulic conductance of mutants described as ABA-insensitive at the guard cell level. We show that foliar transpiration of several ABA-insensitive mutants decreases in response to ABA. We demonstrate that ABA decreases stomatal conductance and down-regulates leaf hydraulic conductance in both the wildtype Col-0 and the ABA-insensitive mutant ost2-2. We propose that ABA promotes stomatal closure in a dual way via its already known biochemical effect on guard cells and a novel, indirect hydraulic effect through a decrease in water permeability within leaf vascular tissues. Variability in sensitivity of leaf hydraulic conductance to ABA among species could provide a physiological basis to the isohydric or anisohydric behaviour.

  16. Distinct light responses of the adaxial and abaxial stomata in intact leaves of Helianthus annuus L.

    PubMed

    Wang, Yin; Noguchi, Ko; Terashima, Ichiro

    2008-09-01

    Using a laboratory-constructed system that can measure the gas exchange rates of two leaf surfaces separately, the light responses of the adaxial and abaxial stomata in intact leaves of sunflower (Helianthus annuus L.) were investigated, keeping the intercellular CO(2) concentration (C(i)) at 300 microL L(-1). When evenly illuminating both sides of the leaf, the stomatal conductance (g(s)) of the abaxial surface was higher than that of the adaxial surface at any light intensity. When each surface of the leaf was illuminated separately, both the adaxial and abaxial stomata were more sensitive to the light transmitted through the leaf (self-transmitted light) than to direct illumination. Relationships between the whole leaf photosynthetic rate (A(n)) and the g(s) for each side highlighted a strong dependence of stomatal opening on mesophyll photosynthesis. Light transmitted through another leaf was more effective than the direct white light for the abaxial stomata, but not for the adaxial stomata. Moreover, green monochromatic light induced an opening of the abaxial stomata, but not of the adaxial stomata. As the proportion of blue light in the transmitted light is less than that in the white light, there may be some uncharacterized light responses, which are responsible for the opening of the abaxial stomata by the transmitted, green light.

  17. Dynamic microtubules at the vegetal cortex predict the embryonic axis in zebrafish.

    PubMed

    Tran, Long Duc; Hino, Hiromu; Quach, Helen; Lim, Shimin; Shindo, Asako; Mimori-Kiyosue, Yuko; Mione, Marina; Ueno, Naoto; Winkler, Christoph; Hibi, Masahiko; Sampath, Karuna

    2012-10-01

    In zebrafish, as in many animals, maternal dorsal determinants are vegetally localized in the egg and are transported after fertilization in a microtubule-dependent manner. However, the organization of early microtubules, their dynamics and their contribution to axis formation are not fully understood. Using live imaging, we identified two populations of microtubules, perpendicular bundles and parallel arrays, which are directionally oriented and detected exclusively at the vegetal cortex before the first cell division. Perpendicular bundles emanate from the vegetal cortex, extend towards the blastoderm, and orient along the animal-vegetal axis. Parallel arrays become asymmetric on the vegetal cortex, and orient towards dorsal. We show that the orientation of microtubules at 20 minutes post-fertilization can predict where the embryonic dorsal structures in zebrafish will form. Furthermore, we find that parallel microtubule arrays colocalize with wnt8a RNA, the candidate maternal dorsal factor. Vegetal cytoplasmic granules are displaced with parallel arrays by ~20°, providing in vivo evidence of a cortical rotation-like process in zebrafish. Cortical displacement requires parallel microtubule arrays, and probably contributes to asymmetric transport of maternal determinants. Formation of parallel arrays depends on Ca(2+) signaling. Thus, microtubule polarity and organization predicts the zebrafish embryonic axis. In addition, our results suggest that cortical rotation-like processes might be more common in early development than previously thought.

  18. Arabidopsis microtubule destabilizing protein40 is involved in brassinosteroid regulation of hypocotyl elongation.

    PubMed

    Wang, Xianling; Zhang, Jin; Yuan, Ming; Ehrhardt, David W; Wang, Zhiyong; Mao, Tonglin

    2012-10-01

    The brassinosteroid (BR) phytohormones play crucial roles in regulating plant cell growth and morphogenesis, particularly in hypocotyl cell elongation. The microtubule cytoskeleton is also known to participate in the regulation of hypocotyl elongation. However, it is unclear if BR regulation of hypocotyl elongation involves the microtubule cytoskeleton. In this study, we demonstrate that BRs mediate hypocotyl cell elongation by influencing the orientation and stability of cortical microtubules. Further analysis identified the previously undiscovered Arabidopsis thaliana microtubule destabilizing protein40 (MDP40) as a positive regulator of hypocotyl cell elongation. Brassinazole-resistant1, a key transcription factor in the BR signaling pathway, directly targets and upregulates MDP40. Overexpression of MDP40 partially rescued the shorter hypocotyl phenotype in BR-deficient mutant de-etiolated-2 seedlings. Reorientation of the cortical microtubules in the cells of MDP40 RNA interference transgenic lines was less sensitive to BR. These findings demonstrate that MDP40 is a key regulator in BR regulation of cortical microtubule reorientation and mediates hypocotyl growth. This study reveals a mechanism involving BR regulation of microtubules through MDP40 to mediate hypocotyl cell elongation.

  19. Involvement of the cytoskeleton in the movement of cortical granules during oocyte maturation, and cortical granule anchoring in mouse eggs.

    PubMed

    Connors, S A; Kanatsu-Shinohara, M; Schultz, R M; Kopf, G S

    1998-08-01

    Exocytosis of cortical granules in mouse eggs is required to produce the zona pellucida block to polyspermy. In this study, we examined the role of microfilaments and microtubules in the regulation of cortical granule movement toward the cortex during oocyte maturation and anchoring of cortical granules in the cortex. Fluorescently labeled cortical granules, microfilaments, and microtubules were visualized using laser-scanning confocal microscopy. It was observed that cortical granules migrate to the periphery of the oocyte during oocyte maturation. This movement is blocked by the treatment of oocytes with cytochalasin D, an inhibitor of microfilament polymerization, but not with nocodazole or colchicine, inhibitors of microtubule polymerization. Cortical granules, once anchored at the cortex, remained in the cortex following treatment of metaphase II-arrested eggs with each of these inhibitors; i.e., there was neither inward movement nor precocious exocytosis. Finally, the single cortical granule-free domain that normally becomes localized over the metaphase II spindle was not observed when the chromosomes become scattered following microtubule disruption with nocodazole or colchicine. In these instances a cortical granule-free domain was observed over each individual chromosome, suggesting that the chromosome or chromosome-associated material, and not the spindle, dictates the localization of the cortical granule-free domain.

  20. Size matters: point pattern analysis biases the estimation of spatial properties of stomata distribution.

    PubMed

    Naulin, Paulette I; Valenzuela, Gerardo; Estay, Sergio A

    2017-03-01

    Stomata distribution is an example of biological patterning. Formal methods used to study stomata patterning are generally based on point-pattern analysis, which assumes that stomata are points and ignores the constraints imposed by size on the placement of neighbors. The inclusion of size in the analysis requires the use of a null model based on finite-size object geometry. In this study, we compare the results obtained by analyzing samples from several species using point and disc null models. The results show that depending on the null model used, there was a 20% reduction in the number of samples classified as uniform; these results suggest that stomata patterning is not as general as currently reported. Some samples changed drastically from being classified as uniform to being classified as clustered. In samples of Arabidopsis thaliana, only the disc model identified clustering at high densities of stomata. This reinforces the importance of selecting an appropriate null model to avoid incorrect inferences about underlying biological mechanisms. Based on the results gathered here, we encourage researchers to abandon point-pattern analysis when studying stomata patterning; more realistic conclusions can be drawn from finite-size object analysis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  1. Transport efficiency through uniformity: organization of veins and stomata in angiosperm leaves.

    PubMed

    Fiorin, Lucia; Brodribb, Timothy J; Anfodillo, Tommaso

    2016-01-01

    Leaves of vascular plants use specific tissues to irrigate the lamina (veins) and to regulate water loss (stomata), to approach homeostasis in leaf hydration during photosynthesis. As both tissues come with attendant costs, it would be expected that the synthesis and spacing of leaf veins and stomata should be coordinated in a way that maximizes benefit to the plant. We propose an innovative geoprocessing method based on image editing and a geographic information system to study the quantitative relationships between vein and stomatal spatial patterns on leaves collected from 31 angiosperm species from different biomes. The number of stomata within each areole was linearly related to the length of the looping vein contour. As a consequence of the presence of free-ending veinlets, the minimum mean distance of stomata from the nearest veins was invariant with areole size in most of the species, and species with smaller distances carried a higher density of stomata. Uniformity of spatial patterning was consistent within leaves and species. Our results demonstrate the existence of an optimal spatial organization of veins and stomata, and suggest their interplay as a key feature for achieving a constant mesophyll hydraulic resistance throughout the leaf.

  2. CLASPs function redundantly to regulate astral microtubules in the C. elegans embryo

    PubMed Central

    Espiritu, Eugenel B.; Krueger, Lori E.; Ye, Anna; Rose, Lesilee S.

    2012-01-01

    Microtubule dynamics are thought to play an important role in regulating microtubule interactions with cortical force generating motor proteins that position the spindle during asymmetric cell division. CLASPs are microtubule-associated proteins that have a conserved role in regulating microtubule dynamics in diverse cell types. Caenorhabditis elegans has three CLASP homologs in its genome. CLS-2 is known to localize to kinetochores and is needed for chromosome segregation at meiosis and mitosis; however CLS-1 and CLS-3 have not been reported to have any role in embryonic development. Here, we show that depletion of CLS-2 in combination with either CLS-1 or CLS-3 results in defects in nuclear rotation, maintenance of spindle length, and spindle displacement in the one-cell embryo. Polarity is normal in these embryos, but reduced numbers of astral microtubules reach all regions of the cortex at the time of spindle positioning. Analysis of the microtubule plus-end tracker EB1 also revealed a reduced number of growing microtubules reaching the cortex in CLASP depleted embryos, but the polymerization rate of astral microtubules was not slower than in wild type. These results indicate that C. elegans CLASPs act partially redundantly to regulate astral microtubules and position the spindle during asymmetric cell division. Further, we show that these spindle pole-positioning roles are independent of the CLS-2 binding proteins HCP-1 and HCP-2. PMID:22613359

  3. Microtubule dynamics in fish melanophores

    PubMed Central

    1994-01-01

    We have studied the dynamics of microtubules in black tetra (Gymnocorymbus ternetzi) melanophores to test the possible correlation of microtubule stability and intracellular particle transport. X- rhodamine-or caged fluorescein-conjugated tubulin were microinjected and visualized by fluorescence digital imaging using a cooled charge coupled device and videomicroscopy. Microtubule dynamics were evaluated by determining the time course of tubulin incorporation after pulse injection, by time lapse observation, and by quantitation of fluorescence redistribution after photobleaching and photoactivation. The time course experiments showed that the kinetics of incorporation of labeled tubulin into microtubules were similar for cells with aggregated or dispersed pigment with most microtubules becoming fully labeled within 15-20 min after injection. Quantitation by fluorescence redistribution after photobleaching and photoactivation confirmed that microtubule turnover was rapid in both states, t1/2 = 3.5 +/- 1.5 and 6.1 +/- 3.0 min for cells with aggregated and dispersed pigment, respectively. In addition, immunostaining with antibodies specific to posttranslationally modified alpha-tubulin, which is usually enriched in stable microtubules, showed that microtubules composed exclusively of detyrosinated tubulin were absent and microtubules containing acetylated tubulin were sparse. We conclude that the microtubules of melanophores are very dynamic, that their dynamic properties do not depend critically on the state of pigment distribution, and that their stabilization is not a prerequisite for intracellular transport. PMID:8089178

  4. Plant microtubule cytoskeleton complexity: microtubule arrays as fractals.

    PubMed

    Gardiner, John; Overall, Robyn; Marc, Jan

    2012-01-01

    Biological systems are by nature complex and this complexity has been shown to be important in maintaining homeostasis. The plant microtubule cytoskeleton is a highly complex system, with contributing factors through interactions with microtubule-associated proteins (MAPs), expression of multiple tubulin isoforms, and post-translational modification of tubulin and MAPs. Some of this complexity is specific to microtubules, such as a redundancy in factors that regulate microtubule depolymerization. Plant microtubules form partial helical fractals that play a key role in development. It is suggested that, under certain cellular conditions, other categories of microtubule fractals may form including isotropic fractals, triangular fractals, and branched fractals. Helical fractal proteins including coiled-coil and armadillo/beta-catenin repeat proteins and the actin cytoskeleton are important here too. Either alone, or in combination, these fractals may drive much of plant development.

  5. Cell Biology: Microtubule Collisions to the Rescue.

    PubMed

    Gardner, Melissa K

    2016-12-19

    The proper regulation of microtubule lengths is fundamental to their cellular function. New work now reports that the collision of a growing microtubule end with another object, such as a microtubule, can contribute to the regulation of microtubule lengths by leaving behind damage that ultimately acts to stabilize the microtubule network.

  6. Role of Ca[sup ++]/calmodulin in the regulation of microtubules in higher plants

    SciTech Connect

    Cyr, R.

    1991-01-01

    This work is aimed at defining the role of calcium/calmodulin in regulating cortical microtubules (MTS) in higher plants. Recent thrust has been to define the effects of calcium upon microtubules in vivo. Using lysed protoplasts, we noted Mts are destabilized by calcium/calmodulin. This effect could be the result of gross depolymerization induced by Ca[sup ++]/calmodulin, or by an increase in the dynamic flux rate. Intact protoplasts exposed to high (10 mM) levels of calcium (which would be expected to increase intercellular calcium levels) contained microtubules that were hypersensitive to Mt inhibitors, compared to control protoplasts exposed to low calcium environments.

  7. Long Astral Microtubules and RACK-1 Stabilize Polarity Domains during Maintenance Phase in Caenorhabditis elegans Embryos

    PubMed Central

    Ai, Erkang; Poole, Daniel S.; Skop, Ahna R.

    2011-01-01

    Cell polarity is a very well conserved process important for cell differentiation, cell migration, and embryonic development. After the establishment of distinct cortical domains, polarity cues have to be stabilized and maintained within a fluid and dynamic membrane to achieve proper cell asymmetry. Microtubules have long been thought to deliver the signals required to polarize a cell. While previous studies suggest that microtubules play a key role in the establishment of polarity, the requirement of microtubules during maintenance phase remains unclear. In this study, we show that depletion of Caenorhabditis elegans RACK-1, which leads to short astral microtubules during prometaphase, specifically affects maintenance of cortical PAR domains and Dynamin localization. We then investigated the consequence of knocking down other factors that also abolish astral microtubule elongation during polarity maintenance phase. We found a correlation between short astral microtubules and the instability of PAR-6 and PAR-2 domains during maintenance phase. Our data support a necessary role for astral microtubules in the maintenance phase of cell polarity. PMID:21533050

  8. 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.

  9. Analysis of Stomata Distribution Patterns for Quantification of the Foliar Plasticity of Tradescantia Zebrina

    NASA Astrophysics Data System (ADS)

    Batista Florindo, Joao; Landini, Gabriel; Almeida Filho, Humberto; Martinez Bruno, Odemir

    2015-09-01

    Here we propose a method for the analysis of the stomata distribution patterns on the surface of plant leaves. We also investigate how light exposure during growth can affect stomata distribution and the plasticity of leaves. Understanding foliar plasticity (the ability of leaves to modify their structural organization to adapt to changing environmental resources) is a fundamental problem in Agricultural and Environmental Sciences. Most published work on quantification of stomata has concentrated on descriptions of their density per unit of leaf area, however density alone does not provide a complete description of the problem and leaves several unanswered questions (e.g. whether the stomata patterns change across various areas of the leaf, or how the patterns change under varying observational scales). We used two approaches here, to know, multiscale fractal dimension and complex networks, as a means to provide a description of the complexity of these distributions. In the experiments, we used 18 samples from the plant Tradescantia Zebrina grown under three different conditions (4 hours of artificial light each day, 24 hours of artificial light each day, and sunlight) for a total of 69 days. The network descriptors were capable of correctly discriminating the different conditions in 88% of cases, while the fractal descriptors discriminated 83% of the samples. This is a significant improvement over the correct classification rates achieved when using only stomata density (56% of the samples).

  10. The evolutionary relations of sunken, covered, and encrypted stomata to dry habitats in Proteaceae.

    PubMed

    Jordan, Gregory J; Weston, Peter H; Carpenter, Raymond J; Dillon, Rebecca A; Brodribb, Timothy J

    2008-05-01

    Sunken, covered, and encrypted stomata have been anecdotally linked with dry climates and reduced transpiration and therefore have been used to infer dry palaeoclimates from fossils. This study assesses the evolutionary and ecological associations of such stomatal protection in a model system-the diverse southern hemisphere family Proteaceae. Analyses were based on the morphology of over 1400 Australian, South African, New Caledonian, New Zealand, and South American species, anatomy of over 300 of these species, and bioclimatic data from all 1109 Australian species. Ancestral state reconstruction revealed that five or six evolutionary transitions explain over 98% of the dry climate species in the family, with a few other, minor invasions of dry climates. Deep encryption, i.e., stomata in deep pits, in grooves, enclosed by tightly revolute margins or strongly overarched by cuticle, evolved at least 11 times in very dry environments. Other forms of stomatal protection (sunken but not closely encrypted stomata, papillae, and layers of hairs covering the stomata) also evolved repeatedly, but had no systematic association with dry climates. These data are evidence for a strong distinction in function, with deep encryption being an adaptation to aridity, whereas broad pits and covered stomata have more complex relations to climate.

  11. Folding of the Tau Protein on Microtubules.

    PubMed

    Kadavath, Harindranath; Jaremko, Mariusz; Jaremko, Łukasz; Biernat, Jacek; Mandelkow, Eckhard; Zweckstetter, Markus

    2015-08-24

    Microtubules are regulated by microtubule-associated proteins. However, little is known about the structure of microtubule-associated proteins in complex with microtubules. Herein we show that the microtubule-associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a β-sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Microtubule arrays of the zebrafish yolk cell: organization and function during epiboly.

    PubMed

    Solnica-Krezel, L; Driever, W

    1994-09-01

    In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover the animal pole of a large yolk cell. At the 500-1000 cell stage, the marginal blastomeres fuse with the yolk cell forming the yolk syncytial layer. During epiboly the blastoderm and the yolk syncytial layer spread toward the vegetal pole. We have studied developmental changes in organization and function during epiboly of two distinct microtubule arrays located in the cortical cytoplasm of the yolk cell. In the anuclear yolk cytoplasmic layer, an array of microtubules extends along the animal-vegetal axis to the vegetal pole. In the early blastula the yolk cytoplasmic layer microtubules appear to originate from the marginal blastomeres. Once formed, the yolk syncytial layer exhibits its own network of intercrossing mitotic or interphase microtubules. The microtubules of the yolk cytoplasmic layer emanate from the microtubule network of the syncytial layer. At the onset of epiboly, the external yolk syncytial layer narrows, the syncytial nuclei become tightly packed and the network of intercrossing microtubules surrounding them becomes denser. Soon after, there is a vegetal expansion of the blastoderm and of the yolk syncytial layer with its network of intercrossing microtubules. Concomitantly, the yolk cytoplasmic layer diminishes and its set of animal-vegetal microtubules becomes shorter. We investigated the involvement of microtubules in epiboly using the microtubule depolymerizing agent nocodazole and a stabilizing agent taxol. In embryos treated with nocodazole, microtubules were absent and epibolic movements of the yolk syncytial nuclei were blocked. In contrast, the vegetal expansion of the enveloping layer and deep cells was only partially inhibited. The process of endocytosis, proposed to play a major role in epiboly of the yolk syncytial layer (Betchaku, T. and Trinkaus, J. P. (1986) Am. Zool. 26, 193-199), was still observed in nocodazole-treated embryos

  13. 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

  14. Active sliding between cytoplasmic microtubules.

    PubMed

    Koonce, M P; Tong, J; Euteneuer, U; Schliwa, M

    Microtubules are versatile cellular polymers that play a role in cell shape determination and mediate various motile processes such as ciliary and flagellar bending, chromosome movements and organelle transport. That a sliding microtubule mechanism can generate force has been demonstrated in highly ordered structures such as axonemes, and microtubule-based force generation almost certainly contributes to the function of mitotic and meiotic spindles. Most cytoplasmic microtubule arrays, however, do not exhibit the structural regularity of axonemes and some spindles, and often appear disorganized. Yet many cellular activities (such as shape changes during morphogenesis, axonal extension and spindle assembly) involve highly coordinated microtubule behaviour and possibly require force generated by an intermicrotubule sliding mechanism, or perhaps use sliding to move microtubules rapidly into a protrusion for stabilization. Here we show that active sliding between cytoplasmic microtubules can occur in microtubule bundles of the amoeba Reticulomyxa. A force-producing mechanism of this sort could be used by this organism to facilitate the extension of cell processes and to generate the dynamic movements of the cytoplasmic network.

  15. Microtubule dynamics in plant cells.

    PubMed

    Buschmann, Henrik; Sambade, Adrian; Pesquet, Edouard; Calder, Grant; Lloyd, Clive W

    2010-01-01

    This chapter describes some of the choices and unavoidable compromises to be made when studying microtubule dynamics in plant cells. The choice of species still depends very much on the ability to produce transgenic plants and most work has been done in the relatively small cells of Arabidopsis plants or in tobacco BY-2 suspension cells. Fluorescence-tagged microtubule proteins have been used to label entire microtubules, or their plus ends, but there are still few minus-end markers for these acentrosomal cells. Pragmatic decisions have to be made about probes, balancing the efficacy of microtubule labeling against a tendency to overstabilize and bundle the microtubules and even induce helical plant growth. A key limitation in visualizing plant microtubules is the ability to keep plants alive for long periods under the microscope and we describe a biochamber that allows for plant cell growth and development while allowing gas exchange and reducing evaporation. Another major difficulty is the limited fluorescence lifetime and we describe imaging strategies to reduce photobleaching in long-term imaging. We also discuss methods of measuring microtubule dynamics, with emphasis on the behavior of plant-specific microtubule arrays. 2010 Elsevier Inc. All rights reserved.

  16. CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells

    PubMed Central

    Toya, Mika; Kobayashi, Saeko; Kawasaki, Miwa; Shioi, Go; Kaneko, Mari; Ishiuchi, Takashi; Misaki, Kazuyo; Meng, Wenxiang; Takeichi, Masatoshi

    2016-01-01

    Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated–spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells. PMID:26715742

  17. 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.

  18. 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.

  19. A "MICROTUBULE" IN A BACTERIUM

    PubMed Central

    van Iterson, Woutera; Hoeniger, Judith F. M.; van Zanten, Eva Nijman

    1967-01-01

    A study of the anchorage of the flagella in swarmers of Proteus mirabilis led to the incidental observation of microtubules. These microtubules were found in thin sections and in whole mount preparations of cells from which most of the content had been released by osmotic shock before staining negatively with potassium phosphotungstate (PTA). The microtubules are in negatively stained preparations about 200 A wide, i.e. somewhat thicker than the flagella (approximately 130 A). They are thus somewhat thinner than most microtubules recorded for other cells. They are referred to as microtubules because of their smooth cylindrical wall, or cortex, surrounding a hollow core which is readily filled with PTA when stained negatively. Since this is probably the first time that such a structure is described inside a bacterium, we do not know for certain whether it represents a normal cell constituent or an abnormality, for instance of the type of "polysheaths" (16). PMID:10976198

  20. Equimolar heterodimers in microtubules

    PubMed Central

    1982-01-01

    Two equimolar beta chains can be resolved from sea urchin sperm flagellar and scallop gill ciliary tubulins, and from certain brain tubulins as well, using the Triton X-100-acid-urea polyacrylamide gel system commonly used for histone analysis. The beta chains are identified as such from their mobility on urea-free SDS PAGE, from amino acid composition, and from tryptic peptide distribution. Scallop beta chains have almost identical amino acid profiles but they differ by one tryptic peptide. Optimal conditions for beta chain resolution are very species-dependent, with some closely related species showing either maximal or no beta chain separation. In addition, beef brain tubulin on Triton X-100-acid-urea electrophoresis and scallop gill ciliary tubulin upon isoelectric focusing in the presence of SDS show two approximately equimolar alpha chains. These data, indicating equimolar amounts of two potentially different tubulin heterodimers from a variety of microtubule types, support a model for microtubule structure wherein protofilaments consist of alternating heterodimers of two kinds, generating a 16-nm (2-dimer) axial repeat. PMID:7202008

  1. Dynamic changes in microtubule configuration correlate with nuclear migration in the preblastoderm Drosophila embryo

    PubMed Central

    1993-01-01

    Drosophila embryogenesis is initiated by a series of syncytial mitotic divisions. The first nine of these divisions are internal, and are accompanied by two temporally distinct nuclear movements that lead to the formation of a syncytial blastoderm with a uniform monolayer of cortical nuclei. The first of these movements, which we term axial expansion, occurs during division cycles 4-6 and distributes nuclei in a hollow ellipsoid underlying the cortex. This is followed by cortical migration, during cycles 7-10, which places the nuclei in a uniform monolayer at the cortex. Here we report that these two movements differ in their geometry, velocity, cell-cycle dependence, and protein synthesis requirement. We therefore conclude that axial expansion and cortical migration are mechanistically distinct, amplifying a similar conclusion based on pharmacological data (Zalokar and Erk, 1976). We have examined microtubule organization during cortical migration and find that a network of interdigitating microtubules connects the migrating nuclei. These anti-parallel microtubule arrays are observed between migrating nuclei and yolk nuclei located deeper in the embryo. These arrays are present during nuclear movement but break down when the nuclei are not moving. We propose that cortical migration is driven by microtubule-dependent forces that repel adjacent nuclei, leading to an expansion of the nuclear ellipsoid established by axial expansion. PMID:8314839

  2. Cuticle Affects Calculations of Internal CO2 in Leaves Closing Their Stomata.

    PubMed

    Tominaga, Jun; Kawamitsu, Yoshinobu

    2015-10-01

    Analyzing the assimilation rate (A) relative to the CO(2) concentration inside leaves (C(i)) has been a useful approach for investigating plant responses to various environments. Nevertheless, there are uncertainties in calculating C(i) when stomata close, restricting the application. Here, A-C(i) curves were traced in sunflower (Helianthus annuus L.) leaves using a method for directly measuring C(i). The method was incorporated into an LI-6400 open gas exchange system, and stomata were closed by feeding 10 µM ABA through petioles. The conductance to CO(2) was derived from the directly measured C(i) and compared with the conductance from the water vapor flux (i.e. the standard calculation). When stomata were open, measured and calculated C(i) gave similar A-C(i) curves. When stomata were closed, the curves differed because measured C(i) departed from the calculated value. This difference caused the calculation to trace an artifactual limitation of photosynthesis. The direct measurement avoided this problem and followed the curve for leaves with open stomata. Largely because of the cuticle, the calculation overestimated CO(2) entry into the leaf because the cuticle transmitted more water vapor than CO(2), and the calculation relied on water vapor. Consequently, the standard calculation gave conductances larger than those from directly measured C(i). Although the cuticle conductance to water vapor remained constant as stomata closed, it increasingly contributed to the overestimation of C(i). The system provided here is not affected by these cuticle properties and thus is expected to open up the opportunity for A-C(i) analysis in plant physiology.

  3. Study on the mechanism of regulation on peritoneal lymphatic stomata with Chinese herbal medicine

    PubMed Central

    Ding, Shi-Ping; Li, Ji-Cheng; Xu, Jian; Mao, Lian-Gen

    2002-01-01

    AIM: To study the mechanism of Chinese herbal medicine (CHM, the prescription consists of Radix Salviae Miltiorrhizae, Radix Codonopsitis Pilosulae, Rhizoma Atractylodis Alba and Rhizoma Alismatis, Leonurus Heterophyllus Sweet, etc) on the regulation of the peritoneal lymphatic stomata and the ascites drainage. METHODS: The mouse model of live fibrosis was established with the application of intragastric installations of carbon tetrachloride once every three days; scanning electron microscope and computer image processing were used to detect the area and the distributive density of the peritoneal lymphatic stomata; and the concentrations of urinary ion and NO in the serum were analyzed in the experiment. RESULTS: Two different doses of CHM could significantly increase the area of the peritoneal lymphatic stomata, promote its distributive density and enhance the drainage of urinary ion such as sodium, potassium and chlorine. Meanwhile, the NO concentration of two different doses of CHM groups was 133.52 ± 23.57 μmol/L, and 137.2 ± 26.79 μmol/L respectively. In comparison with the control group and model groups (48.36 ± 6.83 μmol/L, and 35.22 ± 8.94 μmol/L, P < 0.01), there existed significantly marked difference, this made it clear that Chinese herbal medicine could induce high endogenous NO concentration. The effect of Chinese herbal medicine on the peritoneal lymphatic stomata and the drainage of urinary ion was altered by adding NO donor(sodium nitropurruside, SNP) or NO synthase (NOS) inhibitor (N(G)-monomethyl-L-arginine, L-NMMA) to the peritoneal cavity. CONCLUSION: There existed correlations between high NO concentration and enlargement of the peritoneal lymphatic stomata, which result in enhanced drainage of ascites. These data supported the hypothesis that Chinese herbal medicine could regulate the peritoneal lymphatic stomata by accelerating the synthesis and release of endogenous NO. PMID:11833101

  4. Arabidopsis MICROTUBULE DESTABILIZING PROTEIN40 Is Involved in Brassinosteroid Regulation of Hypocotyl Elongation[C][W][OA

    PubMed Central

    Wang, Xianling; Zhang, Jin; Yuan, Ming; Ehrhardt, David W.; Wang, Zhiyong; Mao, Tonglin

    2012-01-01

    The brassinosteroid (BR) phytohormones play crucial roles in regulating plant cell growth and morphogenesis, particularly in hypocotyl cell elongation. The microtubule cytoskeleton is also known to participate in the regulation of hypocotyl elongation. However, it is unclear if BR regulation of hypocotyl elongation involves the microtubule cytoskeleton. In this study, we demonstrate that BRs mediate hypocotyl cell elongation by influencing the orientation and stability of cortical microtubules. Further analysis identified the previously undiscovered Arabidopsis thaliana MICROTUBULE DESTABILIZING PROTEIN40 (MDP40) as a positive regulator of hypocotyl cell elongation. BRASSINAZOLE-RESISTANT1, a key transcription factor in the BR signaling pathway, directly targets and upregulates MDP40. Overexpression of MDP40 partially rescued the shorter hypocotyl phenotype in BR-deficient mutant de-etiolated-2 seedlings. Reorientation of the cortical microtubules in the cells of MDP40 RNA interference transgenic lines was less sensitive to BR. These findings demonstrate that MDP40 is a key regulator in BR regulation of cortical microtubule reorientation and mediates hypocotyl growth. This study reveals a mechanism involving BR regulation of microtubules through MDP40 to mediate hypocotyl cell elongation. PMID:23115248

  5. Teamwork in microtubule motors.

    PubMed

    Mallik, Roop; Rai, Arpan K; Barak, Pradeep; Rai, Ashim; Kunwar, Ambarish

    2013-11-01

    Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. 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

  7. Stability and function of a putative microtubule organizing center in the human parasite Toxoplasma gondii.

    PubMed

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

    2017-03-22

    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 discovered 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 to 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.

  8. Astral microtubules control redistribution of dynein at the cell cortex to facilitate spindle positioning

    PubMed Central

    Tame, Mihoko A; Raaijmakers, Jonne A; van den Broek, Bram; Lindqvist, Arne; Jalink, Kees; Medema, René H

    2014-01-01

    Cytoplasmic dynein is recruited to the cell cortex in early mitosis, where it can generate pulling forces on astral microtubules to position the mitotic spindle. Recent work has shown that dynein displays a dynamic asymmetric cortical localization, and that dynein recruitment is negatively regulated by spindle pole-proximity. This results in oscillating dynein recruitment to opposite sides of the cortex to center the mitotic spindle. However, although the centrosome-derived signal that promotes displacement of dynein has been identified, it is currently unknown how dynein is re-recruited to the cortex once it has been displaced. Here we show that re-recruitment of cortical dynein requires astral microtubules. We find that microtubules are necessary for the sustained localized enrichment of dynein at the cortex. Furthermore, we show that stabilization of astral microtubules causes spindle misorientation, followed by mispositioning of dynein at the cortex. Thus, our results demonstrate the importance of astral microtubules in the dynamic regulation of cortical dynein recruitment in mitosis. PMID:24553118

  9. Stomata actively regulate internal aeration of the sacred lotus Nelumbo nucifera.

    PubMed

    Matthews, Philip G D; Seymour, Roger S

    2014-02-01

    The sacred lotus Nelumbo nucifera (Gaertn.) possesses a complex system of gas canals that channel pressurized air from its leaves, down through its petioles and rhizomes, before venting this air back to the atmosphere through large stomata found in the centre of every lotus leaf. These central plate stomata (CPS) lie over a gas canal junction that connects with two-thirds of the gas canals within the leaf blade and with the larger of two discrete pairs of gas canals within the petiole that join with those in the rhizome. It is hypothesized that the lotus actively regulates the pressure, direction and rate of airflow within its gas canals by opening and closing these stomata. Impression casting the CPS reveal that they are open in the morning, close at midday and reopen in the afternoon. The periodic closure of the CPS during the day coincides with a temporary reversal in airflow direction within the petiolar gas canals. Experiments show that the conductance of the CPS decreases in response to increasing light level. This behaviour ventilates the rhizome and possibly directs benthic CO2 towards photosynthesis in the leaves. These results demonstrate a novel function for stomata: the active regulation of convective airflow.

  10. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2011-08-20

    electronic properties of a single Microtubule Google Mountain view campus, workshop on quantum biology 22 October 2010 http://www.youtube.com/watch?v...Chemists, Tsukuba, Japan March 1-3, (2011) 3. Quantum aspects of microtubule: Direct experimental evidence for the existence of quantum states in...microtubule, Towards a science of consciousness May 2-8 (2011), Sweden 4. Electromagnetic energy of cells and microtubule: how microtubule research will

  11. Identification and Characterization of a Novel Microtubule-Based Motor Associated with Membranous Organelles in Tobacco Pollen Tubes

    PubMed Central

    Cai, Giampiero; Romagnoli, Silvia; Moscatelli, Alessandra; Ovidi, Elisa; Gambellini, Gabriella; Tiezzi, Antonio; Cresti, Mauro

    2000-01-01

    Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes. PMID:11006343

  12. Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury.

    PubMed

    Tang-Schomer, Min D; Johnson, Victoria E; Baas, Peter W; Stewart, William; Smith, Douglas H

    2012-01-01

    Due to their viscoelastic nature, white matter axons are susceptible to damage by high strain rates produced during traumatic brain injury (TBI). Indeed, diffuse axonal injury (DAI) is one of the most common features of TBI, characterized by the hallmark pathological profiles of axonal bulbs at disconnected terminal ends of axons and periodic swellings along axons, known as "varicosities." Although transport interruption underlies axonal bulb formation, it is unclear how varicosities arise, with multiple sites accumulating transported materials along one axon. Recently, axonal microtubules have been found to physically break during dynamic stretch injury of cortical axons in vitro. Here, the same in vitro model was used in parallel with histopathological analyses of human brains acquired acutely following TBI to examine the potential role of mechanical microtubule damage in varicosity formation post-trauma. Transmission electron microscopy (TEM) following in vitro stretch injury revealed periodic breaks of individual microtubules along axons that regionally corresponded with undulations in axon morphology. However, typically less than a third of microtubules were broken in any region of an axon. Within hours, these sites of microtubule breaks evolved into periodic swellings. This suggests axonal transport may be halted along one broken microtubule, yet can proceed through the same region via other intact microtubules. Similar axonal undulations and varicosities were observed following TBI in humans, suggesting primary microtubule failure may also be a feature of DAI. These data indicate a novel mechanism of mechanical microtubule damage leading to partial transport interruption and varicosity formation in traumatic axonal injury.

  13. Microtubules for Nonlinear Optical Limiting

    DTIC Science & Technology

    1993-12-03

    microtubule alignment and centering was accomplished by attaching a l-mL glass syringe and 26 gauge needle to one or both ends of a glass capillary prefilled ...with liquid crystal or a microtubule/K24 mixture. The capillary ends were epoxy sealed to the syringe needles and then pressure or vacuum was applied...to the capillary by pushing or pulling the syringe plungers. All photographs were taken using a Wild MPS 11 35 mm camera attached to Nikon Optiphot

  14. 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.

  15. Periodic actin structures in neuronal axons are required to maintain microtubules

    PubMed Central

    Qu, Yue; Hahn, Ines; Webb, Stephen E.D.; Pearce, Simon P.; Prokop, Andreas

    2017-01-01

    Axons are cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily conserved, ubiquitous, highly ordered feature of axons, the function of PMS is unknown. Here we studied PMS abundance, organization, and function, combining versatile Drosophila genetics with superresolution microscopy and various functional readouts. Analyses with 11 actin regulators and three actin-targeting drugs suggest that PMS contains short actin filaments that are depolymerization resistant and sensitive to spectrin, adducin, and nucleator deficiency, consistent with microscopy-derived models proposing PMS as specialized cortical actin. Upon actin removal, we observed gaps in microtubule bundles, reduced microtubule polymerization, and reduced axon numbers, suggesting a role of PMS in microtubule organization. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilizing protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerization contributes to their maintenance in axons. We discuss potential implications of this novel PMS function along axon shafts for axon maintenance and regeneration. PMID:27881663

  16. Caenorhabditis elegans EFA-6 limits microtubule growth at the cell cortex.

    PubMed

    O'Rourke, Sean M; Christensen, Sara N; Bowerman, Bruce

    2010-12-01

    Microtubules are polymers of tubulin heterodimers that exhibit dynamic instability: periods of growth followed by periods of shrinkage. However, the molecular regulation of dynamic instability remains elusive. Here, we show that EFA-6, a cortically-localized protein, limits the growth of microtubules near the cell cortex of early embryonic cells from Caenorhabditis elegans, possibly by inducing microtubule catastrophes. Compared with wild type, embryos lacking EFA-6 had abnormally long and dense microtubules at the cell cortex, and growing microtubule plus ends resided at the cortex for up to five-fold longer. Loss of EFA-6 also caused excess centrosome separation and displacement towards the cell cortex early in mitosis, and subsequently a loss of anaphase spindle-pole oscillations and increased rates of spindle elongation. The centrosome separation phenotype was dependent on the motor protein dynein, suggesting a possible link between the modulation of microtubule dynamics at the cortex and dynein-dependent force production. EFA-6 orthologues activate ARF6-type GTPases to regulate vesicle trafficking. However, we show that only the C. elegans EFA-6 amino-terminus is both necessary and sufficient to limit microtubule growth along the cortex, and that this function is independent of ARF-6.

  17. Isolation of a 90-kD Microtubule-Associated Protein from Tobacco Membranes.

    PubMed Central

    Marc, J.; Sharkey, D. E.; Durso, N. A.; Zhang, M.; Cyr, R. J.

    1996-01-01

    The organization and function of microtubules in plant cells are important in key developmental events, including the regulation of directional cellulose deposition. Bridges connecting microtubules to each other and to membranes and other organelles have been documented by electron microscopy; however, the biochemical and molecular nature of these linkages is not known. We have partitioned proteins from a suspension culture of tobacco into cytosolic and membrane fractions, solubilized the membrane fraction with a zwitterionic detergent, and then used affinity chromatography and salt elution to isolate tubulin binding proteins. Dark-field microscopy of in vitro-assembled microtubules showed that the eluted proteins from both fractions induce microtubule bundling and, in the presence of purified tubulin, promote microtubule elongation. Gel electrophoresis of the eluted proteins revealed two distinct sets of polypeptides. Those in the membrane eluate included unique bands with apparent molecular masses of 98, 90, and 75 kD in addition to bands present in both eluates. The cytosolic eluate, in contrast, typically included relatively smaller proteins. The eluted proteins also bound to taxol-stabilized microtubules. Initial immunological characterization using monoclonal antibodies raised against the 90-kD polypeptide showed that it is colocalized in situ with cortical microtubules in tobacco protoplast ghosts. PMID:12239375

  18. Microtubule Dynamics in Living Root Hairs: Transient Slowing by Lipochitin Oligosaccharide Nodulation SignalsW⃞

    PubMed Central

    Vassileva, Valya N.; Kouchi, Hiroshi; Ridge, Robert W.

    2005-01-01

    The incorporation of a fusion of green fluorescent protein and tubulin-α 6 from Arabidopsis thaliana in root hairs of Lotus japonicus has allowed us to visualize and quantify the dynamic parameters of the cortical microtubules in living root hairs. Analysis of individual microtubule turnover in real time showed that only plus polymer ends contributed to overall microtubule dynamicity, exhibiting dynamic instability as the main type of microtubule behavior in Lotus root hairs. Comparison of the four standard parameters of in vivo dynamic instability—the growth rate, the disassembly rate, and the frequency of transitions from disassembly to growth (rescue) and from growth to disassembly (catastrophe)—revealed that microtubules in young root hairs were more dynamic than those in mature root hairs. Either inoculation with Mesorhizobium loti or purified M. loti lipochitin oligosaccharide signal molecules (Nod factors) significantly affected the growth rate and transition frequencies in emerging and growing root hairs, making microtubules less dynamic at a specific window after symbiotic inoculation. This response of root hair cells to rhizobial Nod factors is discussed in terms of the possible biological significance of microtubule dynamics in the early signaling events leading to the establishment and progression of the globally important Rhizobium/legume symbiosis. PMID:15863517

  19. Microtubule bundling and shape transitions: Mechanics, interactions, and self-assemblies

    NASA Astrophysics Data System (ADS)

    Needleman, Daniel Joseph

    Microtubules associate to form bundles in vivo in a wide variety of contexts including the mitotic spindle, neuronal processes, and the cortical array in plant cells. These supramolecular assemblies differ in size and shape, and in their internal structure, but the principles that determine this variation in morphology are not understood. To help elucidate such principals we constructed microtubule bundles in vitro using a variety of bundling agents. We have characterized the structure of these supramolecular assemblies of microtubules from the nanoscale to the mesoscale using synchrotron x-ray scattering and diffraction, video enhanced DIC and fluorescence microscopy, and electron microscopy. In the presence of inert polymers, an osmotic pressure imbalance between the inside and the outside of the microtubules may cause them to buckle to a non-circular cross-section. Depletion effects cause these distorted microtubules to bundle into a lattice with rectangular symmetry. The critical buckling pressure provides a measure of the stiffness of the inter-protofilament bond, and we determined that microtubule associated proteins enhance the strength of this bond, while the chemotherapeutic drug taxol has no effect. Multivalent ions cause microtubules to associate into bundles whose morphology depends on the condensing ion. Tightly packed hexagonal bundles with controllable diameters are observed for large tri-, tetra-, and pentavalent counterions. Unexpectedly, in the presence of small divalent cations, we have discovered a living necklace bundle phase, comprised of dynamical assemblies of MT nematic membranes with linear, branched, and loop topologies. Cations may also cause tubulin to assemble into non-microtubule structures. For example, in the presence of spermine, over time the microtubule bundles transform into a columnar phase of inverted tubules, such that the surface which was facing outside of the microtubules switches to the inside. This rearrangement between

  20. Persistence Length of Stable Microtubules

    NASA Astrophysics Data System (ADS)

    Hawkins, Taviare; Mirigian, Matthew; Yasar, M. Selcuk; Ross, Jennifer

    2011-03-01

    Microtubules are a vital component of the cytoskeleton. As the most rigid of the cytoskeleton filaments, they give shape and support to the cell. They are also essential for intracellular traffic by providing the roadways onto which organelles are transported, and they are required to reorganize during cellular division. To perform its function in the cell, the microtubule must be rigid yet dynamic. We are interested in how the mechanical properties of stable microtubules change over time. Some ``stable'' microtubules of the cell are recycled after days, such as in the axons of neurons or the cilia and flagella. We measured the persistence length of freely fluctuating taxol-stabilized microtubules over the span of a week and analyzed them via Fourier decomposition. As measured on a daily basis, the persistence length is independent of the contour length. Although measured over the span of the week, the accuracy of the measurement and the persistence length varies. We also studied how fluorescently-labeling the microtubule affects the persistence length and observed that a higher labeling ratio corresponded to greater flexibility. National Science Foundation Grant No: 0928540 to JLR.

  1. Unconventional functions of microtubule motors.

    PubMed

    Muresan, Virgil; Muresan, Zoia

    2012-04-01

    With the functional characterization of proteins advancing at fast pace, the notion that one protein performs different functions - often with no relation to each other - emerges as a novel principle of how cells work. Molecular motors are no exception to this new development. Here, we provide an account on recent findings revealing that microtubule motors are multifunctional proteins that regulate many cellular processes, in addition to their main function in transport. Some of these functions rely on their motor activity, but others are independent of it. Of the first category, we focus on the role of microtubule motors in organelle biogenesis, and in the remodeling of the cytoskeleton, especially through the regulation of microtubule dynamics. Of the second category, we discuss the function of microtubule motors as static anchors of the cargo at the destination, and their participation in regulating signaling cascades by modulating interactions between signaling proteins, including transcription factors. We also review atypical forms of transport, such as the cytoplasmic streaming in the oocyte, and the movement of cargo by microtubule fluctuations. Our goal is to provide an overview of these unexpected functions of microtubule motors, and to incite future research in this expanding field.

  2. Microtubules as key cytoskeletal elements in cellular transport and shape changes: their expected responses to space environments

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

    Application of reference standard reagents to alternatively depolymerize or stabilize microtubules in a cell that undergoes very regular cytoskeleton-dependent shape changes provides a model system in which some expected components of the environments of spacecraft and space can be tested on Earth for their effects on the cytoskeleton. The fertilized eggs of Ilyanassa obsoleta undergo polar lobe formation by repeated, dramatic, constriction and relaxation of a microfilamentous band localized in the cortical cytoplasm and activated by microtubules.

  3. Microtubules as key cytoskeletal elements in cellular transport and shape changes: their expected responses to space environments

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

    Application of reference standard reagents to alternatively depolymerize or stabilize microtubules in a cell that undergoes very regular cytoskeleton-dependent shape changes provides a model system in which some expected components of the environments of spacecraft and space can be tested on Earth for their effects on the cytoskeleton. The fertilized eggs of Ilyanassa obsoleta undergo polar lobe formation by repeated, dramatic, constriction and relaxation of a microfilamentous band localized in the cortical cytoplasm and activated by microtubules.

  4. Cell proliferation, cell shape, and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space.

    PubMed

    Sieberer, Björn J; Kieft, Henk; Franssen-Verheijen, Tiny; Emons, Anne Mie C; Vos, Jan W

    2009-11-01

    The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant's final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March-April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.

  5. Microtubules in plants.

    PubMed

    Hashimoto, Takashi

    2015-01-01

    Microtubules (MTs) are highly conserved polar polymers that are key elements of the eukaryotic cytoskeleton and are essential for various cell functions. αβ-tubulin, a heterodimer containing one structural GTP and one hydrolysable and exchangeable GTP, is the building block of MTs and is formed by the sequential action of several molecular chaperones. GTP hydrolysis in the MT lattice is mechanistically coupled with MT growth, thus giving MTs a metastable and dynamic nature. MTs adopt several distinct higher-order organizations that function in cell division and cell morphogenesis. Small molecular weight compounds that bind tubulin are used as herbicides and as research tools to investigate MT functions in plant cells. The de novo formation of MTs in cells requires conserved γ-tubulin-containing complexes and targeting/activating regulatory proteins that contribute to the geometry of MT arrays. Various MT regulators and tubulin modifications control the dynamics and organization of MTs throughout the cell cycle and in response to developmental and environmental cues. Signaling pathways that converge on the regulation of versatile MT functions are being characterized.

  6. Microtubules in Plants

    PubMed Central

    Hashimoto, Takashi

    2015-01-01

    Microtubules (MTs) are highly conserved polar polymers that are key elements of the eukaryotic cytoskeleton and are essential for various cell functions. αβ-tubulin, a heterodimer containing one structural GTP and one hydrolysable and exchangeable GTP, is the building block of MTs and is formed by the sequential action of several molecular chaperones. GTP hydrolysis in the MT lattice is mechanistically coupled with MT growth, thus giving MTs a metastable and dynamic nature. MTs adopt several distinct higher-order organizations that function in cell division and cell morphogenesis. Small molecular weight compounds that bind tubulin are used as herbicides and as research tools to investigate MT functions in plant cells. The de novo formation of MTs in cells requires conserved γ-tubulin-containing complexes and targeting/activating regulatory proteins that contribute to the geometry of MT arrays. Various MT regulators and tubulin modifications control the dynamics and organization of MTs throughout the cell cycle and in response to developmental and environmental cues. Signaling pathways that converge on the regulation of versatile MT functions are being characterized. PMID:26019693

  7. Anti-Microtubule Drugs.

    PubMed

    Florian, Stefan; Mitchison, Timothy J

    2016-01-01

    Small molecule drugs that target microtubules (MTs), many of them natural products, have long been important tools in the MT field. Indeed, tubulin (Tb) was discovered, in part, as the protein binding partner of colchicine. Several anti-MT drug classes also have important medical uses, notably colchicine, which is used to treat gout, familial Mediterranean fever (FMF), and pericarditis, and the vinca alkaloids and taxanes, which are used to treat cancer. Anti-MT drugs have in common that they bind specifically to Tb in the dimer, MT or some other form. However, their effects on polymerization dynamics and on the human body differ markedly. Here we briefly review the most-studied molecules, and comment on their uses in basic research and medicine. Our focus is on practical applications of different anti-MT drugs in the laboratory, and key points that users should be aware of when designing experiments. We also touch on interesting unsolved problems, particularly in the area of medical applications. In our opinion, the mechanism by which any MT drug cures or treats any disease is still unsolved, despite decades of research. Solving this problem for particular drug-disease combinations might open new uses for old drugs, or provide insights into novel routes for treatment.

  8. Self-repair promotes microtubule rescue

    PubMed Central

    Gaillard, Jérémie; John, Karin; Blanchoin, Laurent; Théry, Manuel

    2016-01-01

    Summary The dynamic instability of microtubules is characterised by slow growth phases stochastically interrupted by rapid depolymerisations called catastrophes. Rescue events can arrest the depolymerisation and restore microtubule elongation. However the origin of these rescue events remain unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints. PMID:27617929

  9. Role of Ca{sup ++}/calmodulin in the regulation of microtubules in higher plants. Progress report, FY91

    SciTech Connect

    Cyr, R.

    1991-12-31

    This work is aimed at defining the role of calcium/calmodulin in regulating cortical microtubules (MTS) in higher plants. Recent thrust has been to define the effects of calcium upon microtubules in vivo. Using lysed protoplasts, we noted Mts are destabilized by calcium/calmodulin. This effect could be the result of gross depolymerization induced by Ca{sup ++}/calmodulin, or by an increase in the dynamic flux rate. Intact protoplasts exposed to high (10 mM) levels of calcium (which would be expected to increase intercellular calcium levels) contained microtubules that were hypersensitive to Mt inhibitors, compared to control protoplasts exposed to low calcium environments.

  10. A scanning electron microscopy and computer image processing morphometric study of the pharmacological regulation of patency of the peritoneal stomata.

    PubMed

    Li, J; Lu, Z; Wu, N; Zhou, J; Shi, Y

    1996-10-01

    The experiment on mice was carried out by injecting intraperitoneally Chinese materia medica for treating hepatocirrhosis with ascites. Observations and a quantitative analysis were carried out on the pharmacological regulation of the peritoneal stomata by using a scanning electron microscope (SEM) and a computer image processing system attached to the SEM. There was a significant increase in both the diameter (P < 0.05) and distribution density (P < 0.01) of the peritoneal stomata in the red sage root and alismatis rhizome groups, whereas the effect of poria and poria peel was not significant compared with the control group (P > 0.05). Our findings confirm the effect of red sage root and alismatis rhizome on the regulation of the peritoneal stomata, which can enhance the absorption of ascitic fluid, taking into consideration the absorbent function of these stomata. They indicate that the patency of peritoneal stomata can vary in response to the effect of some Chinese materia. They also suggest that the ascites is drained mainly by means of enhancing the patency of the stomata and lymphatic absorption of the stomata during the process of treatment by traditional Chinese medicine.

  11. The Cotton Kinesin-Like Calmodulin-Binding Protein Associates with Cortical Microtubles in Cotton Fibers

    SciTech Connect

    Preuss, Mary L.; Delmar, Deborah P.; Liu, Bo

    2003-05-01

    Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP was detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus the results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.

  12. Microtubule defects & Neurodegeneration.

    PubMed

    Baird, Fiona J; Bennett, Craig L

    2013-12-06

    One of the major challenges facing the long term survival of neurons is their requirement to maintain efficient axonal transport over long distances. In humans as large, long-lived vertebrates, the machinery maintaining neuronal transport must remain efficient despite the slow accumulation of cell damage during aging. Mutations in genes encoding proteins which function in the transport system feature prominently in neurologic disorders. Genes known to cause such disorders and showing traditional Mendelian inheritance have been more readily identified. It has been more difficult, however, to isolate factors underlying the complex genetics contributing to the more common idiopathic forms of neurodegenerative disease. At the heart of neuronal transport is the rail network or scaffolding provided by neuron specific microtubules (MTs). The importance of MT dynamics and stability is underscored by the critical role tau protein plays in MT-associated stabilization versus the dysfunction seen in Alzheimer's disease, frontotemporal dementia and other tauopathies. Another example of the requirement for tight regulation of MT dynamics is the need to maintain balanced levels of post-translational modification of key MT building-blocks such as α-tubulin. Tubulins require extensive polyglutamylation at their carboxyl-terminus as part of a novel post-translational modification mechanism to signal MT growth versus destabilization. Dramatically, knock-out of a gene encoding a deglutamylation family member causes an extremely rapid cell death of Purkinje cells in the ataxic mouse model, pcd. This review will examine a range of neurodegenerative conditions where current molecular understanding points to defects in the stability of MTs and axonal transport to emphasize the central role of MTs in neuron survival.

  13. Stomata size and spatial pattern effects on leaf gas exchange - a quantitative assessment of plant evolutionary choices

    NASA Astrophysics Data System (ADS)

    Or, Dani; Assouline, Shmuel; Aminzadeh, Milad; Haghighi, Erfan; Schymanski, Stan; Lehmann, Peter

    2014-05-01

    Land plants developed a dynamically gas-permeable layer at their leaf surfaces to allow CO2 uptake for photosynthesis while controlling water vapor loss through numerous adjustable openings (stomata) in the impervious leaf epidermis. Details of stomata structure, density and function may vary greatly among different plant families and respond to local environmental conditions, yet they share basic traits in dynamically controlling gaseous exchange rates by varying stomata apertures. We implement a pore scale gas diffusion model to quantitatively interpret the functionality of different combinations of stomata size and pattern on leaf gas exchange and thermal management based on data from fossil records and contemporary data sets. Considering all available data we draw several general conclusions concerning stomata design considerations: (1) the sizes and densities of stomata in the available fossil record leaves were designed to evaporate at rates in the range 0.75≤e/e0 ≤0.99 (relative to free water evaporation); (2) examination of evaporation curves show that for a given stomata size, the density (jointly defining the leaf evaporating area when fully open) was chosen to enable a high sensitivity in reducing evaporation rate with incremental stomatal closure, nevertheless, results show the design includes safety margins to account for different wind conditions (boundary layer thickness); (3) scaled for mean vapor flux, the size of stomata plays a minor role in the uniformity of leaf thermal field for a given stomata density. These principles enable rationale assessment of plant response to raising CO2, and provide a physical framework for considering the consequences of different stomata patterns (patchy) on leaf gas exchange (and thermal regime). In contrast with present quantitative description of traits and functionality of these dynamic covers in terms of gaseous diffusion resistance (or conductance), where stomata size, density and spatial pattern are

  14. Elevated-CO2 Response of Stomata and Its Dependence on Environmental Factors

    PubMed Central

    Xu, Zhenzhu; Jiang, Yanling; Jia, Bingrui; Zhou, Guangsheng

    2016-01-01

    Stomata control the flow of gases between plants and the atmosphere. This review is centered on stomatal responses to elevated CO2 concentration and considers other key environmental factors and underlying mechanisms at multiple levels. First, an outline of general responses in stomatal conductance under elevated CO2 is presented. Second, stomatal density response, its development, and the trade-off with leaf growth under elevated CO2 conditions are depicted. Third, the molecular mechanism regulating guard cell movement at elevated CO2 is suggested. Finally, the interactive effects of elevated CO2 with other factors critical to stomatal behavior are reviewed. It may be useful to better understand how stomata respond to elevated CO2 levels while considering other key environmental factors and mechanisms, including molecular mechanism, biochemical processes, and ecophysiological regulation. This understanding may provide profound new insights into how plants cope with climate change. PMID:27242858

  15. Removal of benzene from indoor air by Dracaena sanderiana: Effect of wax and stomata

    NASA Astrophysics Data System (ADS)

    Treesubsuntorn, Chairat; Thiravetyan, Paitip

    2012-09-01

    From screening 8 ornamental plants, it was found that Dracaena sanderiana had the highest benzene removal efficiency. In a long-term study, 4 cycles of benzene were studied under both 24 h dark and 24 h light conditions. From the 2nd to 4th cycle, benzene uptake by plants under 24 h light condition had higher intensity than under 24 h dark conditions, and the close of D. sanderiana stomata was found only in 24 h dark condition. At the final cycle, D. sanderiana still survived, and benzene uptake continued. From the calculation, 46% of benzene was taken up by D. sanderiana crude wax, while 54% was predicted to be taken up by the stomata by 72 h.

  16. Elevated-CO2 Response of Stomata and Its Dependence on Environmental Factors.

    PubMed

    Xu, Zhenzhu; Jiang, Yanling; Jia, Bingrui; Zhou, Guangsheng

    2016-01-01

    Stomata control the flow of gases between plants and the atmosphere. This review is centered on stomatal responses to elevated CO2 concentration and considers other key environmental factors and underlying mechanisms at multiple levels. First, an outline of general responses in stomatal conductance under elevated CO2 is presented. Second, stomatal density response, its development, and the trade-off with leaf growth under elevated CO2 conditions are depicted. Third, the molecular mechanism regulating guard cell movement at elevated CO2 is suggested. Finally, the interactive effects of elevated CO2 with other factors critical to stomatal behavior are reviewed. It may be useful to better understand how stomata respond to elevated CO2 levels while considering other key environmental factors and mechanisms, including molecular mechanism, biochemical processes, and ecophysiological regulation. This understanding may provide profound new insights into how plants cope with climate change.

  17. Sensitivity of Stomata to Abscisic Acid (An Effect of the Mesophyll).

    PubMed Central

    Trejo, C. L.; Davies, W. J.; Ruiz, LdMP.

    1993-01-01

    The effects of added abscisic acid (ABA) on the stomatal behavior of Commelina communis L. were tested using three different systems. ABA was applied to isolated epidermis or to leaf pieces incubated in the light in bathing solutions perfused with CO2-free air. ABA was also fed to detached leaves in a transpiration bioassay. The apparent sensitivity of stomata to ABA was highly dependent on the method used to feed ABA. Stomata of isolated epidermis were apparently most sensitive to ABA, such that a concentration of 1 [mu]M caused almost complete stomatal closure. When pieces of whole leaves were floated on solutions of ABA of the same concentration, the stomata were almost completely open. The same concentration of ABA fed through the midrib of transpiring detached leaves caused an intermediate response. These differences in stomatal sensitivity to added ABA were found to be a function of differences in the ABA concentration in the epidermes. Comparison of the three application systems suggested that, when leaf pieces were incubated in ABA or fed with ABA through the midrib, accumulation of ABA in the epidermes was limited by the presence of the mesophyll. Even bare mesophyll incubated in ABA solution did not accumulate ABA. Accumulation of radioactivity by leaf pieces floated on [3H]ABA confirmed ABA uptake in this system. Experiments with tetcyclacis, an inhibitor of phaseic acid formation, suggested that rapid metabolism of ABA in mesophyll can have a controlling influence on ABA concentration in both the mesophyll and the epidermis. Inhibition of ABA catabolism with tetcyclacis allows ABA accumulation and increases the apparent sensitivity of stomata to applied ABA. The results are discussed in the context of an important role for ABA metabolism in the regulation of stomatal behavior. PMID:12231838

  18. Acclimation to humidity modifies the link between leaf size and the density of veins and stomata.

    PubMed

    Carins Murphy, Madeline R; Jordan, Gregory J; Brodribb, Timothy J

    2014-01-01

    The coordination of veins and stomata during leaf acclimation to sun and shade can be facilitated by differential epidermal cell expansion so large leaves with low vein and stomatal densities grow in shade, effectively balancing liquid- and vapour-phase conductances. As the difference in vapour pressure between leaf and atmosphere (VPD) determines transpiration at any given stomatal density, we predict that plants grown under high VPD will modify the balance between veins and stomata to accommodate greater maximum transpiration. Thus, we examined the developmental responses of these traits to contrasting VPD in a woody angiosperm (Toona ciliata M. Roem.) and tested whether the relationship between them was altered. High VPD leaves were one-third the size of low VPD leaves with only marginally greater vein and stomatal density. Transpirational homeostasis was thus maintained by reducing stomatal conductance. VPD acclimation changed leaf size by modifying cell number. Hence, plasticity in vein and stomatal density appears to be generated by plasticity in cell size rather than cell number. Thus, VPD affects cell number and leaf size without changing the relationship between liquid- and vapour-phase conductances. This results in inefficient acclimation to VPD as stomata remain partially closed under high VPD.

  19. Effects of cadmium on photosynthetic oxygen evolution from single stomata in Brassica juncea (L.) Czern.

    PubMed

    Zhu, Renkang; Macfie, Sheila M; Ding, Zhifeng

    2008-12-16

    Scanning electrochemical microscopy (SECM) was utilized to investigate photosynthetic oxygen evolution from single stomata in leaves of live Brassica juncea (L.) Czern cultured in nutrient solution to which 0.2 or 0.01 mM CdC12 had been added. The bulk leaf surface serves as an insulator normally; therefore, a typical negative feedback was observed on the probe approach curves (PACs) when the probe approached epidermal cells. When the probe tip approached an open stoma, a higher tip current was detected due to the O2 release from this stoma. Thus, SECM can be used to map the O2 concentration profile near the leaf surface and study stomatal complex structure size and density. The oxygen release from single stomata was also analyzed by comparison of experimental PACs with those simulated by COMSOL multiphysics software (version 3.4). In addition to an increase in the stomatal complex size and a decrease in the complex density, the Cd accumulation caused up to a 26% decrease in photosynthetic rate determined at the level of a single stoma. The O2 evolution was also monitored by recording the tip current vs time when a tip sat above the center of a stoma. Periodic peaks in O2 release-time curves were observed, varying from 400 to 1600 s. The opening and closing activities of single stomata were also imaged by SECM.

  20. Gamma-aminobutyric acid depletion affects stomata closure and drought tolerance of Arabidopsis thaliana.

    PubMed

    Mekonnen, Dereje Worku; Flügge, Ulf-Ingo; Ludewig, Frank

    2016-04-01

    A rapid accumulation of γ-aminobutyric acid (GABA) during biotic and abiotic stresses is well documented. However, the specificity of the response and the primary role of GABA under such stress conditions are hardly understood. To address these questions, we investigated the response of the GABA-depleted gad1/2 mutant to drought stress. GABA is primarily synthesized from the decarboxylation of glutamate by glutamate decarboxylase (GAD) which exists in five copies in the genome of Arabidopsis thaliana. However, only GAD1 and GAD2 are abundantly expressed, and knockout of these two copies dramatically reduced the GABA content. Phenotypic analysis revealed a reduced shoot growth of the gad1/2 mutant. Furthermore, the gad1/2 mutant was wilted earlier than the wild type following a prolonged drought stress treatment. The early-wilting phenotype was due to an increase in stomata aperture and a defect in stomata closure. The increase in stomata aperture contributed to higher stomatal conductance. The drought oversensitive phenotype of the gad1/2 mutant was reversed by functional complementation that increases GABA level in leaves. The functionally complemented gad1/2 x pop2 triple mutant contained more GABA than the wild type. Our findings suggest that GABA accumulation during drought is a stress-specific response and its accumulation induces the regulation of stomatal opening thereby prevents loss of water.

  1. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

    PubMed

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-10-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation.

  2. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments

    PubMed Central

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C.; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-01-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H+-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation. PMID:26272553

  3. Optimization of isopolar microtubule arrays.

    PubMed

    Agayan, Rodney R; Tucker, Robert; Nitta, Takahiro; Ruhnow, Felix; Walter, Wilhelm J; Diez, Stefan; Hess, Henry

    2013-02-19

    Isopolar arrays of aligned cytoskeletal filaments are components in a number of designs of hybrid nanodevices incorporating biomolecular motors. For example, a combination of filament arrays and motor arrays can form an actuator or a molecular engine resembling an artificial muscle. Here, isopolar arrays of microtubules are fabricated by flow alignment, and their quality is characterized by their degree of alignment. We find, in agreement with our analytical models, that the degree of alignment is ultimately limited by thermal forces, while the kinetics of the alignment process are influenced by the flow strength, the microtubule stiffness, the gliding velocity, and the tip length. Strong flows remove microtubules from the surface and reduce the filament density, suggesting that there is an optimal flow strength for the fabrication of ordered arrays.

  4. Fluctuation Analysis of Centrosomes Reveals a Cortical Function of Kinesin-1

    PubMed Central

    Winkler, Franziska; Gummalla, Maheshwar; Künneke, Lutz; Lv, Zhiyi; Zippelius, Annette; Aspelmeier, Timo; Grosshans, Jörg

    2015-01-01

    The actin and microtubule networks form the dynamic cytoskeleton. Network dynamics is driven by molecular motors applying force onto the networks and the interactions between the networks. Here we assay the dynamics of centrosomes in the scale of seconds as a proxy for the movement of microtubule asters. With this assay we want to detect the role of specific motors and of network interaction. During interphase of syncytial embryos of Drosophila, cortical actin and the microtubule network depend on each other. Centrosomes induce cortical actin to form caps, whereas F-actin anchors microtubules to the cortex. In addition, lateral interactions between microtubule asters are assumed to be important for regular spatial organization of the syncytial embryo. The functional interaction between the microtubule asters and cortical actin has been largely analyzed in a static manner, so far. We recorded the movement of centrosomes at 1 Hz and analyzed their fluctuations for two processes—pair separation and individual movement. We found that F-actin is required for directional movements during initial centrosome pair separation, because separation proceeds in a diffusive manner in latrunculin-injected embryos. For assaying individual movement, we established a fluctuation parameter as the deviation from temporally and spatially slowly varying drift movements. By analysis of mutant and drug-injected embryos, we found that the fluctuations were suppressed by both cortical actin and microtubules. Surprisingly, the microtubule motor Kinesin-1 also suppressed fluctuations to a similar degree as F-actin. Kinesin-1 may mediate linkage of the microtubule (+)-ends to the actin cortex. Consistent with this model is our finding that Kinesin-1-GFP accumulates at the cortical actin caps. PMID:26331244

  5. Rapid Microtubule Self-assembly Kinetics

    PubMed Central

    Gardner, Melissa K.; Charlebois, Blake D.; Jánosi, Imre M.; Howard, Jonathon; Hunt, Alan J.; Odde, David J.

    2011-01-01

    Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit disassociation rate from a microtubule tip is independent of free subunit concentration. Using Total-Internal-Reflection-Fluorescence (TIRF) microscopy and a laser tweezers assay to measure in vitro microtubule assembly with nanometer resolution, we find that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. Because both the association and the dissociation rates increase at higher free subunit concentrations, we find that the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature. PMID:21854983

  6. Rapid microtubule self-assembly kinetics.

    PubMed

    Gardner, Melissa K; Charlebois, Blake D; Jánosi, Imre M; Howard, Jonathon; Hunt, Alan J; Odde, David J

    2011-08-19

    Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit dissociation rate from a microtubule tip is independent of free subunit concentration. Total-Internal-Reflection-Fluorescence (TIRF) microscopy experiments and data from a laser tweezers assay that measures in vitro microtubule assembly with nanometer resolution, provides evidence that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. We find that because both the association and the dissociation rates increase at higher free subunit concentrations, the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. 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

  8. How dynein moves along microtubules

    PubMed Central

    Bhabha, Gira; Johnson, Graham T.; Schroeder, Courtney M.; Vale, Ronald D.

    2015-01-01

    Cytoplasmic dynein, a member of the AAA family of ATPases, drives the processive movement of numerous intracellular cargos towards the minus end of microtubules. Here, we summarize the structural and motile properties of dynein and highlight features that distinguish this motor from kinesin-1 and myosin V, two well-studied transport motors. Integrating information from recent crystal and cryo-EM structures as well as high-resolution single molecule studies, we also discuss models for how dynein biases its movement in one direction along a microtubule track, and present a movie that illustrates these principles. PMID:26678005

  9. Cross-scale modelling of transpiration from stomata via the leaf boundary layer.

    PubMed

    Defraeye, Thijs; Derome, Dominique; Verboven, Pieter; Carmeliet, Jan; Nicolai, Bart

    2014-09-01

    Leaf transpiration is a key parameter for understanding land surface-climate interactions, plant stress and plant structure–function relationships. Transpiration takes place at the microscale level, namely via stomata that are distributed discretely over the leaf surface with a very low surface coverage (approx. 0·2-5%). The present study aims to shed more light on the dependency of the leaf boundary-layer conductance (BLC) on stomatal surface coverage and air speed. An innovative three-dimensional cross-scale modelling approach was applied to investigate convective mass transport from leaves, using computational fluid dynamics. The gap between stomatal and leaf scale was bridged by including all these scales in the same computational model (10⁻⁵-10⁻¹ m), which implies explicitly modelling individual stomata. BLC was strongly dependent on stomatal surface coverage and air speed. Leaf BLC at low surface coverage ratios (CR), typical for stomata, was still relatively high, compared with BLC of a fully wet leaf (hypothetical CR of 100%). Nevertheless, these conventional BLCs (CR of 100%), as obtained from experiments or simulations on leaf models, were found to overpredict the convective exchange. In addition, small variations in stomatal CR were found to result in large variations in BLCs. Furthermore, stomata of a certain size exhibited a higher mass transfer rate at lower CRs. The proposed cross-scale modelling approach allows us to increase our understanding of transpiration at the sub-leaf level as well as the boundary-layer microclimate in a way currently not feasible experimentally. The influence of stomatal size, aperture and surface density, and also flow-field parameters can be studied using the model, and prospects for further improvement of the model are presented. An important conclusion of the study is that existing measures of conductances (e.g. from artificial leaves) can be significantly erroneous because they do not account for microscopic

  10. Cross-scale modelling of transpiration from stomata via the leaf boundary layer

    PubMed Central

    Defraeye, Thijs; Derome, Dominique; Verboven, Pieter; Carmeliet, Jan; Nicolai, Bart

    2014-01-01

    Background and Aims Leaf transpiration is a key parameter for understanding land surface–climate interactions, plant stress and plant structure–function relationships. Transpiration takes place at the microscale level, namely via stomata that are distributed discretely over the leaf surface with a very low surface coverage (approx. 0·2–5 %). The present study aims to shed more light on the dependency of the leaf boundary-layer conductance (BLC) on stomatal surface coverage and air speed. Methods An innovative three-dimensional cross-scale modelling approach was applied to investigate convective mass transport from leaves, using computational fluid dynamics. The gap between stomatal and leaf scale was bridged by including all these scales in the same computational model (10−5–10−1 m), which implies explicitly modelling individual stomata. Key Results BLC was strongly dependent on stomatal surface coverage and air speed. Leaf BLC at low surface coverage ratios (CR), typical for stomata, was still relatively high, compared with BLC of a fully wet leaf (hypothetical CR of 100 %). Nevertheless, these conventional BLCs (CR of 100 %), as obtained from experiments or simulations on leaf models, were found to overpredict the convective exchange. In addition, small variations in stomatal CR were found to result in large variations in BLCs. Furthermore, stomata of a certain size exhibited a higher mass transfer rate at lower CRs. Conclusions The proposed cross-scale modelling approach allows us to increase our understanding of transpiration at the sub-leaf level as well as the boundary-layer microclimate in a way currently not feasible experimentally. The influence of stomatal size, aperture and surface density, and also flow-field parameters can be studied using the model, and prospects for further improvement of the model are presented. An important conclusion of the study is that existing measures of conductances (e.g. from artificial leaves) can be

  11. Fluorometric Measurement of Individual Stomata Activity and Transpiration via a “Brush-on”, Water-Responsive Polymer

    PubMed Central

    Seo, Minjeong; Park, Dong-Hoon; Lee, Chan Woo; Jaworski, Justyn; Kim, Jong-Man

    2016-01-01

    Much of atmospheric water originates from transpiration, the process by which plants release H2O from pores, known as stomata, that simultaneously intake CO2 for photosynthesis. Controlling stomatal aperture can regulate the extent of water transport in response to dynamic environmental factors including osmotic stress, temperature, light, and wind. While larger leaf regions are often examined, the extent of water vapor release from individual stomata remains unexplored. Using a “brush-on” sensing material, we can now assess transpiration using a water-responsive, polydiacetylene-based coating on the leaves surfaces. By eliciting a fluorometric signal to passing water vapor, we obtained information regarding the activity of individual stomata. In this demonstration, our results prove that this coating can identify the proportion of active stomata and the extent of transpirational diffusion of water in response to different conditions. PMID:27578430

  12. Fluorometric Measurement of Individual Stomata Activity and Transpiration via a “Brush-on”, Water-Responsive Polymer

    NASA Astrophysics Data System (ADS)

    Seo, Minjeong; Park, Dong-Hoon; Lee, Chan Woo; Jaworski, Justyn; Kim, Jong-Man

    2016-08-01

    Much of atmospheric water originates from transpiration, the process by which plants release H2O from pores, known as stomata, that simultaneously intake CO2 for photosynthesis. Controlling stomatal aperture can regulate the extent of water transport in response to dynamic environmental factors including osmotic stress, temperature, light, and wind. While larger leaf regions are often examined, the extent of water vapor release from individual stomata remains unexplored. Using a “brush-on” sensing material, we can now assess transpiration using a water-responsive, polydiacetylene-based coating on the leaves surfaces. By eliciting a fluorometric signal to passing water vapor, we obtained information regarding the activity of individual stomata. In this demonstration, our results prove that this coating can identify the proportion of active stomata and the extent of transpirational diffusion of water in response to different conditions.

  13. A viscoelastic model for axonal microtubule rupture.

    PubMed

    Shamloo, Amir; Manuchehrfar, Farid; Rafii-Tabar, Hashem

    2015-05-01

    Axon is an important part of the neuronal cells and axonal microtubules are bundles in axons. In axons, microtubules are coated with microtubule-associated protein tau, a natively unfolded filamentous protein in the central nervous system. These proteins are responsible for cross-linking axonal microtubule bundles. Through complimentary dimerization with other tau proteins, bridges are formed between nearby microtubules creating bundles. Formation of bundles of microtubules causes their transverse reinforcement and has been shown to enhance their ability to bear compressive loads. Though microtubules are conventionally regarded as bearing compressive loads, in certain circumstances during traumatic brain injuries, they are placed in tension. In our model, microtubule bundles were formed from a large number of discrete masses. We employed Standard Linear Solid model (SLS), a viscoelastic model, to computationally simulate microtubules. In this study, we investigated the dynamic responses of two dimensional axonal microtubules under suddenly applied end forces by implementing discrete masses connected to their neighboring masses with a Standard Linear Solid unit. We also investigated the effect of the applied force rate and magnitude on the deformation of bundles. Under tension, a microtubule fiber may rupture as a result of a sudden force. Using the developed model, we could predict the critical regions of the axonal microtubule bundles in the presence of varying end forces. We finally analyzed the nature of microtubular failure under varying mechanical stresses. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. 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.

  15. Microtubule Control of Metabolism in Prostate Cancer

    DTIC Science & Technology

    2013-06-01

    that increase cell death when combined with docetaxel.Here we tested whether two metabolic inhibitors, metformin or 2- deoxy-glucose, function...microtubule cytoskeleton (docetaxel, paclitaxel, or nocodazole) singly, or in combination with metabolic inhibitors ( metformin or 2-deoxy-glucose...Microtubule-targeted drugs, which either stabilize or destabilize microtubules, acted synergistically with either metformin or 2-deoxy-glucose to

  16. On and around microtubules: an overview.

    PubMed

    Wade, Richard H

    2009-10-01

    Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from alphabeta-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the beta-tubulin subunit facing the microtubule plus end conferring a structural polarity. The alpha- and beta-tubulins are highly conserved. A third member of the tubulin family, gamma-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with beta-tubulin so that microtubules consist principally of 'GDP-tubulin' stabilized at the plus end by a short 'cap'. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins.

  17. 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

  18. Increased lateral microtubule contact at the cell cortex is sufficient to drive mammalian spindle elongation

    PubMed Central

    Guild, Joshua; Ginzberg, Miriam B.; Hueschen, Christina L.; Mitchison, Timothy J.; Dumont, Sophie

    2017-01-01

    The spindle is a dynamic structure that changes its architecture and size in response to biochemical and physical cues. For example, a simple physical change, cell confinement, can trigger centrosome separation and increase spindle steady-state length at metaphase. How this occurs is not understood, and is the question we pose here. We find that metaphase and anaphase spindles elongate at the same rate when confined, suggesting that similar elongation forces can be generated independent of biochemical and spindle structural differences. Furthermore, this elongation does not require bipolar spindle architecture or dynamic microtubules. Rather, confinement increases numbers of astral microtubules laterally contacting the cortex, shifting contact geometry from “end-on” to “side-on.” Astral microtubules engage cortically anchored motors along their length, as demonstrated by outward sliding and buckling after ablation-mediated release from the centrosome. We show that dynein is required for confinement-induced spindle elongation, and both chemical and physical centrosome removal demonstrate that astral microtubules are required for such spindle elongation and its maintenance. Together the data suggest that promoting lateral cortex–microtubule contacts increases dynein-mediated force generation and is sufficient to drive spindle elongation. More broadly, changes in microtubule-to-cortex contact geometry could offer a mechanism for translating changes in cell shape into dramatic intracellular remodeling. PMID:28468979

  19. Bottle gourd rootstock-grafting promotes photosynthesis by regulating the stomata and non-stomata performances in leaves of watermelon seedlings under NaCl stress.

    PubMed

    Yang, Yanjuan; Yu, Li; Wang, Liping; Guo, Shirong

    2015-08-15

    Previously, we found that the amelioration of photosynthetic capacity by bottle gourd (Lagenaria siceraria Standl.) rootstock in watermelon seedlings (Citrullus lanatus [Thunb.] Mansf.) with salt treatment might be closely related to the enzymes in Calvin cycle such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Yang et al., 2012). We confirmed this and showed more details in this study that improved photosynthesis of watermelon plants by bottle gourd rootstock was associated with the decreased stomata resistance and the increased photochemical activity and photosynthetic metabolism with or without 100mM NaCl stress for 3 days. The analysis of gas exchange parameters showed that self-grafted plants suffered serious non-stomatal limitation to photosynthesis under salt stress while rootstock-grafted plants were mainly affected by stomata limitation in stress conditions. Further, results showed that NaCl stress markedly reduced the chlorophyll content, damaged the structure of photosynthetic apparatus, and inhibited photochemical activity and CO2 assimilation in self-grafted plants. In contrast, rootstock-grafting increased the chlorophyll content, especially chlorophyll b, and minimized the harmful effects on photosystem II (PSII) reaction center and the thylakoids structure induced by NaCl stress. Furthermore, rootstock-grafting enhanced the content and activity of Rubisco and thus elevated carbon fixation in the leaves of watermelon scions under salt stress. The gene expressions of enzymes related to ribulose-1,5-bisphosphate (RuBP) regeneration were also up-regulated by rootstock and this probably guaranteed the sufficient supply of RuBP for the operation of Calvin cycle in watermelon scions under salt stress. Thus, bottle gourd rootstock promoted photosynthesis by the activation of stomatal and non-stomatal abilities, especially the regulation of a variety of photosynthetic enzymes, including Rubisco in grafted watermelon plants under NaCl stress

  20. 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.

  1. Dear microtubule, I see you

    PubMed Central

    Nogales, Eva

    2016-01-01

    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. PMID:27799495

  2. Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

    PubMed Central

    Goldspink, Deborah A.; Rookyard, Chris; Tyrrell, Benjamin J.; Perkins, James; Lund, Elizabeth K.; Galjart, Niels; Thomas, Paul; Wileman, Tom

    2017-01-01

    Differentiation of columnar epithelial cells involves a dramatic reorganization of the microtubules (MTs) and centrosomal components into an apico-basal array no longer anchored at the centrosome. Instead, the minus-ends of the MTs become anchored at apical non-centrosomal microtubule organizing centres (n-MTOCs). Formation of n-MTOCs is critical as they determine the spatial organization of MTs, which in turn influences cell shape and function. However, how they are formed is poorly understood. We have previously shown that the centrosomal anchoring protein ninein is released from the centrosome, moves in a microtubule-dependent manner and accumulates at n-MTOCs during epithelial differentiation. Here, we report using depletion and knockout (KO) approaches that ninein expression is essential for apico-basal array formation and epithelial elongation and that CLIP-170 is required for its redeployment to n-MTOCs. Functional inhibition also revealed that IQGAP1 and active Rac1 coordinate with CLIP-170 to facilitate microtubule plus-end cortical targeting and ninein redeployment. Intestinal tissue and in vitro organoids from the Clip1/Clip2 double KO mouse with deletions in the genes encoding CLIP-170 and CLIP-115, respectively, confirmed requirement of CLIP-170 for ninein recruitment to n-MTOCs, with possible compensation by other anchoring factors such as p150Glued and CAMSAP2 ensuring apico-basal microtubule formation despite loss of ninein at n-MTOCs. PMID:28179500

  3. Microtubule catastrophe from protofilament dynamics.

    PubMed

    Jemseena, V; Gopalakrishnan, Manoj

    2013-09-01

    The disappearance of the guanosine triphosphate- (GTP) tubulin cap is widely believed to be the forerunner event for the growth-shrinkage transition ("catastrophe") in microtubule filaments in eukaryotic cells. We study a discrete version of a stochastic model of the GTP cap dynamics, originally proposed by Flyvbjerg, Holy, and Leibler [Phys. Rev. Lett. 73, 2372 (1994)]. Our model includes both spontaneous and vectorial hydrolysis, as well as dissociation of a nonhydrolyzed dimer from the filament after incorporation. In the first part of the paper, we apply this model to a single protofilament of a microtubule. A catastrophe transition is defined for each protofilament, similarly to the earlier one-dimensional models, the frequency of occurrence of which is then calculated under various conditions but without explicit assumption of steady-state conditions. Using a perturbative approach, we show that the leading asymptotic behavior of the protofilament catastrophe in the limit of large growth velocities is remarkably similar across different models. In the second part of the paper, we extend our analysis to the entire filament by making a conjecture that a minimum number of such transitions are required to occur for the onset of microtubule catastrophe. The frequency of microtubule catastrophe is then determined using numerical simulations and compared with analytical and semianalytical estimates made under steady-state and quasi-steady-state assumptions, respectively, for the protofilament dynamics. A few relevant experimental results are analyzed in detail and compared with predictions from the model. Our results indicate that loss of GTP cap in two to three protofilaments is necessary to trigger catastrophe in a microtubule.

  4. Microtubule alignment and manipulation using AC electrokinetics.

    PubMed

    Uppalapati, Maruti; Huang, Ying-Ming; Jackson, Thomas N; Hancock, William O

    2008-09-01

    The kinesin-microtubule system plays an important role in intracellular transport and is a model system for integrating biomotor-driven transport into microengineered devices. AC electrokinetics provides a novel tool for manipulating and organizing microtubules in solution, enabling new experimental geometries for investigating and controlling the interactions of microtubules and microtubule motors in vitro. By fabricating microelectrodes on glass substrates and generating AC electric fields across solutions of microtubules in low-ionic-strength buffers, bundles of microtubules are collected and aligned and the electrical properties of microtubules in solution are measured. The AC electric fields result in electro-osmotic flow, electrothermal flow, and dielectrophoresis of microtubules, which can be controlled by varying the solution conductivity, AC frequency, and electrode geometry. By mapping the solution conductivity and AC frequency over which positive dielectrophoresis occurs, the apparent conductivity of taxol-stabilized bovine-brain microtubules in PIPES buffer is measured to be 250 mS m(-1). By maximizing dielectrophoretic forces and minimizing electro-osmotic and electrothermal flow, microtubules are assembled into opposed asters. These experiments demonstrate that AC electrokinetics provides a powerful new tool for kinesin-driven transport applications and for investigating the role of microtubule motors in development and maintenance of the mitotic spindle.

  5. Characterization of microtubule buckling in living cells.

    PubMed

    Pallavicini, Carla; Monastra, Alejandro; Bardeci, Nicolás González; Wetzler, Diana; Levi, Valeria; Bruno, Luciana

    2017-09-01

    Microtubules are filamentous biopolymers involved in essential biological processes. They form key structures in eukaryotic cells, and thus it is very important to determine the mechanisms involved in the formation and maintenance of the microtubule network. Microtubule bucklings are transient and localized events commonly observed in living cells and characterized by a fast bending and its posterior relaxation. Active forces provided by molecular motors have been indicated as responsible for most of these rapid deformations. However, the factors that control the shape amplitude and the time scales of the rising and release stages remain unexplored. In this work, we study microtubule buckling in living cells using Xenopus laevis melanophores as a model system. We tracked single fluorescent microtubules from high temporal resolution (0.3-2 s) confocal movies. We recovered the center coordinates of the filaments with 10-nm precision and analyzed the amplitude of the deformation as a function of time. Using numerical simulations, we explored different force mechanisms resulting in microtubule bending. The simulated events reproduce many features observed for microtubules, suggesting that a mechanistic model captures the essential processes underlying microtubule buckling. Also, we studied the interplay between actively transported vesicles and the microtubule network using a two-color technique. Our results suggest that microtubules may affect transport indirectly besides serving as tracks of motor-driven organelles. For example, they could obstruct organelles at microtubule intersections or push them during filament mechanical relaxation.

  6. 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.

  7. OsKinesin-13A Is an Active Microtubule Depolymerase Involved in Glume Length Regulation via Affecting Cell Elongation

    PubMed Central

    Deng, Zhu Yun; Liu, Ling Tong; Li, Tang; Yan, Song; Kuang, Bai Jian; Huang, Shan Jin; Yan, Chang Jie; Wang, Tai

    2015-01-01

    Grain size is an important trait influencing both the yield and quality of rice and its major determinant is glume size. However, how glume size is regulated remains largely unknown. Here, we report the characterization of OsKinesin-13A, which regulates cell elongation and glume length in rice. The mutant of OsKinesin-13A, sar1, displayed length reduction in grains and other organs including internodes, leaves and roots. The grain phenotype in sar1 was directly caused by reduction in glume length, which in turn restricted caryopsis size. Histological results revealed that length decrease in sar1 organs resulted from abnormalities in cell elongation. The orientation of cellulose microfibrils was defective in sar1. Consistently, sar1 showed reduced transverse orientation of cortical microtubules. Further observations demonstrated that microtubule turnover was decreased in sar1. OsKinesin-13A was shown to be an active microtubule depolymerase and mainly distributed on vesicles derived from the Golgi apparatus and destined for the cell surface. Thus, our results suggest that OsKinesin-13A utilizes its microtubule depolymerization activity to promote microtubule turnover, which may not only influence transverse orientation of cortical microtubules but also facilitate vesicle transport from the Golgi apparatus to the cell surface, and thus affects cellulose microfibril orientation and cell elongation. PMID:25807460

  8. 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.

  9. 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.

  10. Controls on the emission of plant volatiles through stomata: A sensitivity analysis

    NASA Astrophysics Data System (ADS)

    Niinemets, ÜLo; Reichstein, Markus

    2003-04-01

    According to experimental studies, plant emissions of volatile organic compounds (VOC) are controlled by stomata to a varying extent, but the differing responses could not be explained so far. A dynamic emission model developed in a previous study indicated that stomata may limit the emission rate in a nonsteady state conditions, whereas the rate of increase of liquid-phase volatile concentrations controls the degree to which stomata temporarily curtail the emission. Despite its large predictive capability, potentially large number of volatile physico-chemical and leaf structural variables are needed for parameterization of such dynamic models, limiting the usefulness of the approach. We conducted a sensitivity analysis to determine the effect of varying VOC distribution between gas- and liquid-phases (Henry's law constant, H, Pa m3 mol-1) and varying internal diffusion conductances in the liquid- and gas-phases. The model was parameterized for three contrasting leaf architectures (conifer, sclerophyll, and mesophytic leaves). The sensitivity analysis indicated that the volatile H value is the key variable affecting the stomatal sensitivity of VOC emissions. Differences in leaf architecture, in particular in leaf liquid volume to area ratio, also modified the emission responses to changes in stomatal aperture, but these structural effects were superimposed by compound gas/liquid phase partitioning. The results of this analysis indicate that major effort in parameterization of dynamic VOC emission models should be directed toward obtaining reliable gas/liquid-phase equilibria for various plant volatiles, and that these models may readily be applied for leaves with contrasting architecture.

  11. Tomato–Pseudomonas syringae interactions under elevated CO2 concentration: the role of stomata

    PubMed Central

    Li, Xin; Sun, Zenghui; Shao, Shujun; Zhang, Shuai; Ahammed, Golam Jalal; Zhang, Guanqun; Jiang, Yuping; Zhou, Jie; Xia, Xiaojian; Zhou, Yanhong; Yu, Jingquan; Shi, Kai

    2015-01-01

    Increasing atmospheric CO2 concentrations ([CO2]) in agricultural and natural ecosystems is known to reduce plant stomatal opening, but it is unclear whether these CO2-induced stomatal alterations are associated with foliar pathogen infections. In this study, tomato plants were grown under ambient and elevated [CO2] and inoculated with Pseudomonas syringae pv. tomato strain DC3000, a strain that is virulent on tomato plants. We found that elevated [CO2] enhanced tomato defence against P. syringae. Scanning electron microscopy analysis revealed that stomatal aperture of elevated [CO2] plants was considerably smaller than their ambient counterparts, which affected the behaviour of P. syringae bacteria on the upper surface of epidermal peels. Pharmacological experiments revealed that nitric oxide (NO) played a role in elevated [CO2]-induced stomatal closure. Silencing key genes involved in NO generation and stomatal closing, nitrate reductase (NR) and guard cell slow-type anion channel 1 (SLAC1), blocked elevated [CO2]-induced stomatal closure and resulted in significant increases in P. syringae infection. However, the SLAC1-silenced plants, but not the NR-silenced plants, still had significantly higher defence under elevated [CO2] compared with plants treated with ambient [CO2]. Similar results were obtained when the stomata-limiting factor for P. syringae entry was excluded by syringe infiltration inoculation. These results indicate that elevated [CO2] induces defence against P. syringae in tomato plants, not only by reducing the stomata-mediated entry of P. syringae but also by invoking a stomata-independent pathway to counteract P. syringae. This information is valuable for designing proper strategies against bacterial pathogens under changing agricultural and natural ecosystems. PMID:25336683

  12. Pollen and stomata morphometrics and polyploidy in Eriotheca (Malvaceae-Bombacoideae).

    PubMed

    Marinho, R C; Mendes-Rodrigues, C; Bonetti, A M; Oliveira, P E

    2014-03-01

    Approximately 70% of the angiosperm species are polyploid, an important phenomenon in the evolution of those plants. But ploidy estimates have often been hindered because of the small size and large number of chromosomes in many tropical groups. Since polyploidy affects cell size, morphometric analyses of pollen grains and stomata have been used to infer ploidy level. Polyploidy is present in many species of the Cerrado, the Neotropical savanna region in Central Brazil, and has been linked to apomixis in some taxa. Eriotheca gracilipes and Eriotheca pubescens are common tree species in this region, and present cytotypes that form reproductive mosaics. Hexaploid individuals (2n = 6x = 276) are polyembryonic and apomictic, while tetraploid and diploid individuals (2n = 2x = 92, 2n = 4x = 184) are sexual and monoembryonic. We tested whether morphometric analysis can be used to estimate ploidy levels in E. gracilipes and E. pubescens individuals. Pollen material from diploid and hexaploid individuals of E. gracilipes, and tetraploid and hexaploid individuals of E. pubescens, were fixed in 50% FAA, and expanded leaves were dried in silica gel. Pollen grains and stomata of at least five individuals from each population were measured. The results demonstrate that all measures were significantly different among cytotypes. Individuals with higher levels of ploidy (hexaploid) all presented measurements that were higher than those with lower levels (diploid and tetraploid). There was no overlap between ploidy levels in each species at 95% confidence interval. Thus, the size of the pollen grains and stomata are effective parameters for analysis of ploidy levels in E. gracilipes and E. pubescens.

  13. Linking stomata geometries and densities to leaf gas exchange - new opportunities and old pitfalls

    NASA Astrophysics Data System (ADS)

    Schymanski, Stanislaus; Singer, Thomas; Or, Dani

    2017-04-01

    Historical trends in stomatal sizes and numbers are believed to be directly related to trends in atmospheric CO2 concentrations, where lower atmospheric CO2 concentrations selected for larger leaf conductance to achieve adequate CO2 assimilation rates. In addition to affecting maximum stomatal conductance, stomatal size is considered to affect transition time between full opening and full closure with smaller stomata responding faster. Stomatal sizes and numbers are often deduced by direct microscopy of leaf surfaces (fresh or fossil), or from nail polish imprints obtained from leaf epidermis. The maximum stomatal conductance is then calculated for fully open stomata of assumed aperture shape based on gas diffusion from within the leaf across a leaf boundary layer. Direct microscopic observations of leaves in specialised gas exchange chambers or snap-frozen leaves right after removal from a gas exchange chamber enabled correlation of actual stomatal apertures with directly measured bulk stomatal conductance. We combined systematic analyses of stomatal conductance and response times with laser scanning microscopy of epidermis imprints using fast setting dental imprint that preserve highly resolved stomatal apertures after removal from a gas exchange chamber. The simplicity of data collection relative to previous approaches enables data collection across a range of species with different stomatal sizes and numbers. The dataset was used to evaluate the adequacy of different physically-based stomatal conductance formulas based on geometrical attributes relative to measured conductance for a range of external CO2 concentrations. We also investigated the link between stomata size and response time to environmental perturbation. Results point to uncertainties in inferred geometrical attributes and suggest highly patchy stomatal opening that complicates links between stomata aperture and density for estimation of actual stomatal conductance. Additionally, we identified

  14. 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

  15. Novel insights into mammalian embryonic neural stem cell division: focus on microtubules.

    PubMed

    Mora-Bermúdez, Felipe; Huttner, Wieland B

    2015-12-01

    During stem cell divisions, mitotic microtubules do more than just segregate the chromosomes. They also determine whether a cell divides virtually symmetrically or asymmetrically by establishing spindle orientation and the plane of cell division. This can be decisive for the fate of the stem cell progeny. Spindle defects have been linked to neurodevelopmental disorders, yet the role of spindle orientation for mammalian neurogenesis has remained controversial. Here we explore recent advances in understanding how the microtubule cytoskeleton influences mammalian neural stem cell division. Our focus is primarily on the role of spindle microtubules in the development of the cerebral cortex. We also highlight unique characteristics in the architecture and dynamics of cortical stem cells that are tightly linked to their mode of division. These features contribute to setting these cells apart as mitotic "rule breakers," control how asymmetric a division is, and, we argue, are sufficient to determine the fate of the neural stem cell progeny in mammals.

  16. CYLD regulates spindle orientation by stabilizing astral microtubules and promoting dishevelled-NuMA-dynein/dynactin complex formation

    PubMed Central

    Yang, Yunfan; Liu, Min; Li, Dengwen; Ran, Jie; Gao, Jinmin; Suo, Shaojun; Sun, Shao-Cong; Zhou, Jun

    2014-01-01

    Oriented cell division is critical for cell fate specification, tissue organization, and tissue homeostasis, and relies on proper orientation of the mitotic spindle. The molecular mechanisms underlying the regulation of spindle orientation remain largely unknown. Herein, we identify a critical role for cylindromatosis (CYLD), a deubiquitinase and regulator of microtubule dynamics, in the control of spindle orientation. CYLD is highly expressed in mitosis and promotes spindle orientation by stabilizing astral microtubules and deubiquitinating the cortical polarity protein dishevelled. The deubiquitination of dishevelled enhances its interaction with nuclear mitotic apparatus, stimulating the cortical localization of nuclear mitotic apparatus and the dynein/dynactin motor complex, a requirement for generating pulling forces on astral microtubules. These findings uncover CYLD as an important player in the orientation of the mitotic spindle and cell division and have important implications in health and disease. PMID:24469800

  17. Microtubule-associated proteins from Antarctic fishes.

    PubMed

    Detrich, H W; Neighbors, B W; Sloboda, R D; Williams, R C

    1990-01-01

    Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.

  18. 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.

  19. End-binding proteins sensitize microtubules to the action of microtubule-targeting agents.

    PubMed

    Mohan, Renu; Katrukha, Eugene A; Doodhi, Harinath; Smal, Ihor; Meijering, Erik; Kapitein, Lukas C; Steinmetz, Michel O; Akhmanova, Anna

    2013-05-28

    Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert on microtubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process.

  20. Regulating effect of Chinese herbal medicine on the peritoneal lymphatic stomata in enhancing ascites absorption of experimental hepatofibrotic mice

    PubMed Central

    Li, Ji-Cheng; Ding, Shi-Ping; Xu, Jian

    2002-01-01

    AIM: To observe the regulatory effect of Chinese herbal medicine on peritoneal lymphatic stomata and its significance in treating ascites in liver fibrosis model mice. METHODS: Two Chinese herbal composite prescriptions were used separately to treat the carbon tetrachloride-induced mouse model of liver fibrosis. The histo-pathologic changes of the liver sections (HE and VG stainings) were observed. The peritoneal lymphatic stomata was detected by scanning electron microscopy and computer image processing. The changes of urinary volume and sodium ion concentration were measured. RESULTS: In the model group, lots of fibrous tissue formed in liver and extended into the hepatic lobules to separate them incompletely. In the treated and prevention groups, the histo-pathologic changes of liver was rather milder, only showed much less fibrous tissue proliferation in the hepatic lobules. The peritoneal lymphatic stomata enlarged with increased density in the experimental groups (diameter: PA, 3.07 ± 0.69 µm; PB, 2.82 ± 0.37 µm; TA, 3.25 ± 0.82 µm and TB, 2.82 ± 0.56 µm; density: PA, 7.11 ± 1.90 stomata·1000 µm-2; PB, 8.76 ± 1.45 stomata·1000 µm-1; TA, 6.55 ± 1.44 stomata·1000 µm-2 and TB, 8.76 ± 1.79 stomata·1000 µm-2), as compared with the model group (diameter: 2.00 ± 0.52 µm; density: 4.45 ± 1.05 stomata·1000 µm-2). After treatment, the urinary volume and sodium ion excretion increased in the experimental groups (PA, 231.28 ± 41.09 mmol·L-1; PB, 171.69 ± 27.48 mmol·L-1 and TA, 231.44 ± 34.12 mmol·L-1), which were significantly different with those in the model group (129.33 ± 36.75 mmol·L-1). CONCLUSION: Chinese herbal medicine has marked effects in alleviating liver fibrosis, regulating peritoneal lymphatic stomata, improving the drainage of ascites from peritoneal cavity and causing increase of urinary volume and sodium ion excretion to reduce the water and sodium retention, and thus have favorable therapeutic effect in treating ascites

  1. A Rapid and Simple Method for Microscopy-Based Stomata Analyses

    PubMed Central

    Eisele, Jochen F.; Fäßler, Florian; Bürgel, Patrick F.; Chaban, Christina

    2016-01-01

    There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures. Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results. We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli. We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis. The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel. PMID:27732636

  2. Plant water use efficiency shapes co-evolution of stomata size and density over geologic time

    NASA Astrophysics Data System (ADS)

    Assouline, S.; Or, D.

    2010-12-01

    The appearance of stomata and formation of impervious leaf cuticle are key elements in terrestrial plant evolution allowing plants to control gaseous diffusion and regulate water loss during simultaneous carbon dioxide uptake. An important plant gaseous diffusion adaptation was achieved by co-evolution of stomata density (D) and maximum aperture size (S), whose product α=S.D defines the evaporating fraction of the leaf surface and determines gaseous conductance. The plant leaf fossil record reveals significant variations in D and S over the 400 Myrs of the Phanerozoic eon, whose impact on gas-exchange capacity and on plant evolution are not fully understood. Characteristics of evaporation suppression from perforated diffusion barriers deduced from plant fossil record delineate the evolution of α as atmospheric CO2 declined from 4000 ppm to present day values. Surprisingly, despite non monotonous variations in α during plant evolution, plant water use efficiency (WUE) has improved systematically during the decrease in atmospheric CO2 over the Phanerozoic, at variance with conventional predictions. The new WUE trend is based on physical relations between α and evaporation suppression by perforated diffusion barriers, providing new insights on the dominance of water related regulatory function and on consequences of future CO2 enriched atmosphere on plant function and hydrologic cycle such as continental runoff scenarios.

  3. Microtubule Bundling and Shape Transitions

    NASA Astrophysics Data System (ADS)

    Needleman, Daniel

    2005-03-01

    Microtubules (MTs) are hollow cylindrical polymers composed of heterodimers of the protein tubulin that align end-to-end in the MT wall, forming linear protofilaments that interact laterally. Placing MTs under osmotic pressure causes them to reversibly buckle to a noncircular shape and pack into rectangular bundles at a critical osmotic pressure; further increases in pressure continue to distort MTs elastically. At higher osmotic pressures stressing polymers may be forced into the MT lumen causing the MTs to revert to a circle cross-section and pack into hexagonal bundles. This SAXRD-osmotic stress study provides a probe of the inter-protofilament bond strength and gives insight into the mechanisms by which microtubule associated proteins and the cancer chemotherapeutic drug Taxol stabilize MTs. We present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the microtubule lumen. Supported by NSF DMR- 0203755, NIH GM-59288 and NS-13560, and CTS-0103516. SSRL is supported by the U.S. DOE.

  4. Autophagy induction stabilizes microtubules and promotes axon regeneration after spinal cord injury

    PubMed Central

    He, Miao; Ding, Yuetong; Chu, Chen; Tang, Jing; Xiao, Qi; Luo, Zhen-Ge

    2016-01-01

    Remodeling of cytoskeleton structures, such as microtubule assembly, is believed to be crucial for growth cone initiation and regrowth of injured axons. Autophagy plays important roles in maintaining cellular homoeostasis, and its dysfunction causes neuronal degeneration. The role of autophagy in axon regeneration after injury remains speculative. Here we demonstrate a role of autophagy in regulating microtubule dynamics and axon regeneration. We found that autophagy induction promoted neurite outgrowth, attenuated the inhibitory effects of nonpermissive substrate myelin, and decreased the formation of retraction bulbs following axonal injury in cultured cortical neurons. Interestingly, autophagy induction stabilized microtubules by degrading SCG10, a microtubule disassembly protein in neurons. In mice with spinal cord injury, local administration of a specific autophagy-inducing peptide, Tat-beclin1, to lesion sites markedly attenuated axonal retraction of spinal dorsal column axons and cortical spinal tract and promoted regeneration of descending axons following long-term observation. Finally, administration of Tat-beclin1 improved the recovery of motor behaviors of injured mice. These results show a promising effect of an autophagy-inducing reagent on injured axons, providing direct evidence supporting a beneficial role of autophagy in axon regeneration. PMID:27638205

  5. The preprophase band of microtubules controls the robustness of division orientation in plants.

    PubMed

    Schaefer, Estelle; Belcram, Katia; Uyttewaal, Magalie; Duroc, Yann; Goussot, Magali; Legland, David; Laruelle, Elise; de Tauzia-Moreau, Marie-Ludivine; Pastuglia, Martine; Bouchez, David

    2017-04-14

    Controlling cell division plane orientation is essential for morphogenesis in multicellular organisms. In plant cells, the future cortical division plane is marked before mitotic entry by the preprophase band (PPB). Here, we characterized an Arabidopsis trm (TON1 Recruiting Motif) mutant that impairs PPB formation but does not affect interphase microtubules. Unexpectedly, PPB disruption neither abolished the capacity of root cells to define a cortical division zone nor induced aberrant cell division patterns but rather caused a loss of precision in cell division orientation. Our results advocate for a reassessment of PPB function and division plane determination in plants and show that a main output of this microtubule array is to limit spindle rotations in order to increase the robustness of cell division.

  6. Stochastic Optical Reconstruction Microscopy Imaging of Microtubule Arrays in Intact Arabidopsis thaliana Seedling Roots

    PubMed Central

    Dong, Bin; Yang, Xiaochen; Zhu, Shaobin; Bassham, Diane C.; Fang, Ning

    2015-01-01

    Super-resolution fluorescence microscopy has generated tremendous success in revealing detailed subcellular structures in animal cells. However, its application to plant cell biology remains extremely limited due to numerous technical challenges, including the generally high fluorescence background of plant cells and the presence of the cell wall. In the current study, stochastic optical reconstruction microscopy (STORM) imaging of intact Arabidopsis thaliana seedling roots with a spatial resolution of 20–40 nm was demonstrated. Using the super-resolution images, the spatial organization of cortical microtubules in different parts of a whole Arabidopsis root tip was analyzed quantitatively, and the results show the dramatic differences in the density and spatial organization of cortical microtubules in cells of different differentiation stages or types. The method developed can be applied to plant cell biological processes, including imaging of additional elements of the cytoskeleton, organelle substructure, and membrane domains. PMID:26503365

  7. Microtubule detyrosination guides chromosomes during mitosis

    PubMed Central

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K.; Magiera, Maria M.; Zaytsev, Anatoly V.; Pereira, Ana L.; Janke, Carsten; Grishchuk, Ekaterina L.; Maiato, Helder

    2015-01-01

    Before chromosomes segregate into daughter cells they align at the mitotic spindle equator, a process known as chromosome congression. CENP-E/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically towards the equator. Here we found that congression of pole-proximal chromosomes depended on specific post-translational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. PMID:25908662

  8. Microtubule dynamics of the centrosome-like polar organizers from the basal land plant Marchantia polymorpha.

    PubMed

    Buschmann, Henrik; Holtmannspötter, Michael; Borchers, Agnes; O'Donoghue, Martin-Timothy; Zachgo, Sabine

    2016-02-01

    The liverwort Marchantia employs both modern and ancestral devices during cell division: it forms preprophase bands and in addition it shows centrosome-like polar organizers. We investigated whether polar organizers and preprophase bands cooperate to set up the division plane. To this end, two novel green fluorescent protein-based microtubule markers for dividing cells of Marchantia were developed. Cells of the apical notch formed polar organizers first and subsequently assembled preprophase bands. Polar organizers were formed de novo from multiple mobile microtubule foci localizing to the nuclear envelope. The foci then became concentrated by bipolar aggregation. We determined the comet production rate of polar organizers and show that microtubule plus ends of astral microtubules polymerize faster than those found on cortical microtubules. Importantly, it was observed that conditions increasing polar organizer numbers interfere with preprophase band formation. The data show that polar organizers have much in common with centrosomes, but that they also have specialized features. The results suggest that polar organizers contribute to preprophase band formation and in this way are involved in controlling the division plane. Our analyses of the basal land plant Marchantia shed new light on the evolution of plant cell division.

  9. “CLASPing” tungsten's effects on microtubules with “PINs”

    PubMed Central

    Adamakis, Ioannis Dimosthenis S; Panteris, Emmanuel; Eleftheriou, Eleftherios P

    2015-01-01

    Tungsten, supplied as sodium tungstate, inhibits root elongation in Arabidopsis thaliana, which has been attributed to a diminishing of PIN2 and PIN3 auxin efflux carriers. In this work, we sought to analyze the effect of tungsten on cortical microtubules and CLASP (Cytoplasmic Linker Associated Protein), which are also involved in the anisotropic cell expansion of root cells. Seedlings grown in a tungsten-free substrate for 4 d and then transplanted into a tungsten-containing substrate exhibited randomly oriented microtubules in a time-dependent manner. While tungsten had no effect on roots treated for 3 h, microtubule alignment was obviously affected in the transition and elongation zones after a 6, 12, 24, 48 h tungsten treatment, at prolonged tungsten administrations and in seedlings grown directly in the presence of tungsten. This change in microtubule orientation may be associated with the reduction of CLASP protein expression induced by tungsten, as evidenced in experiments with plants expressing the CLASP-GFP protein. A possible mechanism, by which the coordinated functions of CLASP, PIN2 and microtubules are affected, as revealed by inhibited root growth, is discussed. PMID:26313814

  10. 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

  11. SDF1 reduces interneuron leading process branching through dual regulation of actin and microtubules.

    PubMed

    Lysko, Daniel E; Putt, Mary; Golden, Jeffrey A

    2014-04-02

    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.

  12. Spatio-temporal orientation of microtubules controls conical cell shape in Arabidopsis thaliana petals.

    PubMed

    Ren, Huibo; Dang, Xie; Cai, Xianzhi; Yu, Peihang; Li, Yajun; Zhang, Shanshan; Liu, Menghong; Chen, Binqing; Lin, Deshu

    2017-06-01

    The physiological functions of epidermal cells are largely determined by their diverse morphologies. Most flowering plants have special conical-shaped petal epidermal cells that are thought to influence light capture and reflectance, and provide pollinator grips, but the molecular mechanisms controlling conical cell shape remain largely unknown. Here, we developed a live-confocal imaging approach to quantify geometric parameters of conical cells in Arabidopsis thaliana (A. thaliana). Through genetic screens, we identified katanin (KTN1) mutants showing a phenotype of decreased tip sharpening of conical cells. Furthermore, we demonstrated that SPIKE1 and Rho of Plants (ROP) GTPases were required for the final shape formation of conical cells, as KTN1 does. Live-cell imaging showed that wild-type cells exhibited random orientation of cortical microtubule arrays at early developmental stages but displayed a well-ordered circumferential orientation of microtubule arrays at later stages. By contrast, loss of KTN1 prevented random microtubule networks from shifting into well-ordered arrays. We further showed that the filamentous actin cap, which is a typical feature of several plant epidermal cell types including root hairs and leaf trichomes, was not observed in the growth apexes of conical cells during cell development. Moreover, our genetic and pharmacological data suggested that microtubules but not actin are required for conical cell shaping. Together, our results provide a novel imaging approach for studying petal conical cell morphogenesis and suggest that the spatio-temporal organization of microtubule arrays plays crucial roles in controlling conical cell shape.

  13. Mechanical stress in Arabidopsis leaves orients microtubules in a 'continuous’ supracellular pattern

    PubMed Central

    2013-01-01

    Background Cortical microtubules form a dynamic network and continuously undergo shrinking (catastrophe), pausing and rebuilding (rescue). The advantage of such a dynamic system is that it may mediate appropriate responses in a short time span. Microtubules are known to play a pivotal role in determining the orientation of the cellulose microfibril deposition in the plant cell wall. The latter is a solid exoskeleton surrounding the protoplast. It forms the physical framework that interconnects most cells and has to bear the tensile stresses within the tissue. Here we describe the effect of externally applied pressure on microtubule organization in growing Arabidopsis leaves. Results Confocal microscopy examination of transgenic plants bearing GFP-tagged TUA6 proteins led to the observation that application of an additional mechanical pressure on growing Arabidopsis leaves triggers an excessive bundling of microtubules within the individual cell. Besides, the microtubules seem to align in neighboring cells, creating a 'continuous’ supracellular pattern. This effect occurs within 3 hours after applied external force and is age-dependent, whereby only cells of leaves up to 19 days after sowing (DAS) are susceptible to the applied pressure. Conclusions Upon externally applied pressure on developing Arabidopsis leaves, microtubules bundle and rearrange to form seemingly continuous supracellular patterns. As microtubules guide the cellulose synthase complexes, this observed reorganisation pattern probably affects the cellulose deposition, contributing to the reinforcement of the cell wall in a particular position to cope with the extra-applied pressure. The age-effect is reasonable, since younger cells, which are actively shaping their cell walls, are more vulnerable to altered mechanical stresses while in leaves older than 19 DAS, the walls are more robust and therefore can sustain the applied forces. PMID:24138025

  14. Periodic actin structures in neuronal axons are required to maintain microtubules.

    PubMed

    Qu, Yue; Hahn, Ines; Webb, Stephen E D; Pearce, Simon P; Prokop, Andreas

    2017-01-15

    Axons are cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily conserved, ubiquitous, highly ordered feature of axons, the function of PMS is unknown. Here we studied PMS abundance, organization, and function, combining versatile Drosophila genetics with superresolution microscopy and various functional readouts. Analyses with 11 actin regulators and three actin-targeting drugs suggest that PMS contains short actin filaments that are depolymerization resistant and sensitive to spectrin, adducin, and nucleator deficiency, consistent with microscopy-derived models proposing PMS as specialized cortical actin. Upon actin removal, we observed gaps in microtubule bundles, reduced microtubule polymerization, and reduced axon numbers, suggesting a role of PMS in microtubule organization. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilizing protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerization contributes to their maintenance in axons. We discuss potential implications of this novel PMS function along axon shafts for axon maintenance and regeneration. © 2017 Qu, Hahn, 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).

  15. Damage to stomata and inhibition of photosynthesis by toxic pollutants in Pinus sylvestris needles as affected by the exposure time

    SciTech Connect

    Kaipiainen, L.K.; Sofronova, G.I.; Hari, P.

    1995-11-01

    The impact of persistent exposure of Pinus sylvestris L. trees of various ages to industrial emissions on stomata and photosynthesis of needles was studied in relation to the exposure time. The electron microscopic examination of the needles revealed an erosion of the epicuticular wax and damage to stomata, which increased with needle age until stomata were completely occluded by polymetallic dust. Pollutant particles wee found to contain S, Cl, Ca, K, Mg, Mn, Al, Ni, Fe, Cu, Co, Ti, and Zn. Photosynthetic rates were inhibited by 20-60%, depending on the needle age and tree condition. It is concluded that a nonuniformity in the toxicant distribution over the forest canopy and the age-dependent changes in the state of the cuticular wax layer are the most likely causes of variability in the extent to which individual trees were damaged by the toxicants.

  16. Ectopic A-lattice seams destabilize microtubules.

    PubMed

    Katsuki, Miho; Drummond, Douglas R; Cross, Robert A

    2014-01-01

    Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

  17. Ectopic A-lattice seams destabilize microtubules

    PubMed Central

    Katsuki, Miho; Drummond, Douglas R.; Cross, Robert A.

    2014-01-01

    Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe. PMID:24463734

  18. Mechanical stress induced mechanism of microtubule catastrophes.

    PubMed

    Hunyadi, Viktória; Chrétien, Denis; Jánosi, Imre M

    2005-05-13

    Microtubules assembled in vitro from pure tubulin can switch occasionally from growing to shrinking states or resume assembly, an unusual behavior termed "dynamic instability of microtubule growth". Its origin remains unclear and several models have been proposed, including occasional switching of the microtubules into energetically unfavorable configurations during assembly. In this study, we have asked whether the excess energy accumulated in these configurations would be of sufficient magnitude to destabilize the capping region that must exist at the end of growing microtubules. For this purpose, we have analyzed the frequency distribution of microtubules assembled in vitro from pure tubulin, and modeled the different mechanical constraints accumulated in their wall. We find that the maximal excess energy that the microtubule lattice can store is in the order of 11 kBT per dimer. Configurations that require distortions up to approximately 20 kBT are allowed at the expense of a slight conformational change, and larger distortions are not observed. Modeling of the different elastic deformations suggests that the excess energy is essentially induced by protofilament skewing, microtubule radial curvature change and inter-subunit shearing, distortions that must destabilize further the tubulin subunits interactions. These results are consistent with the hypothesis that unfavorable closure events may trigger the catastrophes observed at low tubulin concentration in vitro. In addition, we propose a novel type of representation that describes the stability of microtubule assembly systems, and which might be of considerable interest to study the effects of stabilizing and destabilizing factors on microtubule structure and dynamics.

  19. Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

    PubMed Central

    Patel-Hett, Sunita; Richardson, Jennifer L.; Schulze, Harald; Drabek, Ksenija; Isaac, Natasha A.; Hoffmeister, Karin; Shivdasani, Ramesh A.; Bulinski, J. Chloë; Galjart, Niels; Hartwig, John H.

    2008-01-01

    The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (± 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (± 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP–expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states. PMID:18230754

  20. Depletion of JMJD5 sensitizes tumor cells to microtubule-destabilizing agents by altering microtubule stability.

    PubMed

    Wu, Junyu; He, Zhimin; Wang, Da-Liang; Sun, Fang-Lin

    2016-11-01

    Microtubules play essential roles in mitosis, cell migration, and intracellular trafficking. Drugs that target microtubules have demonstrated great clinical success in cancer treatment due to their capacity to impair microtubule dynamics in both mitotic and interphase stages. In a previous report, we demonstrated that JMJD5 associated with mitotic spindle and was required for proper mitosis. However, it remains elusive whether JMJD5 could regulate the stability of cytoskeletal microtubules and whether it affects the efficacy of microtubule-targeting agents. In this study, we find that JMJD5 localizes not only to the nucleus, a fraction of it also localizes to the cytoplasm. JMJD5 depletion decreases the acetylation and detyrosination of α-tubulin, both of which are markers of microtubule stability. In addition, microtubules in JMJD5-depleted cells are more sensitive to nocodazole-induced depolymerization, whereas JMJD5 overexpression increases α-tubulin detyrosination and enhances the resistance of microtubules to nocodazole. Mechanistic studies revealed that JMJD5 regulates MAP1B protein levels and that MAP1B overexpression rescued the microtubule destabilization induced by JMJD5 depletion. Furthermore, JMJD5 depletion significantly promoted apoptosis in cancer cells treated with the microtubule-targeting anti-cancer drugs vinblastine or colchicine. Together, these findings suggest that JMJD5 is required to regulate the stability of cytoskeletal microtubules and that JMJD5 depletion increases the susceptibility of cancer cells to microtubule-destabilizing agents.

  1. MAPK Phosphatase AP2C3 Induces Ectopic Proliferation of Epidermal Cells Leading to Stomata Development in Arabidopsis

    PubMed Central

    Kazanaviciute, Vaiva; Magyar, Zoltan; Ayatollahi, Zahra; Unterwurzacher, Verena; Choopayak, Chonnanit; Boniecka, Justyna; Murray, James A. H.; Bogre, Laszlo; Meskiene, Irute

    2010-01-01

    In plant post-embryonic epidermis mitogen-activated protein kinase (MAPK) signaling promotes differentiation of pavement cells and inhibits initiation of stomata. Stomata are cells specialized to modulate gas exchange and water loss. Arabidopsis MAPKs MPK3 and MPK6 are at the core of the signaling cascade; however, it is not well understood how the activity of these pleiotropic MAPKs is constrained spatially so that pavement cell differentiation is promoted only outside the stomata lineage. Here we identified a PP2C-type phosphatase termed AP2C3 (Arabidopsis protein phosphatase 2C) that is expressed distinctively during stomata development as well as interacts and inactivates MPK3, MPK4 and MPK6. AP2C3 co-localizes with MAPKs within the nucleus and this localization depends on its N-terminal extension. We show that other closely related phosphatases AP2C2 and AP2C4 are also MAPK phosphatases acting on MPK6, but have a distinct expression pattern from AP2C3. In accordance with this, only AP2C3 ectopic expression is able to stimulate cell proliferation leading to excess stomata development. This function of AP2C3 relies on the domains required for MAPK docking and intracellular localization. Concomitantly, the constitutive and inducible AP2C3 expression deregulates E2F-RB pathway, promotes the abundance and activity of CDKA, as well as changes of CDKB1;1 forms. We suggest that AP2C3 downregulates the MAPK signaling activity to help maintain the balance between differentiation of stomata and pavement cells. PMID:21203456

  2. Transcriptional profiles of Arabidopsis stomataless mutants reveal developmental and physiological features of life in the absence of stomata

    PubMed Central

    de Marcos, Alberto; Triviño, Magdalena; Pérez-Bueno, María Luisa; Ballesteros, Isabel; Barón, Matilde; Mena, Montaña; Fenoll, Carmen

    2015-01-01

    Loss of function of the positive stomata development regulators SPCH or MUTE in Arabidopsis thaliana renders stomataless plants; spch-3 and mute-3 mutants are extreme dwarfs, but produce cotyledons and tiny leaves, providing a system to interrogate plant life in the absence of stomata. To this end, we compared their cotyledon transcriptomes with that of wild-type plants. K-means clustering of differentially expressed genes generated four clusters: clusters 1 and 2 grouped genes commonly regulated in the mutants, while clusters 3 and 4 contained genes distinctively regulated in mute-3. Classification in functional categories and metabolic pathways of genes in clusters 1 and 2 suggested that both mutants had depressed secondary, nitrogen and sulfur metabolisms, while only a few photosynthesis-related genes were down-regulated. In situ quenching analysis of chlorophyll fluorescence revealed limited inhibition of photosynthesis. This and other fluorescence measurements matched the mutant transcriptomic features. Differential transcriptomes of both mutants were enriched in growth-related genes, including known stomata development regulators, which paralleled their epidermal phenotypes. Analysis of cluster 3 was not informative for developmental aspects of mute-3. Cluster 4 comprised genes differentially up−regulated in mute−3, 35% of which were direct targets for SPCH and may relate to the unique cell types of mute−3. A screen of T-DNA insertion lines in genes differentially expressed in the mutants identified a gene putatively involved in stomata development. A collection of lines for conditional overexpression of transcription factors differentially expressed in the mutants rendered distinct epidermal phenotypes, suggesting that these proteins may be novel stomatal development regulators. Thus, our transcriptome analysis represents a useful source of new genes for the study of stomata development and for characterizing physiology and growth in the absence of

  3. Transcriptional profiles of Arabidopsis stomataless mutants reveal developmental and physiological features of life in the absence of stomata.

    PubMed

    de Marcos, Alberto; Triviño, Magdalena; Pérez-Bueno, María Luisa; Ballesteros, Isabel; Barón, Matilde; Mena, Montaña; Fenoll, Carmen

    2015-01-01

    Loss of function of the positive stomata development regulators SPCH or MUTE in Arabidopsis thaliana renders stomataless plants; spch-3 and mute-3 mutants are extreme dwarfs, but produce cotyledons and tiny leaves, providing a system to interrogate plant life in the absence of stomata. To this end, we compared their cotyledon transcriptomes with that of wild-type plants. K-means clustering of differentially expressed genes generated four clusters: clusters 1 and 2 grouped genes commonly regulated in the mutants, while clusters 3 and 4 contained genes distinctively regulated in mute-3. Classification in functional categories and metabolic pathways of genes in clusters 1 and 2 suggested that both mutants had depressed secondary, nitrogen and sulfur metabolisms, while only a few photosynthesis-related genes were down-regulated. In situ quenching analysis of chlorophyll fluorescence revealed limited inhibition of photosynthesis. This and other fluorescence measurements matched the mutant transcriptomic features. Differential transcriptomes of both mutants were enriched in growth-related genes, including known stomata development regulators, which paralleled their epidermal phenotypes. Analysis of cluster 3 was not informative for developmental aspects of mute-3. Cluster 4 comprised genes differentially up-regulated in mute-3, 35% of which were direct targets for SPCH and may relate to the unique cell types of mute-3. A screen of T-DNA insertion lines in genes differentially expressed in the mutants identified a gene putatively involved in stomata development. A collection of lines for conditional overexpression of transcription factors differentially expressed in the mutants rendered distinct epidermal phenotypes, suggesting that these proteins may be novel stomatal development regulators. Thus, our transcriptome analysis represents a useful source of new genes for the study of stomata development and for characterizing physiology and growth in the absence of stomata.

  4. Separation and Measurement of Direct and Indirect Effects of Light on Stomata 1

    PubMed Central

    Sharkey, Thomas D.; Raschke, Klaus

    1981-01-01

    Conductance for water vapor, assimilation of CO2, and intercellular CO2 concentration of leaves of five species were determined at various irradiances and ambient CO2 concentrations. Conductance and assimilation were then plotted as functions of irradiance and intercellular CO2 concentration. The slopes of these curves allowed us to estimate infinitesimal changes in conductance (and assimilation) that occurred when irradiance changed and intercellular CO2 concentration was constant, and when CO2 concentration changed and irradiance was constant. On leaves of Xanthium strumarium L., Gossypium hirsutum L., Phaseolus vulgaris L., and Perilla frutescens (L.), Britt., the stomatal response to light was determined to be mainly a direct response to light and to a small extent only a response to changes in intercellular CO2 concentration. This was also true for stomata of Zea mays L., except at irradiances < 150 watts per square meter, when stomata responded primarily to the depletion of the intercellular spaces of CO2 which in turn was caused by changes in the assimilation of CO2. Stomata responded to light even in leaves whose net exchange of CO2 was reduced to zero through application of the inhibitor of photosynthetic electron transport, cyanazine (2-chloro-4[1-cyano-1-methylethylamino]-6-ethylamino-S-triazine). When leaves were inverted and irradiated on the abaxial surface, conductance decreased in the shaded and increased in the illuminated epidermis, indicating that the photoreceptor pigment(s) involved are located in the epidermis (presumably in the guard cells). In leaves of X. strumarium, the direct effect of light on conductance is primarily a response to blue light. Stomatal responses to CO2 and to light opposed each other. In X. strumarium, stomatal opening in response to light was strongest in CO2 free air and saturated at lower irradiances than in CO2 containing air. Conversely, stomatal closure in response to CO2 was strongest in darkness and it decreased

  5. Partial Interruption of Axonal Transport Due to Microtubule Breakage Accounts for the Formation of Periodic Varicosities after Traumatic Axonal Injury

    PubMed Central

    Tang-Schomer, Min D.; Johnson, Victoria E.; Baas, Peter W.; Stewart, William; Smith, Douglas H.

    2012-01-01

    Due to their viscoelastic nature, white matter axons are susceptible to damage by high strain rates produced during traumatic brain injury (TBI). Indeed, diffuse axonal injury (DAI) is one of the most common features of TBI, characterized by the hallmark pathological profiles of axonal bulbs at disconnected terminal ends of axons and periodic swellings along axons, known as “varicosities.” Although transport interruption underlies axonal bulb formation, it is unclear how varicosities arise, with multiple sites accumulating transported materials along one axon. Recently, axonal microtubules have been found to physically break during dynamic stretch-injury of cortical axons in vitro. Here, the same in vitro model was used in parallel with histopathological analyses of human brains acquired acutely following TBI to examine the potential role of mechanical microtubule damage in varicosity formation post-trauma. Transmission electron microscopy (TEM) following in vitro stretch-injury revealed periodic breaks of individual microtubules along axons that regionally corresponded with undulations in axon morphology. However, typically less than a third of microtubules were broken in any region of an axon. Within hours, these sites of microtubule breaks evolved into periodic swellings. This suggests axonal transport may be halted along one broken microtubule, yet can proceed through the same region via other intact microtubules. Similar axonal undulations and varicosities were observed following TBI in humans, suggesting primary microtubule failure may also be a feature of DAI. These data indicate a novel mechanism of mechanical microtubule damage leading to partial transport interruption and varicosity formation in traumatic axonal injury. PMID:22079153

  6. Fabrication of Triple-parted Stomata-inspired Membrane with Stimulus-responsive Functions

    PubMed Central

    Kim, Hyejeong; Lee, Sang-Joon

    2016-01-01

    Hydrogels with controllable morphologies and functional movements present a wide range of practical applications. In this work, a triple-parted stomata-inspired membrane (SIM) was fabricated using a UV light cured hydrogel by polymerization-induced diffusion of reactants. A single UV light illumination yielded the SIM that has completely-penetrating pores and semi-penetrated parts. Membranes of various shapes can be easily fabricated within a few minutes by changing the photomask design and composition of the pre-gel solution. Similar to stomatal movement, pores in the fabricated SIM open and close their aperture in response to thermal stimuli. The deformability and transparency of the SIM can be easily controlled for a given application. This SIM exhibits stimulus-response, and therefore has numerous practical applications, such as filter membranes with self-adjustable pores, membrane-based sensors, and functional smart membranes. PMID:26887794

  7. Isolation and characterization of a barley mutant with abscisic-acid-insensitive stomata.

    PubMed

    Raskin, I; Ladyman, J A

    1988-01-01

    A barley (Hordeum vulgare L.) mutant ("cool") with leaf transpiration unaffected by the application of 1 mM abscisic acid (ABA) was isolated from the population of M2 seedlings using thermography (electronic visualization, and quantitation of the temperature profiles on the surface of the leaves). Stomata of the mutant plants were insensitive to exogenously applied ABA, darkness, and such desiccation treatments as leaf excision and drought stress. The evaporative cooling of the leaves of the "cool" barley was always higher than that of the wild-type barley, even without ABA application, indicating that the diffusive resistance of the mutant leaves to water loss was always lower. Guard-cell morphology and stomatal density as well as ABA level and metabolism were seemingly unaltered in the mutant plants. In addition, gibberellin-induced α-amylase secretion and precocious embryo germination in the mutant barley was inhibited by ABA to the same extent as in the wild-type barley.

  8. Closing Plant Stomata Requires a Homolog of an Aluminum-Activated Malate Transporter

    PubMed Central

    Sasaki, Takayuki; Mori, Izumi C.; Furuichi, Takuya; Munemasa, Shintaro; Toyooka, Kiminori; Matsuoka, Ken; Murata, Yoshiyuki; Yamamoto, Yoko

    2010-01-01

    Plant stomata limit both carbon dioxide uptake and water loss; hence, stomatal aperture is carefully set as the environment fluctuates. Aperture area is known to be regulated in part by ion transport, but few of the transporters have been characterized. Here we report that AtALMT12 (At4g17970), a homolog of the aluminum-activated malate transporter (ALMT) of wheat, is expressed in guard cells of Arabidopsis thaliana. Loss-of-function mutations in AtALMT12 impair stomatal closure induced by ABA, calcium and darkness, but do not abolish either the rapidly activated or the slowly activated anion currents previously identified as being important for stomatal closure. Expressed in Xenopus oocytes, AtALMT12 facilitates chloride and nitrate currents, but not those of organic solutes. Therefore, we conclude that AtALMT12 is a novel class of anion transporter involved in stomatal closure. PMID:20154005

  9. Effects of phaseic acid and dihydrophaseic acid on stomata and the photosynthetic apparatus

    SciTech Connect

    Sharkey, T.D.; Raschke, K.

    1980-02-01

    Plant extracts containing phaseic acid (PA), as well as solutions of purified PA and dihydrophaseic acid (DPA) were applied to leaves, isolated mesophyll cells, and isolated epidermal strips. In Commelina communis, stomatal closure began 4 minutes after the addition of either 20 micromolar (+-)-abscisic acid or 10 micromolar PA. Stomata closed less rapidly after treatment with 10 micromolar PA than after treatment with 10 micromolar (+-)-abscisic acid in Amaranthus powelli, Hordeum vulgare, Xanthium strumarium, and Zea mays and did not respond at all to PA in Vicia faba. DPA (10 micromolar) did not cause stomatal closure in any species. Plant extracts containing PA reduced photosynthesis. Subsequent experiments with PA purified by crystallization and with residues of solvents employed in the extraction of PA proved that it was not PA that impaired photosynthetic O/sub 2/ evolution or CO/sub 2/ uptake but unidentified contaminants of the allegedly pure solvents.

  10. Homologue Structure of the SLAC1 Anion Channel for Closing Stomata in Leaves

    SciTech Connect

    Y Chen; L Hu; M Punta; R Bruni; B Hillerich; B Kloss; B Rost; J Love; S Siegelbaum; W Hendrickson

    2011-12-31

    The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 {angstrom} resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

  11. Fabrication of Triple-parted Stomata-inspired Membrane with Stimulus-responsive Functions

    NASA Astrophysics Data System (ADS)

    Kim, Hyejeong; Lee, Sang-Joon

    2016-02-01

    Hydrogels with controllable morphologies and functional movements present a wide range of practical applications. In this work, a triple-parted stomata-inspired membrane (SIM) was fabricated using a UV light cured hydrogel by polymerization-induced diffusion of reactants. A single UV light illumination yielded the SIM that has completely-penetrating pores and semi-penetrated parts. Membranes of various shapes can be easily fabricated within a few minutes by changing the photomask design and composition of the pre-gel solution. Similar to stomatal movement, pores in the fabricated SIM open and close their aperture in response to thermal stimuli. The deformability and transparency of the SIM can be easily controlled for a given application. This SIM exhibits stimulus-response, and therefore has numerous practical applications, such as filter membranes with self-adjustable pores, membrane-based sensors, and functional smart membranes.

  12. Signaling Scaffold Protein IQGAP1 Interacts with Microtubule Plus-end Tracking Protein SKAP and Links Dynamic Microtubule Plus-end to Steer Cell Migration*

    PubMed Central

    Cao, Dan; Su, Zeqi; Wang, Wenwen; Wu, Huihui; Liu, Xing; Akram, Saima; Qin, Bo; Zhou, Jiajia; Zhuang, Xiaoxuan; Adams, Gregory; Jin, Changjiang; Wang, Xiwei; Liu, Lifang; Hill, Donald L.; Wang, Dongmei; Ding, Xia; Yao, Xuebiao

    2015-01-01

    Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex. PMID:26242911

  13. Long astral microtubules uncouple mitotic spindles from the cytokinetic furrow

    PubMed Central

    Rankin, Kathleen E.

    2010-01-01

    Astral microtubules (MTs) are known to be important for cleavage furrow induction and spindle positioning, and loss of astral MTs has been reported to increase cortical contractility. To investigate the effect of excess astral MT activity, we depleted the MT depolymerizer mitotic centromere-associated kinesin (MCAK) from HeLa cells to produce ultra-long, astral MTs during mitosis. MCAK depletion promoted dramatic spindle rocking in early anaphase, wherein the entire mitotic spindle oscillated along the spindle axis from one proto-daughter cell to the other, driven by oscillations of cortical nonmuscle myosin II. The effect was phenocopied by taxol treatment. Live imaging revealed that cortical actin partially vacates the polar cortex in favor of the equatorial cortex during anaphase. We propose that this renders the polar actin cortex vulnerable to rupture during normal contractile activity and that long astral MTs enlarge the blebs. Excessively large blebs displace mitotic spindle position by cytoplasmic flow, triggering the oscillations as the blebs resolve. PMID:20603328

  14. Stochastic Model of Microtubule Dynamics

    NASA Astrophysics Data System (ADS)

    Hryniv, Ostap; Martínez Esteban, Antonio

    2017-10-01

    We introduce a continuous time stochastic process on strings made of two types of particle, whose dynamics mimics that of microtubules in a living cell. The long term behaviour of the system is described in terms of the velocity v of the string end. We show that v is an analytic function of its parameters and study its monotonicity properties. We give a complete characterisation of the phase diagram of the model and derive several criteria of the growth (v>0) and the shrinking (v<0) regimes of the dynamics.

  15. Tubers from patients with tuberous sclerosis complex are characterized by changes in microtubule biology through ROCK2 signalling.

    PubMed

    Ferrer, Isidre; Mohan, Pooja; Chen, Helen; Castellsague, Joan; Gómez-Baldó, Laia; Carmona, Marga; García, Nadia; Aguilar, Helena; Jiang, Jihong; Skowron, Margaretha; Nellist, Mark; Ampuero, Israel; Russi, Antonio; Lázaro, Conxi; Maxwell, Christopher A; Pujana, Miguel Angel

    2014-07-01

    Most patients with tuberous sclerosis complex (TSC) develop cortical tubers that cause severe neurological disabilities. It has been suggested that defects in neuronal differentiation and/or migration underlie the appearance of tubers. However, the precise molecular alterations remain largely unknown. Here, by combining cytological and immunohistochemical analyses of tubers from nine TSC patients (four of them diagnosed with TSC2 germline mutations), we show that alteration of microtubule biology through ROCK2 signalling contributes to TSC neuropathology. All tubers showed a larger number of binucleated neurons than expected relative to control cortex. An excess of normal and altered cytokinetic figures was also commonly observed. Analysis of centrosomal markers suggested increased microtubule nucleation capacity, which was supported by the analysis of an expression dataset from cortical tubers and control cortex, and subsequently linked to under-expression of Rho-associated coiled-coil containing kinase 2 (ROCK2). Thus, augmented microtubule nucleation capacity was observed in mouse embryonic fibroblasts and human fibroblasts deficient in the Tsc2/TSC2 gene product, tuberin. Consistent with ROCK2 under-expression, microtubule acetylation was found to be increased with tuberin deficiency; this alteration was abrogated by rapamycin treatment and mimicked by HDAC6 inhibition. Together, the results of this study support the hypothesis that loss of TSC2 expression can alter microtubule organization and dynamics, which, in turn, deregulate cell division and potentially impair neuronal differentiation. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  16. 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

  17. Separation and measurement of direct and indirect effects of light on stomata

    SciTech Connect

    Sharkey, T.D.; Raschke, K.

    1981-07-01

    Conductance for water vapor, assimilation of CO/sub 2/, and intercellular CO/sub 2/ concentration of leaves of five species were determined at various irradiances and ambient CO/sub 2/ concentrations. Conductance and assimilation were then plotted as functions of irradiance and intercellular CO/sub 2/ concentration. On leaves of Xanthium strumarium L., Gossypium hirsutum L., Phaseolus vulgaris L., and Perilla frutescens (L.), Britt., the stomatal response to light was mainly a direct response to light and to a small extent only a response to changes in intercellular CO/sub 2/ concentration. This was also true for stomata of Zea mays L., except at irradiances <150 watts per square meter. Stomata responded to light even in leaves whose net exchange of CO/sub 2/ was reduced to zero. When leaves were inverted and irradiated on the abaxial surface, conductance decreased in the shaded and increased in the illuminated epidermis, indicating that the photoreceptor pigment(s) involved are located in the epidermis. In leaves of X. strumarium, the direct effect of light on conductance is primarily a response to blue light. Stomatal responses to CO/sub 2/ and to light opposed each other. In X. strumarium, stomatal opening in response to light was strongest in CO/sub 2/-free air and saturated at lower irradiances than in CO/sub 2/-containing air. Conversely, stomatal closure in response to CO/sub 2/ was strongest in darkness and it decreased as irradiance increased. In X. strumarium, P. vulgaris, and P. frutescens, an irradiance of 300 watts per square meter was sufficient to eliminate the stomatal response to CO/sub 2/ altogether. Application of abscisic acid, or an increase in vapor pressure deficit, or a decrease in leaf temperature reduced the stomatal conductance at light saturation.

  18. Smaller stomata require less severe leaf drying to close: a case study in Rosa hydrida.

    PubMed

    Giday, Habtamu; Kjaer, Katrine H; Fanourakis, Dimitrios; Ottosen, Carl-Otto

    2013-10-15

    Stomata formed at high relative air humidity (RH) close less as leaf dries; an effect that varies depending on the genotype. We here quantified the contribution of each stomatal response characteristic to the higher water loss of high RH-grown plants, and assessed the relationship between response characteristics and intraspecific variation in stomatal size. Stomatal size (length multiplied by width), density and responsiveness to desiccation, as well as pore dimensions were analyzed in ten rose cultivars grown at moderate (60%) or high (85%) RH. Leaf morphological components and transpiration at growth conditions were also assessed. High growth RH resulted in thinner (11%) leaves with larger area. A strong positive genetic correlation of daytime and nighttime transpiration at either RH was observed. Stomatal size determined pore area (r=0.7) and varied by a factor of two, as a result of proportional changes in length and width. Size and density of stomata were not related. Following desiccation, high RH resulted in a significantly lower (6-19%) decline of transpiration in three cultivars, whereas the relative water content (RWC) of high RH-expanded leaflets was lower (29-297%) in seven cultivars. The lower RWC of these leaflets was caused by (a) higher (33-72%) stable transpiration and/or (b) lower (12-143%) RWC at which this stable transpiration occurred, depending on the cultivar. Stomatal size was significantly correlated with both characteristics (r=0.5 and -0.7, respectively). These results indicate that stomatal size explains much of the intraspecific variation in the regulation of transpiration upon water deprivation on rose.

  19. Nectar-secreting Floral Stomata in Maxillaria anceps Ames & C. Schweinf. (Orchidaceae)

    PubMed Central

    DAVIES, K. L.; STPICZYŃSKA, M.; GREGG, A.

    2005-01-01

    • Background and Aims Although it was generally assumed that Maxillaria spp. do not produce nectar, in recent years, nectar has been reported for a number of these orchids. Nevertheless, our current understanding of nectary structure and nectar secretion in Maxillaria is based solely on M. coccinea (Jacq.) L.O. Williams ex Hodge, which, since it shows many features characteristic of ornithophilous flowers, is atypical of this largely entomophilous genus. The aim of the present paper is to describe, for the first time, nectar secretion in a presumed entomophilous species of Maxillaria. • Methods The structure of the nectary of M. anceps Ames & C. Schweinf., nectar composition and the process of nectar secretion were investigated using light microscopy, scanning electron microscopy, transmission electron microscopy, histochemistry, refractometry and high performance liquid chromatography. • Key Results and Conclusions Nectar appears as droplets that are exuded by modified stomata borne upon the labellar callus and collects upon the labellum and at the base of the column-foot. Although such stomata are known to occur in a number of angiosperm families, this is the first time for them to be observed in orchids. The callus consists largely of parenchyma with raphides and is supplied by eight to ten collateral bundles. This tissue, together with the single-layered epidermis, seemingly contains terpenoids. During the bud stage, the callus cells contain an organelle complement consistent with secretory cells whereas by day 4 of anthesis, much of the cell is occupied by a vacuole. The nectar is sucrose-dominant but also contains low concentrations of glucose, fructose, free amino acids and possibly terpenoids. The high sugar concentration (approx. 66 %) is consistent with melittophily and may indicate that, like the majority of Maxillaria spp., M. anceps is visited by stingless bees (Meliponini). PMID:15953790

  20. MCF7 microtubules: Cancer microtubules with relatively slow and stable dynamic in vitro.

    PubMed

    Feizabadi, Mitra Shojania; Rosario, Brandon

    2017-03-04

    There is known to be significant diversity of β-tubulin isoforms in cells. However, whether the functions of microtubules that are polymerized from different distributions of beta isotypes become distinct from one another are still being explored. Of particular interest, recent studies have identified the role that different beta tubulin isotypes carry in regulating the functions of some of the molecular motors along MCF7, or breast cancer, microtubules. That being said, how the specific distribution of beta tubulin isotypes impacts the MCF7 microtubules' dynamic is not well understood. The current study was initiated to directly quantify the in vitro dynamic and polymerization parameters of single MCF7 microtubules and then compare them with those obtained from neuronal microtubules polymerized from porcine brain tubulin. Surprisingly, unlike porcine brain microtubules, this type of cancer microtubule showed a relatively stable and slow dynamic. The comparison between the subsequently fast and unstable dynamic of porcine brain microtubules with the significantly slow and relatively stable dynamic of MCF7 microtubules suggests that beta tubulin isotypes may not only influence the microtubule based functionalities of some molecular motors, but also may change the microtubule's intrinsic dynamic.

  1. Mechanical Properties of Doubly Stabilized Microtubule Filaments

    PubMed Central

    Hawkins, Taviare L.; Sept, David; Mogessie, Binyam; Straube, Anne; Ross, Jennifer L.

    2013-01-01

    Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5′ [(α, β)-methyleno] triphosphate, guanosine-5′-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules. PMID:23561528

  2. Kinesin-5 is a microtubule polymerase

    PubMed Central

    Chen, Yalei; Hancock, William O

    2015-01-01

    Kinesin-5 slides antiparallel microtubules during spindle assembly, and regulates the branching of growing axons. Besides the mechanical activities enabled by its tetrameric configuration, the specific motor properties of kinesin-5 that underlie its cellular function remain unclear. Here by engineering a stable kinesin-5 dimer and reconstituting microtubule dynamics in vitro, we demonstrate that kinesin-5 promotes microtubule polymerization by increasing the growth rate and decreasing the catastrophe frequency. Strikingly, microtubules growing in the presence of kinesin-5 have curved plus ends, suggesting that the motor stabilizes growing protofilaments. Single-molecule fluorescence experiments reveal that kinesin-5 remains bound to the plus ends of static microtubules for 7 s, and tracks growing microtubule plus ends in a manner dependent on its processivity. We propose that kinesin-5 pauses at microtubule plus ends and enhances polymerization by stabilizing longitudinal tubulin–tubulin interactions, and that these activities underlie the ability kinesin-5 to slide and stabilize microtubule bundles in cells. PMID:26437877

  3. Microtubule motors: moving forward on many fronts.

    PubMed

    Allan, Viki

    2009-07-08

    Microtubule motors drive the movement of many different cargoes in eukaryotic cells. A combination of in vitro and in vivo approaches has led to a better understanding of their mechanism of action and function and are also revealing that the microtubule track itself may have an important role to play in directing cargo movement within the cell.

  4. Microtubule stabilization: formins assert their independence.

    PubMed

    DeWard, Aaron D; Alberts, Arthur S

    2008-07-22

    Mammalian Diaphanous-related (mDia) formins are well known for their actin nucleation and filament elongation activities. They have since emerged as microtubule-binding proteins, and a recent study shows that mDia2 stabilizes microtubules independently of its actin nucleation activity.

  5. Mechanical properties of doubly stabilized microtubule filaments.

    PubMed

    Hawkins, Taviare L; Sept, David; Mogessie, Binyam; Straube, Anne; Ross, Jennifer L

    2013-04-02

    Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5' [(α, β)-methyleno] triphosphate, guanosine-5'-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules.

  6. Organization of microtubules in cochlear hair cells.

    PubMed

    Furness, D N; Hackney, C M; Steyger, P S

    1990-07-01

    The organization of microtubules in hair cells of the guinea-pig cochlea has been investigated using transmission electron microscopy and correlated with the location of tubulin-associated immunofluorescence in surface preparations of the organ of Corti. Results from both techniques reveal consistent distributions of microtubules in inner and outer hair cells. In the inner hair cells, microtubules are most concentrated in the apex. Reconstruction from serial sections shows three main groups: firstly, in channels through the cuticular plate and in a discontinuous belt around its upper perimeter; secondly, forming a ring inside a rim extending down from the lower perimeter of the plate; and thirdly, in a meshwork underlying the main body of the plate. In the cell body, microtubules line the inner face of the subsurface cistern and extend longitudinally through a tubulo-vesicular track between the apex and base. In outer hair cells, the pattern of microtubules associated with the cuticular plate is similar, although there are fewer present than in inner hair cells. In outer hair cells from the apex of the cochlea, microtubules occur around an infracuticular protrusion of cuticular plate material. In the cell body, many more microtubules occur in the region below the nucleus compared with inner hair cells. The possible functions of microtubules in hair cells are discussed by comparison with those found in other systems. These include morphogenesis and maintenance of cell shape; intracellular transport, e.g., of neurotransmitter vesicles; providing a possible substrate for motility; mechanical support of structures associated with sensory transduction.

  7. A role for katanin in plant cell division: microtubule organization in dividing root cells of fra2 and lue1Arabidopsis thaliana mutants.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Voulgari, Georgia; Papadopoulou, Galini

    2011-07-01

    Severing of microtubules by katanin has proven to be crucial for cortical microtubule organization in elongating and differentiating plant cells. On the contrary, katanin is currently not considered essential during cell division in plants as it is in animals. However, defects in cell patterning have been observed in katanin mutants, implying a role for it in dividing plant cells. Therefore, microtubule organization was studied in detail by immunofluorescence in dividing root cells of fra2 and lue1 katanin mutants of Arabidopsis thaliana. In both, early preprophase bands consisted of poorly aligned microtubules, prophase spindles were multipolar, and the microtubules of expanding phragmoplasts were elongated, bended toward and connected to the surface of daughter nuclei. Accordingly, severing by katanin seems to be necessary for the proper organization of these microtubule arrays. In both fra2 and lue1, metaphase/anaphase spindles and initiating phragmoplasts exhibited typical organization. However, they were obliquely oriented more frequently than in the wild type. It is proposed that this oblique orientation may be due to prophase spindle multipolarity and results in a failure of the cell plate to follow the predetermined division plane, during cytokinesis, producing oblique cell walls in the roots of both mutants. It is therefore concluded that, like in animal cells, katanin is important for plant cell division, influencing the organization of several microtubule arrays. Moreover, failure in microtubule severing indirectly affects the orientation of the division plane.

  8. Kinesin-12 motors cooperate to suppress microtubule catastrophes and drive the formation of parallel microtubule bundles

    PubMed Central

    Drechsler, Hauke; McAinsh, Andrew D.

    2016-01-01

    Human Kinesin-12 (hKif15) plays a crucial role in assembly and maintenance of the mitotic spindle. These functions of hKif15 are partially redundant with Kinesin-5 (Eg5), which can cross-link and drive the extensile sliding of antiparallel microtubules. Although both motors are known to be tetramers, the functional properties of hKif15 are less well understood. Here we reveal how single or multiple Kif15 motors can cross-link, transport, and focus the plus-ends of intersecting microtubules. During transport, Kif15 motors step simultaneously along both microtubules with relative microtubule transport driven by a velocity differential between motor domain pairs. Remarkably, this differential is affected by the underlying intersection geometry: the differential is low on parallel and extreme on antiparallel microtubules where one motor domain pair becomes immobile. As a result, when intersecting microtubules are antiparallel, canonical transport of one microtubule along the other is allowed because one motor is firmly attached to one microtubule while it is stepping on the other. When intersecting microtubules are parallel, however, Kif15 motors can drive (biased) parallel sliding because the motor simultaneously steps on both microtubules that it cross-links. These microtubule rearrangements will focus microtubule plus-ends and finally lead to the formation of parallel bundles. At the same time, Kif15 motors cooperate to suppress catastrophe events at polymerizing microtubule plus-ends, raising the possibility that Kif15 motors may synchronize the dynamics of bundles that they have assembled. Thus, Kif15 is adapted to operate on parallel microtubule substrates, a property that clearly distinguishes it from the other tetrameric spindle motor, Eg5. PMID:26969727

  9. Kinesin-12 motors cooperate to suppress microtubule catastrophes and drive the formation of parallel microtubule bundles.

    PubMed

    Drechsler, Hauke; McAinsh, Andrew D

    2016-03-22

    Human Kinesin-12 (hKif15) plays a crucial role in assembly and maintenance of the mitotic spindle. These functions of hKif15 are partially redundant with Kinesin-5 (Eg5), which can cross-link and drive the extensile sliding of antiparallel microtubules. Although both motors are known to be tetramers, the functional properties of hKif15 are less well understood. Here we reveal how single or multiple Kif15 motors can cross-link, transport, and focus the plus-ends of intersecting microtubules. During transport, Kif15 motors step simultaneously along both microtubules with relative microtubule transport driven by a velocity differential between motor domain pairs. Remarkably, this differential is affected by the underlying intersection geometry: the differential is low on parallel and extreme on antiparallel microtubules where one motor domain pair becomes immobile. As a result, when intersecting microtubules are antiparallel, canonical transport of one microtubule along the other is allowed because one motor is firmly attached to one microtubule while it is stepping on the other. When intersecting microtubules are parallel, however, Kif15 motors can drive (biased) parallel sliding because the motor simultaneously steps on both microtubules that it cross-links. These microtubule rearrangements will focus microtubule plus-ends and finally lead to the formation of parallel bundles. At the same time, Kif15 motors cooperate to suppress catastrophe events at polymerizing microtubule plus-ends, raising the possibility that Kif15 motors may synchronize the dynamics of bundles that they have assembled. Thus, Kif15 is adapted to operate on parallel microtubule substrates, a property that clearly distinguishes it from the other tetrameric spindle motor, Eg5.

  10. 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.

  11. Tubulin bistability and polymorphic dynamics of microtubules.

    PubMed

    Mohrbach, Hervé; Johner, Albert; Kulić, Igor M

    2010-12-31

    Based on the hypothesis that the GDP-tubulin dimer is a conformationally bistable molecule-rapidly fluctuating between a discrete curved and a straight state-we develop a model for polymorphic dynamics of the microtubule lattice. We show that GDP-tubulin bistability consistently explains unusual dynamic fluctuations, the apparent length-stiffness relation of grafted taxol-stabilized microtubules, and the curved-helical appearance of microtubules in general. When clamped by one end the microtubules undergo an unusual zero energy motion-in its effect reminiscent of a limited rotational hinge. We conclude that microtubules exist in highly cooperative energy-degenerate helical states and discuss possible implications in vivo.

  12. On complex, curved trajectories in microtubule gliding

    NASA Astrophysics Data System (ADS)

    Gosselin, Pierre; Mohrbach, Hervé; Kulić, Igor M.; Ziebert, Falko

    2016-04-01

    We study the dynamics of microtubules in gliding assays. These biofilaments are typically considered as purely semiflexible, hence their trajectories under the action of motors covering the substrate have been regarded so far as straight, modulo fluctuations. However, this is not always the case experimentally, where microtubules are known to move on large scale circles or spirals, or even display quite regular wavy trajectories and more complex dynamics. Incorporating recent experimental evidence for a (small) preferred curvature as well as the microtubules' well established lattice twist into a dynamic model for microtubule gliding, we could reproduce both types of trajectories. Interestingly, as a function of the microtubules' length we found length intervals of stable rings alternating with regions where wavy and more complex dynamics prevails. Finally, both types of dynamics (rings and waves) can be rationalized by considering simple limits of the full model.

  13. Tubulin Bistability and Polymorphic Dynamics of Microtubules

    NASA Astrophysics Data System (ADS)

    Mohrbach, Hervé; Johner, Albert; Kulić, Igor M.

    2010-12-01

    Based on the hypothesis that the GDP-tubulin dimer is a conformationally bistable molecule—rapidly fluctuating between a discrete curved and a straight state—we develop a model for polymorphic dynamics of the microtubule lattice. We show that GDP-tubulin bistability consistently explains unusual dynamic fluctuations, the apparent length-stiffness relation of grafted taxol-stabilized microtubules, and the curved-helical appearance of microtubules in general. When clamped by one end the microtubules undergo an unusual zero energy motion—in its effect reminiscent of a limited rotational hinge. We conclude that microtubules exist in highly cooperative energy-degenerate helical states and discuss possible implications in vivo.

  14. FLUCTUATING MOTOR FORCES BEND GROWING MICROTUBULES

    PubMed Central

    Shekhar, Nandini; Neelam, Srujana; Wu, Jun; Ladd, Anthony JC; Dickinson, Richard B.; Lele, Tanmay P.

    2013-01-01

    Despite their rigidity, microtubules in living cells bend significantly during polymerization resulting in greater curvature than can be explained by thermal forces alone. However, the source of the non-thermal forces that bend growing microtubules remains obscure. We analyzed the motion of microtubule tips in NIH-3T3 fibroblasts expressing EGFP-EB1, a fluorescent +TIP protein that specifically binds to the growing ends of microtubules. We found that dynein inhibition significantly reduced the deviation of the growing tip from its initial trajectory. Inhibiting myosin modestly reduced tip fluctuations, while simultaneous myosin and dynein inhibition caused no further decrease in fluctuations compared to dynein inhibition alone. Our results can be interpreted with a model in which dynein linkages play a key role in generating and transmitting fluctuating forces that bend growing microtubules. PMID:24039637

  15. Active contraction of microtubule networks

    PubMed Central

    Foster, Peter J; Fürthauer, Sebastian; Shelley, Michael J; Needleman, Daniel J

    2015-01-01

    Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction. DOI: http://dx.doi.org/10.7554/eLife.10837.001 PMID:26701905

  16. Targeting Microtubules for Wound Repair

    PubMed Central

    Charafeddine, Rabab A.; Nosanchuk, Joshua D.; Sharp, David J.

    2016-01-01

    Significance: Fast and seamless healing is essential for both deep and chronic wounds to restore the skin and protect the body from harmful pathogens. Thus, finding new targets that can both expedite and enhance the repair process without altering the upstream signaling milieu and causing serious side effects can improve the way we treat wounds. Since cell migration is key during the different stages of wound healing, it presents an ideal process and intracellular structural machineries to target. Recent Advances and Critical Issues: The microtubule (MT) cytoskeleton is rising as an important structural and functional regulator of wound healing. MTs have been reported to play different roles in the migration of the various cell types involved in wound healing. Specific microtubule regulatory proteins (MRPs) can be targeted to alter a section or subtype of the MT cytoskeleton and boost or hinder cell motility. However, inhibiting intracellular components can be challenging in vivo, especially using unstable molecules, such as small interfering RNA. Nanoparticles can be used to protect these unstable molecules and topically deliver them to the wound. Utilizing this approach, we recently showed that fidgetin-like 2, an uncharacterized MRP, can be targeted to enhance cell migration and wound healing. Future Directions: To harness the full potential of the current MRP therapeutic targets, studies should test them with different delivery platforms, dosages, and skin models. Screening for new MT effectors that boost cell migration in vivo would also help find new targets for skin repair. PMID:27785378

  17. Stomata open at night in pole-sized and mature ponderosa pine: implications for O3 exposure metrics

    Treesearch

    Nancy Grulke; R. Alonso; T. Nguyen; C. Cascio; W. Dobrowolski

    2004-01-01

    Ponderosa pine (Pinus ponderosa Dougl. exLaws.) is widely distributed in the western USA.We report the lack of stomatal closure at night in early summer for ponderosa pine at two of three sites investigated. Trees at a third site with lower nitrogen dioxide and nitric acid exposure, but greater drought stress, had slightly open stomata at night in...

  18. NITRIC OXIDE SYNTHASE INHIBITOR L-NAMEAFFECTS ARABIDOPSIS ROOT GROWTH, MORPHOLOGY AND MICROTUBULE ORGANIZATION.

    PubMed

    Yu A, Krasylenko; A I, Yemets; Ya B, Blume

    2017-10-05

    The presence of evolutionarily conserved NOS or NOS-like enzymes in land plants different than those in animals is still unclear, despite their activity has been revealed in cytosol and some organelles. At the same time, the emerging evidence for the importance of L-arginine-dependent pathways of NO synthesis in plant cells is still accumulating. The aim of our study was to reveal physiological effects on growth and differentiation processes and microtubular cytoskeleton organization of the competitive mammalian NO synthase inhibitor L-NAME. Thus, the treatment of Arabidopsis with L-NAME (50 μM-1 mM) caused dose- and time-dependent inhibition of primary roots growth. Moreover, the morphology of primary roots under the influence of L-NAME also underwent changes. L-NAME (>100 μM) induced the formation of novel over-elongated root hairs in shortened elongation zone, while in higher concentrations (500 μM) it caused a slight swelling of epidermal cells in differentiation zone. L-NAME also provoked microtubule reorganization in epidermal cells of different root growth zones. Thus, L-NAME at concentrations of 50 μM-1 mM induces cortical microtubules randomization and/or depolymerization in epidermal cells of the root apex, meristem, transition, elongation and differentiation zones after 2 h of treatment. Disordered microtubules in trichoblasts could initiate the formation of actively elongating root hairs that reveals longitudinal microtubules ensuring their active growth at 24 h of treatment. Therefore, L-NAME inhibits primary root growth, induces the differentiation processes in roots and reorganizes cortical microtubules in epidermal root cells suggesting the importance of L-arginine-dependent pathways of NO synthesis in plants. This article is protected by copyright. All rights reserved.

  19. Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis[OPEN

    PubMed Central

    Buschmann, Henrik; Lloyd, Clive W.

    2015-01-01

    Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric 14N/15N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning. PMID:26432860

  20. NIMA-related kinases regulate directional cell growth and organ development through microtubule function in Arabidopsis thaliana.

    PubMed

    Motose, Hiroyasu; Takatani, Shogo; Ikeda, Tatsuya; Takahashi, Taku

    2012-12-01

    NIMA-related kinase 6 (NEK6) regulates cellular expansion and morphogenesis through microtubule organizaiton in Arabidopsis thaliana. Loss-of-function mutations in NEK6 (nek6/ibo1) cause ectopic outgrowth and microtubule disorganization in epidermal cells. We recently found that NEK6 forms homodimers and heterodimers with NEK4 and NEK5 to destabilize cortical microtubules possibly by direct binding to microtubules and the β-tubulin phosphorylation. Here, we identified a new allele of NEK6 and further analyzed the morphological phenotypes of nek6/ibo1 mutants, along with alleles of nek4 and nek5 mutants. Phenotypic analysis demonstrated that NEK6 is required for the directional growth of roots and hypocotyls, petiole elongation, cell file formation, and trichome morphogenesis. In addition, nek4, nek5, and nek6/ibo1 mutants were hypersensitive to microtubule inhibitors such as propyzamide and taxol. These results suggest that plant NEKs function in directional cell growth and organ development through the regulation of microtubule organization.

  1. MicroFilament Analyzer, an image analysis tool for quantifying fibrillar orientation, reveals changes in microtubule organization during gravitropism.

    PubMed

    Jacques, Eveline; Buytaert, Jan; Wells, Darren M; Lewandowski, Michal; Bennett, Malcolm J; Dirckx, Joris; Verbelen, Jean-Pierre; Vissenberg, Kris

    2013-06-01

    Image acquisition is an important step in the study of cytoskeleton organization. As visual interpretations and manual measurements of digital images are prone to errors and require a great amount of time, a freely available software package named MicroFilament Analyzer (MFA) was developed. The goal was to provide a tool that facilitates high-throughput analysis to determine the orientation of filamentous structures on digital images in a more standardized, objective and repeatable way. Here, the rationale and applicability of the program is demonstrated by analyzing the microtubule patterns in epidermal cells of control and gravi-stimulated Arabidopsis thaliana roots. Differential expansion of cells on either side of the root results in downward bending of the root tip. As cell expansion depends on the properties of the cell wall, this may imply a differential orientation of cellulose microfibrils. As cellulose deposition is orchestrated by cortical microtubules, the microtubule patterns were analyzed. The MFA program detects the filamentous structures on the image and identifies the main orientation(s) within individual cells. This revealed four distinguishable microtubule patterns in root epidermal cells. The analysis indicated that gravitropic stimulation and developmental age are both significant factors that determine microtubule orientation. Moreover, the data show that an altered microtubule pattern does not precede differential expansion. Other possible applications are also illustrated, including field emission scanning electron micrographs of cellulose microfibrils in plant cell walls and images of fluorescent actin. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  2. Ethylene Regulates the Arabidopsis Microtubule-Associated Protein WAVE-DAMPENED2-LIKE5 in Etiolated Hypocotyl Elongation1[OPEN

    PubMed Central

    Sun, Jingbo; Ma, Qianqian; Mao, Tonglin

    2015-01-01

    The phytohormone ethylene plays crucial roles in the negative regulation of plant etiolated hypocotyl elongation. The microtubule cytoskeleton also participates in hypocotyl cell growth. However, it remains unclear if ethylene signaling-mediated etiolated hypocotyl elongation involves the microtubule cytoskeleton. In this study, we functionally identified the previously uncharacterized microtubule-associated protein WAVE-DAMPENED2-LIKE5 (WDL5) as a microtubule-stabilizing protein that plays a positive role in ethylene-regulated etiolated hypocotyl cell elongation in Arabidopsis (Arabidopsis thaliana). ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, directly targets and up-regulates WDL5. Etiolated hypocotyls from a WDL5 loss-of-function mutant (wdl5-1) were more insensitive to 1-aminocyclopropane-1-carboxylic acid treatment than the wild type. Decreasing WDL5 expression partially rescued the shorter etiolated hypocotyl phenotype in the ethylene overproduction mutant eto1-1. Reorganization of cortical microtubules in etiolated hypocotyl cells from the wdl5-1 mutant was less sensitive to 1-aminocyclopropane-1-carboxylic acid treatment. These findings indicate that WDL5 is an important participant in ethylene signaling inhibition of etiolated hypocotyl growth. This study reveals a mechanism involved in the ethylene regulation of microtubules through WDL5 to inhibit etiolated hypocotyl cell elongation. PMID:26134166

  3. Ethylene Regulates the Arabidopsis Microtubule-Associated Protein WAVE-DAMPENED2-LIKE5 in Etiolated Hypocotyl Elongation.

    PubMed

    Sun, Jingbo; Ma, Qianqian; Mao, Tonglin

    2015-09-01

    The phytohormone ethylene plays crucial roles in the negative regulation of plant etiolated hypocotyl elongation. The microtubule cytoskeleton also participates in hypocotyl cell growth. However, it remains unclear if ethylene signaling-mediated etiolated hypocotyl elongation involves the microtubule cytoskeleton. In this study, we functionally identified the previously uncharacterized microtubule-associated protein WAVE-DAMPENED2-LIKE5 (WDL5) as a microtubule-stabilizing protein that plays a positive role in ethylene-regulated etiolated hypocotyl cell elongation in Arabidopsis (Arabidopsis thaliana). ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, directly targets and up-regulates WDL5. Etiolated hypocotyls from a WDL5 loss-of-function mutant (wdl5-1) were more insensitive to 1-aminocyclopropane-1-carboxylic acid treatment than the wild type. Decreasing WDL5 expression partially rescued the shorter etiolated hypocotyl phenotype in the ethylene overproduction mutant eto1-1. Reorganization of cortical microtubules in etiolated hypocotyl cells from the wdl5-1 mutant was less sensitive to 1-aminocyclopropane-1-carboxylic acid treatment. These findings indicate that WDL5 is an important participant in ethylene signaling inhibition of etiolated hypocotyl growth. This study reveals a mechanism involved in the ethylene regulation of microtubules through WDL5 to inhibit etiolated hypocotyl cell elongation.

  4. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

    PubMed

    Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

    2017-04-01

    Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

  5. Distribution and ultrastructure of the stomata connecting the pleural cavity with lymphatics in the rat costal pleura.

    PubMed

    Wang, Q X; Ohtani, O; Saitoh, M; Ohtani, Y

    1997-01-01

    We investigated the detailed distribution and ultrastructure of the stomata connecting the pleural cavity and the lymphatics in the rat costal pleura by scanning electron, transmission electron and light microscopy. The mesothelial cells lining the costal pleura appeared as both flattened and thick cell bodies. The thick cells possessed more rough endoplasmic reticula, Golgi complexes, mitochondria, and free ribosomes than the flattened cells. The thick cells were distributed in the intercostal regions each cephalic to the junction of the costal cartilage and bone, and in the band-like regions along the cephalic and caudal sides of each rib in the lateral and dorsal thoracic walls. In the regions lined with thick cells, there were stomata [12.9 +/- 10.3 microns2 (mean +/- SD) in area] consisting of prolongations of thick mesothelial cells and funnel-like projections of lymphatic endothelial cells that came up along the rims of the pores (5.9 +/- 3.2 microns2 in average area) in the submesothelial collagen fiber network. At the stomata, the basal lamina of the mesothelium was continuous with that of the endothelium. The mesothelial cells forming the stomata were mostly in close contact with the endothelial cells, but some gaps also existed between them. Valve-like endothelial flaps were frequently observed wherever endothelial cells constituting the stomata merged into the submesothelial lymphatics. Also present were lymphatic bulges that were either in close contact with the base of the thick mesothelial cells or exposed through the mesothelial pores. The lymphatic network was especially well developed in the submesothelial layer at and around the thick-cell regions. The initial lymphatics drained into the intercostal collecting lymphatics, which in turn led into either the parasternal or paravertebral lymphatic trunk. Our results suggest that the stomata play a major role in absorbing fluids and particulates in the pleural cavity. The thick mesothelial cells

  6. A Mutation in the Catalytic Subunit of the Glycosylphosphatidylinositol Transamidase Disrupts Growth, Fertility, and Stomata Formation1[OPEN

    PubMed Central

    2016-01-01

    GPI-anchored proteins (GPI-APs) are essential for plant growth and development; knockout mutations in enzymes responsible for anchor biosynthesis or attachment are gametophyte or embryo lethal. In a genetic screen targeted to identify genes regulating stomata formation, we discovered a missense mutation in the Arabidopsis (Arabidopsis thaliana) homolog of GPI8/PIG-K, a Cys protease that transfers an assembled GPI anchor to proteins. The Arabidopsis genome has a single copy of AtGPI8, and the atgpi8-1 mutation reduces the efficiency of this enzyme, leading to reduced accumulation of GPI-anchored proteins. While the atgpi8-1 mutation strongly disrupts plant growth, it is not lethal. Phenotypic analysis of atgpi8-1 mutants suggests that GPI-APs are important for root and shoot growth, stomata formation, apical dominance, transition to flowering, and male gametophyte viability. In addition, atgpi8-1 mutants accumulate higher levels of callose and have reduced plasmodesmata permeability. Genetic interactions of atgpi8-1 with mutations in ERECTA family (ERf) genes suggest the existence of a GPI-AP in a branch of the ERf signaling pathway that regulates stomata formation. Activation of the ERf signal transduction cascade by constitutively active YODA rescues stomata clustering in atgpi8-1, indicating that a GPI-AP functions upstream of the MAP kinase cascade. TOO MANY MOUTHS (TMM) is a receptor-like protein that is able to form heterodimers with ERfs. Our analysis demonstrates that tmm-1 is epistatic to atgpi8-1, indicating that either TMM is a GPI-AP or there is another GPI-AP regulating stomata development whose function is dependent upon TMM. PMID:27208238

  7. Microtubule organization by kinesin motors and microtubule crosslinking protein MAP65

    NASA Astrophysics Data System (ADS)

    Pringle, Joshua; Muthukumar, Amutha; Tan, Amanda; Crankshaw, Laura; Conway, Leslie; Ross, Jennifer L.

    2013-09-01

    Microtubules are rigid, proteinaceous filaments required to organize and rearrange the interior of cells. They organize space by two mechanisms, including acting as the tracks for long-distance cargo transporters, such as kinesin-1, and by forming a network that supports the shape of the cell. The microtubule network is composed of microtubules and a bevy of associated proteins and enzymes that self-organize using non-equilibrium dynamic processes. In order to address the effects of self-organization of microtubules, we have utilized the filament-gliding assay with kinesin-1 motors driving microtubule motion. To further enhance the complexity of the system and determine if new patterns are formed, we added the microtubule crosslinking protein MAP65-1. MAP65-1 is a microtubule-associated protein from plants that crosslinks antiparallel microtubules, similar to mammalian PRC1 and fission yeast Ase1. We find that MAP65 can slow and halt the velocity of microtubules in gliding assays, but when pre-formed microtubule bundles are added to gliding assays, kinesin-1 motors can pull apart the bundles and reconstitute cell-like protrusions.

  8. Cross-linking of microtubules by microtubule-associated proteins (MAPs) from the brine shrimp, Artemia.

    PubMed

    Campbell, E J; MacKinlay, S A; MacRae, T H

    1989-05-01

    Microtubules induced with taxol to assemble in cell-free extracts of the brine shrimp, Artemia, are cross-linked by microtubule-associated proteins (MAPs). When the MAPs, extracted from taxol-stabilized microtubules with 1 M-NaCl are co-assembled with purified Artemia or mammalian neural tubulin, reconstitution of cross-linking between microtubules occurs. The most prominent non-tubulin protein associated with reconstituted cross-linked microtubules has a molecular weight of 49,000 but we cannot yet exclude the possibility that other proteins may be responsible for the cross-linking. Cross-linkers are separated by varying distances while cross-linked microtubules, prepared under different conditions, are 6.9-7.7 nm apart. Cross-linking of microtubules by MAPs occurs whether MAPs are added to assembling tubulin or to microtubules, and it is not disrupted by ATP. The MAPs are heat-sensitive and do not stabilize microtubules to cold. Immunological characterization of Artemia MAPs on Western blots indicates that Artemia lack MAP 1, MAP 2 and tau. Our results clearly demonstrate that Artemia contain novel MAPs with the ability to cross-link microtubules from phylogenetically disparate organisms in an ATP-independent manner.

  9. Reovirus Cell Entry Requires Functional Microtubules

    PubMed Central

    Mainou, Bernardo A.; Zamora, Paula F.; Ashbrook, Alison W.; Dorset, Daniel C.; Kim, Kwang S.; Dermody, Terence S.

    2013-01-01

    ABSTRACT Mammalian reovirus binds to cell-surface glycans and junctional adhesion molecule A and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within the endocytic compartment, reovirus undergoes stepwise disassembly, allowing release of the transcriptionally active viral core into the cytoplasm. To identify cellular mediators of reovirus infectivity, we screened a library of small-molecule inhibitors for the capacity to block virus-induced cytotoxicity. In this screen, reovirus-induced cell killing was dampened by several compounds known to impair microtubule dynamics. Microtubule inhibitors were assessed for blockade of various stages of the reovirus life cycle. While these drugs did not alter reovirus cell attachment or internalization, microtubule inhibitors diminished viral disassembly kinetics with a concomitant decrease in infectivity. Reovirus virions colocalize with microtubules and microtubule motor dynein 1 during cell entry, and depolymerization of microtubules results in intracellular aggregation of viral particles. These data indicate that functional microtubules are required for proper sorting of reovirus virions following internalization and point to a new drug target for pathogens that use the endocytic pathway to invade host cells. PMID:23820395

  10. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

    Nejedla, Michaela; Sadi, Sara; Sulimenko, Vadym; de Almeida, Francisca Nunes; Blom, Hans; Draber, Pavel; Aspenström, Pontus; Karlsson, Roger

    2016-01-01

    Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element. PMID:27307590

  11. Microtubules regulate disassembly of epithelial apical junctions

    PubMed Central

    Ivanov, Andrei I; McCall, Ingrid C; Babbin, Brian; Samarin, Stanislav N; Nusrat, Asma; Parkos, Charles A

    2006-01-01

    Background Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion. Results Calcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex. Conclusion Our data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins. PMID:16509970

  12. Insights into Antiparallel Microtubule Crosslinking by PRC1, a Conserved Nonmotor Microtubule Binding Protein

    SciTech Connect

    Subramanian, Radhika; Wilson-Kubalek, Elizabeth M.; Arthur, Christopher P.; Bick, Matthew J.; Campbell, Elizabeth A.; Darst, Seth A.; Milligan, Ronald A.; Kapoor, Tarun M.

    2010-09-03

    Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks are compliant and do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish crosslinks that selectively mark antiparallel overlap in dynamic cytoskeletal networks.

  13. The microtubule catastrophe promoter Sentin delays stable kinetochore–microtubule attachment in oocytes

    PubMed Central

    Głuszek, A. Agata; Cullen, C. Fiona; Li, Wenjing; Battaglia, Rachel A.; Radford, Sarah J.; Costa, Mariana F.; McKim, Kim S.; Goshima, Gohta

    2015-01-01

    The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore–microtubule attachment in oocytes. PMID:26668329

  14. Dynamic response of axonal microtubules under suddenly applied end forces.

    PubMed

    Manuchehrfar, Farid; Shamloo, Amir; Mehboudi, Nastaran

    2014-01-01

    Axon is a filament in neuronal system and axonal microtubules are bundles in axons. In axons, microtubules are coated with microtubule-associated protein tau, a natively unfolded profuse filamentous protein in the central nervous system. These proteins are responsible for the cross-linked structure of the axonal microtubule bundles. Through complimentary dimerization with other tau proteins, bridges are formed to nearby microtubules to create bundles. The transverse reinforcement of microtubules by cross-linking to the cytoskeleton has been shown to enhance their ability to bear compressive loads. Though microtubules are conventionally regarded as bearing compressive loads, in certain circumstances such as in traumatic stretch injury, they are placed in tension. We employ Standard Linear Solid, a viscoelastic model, to computationally simulate microtubules. This study investigates the dynamic response of two dimensional axonal microtubules under suddenly applied end forces. We obtain the results for steady state behavior of axonal microtubule for different forces.

  15. Centriolar CPAP/SAS-4 Imparts Slow Processive Microtubule Growth.

    PubMed

    Sharma, Ashwani; Aher, Amol; Dynes, Nicola J; Frey, Daniel; Katrukha, Eugene A; Jaussi, Rolf; Grigoriev, Ilya; Croisier, Marie; Kammerer, Richard A; Akhmanova, Anna; Gönczy, Pierre; Steinmetz, Michel O

    2016-05-23

    Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and "caps" microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control.

  16. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

    Elie, Auréliane; Prezel, Elea; Guérin, Christophe; Denarier, Eric; Ramirez-Rios, Sacnicte; Serre, Laurence; Andrieux, Annie; Fourest-Lieuvin, Anne; Blanchoin, Laurent; Arnal, Isabelle

    2015-01-01

    The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts. PMID:25944224

  17. Live Cell Imaging Reveals Structural Associations between the Actin and Microtubule Cytoskeleton in Arabidopsis [W] [OA

    PubMed Central

    Sampathkumar, Arun; Lindeboom, Jelmer J.; Debolt, Seth; Gutierrez, Ryan; Ehrhardt, David W.; Ketelaar, Tijs; Persson, Staffan

    2011-01-01

    In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells. PMID:21693695

  18. Cortical Visual Impairment

    MedlinePlus

    ... Frequently Asked Questions Español Condiciones Chinese Conditions Cortical Visual Impairment En Español Read in Chinese What is cortical visual impairment? Cortical visual impairment (CVI) is a decreased ...

  19. Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules.

    PubMed

    Kwiatkowska, M; Popłońska, K; Stepiński, D

    2005-12-01

    Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B.

  20. Progressive Transverse Microtubule Array Organization in Hormone-Induced Arabidopsis Hypocotyl Cells[W

    PubMed Central

    Vineyard, Laura; Elliott, Andrew; Dhingra, Sonia; Lucas, Jessica R.; Shaw, Sidney L.

    2013-01-01

    The acentriolar cortical microtubule arrays in dark-grown hypocotyl cells organize into a transverse coaligned pattern that is critical for axial plant growth. In light-grown Arabidopsis thaliana seedlings, the cortical array on the outer (periclinal) cell face creates a variety of array patterns with a significant bias (>3:1) for microtubules polymerizing edge-ward and into the side (anticlinal) faces of the cell. To study the mechanisms required for creating the transverse coalignment, we developed a dual-hormone protocol that synchronously induces ∼80% of the light-grown hypocotyl cells to form transverse arrays over a 2-h period. Repatterning occurred in two phases, beginning with an initial 30 to 40% decrease in polymerizing plus ends prior to visible changes in the array pattern. Transverse organization initiated at the cell’s midzone by 45 min after induction and progressed bidirectionally toward the apical and basal ends of the cell. Reorganization corrected the edge-ward bias in polymerization and proceeded without transiting through an obligate intermediate pattern. Quantitative comparisons of uninduced and induced microtubule arrays showed a limited deconstruction of the initial periclinal array followed by a progressive array reorganization to transverse coordinated between the anticlinal and periclinal cell faces. PMID:23444330

  1. The smallest active fragment of microtubule-associated protein 4 and its interaction with microtubules in phosphate buffer.

    PubMed

    Hashi, Yurika; Nagase, Lisa; Matsushima, Kazuyuki; Kotani, Susumu

    2012-01-01

    To analyze the interaction between microtubule-associated protein (MAP) 4 and microtubules physicochemically, a MAP4 active site fragment was designed for nuclear magnetic resonance (NMR) use. The fragment was bacterially expressed and purified to homogeneity. The buffer conditions for NMR were optimized to support microtubule assembly. The fragment was found to bind to microtubules under the optimized buffer conditions.

  2. Physiological framework for adaptation of stomata to CO2 from glacial to future concentrations

    PubMed Central

    Franks, Peter J.; Leitch, Ilia J.; Ruszala, Elizabeth M.; Hetherington, Alistair M.; Beerling, David J.

    2012-01-01

    In response to short-term fluctuations in atmospheric CO2 concentration, ca, plants adjust leaf diffusive conductance to CO2, gc, via feedback regulation of stomatal aperture as part of a mechanism for optimizing CO2 uptake with respect to water loss. The operational range of this elaborate control mechanism is determined by the maximum diffusive conductance to CO2, gc(max), which is set by the size (S) and density (number per unit area, D) of stomata on the leaf surface. Here, we show that, in response to long-term exposure to elevated or subambient ca, plants alter gc(max) in the direction of the short-term feedback response of gc to ca via adjustment of S and D. This adaptive feedback response to ca, consistent with long-term optimization of leaf gas exchange, was observed in four species spanning a diverse taxonomic range (the lycophyte Selaginella uncinata, the fern Osmunda regalis and the angiosperms Commelina communis and Vicia faba). Furthermore, using direct observation as well as flow cytometry, we observed correlated increases in S, guard cell nucleus size and average apparent 1C DNA amount in epidermal cell nuclei with increasing ca, suggesting that stomatal and leaf adaptation to ca is linked to genome scaling. PMID:22232765

  3. Physiological framework for adaptation of stomata to CO2 from glacial to future concentrations.

    PubMed

    Franks, Peter J; Leitch, Ilia J; Ruszala, Elizabeth M; Hetherington, Alistair M; Beerling, David J

    2012-02-19

    In response to short-term fluctuations in atmospheric CO(2) concentration, c(a), plants adjust leaf diffusive conductance to CO(2), g(c), via feedback regulation of stomatal aperture as part of a mechanism for optimizing CO(2) uptake with respect to water loss. The operational range of this elaborate control mechanism is determined by the maximum diffusive conductance to CO(2), g(c(max)), which is set by the size (S) and density (number per unit area, D) of stomata on the leaf surface. Here, we show that, in response to long-term exposure to elevated or subambient c(a), plants alter g(c(max)) in the direction of the short-term feedback response of g(c) to c(a) via adjustment of S and D. This adaptive feedback response to c(a), consistent with long-term optimization of leaf gas exchange, was observed in four species spanning a diverse taxonomic range (the lycophyte Selaginella uncinata, the fern Osmunda regalis and the angiosperms Commelina communis and Vicia faba). Furthermore, using direct observation as well as flow cytometry, we observed correlated increases in S, guard cell nucleus size and average apparent 1C DNA amount in epidermal cell nuclei with increasing c(a), suggesting that stomatal and leaf adaptation to c(a) is linked to genome scaling.

  4. Fossil plant stomata indicate decreasing atmospheric CO2 prior to the Eocene-Oligocene boundary

    NASA Astrophysics Data System (ADS)

    Steinthorsdottir, Margret; Porter, Amanda S.; Holohan, Aidan; Kunzmann, Lutz; Collinson, Margaret; McElwain, Jennifer C.

    2016-02-01

    A unique stratigraphic sequence of fossil leaves of Eotrigonobalanus furcinervis (extinct trees of the beech family, Fagaceae) from central Germany has been used to derive an atmospheric pCO2 record with multiple data points spanning the late middle to late Eocene, two sampling levels which may be earliest Oligocene, and two samples from later in the Oligocene. Using the inverse relationship between the density of stomata and pCO2, we show that pCO2 decreased continuously from the late middle to late Eocene, reaching a relatively stable low value before the end of the Eocene. Based on the subsequent records, pCO2 in parts of the Oligocene was similar to latest Eocene values. These results suggest that a decrease in pCO2 preceded the large shift in marine oxygen isotope records that characterizes the Eocene-Oligocene transition and that when a certain threshold of pCO2 change was crossed, the cumulative effects of this and other factors resulted in rapid temperature decline, ice build up on Antarctica and hence a change of climate mode.

  5. Fossil plant stomata indicate decreasing atmospheric CO2 prior to the Eocene-Oligocene boundary

    NASA Astrophysics Data System (ADS)

    Steinthorsdottir, M.; Porter, A. S.; Holohan, A.; Kunzmann, L.; Collinson, M.; McElwain, J. C.

    2015-10-01

    A unique stratigraphic sequence of fossil leaves of Eotrigonobalanus furcinervis (extinct trees of the beech family, Fagaceae) from central Germany has been used to derive an atmospheric pCO2 record with multiple data points spanning the late middle to late Eocene, two sampling levels which may be earliest Oligocene, and two samples from later in the Oligocene. Using the inverse relationship between the density of stomata and pCO2, we show that pCO2 decreased continuously from the late middle to late Eocene, reaching a relatively stable low value before the end of the Eocene. Based on the subsequent records, pCO2 in parts of the Oligocene was similar to latest Eocene values. These results show that a decrease in pCO2 preceded the large shift in marine oxygen isotope records that characterizes the Eocene-Oliogocene transition. This may be related to the "hysteresis effect" previously proposed - where a certain threshold of pCO2 change was crossed before the cumulative effects of this and other factors resulted in rapid temperature decline, ice build up on Antarctica and hence a change of climate mode.

  6. Smaller, faster stomata: scaling of stomatal size, rate of response, and stomatal conductance

    PubMed Central

    Franks, Peter J.

    2013-01-01

    Maximum and minimum stomatal conductance, as well as stomatal size and rate of response, are known to vary widely across plant species, but the functional relationship between these static and dynamic stomatal properties is unknown. The objective of this study was to test three hypotheses: (i) operating stomatal conductance under standard conditions (g op) correlates with minimum stomatal conductance prior to morning light [g min(dawn)]; (ii) stomatal size (S) is negatively correlated with g op and the maximum rate of stomatal opening in response to light, (dg/dt)max; and (iii) g op correlates negatively with instantaneous water-use efficiency (WUE) despite positive correlations with maximum rate of carboxylation (Vc max) and light-saturated rate of electron transport (J max). Using five closely related species of the genus Banksia, the above variables were measured, and it was found that all three hypotheses were supported by the results. Overall, this indicates that leaves built for higher rates of gas exchange have smaller stomata and faster dynamic characteristics. With the aid of a stomatal control model, it is demonstrated that higher g op can potentially expose plants to larger tissue water potential gradients, and that faster stomatal response times can help offset this risk. PMID:23264516

  7. Open Stomata 1 Kinase is Essential for Yeast Elicitor-Induced Stomatal Closure in Arabidopsis.

    PubMed

    Ye, Wenxiu; Adachi, Yuji; Munemasa, Shintaro; Nakamura, Yoshimasa; Mori, Izumi C; Murata, Yoshiyuki

    2015-06-01

    We recently demonstrated that yeast elicitor (YEL)-induced stomatal closure requires a Ca(2+)-dependent kinase, CPK6. A Ca(2+)-independent kinase, Open Stomata 1 (OST1), is involved in stomatal closure induced by various stimuli including ABA. In the present study, we investigated the role of OST1 in YEL-induced stomatal closure in Arabidopsis using a knock-out mutant, ost1-3, and a kinase-deficient mutant, ost1-2. YEL did not induce stomatal closure or activation of guard cell S-type anion channels in the ost1 mutants unlike in wild-type plants. However, YEL did not increase OST1 kinase activity in wild-type guard cells. The YEL-induced stomatal closure and activation of S-type anion channels were also impaired in a gain-of-function mutant of a clade A type 2C protein phosphatase (ABA INSENSITIVE 1), abi1-1C. In the ost1 mutants like in the wild type, YEL induced H2O2 accumulation, activation of non-selective Ca(2+)-permeable cation (ICa) channels and transient elevations in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in guard cells. These results suggest that OST1 kinase is essential for stomatal closure and activation of S-type anion channels induced by YEL and that OST1 is not involved in H2O2 accumulation, ICa channel activation or [Ca(2+)]cyt elevations in guard cells induced by YEL.

  8. Kinesins to the core: the role of microtubule-based motor proteins in building the mitotic spindle midzone

    PubMed Central

    Hornick, Jessica E.; Karanjeet, Kul; Collins, Elizabeth S.; Hinchcliffe, Edward H.

    2013-01-01

    In mammalian cultured cells the initiation of cytokinesis is regulated – both temporally and spatially – by the overlapping, anti-parallel microtubules of the spindle midzone. This region recruits several key central spindle components: PRC-1, Polo-like kinase 1 (Plk-1), the centralspindlin complex, and the Chromosome Passenger Complex (CPC), which together serve to stabilize the microtubule overlap, and also to coordinate the assembly of the cortical actin/myosin cytoskeleton necessary to physically cleave the cell in two. The localization of these crucial elements to the spindle midzone requires members of the kinesin superfamily of microtubule-based motor proteins. Here we focus on reviewing the role played by a variety of kinesins in both building and operating the spindle midzone machinery during cytokinesis. PMID:20109573

  9. Measuring kinetochore-microtubule interaction in vitro

    PubMed Central

    Driver, Jonathan W.; Powers, Andrew F.; Sarangapani, Krishna K.; Biggins, Sue; Asbury, Charles L.

    2014-01-01

    Many proteins and protein complexes perform sophisticated, regulated functions in vivo. Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause-effect relationships, for example between a protein and its phosphorylating kinase. Here, we describe a protocol to purify kinetochores, the protein complexes that attach chromosomes to microtubules during mitosis, and quantitatively assay their microtubule binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of microtubules to kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of kinetochore-microtubule attachments, which is believed to be critical for correcting erroneous kinetochore-spindle attachments and thereby avoiding chromosome mis-segregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology. PMID:24630115

  10. The microtubule as a breast cancer target.

    PubMed

    Higa, Gerald M

    2011-04-01

    Manifestations of non-equilibrium polarity, random transgressions, and catastrophes are not conditions usually associated with a sense of normalcy. Yet these disquieting features distinguish a utilitarian behavior known as dynamic instability, the signature characteristic of the microtubule. Long known to be a tumor target, disruption of this fragile attribute is associated with some of the most effective agents used to treat breast cancer today. Although the biology of the microtubule is under intense investigation much still remains unknown. As such, our understanding of regulatory molecules and resistance mechanisms are still rudimentary, further compromising our ability to develop novel therapeutic strategies to improve microtubule inhibitors. This review focuses on several classes of anti-microtubule agents and their effects on the functional dynamics of the targeted polymer. The primary objective is to critically examine the molecular mechanisms that contribute to tumor cell death, tumor-resistance, and incident neurotoxicity.

  11. Rigidity of microtubules is increased by stabilizing agents

    PubMed Central

    1995-01-01

    Microtubules are rigid polymers that contribute to the static mechanical properties of cells. Because microtubules are dynamic structures whose polymerization is regulated during changes in cell shape, we have asked whether the mechanical properties of microtubules might also be modulated. We measured the flexural rigidity, or bending stiffness, of individual microtubules under a number of different conditions that affect the stability of microtubules against depolymerization. The flexural rigidity of microtubules polymerized with the slowly hydrolyzable nucleotide analogue guanylyl-(alpha, beta)- methylene-diphosphonate was 62 +/- 9 x 10(-24) Nm2 (weighted mean +/- SEM); that of microtubules stabilized with tau protein was 34 +/- 3 x 10(-24) Nm2; and that of microtubules stabilized with the antimitotic drug taxol was 32 +/- 2 x 10(-24) Nm2. For comparison, microtubules that were capped to prevent depolymerization, but were not otherwise stabilized, had a flexural rigidity of 26 +/- 2 x 10(-24) Nm2. Decreasing the temperature from 37 degrees C to approximately 25 degrees C, a condition that makes microtubules less stable, decreased the stiffness of taxol-stabilized microtubules by one-third. We thus find that the more stable a microtubule, the higher its flexural rigidity. This raises the possibility that microtubule rigidity may be regulated in vivo. In addition, the high rigidity of an unstabilized, GDP-containing microtubule suggests that a large amount of energy could be stored as mechanical strain energy in the protein lattice for subsequent force generation during microtubule depolymerization. PMID:7642706

  12. Microtubules, polarity and vertebrate neural tube morphogenesis

    PubMed Central

    Cearns, Michael D.; Escuin, Sarah; Alexandre, Paula; Greene, Nicholas D. E.; Copp, Andrew J.

    2016-01-01

    Microtubules are key cellular components, long known to participate in morphogenetic events that shape the developing embryo. However, the links between the cellular functions of microtubules, their effects on cell shape and polarity and their role in large-scale morphogenesis remain poorly understood. Here, we examine these relationships with respect to two strategies for generating the vertebrate neural tube: bending and closure of the mammalian neural plate, and cavitation of the teleost neural rod. The latter process has been compared to ‘secondary’ neurulation that generates the caudal spinal cord in mammals. Microtubules align along the apico-basal axis of the mammalian neuroepithelium early in neural tube closure, participating functionally in interkinetic nuclear migration which indirectly impacts on cell shape. Whether microtubules play other functional roles in mammalian neurulation remains unclear. In the zebrafish, microtubules are important for defining the neural rod midline prior to its cavitation, both by localizing apical proteins at the tissue midline and by orienting cell division through a mirror-symmetric microtubule apparatus that helps to further define the medial localization of apical polarity proteins. Par proteins have been implicated in centrosome positioning in neuroepithelia as well as in the control of polarized morphogenetic movements in the neural rod. Understanding of microtubule functions during early nervous system development has so far been limited, partly by techniques that fail to distinguish ‘cause’ from ‘effect’. Future developments will likely rely on novel ways to selectively impair microtubule function in order to investigate the roles they play. PMID:27025884

  13. Polar organizers and girdling bands of microtubules are associated with gamma-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta).

    PubMed

    Brown, R C; Lemmon, B E

    2006-05-01

    Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of gamma-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants.

  14. Kinetochore-microtubule interactions during cell division.

    PubMed

    Maiato, Helder; Sunkel, Claudio E

    2004-01-01

    Proper segregation of chromosomes during cell division is essential for the maintenance of genetic stability. During this process chromosomes must establish stable functional interactions with microtubules through the kinetochore, a specialized protein structure located on the surface of the centromeric heterochromatin. Stable attachment of kinetochores to a number of microtubules results in the formation of a kinetochore fibre that mediates chromosome movement. How the kinetochore fibre is formed and how chromosome motion is produced and regulated remain major questions in cell biology. Here we look at some of the history of research devoted to the study of kinetochore-microtubule interaction and attempt to identify significant advances in the knowledge of the basic processes. Ultrastructural work has provided substantial insights into the structure of the kinetochore and associated microtubules during different stages of mitosis. Also, recent in-vivo studies have probed deep into the dynamics of kinetochore-attached microtubules suggesting possible models for the way in which kinetochores harness the capacity of microtubules to do work and turn it into chromosome motion. Much of the research in recent years suggests that indeed multiple mechanisms are involved in both formation of the k-fibre and chromosome motion. Thus, rather than moving to a unified theory, it has become apparent that most cell types have the capacity to build the spindle using multiple and probably redundant mechanisms.

  15. Harnessing microtubule dynamic instability for nanostructure assembly

    NASA Astrophysics Data System (ADS)

    Bouchard, Ann M.; Warrender, Christina E.; Osbourn, Gordon C.

    2006-10-01

    Intracellular molecular machines synthesize molecules, tear apart others, transport materials, transform energy into different forms, and carry out a host of other coordinated processes. Many molecular processes have been shown to work outside of cells, and the idea of harnessing these molecular machines to build nanostructures is attractive. Two examples are microtubules and motor proteins, which aid cell movement, help determine cell shape and internal structure, and transport vesicles and organelles within the cell. These molecular machines work in a stochastic, noisy fashion: microtubules switch randomly between growing and shrinking in a process known as dynamic instability; motor protein movement along microtubules is randomly interrupted by the motor proteins falling off. A common strategy in attempting to gain control over these highly dynamic, stochastic processes is to eliminate some processes (e.g., work with stabilized microtubules) in order to focus on others (interaction of microtubules with motor proteins). In this paper, we illustrate a different strategy for building nanostructures, which, rather than attempting to control or eliminate some dynamic processes, uses them to advantage in building nanostructures. Specifically, using stochastic agent-based simulations, we show how the natural dynamic instability of microtubules can be harnessed in building nanostructures, and discuss strategies for ensuring that “unreliable” stochastic processes yield a robust outcome.

  16. Harnessing microtubule dynamic instability for nanostructure assembly.

    SciTech Connect

    Bouchard, Ann Marie; Osbourn, Gordon Cecil

    2004-06-01

    Intracellular molecular machines synthesize molecules, tear apart others, transport materials, transform energy into different forms, and carry out a host of other coordinated processes. Many molecular processes have been shown to work outside of cells, and the idea of harnessing these molecular machines to build nanostructures is attractive. Two examples are microtubules and motor proteins, which aid cell movement, help determine cell shape and internal structure, and transport vesicles and organelles within the cell. These molecular machines work in a stochastic, noisy fashion: microtubules switch randomly between growing and shrinking in a process known as dynamic instability; motor protein movement along microtubules is randomly interrupted by the motor proteins falling off. A common strategy in attempting to gain control over these highly dynamic, stochastic processes is to eliminate some processes (e.g., work with stabilized microtubules) in order to focus on others (interaction of microtubules with motor proteins). In this paper, we illustrate a different strategy for building nanostructures, which, rather than attempting to control or eliminate some dynamic processes, uses them to advantage in building nanostructures. Specifically, using stochastic agent-based simulations, we show how the natural dynamic instability of microtubules can be harnessed in building nanostructures, and discuss strategies for ensuring that 'unreliable' stochastic processes yield a robust outcome.

  17. Mobility of Taxol in Microtubule Bundles

    NASA Astrophysics Data System (ADS)

    Ross, J.

    2003-06-01

    Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching (FRAP) on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With > 63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, k_{off}. It was found that 10*k_{off} (GMPCPP) ~ k_{off} (GTP), consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With < 63% sites occupied, recovery times decreased as ~ [Tax]^{-1/5} for both types of microtubules. We conclude that the diffusion of taxol along the microtubule interior is hindered by rebinding events when open sites are within ~7 nm of each other.

  18. Mitosis. Microtubule detyrosination guides chromosomes during mitosis.

    PubMed

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K; Magiera, Maria M; Zaytsev, Anatoly V; Pereira, Ana L; Janke, Carsten; Grishchuk, Ekaterina L; Maiato, Helder

    2015-05-15

    Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. Copyright © 2015, American Association for the Advancement of Science.

  19. Discovery of synthetic small molecules that enhance the number of stomata: C-H functionalization chemistry for plant biology.

    PubMed

    Ziadi, Asraa; Uchida, Naoyuki; Kato, Hiroe; Hisamatsu, Rina; Sato, Ayato; Hagihara, Shinya; Itami, Kenichiro; Torii, Keiko U

    2017-08-24

    The increasing climate changes and global warming are leading to colossal agricultural problems such as abatement of food production and quality. As stomatal development is considered to play a key role in crop plant productivity and water-use efficiency, studying stomatal development is useful for understanding the productivity of plant systems for both natural and agricultural systems. Herein, we report the first-in-class synthetic small molecules enhancing the number of stomata in Arabidopsis thaliana that have been discovered by screening of the chemical library and further optimized by the Pd-catalyzed C-H arylation reaction. The present study shows not only huge potential of small molecules to control the cellular and developmental processes of stomata without using genetically modified plants, but also the power of C-H functionalization chemistry to rapidly identify the optimized compounds.

  20. Dynein Light Chain 1 (LC8) Association Enhances Microtubule Stability and Promotes Microtubule Bundling*

    PubMed Central

    Asthana, Jayant; Kuchibhatla, Anuradha; Jana, Swadhin Chandra; Ray, Krishanu; Panda, Dulal

    2012-01-01

    Dynein light chain 1 (LC8), a highly conserved protein, is known to bind to a variety of different polypeptides. It functions as a dimer, which is inactivated through phosphorylation at the Ser-88 residue. A loss of LC8 function causes apoptosis in Drosophila embryos, and its overexpression induces malignant transformation of breast cancer cells. Here we show that LC8 binds to tubulin, promotes microtubule assembly, and induces the bundling of reconstituted microtubules in vitro. Furthermore, LC8 decorates microtubules both in Drosophila embryos and in HeLa cells, increases the microtubule stability, and promotes microtubule bundling in these cells. Microtubule stability influences a number of different cellular functions including mitosis and cell differentiation. The LC8 overexpression reduces the susceptibility of microtubules to cold and nocodazole-induced depolymerization in tissue-cultured cells and increases microtubule acetylation, suggesting that LC8 stabilizes microtubules. We also show that LC8 knockdown or transfection with inhibitory peptides destabilizes microtubules and inhibits bipolar spindle assembly in HeLa cells. In addition, LC8 knockdown leads to the mitotic block in HeLa cells. Furthermore, molecular docking analysis using the crystal structures of tubulin and LC8 dimer indicated that the latter may bind at α-β tubulin junction in a protofilament at sites distinct from the kinesin and dynein binding sites. Together, we provide the first evidence of a novel microtubule-associated protein-like function of LC8 that could explain its reported roles in cellular metastasis and differentiation. PMID:23038268

  1. Surface Structures Involved in Plant Stomata and Leaf Colonization by Shiga-Toxigenic Escherichia Coli O157:H7

    PubMed Central

    Saldaña, Zeus; Sánchez, Ethel; Xicohtencatl-Cortes, Juan; Puente, Jose Luis; Girón, Jorge A.

    2011-01-01

    Shiga-toxigenic Escherichia coli (STEC) O157:H7 uses a myriad of surface adhesive appendages including pili, flagella, and the type 3 secretion system (T3SS) to adhere to and inflict damage to the human gut mucosa. Consumption of contaminated ground beef, milk, juices, water, or leafy greens has been associated with outbreaks of diarrheal disease in humans due to STEC. The aim of this study was to investigate which of the known STEC O157:H7 adherence factors mediate colonization of baby spinach leaves and where the bacteria reside within tainted leaves. We found that STEC O157:H7 colonizes baby spinach leaves through the coordinated production of curli, the E. coli common pilus, hemorrhagic coli type 4 pilus, flagella, and T3SS. Electron microscopy analysis of tainted leaves revealed STEC bacteria in the internal cavity of the stomata, in intercellular spaces, and within vascular tissue (xylem and phloem), where the bacteria were protected from the bactericidal effect of gentamicin, sodium hypochlorite or ozonated water treatments. We confirmed that the T3S escN mutant showed a reduced number of bacteria within the stomata suggesting that T3S is required for the successful colonization of leaves. In agreement, non-pathogenic E. coli K-12 strain DH5α transformed with a plasmid carrying the locus of enterocyte effacement (LEE) pathogenicity island, harboring the T3SS and effector genes, internalized into stomata more efficiently than without the LEE. This study highlights a role for pili, flagella, and T3SS in the interaction of STEC with spinach leaves. Colonization of plant stomata and internal tissues may constitute a strategy by which STEC survives in a nutrient-rich microenvironment protected from external foes and may be a potential source for human infection. PMID:21887151

  2. Surface structures involved in plant stomata and leaf colonization by shiga-toxigenic Escherichia coli o157:h7.

    PubMed

    Saldaña, Zeus; Sánchez, Ethel; Xicohtencatl-Cortes, Juan; Puente, Jose Luis; Girón, Jorge A

    2011-01-01

    Shiga-toxigenic Escherichia coli (STEC) O157:H7 uses a myriad of surface adhesive appendages including pili, flagella, and the type 3 secretion system (T3SS) to adhere to and inflict damage to the human gut mucosa. Consumption of contaminated ground beef, milk, juices, water, or leafy greens has been associated with outbreaks of diarrheal disease in humans due to STEC. The aim of this study was to investigate which of the known STEC O157:H7 adherence factors mediate colonization of baby spinach leaves and where the bacteria reside within tainted leaves. We found that STEC O157:H7 colonizes baby spinach leaves through the coordinated production of curli, the E. coli common pilus, hemorrhagic coli type 4 pilus, flagella, and T3SS. Electron microscopy analysis of tainted leaves revealed STEC bacteria in the internal cavity of the stomata, in intercellular spaces, and within vascular tissue (xylem and phloem), where the bacteria were protected from the bactericidal effect of gentamicin, sodium hypochlorite or ozonated water treatments. We confirmed that the T3S escN mutant showed a reduced number of bacteria within the stomata suggesting that T3S is required for the successful colonization of leaves. In agreement, non-pathogenic E. coli K-12 strain DH5α transformed with a plasmid carrying the locus of enterocyte effacement (LEE) pathogenicity island, harboring the T3SS and effector genes, internalized into stomata more efficiently than without the LEE. This study highlights a role for pili, flagella, and T3SS in the interaction of STEC with spinach leaves. Colonization of plant stomata and internal tissues may constitute a strategy by which STEC survives in a nutrient-rich microenvironment protected from external foes and may be a potential source for human infection.

  3. Evidence for dynein and astral microtubule–mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells

    PubMed Central

    Zheng, Zhen; Wan, Qingwen; Liu, Jing; Zhu, Huabin; Chu, Xiaogang; Du, Quansheng

    2013-01-01

    Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gαi-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gαi-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule–mediated transport of Gαi/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gαi/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells. PMID:23389635

  4. Single file diffusion in microtubules

    NASA Astrophysics Data System (ADS)

    Rutenberg, Andrew; Farrell, Spencer; Brown, Aidan

    2015-03-01

    We investigate the single file diffusion (SFD) of large particles entering a confined tubular geometry, such as luminal diffusion of proteins inside microtubules or flagella. While single-file effects have no effect on particle density, we report significant single-file effects for individually-tracked tracer particle motion. Both exact and approximate ordering statistics of particles entering semi-infinite tubes agree well with our stochastic simulations. Considering initially empty semi-infinite tubes, with particles entering at one end starting from an initial time t = 0 , tracked particles display super-diffusive effective exponents just after they enter the system and trends towards diffusive exponents at later times. Equivalently, if diffusive exponents are assumed the effective diffusivity is reduced at early times and enhanced at later times through a logarithmic factor logN , where N is the number of particles in the tube. When we number each particle from the first (n = 1) to the most recent (n = N), we find good scaling collapse of the effective diffusivity for all n. Techniques that track individual particles, or local groups of particles, such as photo-activation or photobleaching, will exhibit single-file effects.

  5. Microtubule organization is determined by the shape of epithelial cells

    PubMed Central

    Gomez, Juan Manuel; Chumakova, Lyubov; Bulgakova, Natalia A.; Brown, Nicholas H.

    2016-01-01

    Interphase microtubule organization is critical for cell function and tissue architecture. In general, physical mechanisms are sufficient to drive microtubule organization in single cells, whereas cells within tissues are thought to utilize signalling mechanisms. By improving the imaging and quantitation of microtubule alignment within developing Drosophila embryos, here we demonstrate that microtubule alignment underneath the apical surface of epithelial cells follows cell shape. During development, epidermal cell elongation and microtubule alignment occur simultaneously, but by perturbing cell shape, we discover that microtubule organization responds to cell shape, rather than the converse. A simple set of microtubule behaviour rules is sufficient for a computer model to mimic the observed responses to changes in cell surface geometry. Moreover, we show that microtubules colliding with cell boundaries zip-up or depolymerize in an angle-dependent manner, as predicted by the model. Finally, we show microtubule alignment responds to cell shape in diverse epithelia. PMID:27779189

  6. [Microtubules in the nerve cells: morphological and functional aspects].

    PubMed

    Vorob'ev, V S; Portuganov, V V

    1980-10-01

    The modern literature concerning ultrastructure and cytochemistry of microtubules in the nervous tissue is reviewed. Common features of cytological and biochemical organization of microtubules in different parts of the nervous system of the vertebrates and invertebrates are analysed: the similarity of ultrastructure of microtubules and their molecular organization (tubulin and its alpha- and beta-monomeres), the ability of microtubules to assemble and disassemble, to bind specifically with poisons--colchicine and vinblastine, participation of microtubules in the neuroplastic transport. The authors' data on space arrangement of microtubules within cytoplasm of the neuronal processes (dendrites and unmyelinated axons in the central and peripheral nevous system) are presented. Some literature and personal results concerning ultrastructure of neurofilaments and microtubules in the myelinated nerve fibres are also considered. The functional significance of microtubules in the nervous system is discussed with special reference to facts and hypotheses on a possible role of microtubules in the propagation of nerve impulse.

  7. Organization of neuronal microtubules in the nematode Caenorhabditis elegans

    PubMed Central

    1979-01-01

    We have studied the organization of microtubules in neurons of the nematode Caenorhabditis elegans. Six neurons, which we call the microtubule cells, contain bundles of darkly staining microtubules which can be followed easily in serial-section electron micrographs. Reconstruction of individual microtubules in these cells indicate that most, if not all, microtubules are short compared with the length of the cell process. Average microtubule length varies characteristically with cell type. The arrangement of microtubules gives an overall polarity to each bundle: the distal ends of the microtubles are on the outside of the bundle, whereas the proximal ends are preferentially inside. The distal and proximal ends each have a characteristic appearance indicating that these microtubules may have a polarity of their own. Short microtubules in processes of other neurons in C. elegans have also been observed. PMID:479300

  8. 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.

  9. 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

  10. 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.

  11. History-dependent catastrophes regulate axonal microtubule behavior.

    PubMed

    Stepanova, Tatiana; Smal, Ihor; van Haren, Jeffrey; Akinci, Umut; Liu, Zhe; Miedema, Marja; Limpens, Ronald; van Ham, Marco; van der Reijden, Michael; Poot, Raymond; Grosveld, Frank; Mommaas, Mieke; Meijering, Erik; Galjart, Niels

    2010-06-08

    In Chinese hamster ovary cells, microtubules originate at the microtubule organizing center (MTOC) and grow persistently toward the cell edge, where they undergo catastrophe. In axons, microtubule dynamics must be regulated differently because microtubules grow parallel to the plasma membrane and there is no MTOC. GFP-tagged microtubule plus end tracking proteins (+TIPs) mark the ends of growing neuronal microtubules. Their fluorescent "comet-like" pattern reflects turnover of +TIP binding sites. Using GFP-tagged +TIPs and fluorescence-based segmentation and tracking tools, we show that axonal microtubules grow with a constant average velocity and that they undergo catastrophes at random positions, yet in a programmed fashion. Using protein depletion approaches, we find that the +TIPs CLIP-115 and CLIP-170 affect average microtubule growth rate and growth distance in neurons but not the duration of a microtubule growth event. In N1E-115 neuroblastoma cells, we find that EB1, the core +TIP, regulates microtubule growth rate, growth distance, and duration, consistent with in vitro data. Combined, our data suggest that CLIPs influence the axonal microtubule/tubulin ratio, whereas EB1 stimulates microtubule growth and structural transitions at microtubule ends, thereby regulating microtubule catastrophes and the turnover of +TIP binding sites.

  12. PKA antagonizes CLASP-dependent microtubule stabilization to re-localize Pom1 and buffer cell size upon glucose limitation

    PubMed Central

    Kelkar, Manasi; Martin, Sophie G.

    2015-01-01

    Cells couple growth with division and regulate size in response to nutrient availability. In rod-shaped fission yeast, cell-size control occurs at mitotic commitment. An important regulator is the DYRK-family kinase Pom1, which forms gradients from cell poles and inhibits the mitotic activator Cdr2, itself localized at the medial cortex. Where and when Pom1 modulates Cdr2 activity is unclear as Pom1 medial cortical levels remain constant during cell elongation. Here we show that Pom1 re-localizes to cell sides upon environmental glucose limitation, where it strongly delays mitosis. This re-localization is caused by severe microtubule destabilization upon glucose starvation, with microtubules undergoing catastrophe and depositing the Pom1 gradient nucleator Tea4 at cell sides. Microtubule destabilization requires PKA/Pka1 activity, which negatively regulates the microtubule rescue factor CLASP/Cls1/Peg1, reducing CLASP's ability to stabilize microtubules. Thus, PKA signalling tunes CLASP's activity to promote Pom1 cell side localization and buffer cell size upon glucose starvation. PMID:26443240

  13. The effects of the phospholipase D-antagonist 1-butanol on seedling development and microtubule organisation in Arabidopsis.

    PubMed

    Gardiner, John; Collings, David A; Harper, John D I; Marc, Jan

    2003-07-01

    The organisation of plant microtubules into distinct arrays during the cell cycle requires interactions with partner proteins. Having recently identified a 90-kDa phospholipase D (PLD) that associates with microtubules and the plasma membrane [Gardiner et al. (2001) Plant Cell 13: 2143], we exposed seeds and young seedlings of Arabidopsis to 1-butanol, a specific inhibitor of PLD-dependent production of the signalling molecule phosphatidic acid (PA). When added to agar growth media, 0.2% 1-butanol strongly inhibited the emergence of the radicle and cotyledons, while 0.4% 1-butanol effectively blocked germination. When normal seedlings were transferred onto media containing 0.2% and 0.4% 1-butanol, the inhibitor retarded root growth by about 40% and 90%, respectively, by reducing cell elongation. Inhibited plants showed significant swelling in the root elongation zone, bulbous or branched root hairs, and modified cotyledon morphology. Confocal immunofluorescence microscopy of root tips revealed that 1-butanol disrupted the organisation of interphase cortical microtubules. Butanol isomers that do not inhibit PLD-dependent PA production, 2- and 3-butanol, had no effect on seed germination, seedling growth, or microtubule organisation. We propose that production of PA by PLD may be required for normal microtubule organisation and hence normal growth in Arabidopsis.

  14. YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

    PubMed Central

    Chernov, Konstantin G; Mechulam, Alain; Popova, Nadezhda V; Pastre, David; Nadezhdina, Elena S; Skabkina, Olga V; Shanina, Nina A; Vasiliev, Victor D; Tarrade, Anne; Melki, Judith; Joshi, Vandana; Baconnais, Sonia; Toma, Flavio; Ovchinnikov, Lev P; Curmi, Patrick A

    2008-01-01

    Background YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. Results We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. Conclusion These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation. PMID:18793384

  15. Real-time monitoring of changes in microtubule mechanical properties in response to microtubule-destabilizing drug treatment.

    PubMed

    Han, Sung-Woong; Simona, Patriche; Banu, Mihaela; Adachi, Taiji

    2013-03-01

    Microtubules are cylindrical protein polymers that play important roles in a number of cellular functions. The properties of microtubules are dynamically changed by interacting with many microtubule-related proteins and drugs. In this study, we used atomic force microscopy to evaluate the changes in microtubule mechanical properties induced by treatment with nocodazole, which is a microtubule-destabilizing drug. The average spring constant of the microtubules, which was used as a measure of microtubule lateral stiffness, was drastically decreased by treatment with nocodazole within 30 min from 0.052 +/- 0.014 N/m to 0.029 +/- 0.015 N/m. Our findings will aid in the understanding of microtubule dynamics, protein interactions in response to drug treatment, microtubule-related diseases, and drug development.

  16. Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

    PubMed

    Margolis, R L; Rauch, C T; Pirollet, F; Job, D

    1990-12-01

    STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.

  17. Non-centrosomal nucleation mediated by augmin organizes microtubules in post-mitotic neurons and controls axonal microtubule polarity

    PubMed Central

    Sánchez-Huertas, Carlos; Freixo, Francisco; Viais, Ricardo; Lacasa, Cristina; Soriano, Eduardo; Lüders, Jens

    2016-01-01

    Neurons display a highly polarized microtubule network that mediates trafficking throughout the extensive cytoplasm and is crucial for neuronal differentiation and function. In newborn migrating neurons, the microtubule network is organized by the centrosome. During neuron maturation, however, the centrosome gradually loses this activity, and how microtubules are organized in more mature neurons remains poorly understood. Here, we demonstrate that microtubule organization in post-mitotic neurons strongly depends on non-centrosomal nucleation mediated by augmin and by the nucleator γTuRC. Disruption of either complex not only reduces microtubule density but also microtubule bundling. These microtubule defects impair neurite formation, interfere with axon specification and growth, and disrupt axonal trafficking. In axons augmin does not merely mediate nucleation of microtubules but ensures their uniform plus end-out orientation. Thus, the augmin-γTuRC module, initially identified in mitotic cells, may be commonly used to generate and maintain microtubule configurations with specific polarity. PMID:27405868

  18. Identification of novel microtubule-binding proteins by taxol-mediated microtubule stabilization and mass spectrometry analysis

    PubMed Central

    He, Xianfei; Liu, Zhu; He, Qianqian; Qin, Juan; Liu, Ningning; Zhang, Linlin; Li, Dengwen; Zhou, Jun; Shui, Wenqing; Liu, Min

    2015-01-01

    Microtubule-binding proteins (MBPs) are structurally and functionally diverse regulators of microtubule-mediated cellular processes. Alteration of MBPs has been implicated in the pathogenesis of human diseases, including cancer. MBPs can stabilize or destabilize microtubules or move along microtubules to transport various cargoes. In addition, MBPs can control microtubule dynamics through direct interaction with microtubules or coordination with other proteins. To better understand microtubule structure and function, it is necessary to identify additional MBPs. In this study, we isolated microtubules and MBPs from mammalian cells by a taxol-based method and then profiled a panel of MBPs by mass spectrometry. We discovered a number of previously uncharacterized MBPs, including several membrane-associated proteins and proteins involved in post-translational modifications, in addition to several structural components. These results support the notion that microtubules have a wide range of functions and may undergo more exquisite regulation than previously recognized. PMID:26445615

  19. Spatio-temporal orientation of microtubules controls conical cell shape in Arabidopsis thaliana petals

    PubMed Central

    Cai, Xianzhi; Yu, Peihang; Li, Yajun; Zhang, Shanshan; Liu, Menghong; Chen, Binqing

    2017-01-01

    The physiological functions of epidermal cells are largely determined by their diverse morphologies. Most flowering plants have special conical-shaped petal epidermal cells that are thought to influence light capture and reflectance, and provide pollinator grips, but the molecular mechanisms controlling conical cell shape remain largely unknown. Here, we developed a live-confocal imaging approach to quantify geometric parameters of conical cells in Arabidopsis thaliana (A. thaliana). Through genetic screens, we identified katanin (KTN1) mutants showing a phenotype of decreased tip sharpening of conical cells. Furthermore, we demonstrated that SPIKE1 and Rho of Plants (ROP) GTPases were required for the final shape formation of conical cells, as KTN1 does. Live-cell imaging showed that wild-type cells exhibited random orientation of cortical microtubule arrays at early developmental stages but displayed a well-ordered circumferential orientation of microtubule arrays at later stages. By contrast, loss of KTN1 prevented random microtubule networks from shifting into well-ordered arrays. We further showed that the filamentous actin cap, which is a typical feature of several plant epidermal cell types including root hairs and leaf trichomes, was not observed in the growth apexes of conical cells during cell development. Moreover, our genetic and pharmacological data suggested that microtubules but not actin are required for conical cell shaping. Together, our results provide a novel imaging approach for studying petal conical cell morphogenesis and suggest that the spatio-temporal organization of microtubule arrays plays crucial roles in controlling conical cell shape. PMID:28644898

  20. Pinus Monophylla (Single Needled Pinyon Pine) show morphological changes in needle cell size and stomata over the past 100 years of rising CO2 in Western Arid Ecosystems.

    NASA Astrophysics Data System (ADS)

    Van De Water, P. K.

    2016-12-01

    The size, frequency, and morphology of leaf surface stomata is used to reconstruct past levels of atmospheric carbon dioxide over geologic time. This technique relies on measuring cell and cell-clusters to correlate with changes of known carbon dioxide levels in the atmosphere. Unfortunately, not all plants are suitable because the occurrence and placement of stomatal cell-complexes differ significantly between plant families. Monocot and dicot angiosperms exhibit different types of stomata and stomatal complexes that lack order and thus are unsuitable. But, in gymnosperms, the number and distribution of stomata and pavement cells is formalized and can be used to reconstruct past atmospheric carbon dioxide levels. However, characteristic of each plant species must still be considered. For example, conifers are useful but are divided into two-needle to five-needle pines, or have irregular surface morphology (Pseudotsuga sp. and Tsuga sp. needles). This study uses Pinus monophylla an undivided needle morphology, that being a cylinder has no interior surface cells. Pinus monophylla (single needle pinyon) needles were collected along Geiger Grade (Nevada State Highway 341, Reno) in 2005 and 2013 from 1500m to 2195m. Herbarium samples were also collected from 13 historic collections made between 1911 and 1994. The study determined changes with elevation and/or over time using in these populations. Using Pinus monophylla, insured needles represented a single surface with stomata, stomatal complex cells, and co-occurring pavement cell types. Results show decreased stomatal densities (stomata/area), stomatal index (stomata/stomata + epidermal cells) and stable stomata per row (stomata/row) . Epidermal cell density (Epidermal Cells /Area), and Pavement cell density (Pavement cell/area) track stomatal density similarly. Data comparison, using elevation in the 2005 and 2013 collections showed no-significant trends. Individual stomatal complexes show no differences in the size

  1. Microtubules Contribute to Tubule Elongation and Anchoring of Endoplasmic Reticulum, Resulting in High Network Complexity in Arabidopsis1[W][OPEN

    PubMed Central

    Hamada, Takahiro; Ueda, Haruko; Kawase, Takashi; Hara-Nishimura, Ikuko

    2014-01-01

    The endoplasmic reticulum (ER) is a network of tubules and sheet-like structures in eukaryotic cells. Some ER tubules dynamically change their morphology, and others form stable structures. In plants, it has been thought that the ER tubule extension is driven by the actin-myosin machinery. Here, we show that microtubules also contribute to the ER tubule extension with an almost 20-fold slower rate than the actin filament-based ER extension. Treatment with the actin-depolymerizing drug Latrunculin B made it possible to visualize the slow extension of the ER tubules in transgenic Arabidopsis (Arabidopsis thaliana) plants expressing ER-targeted green fluorescent protein. The ER tubules elongated along microtubules in both directions of microtubules, which have a distinct polarity. This feature is similar to the kinesin- or dynein-driven ER tubule extension in animal cells. In contrast to the animal case, ER tubules elongating with the growing microtubule ends were not observed in Arabidopsis. We also found the spots where microtubules are stably colocalized with the ER subdomains during long observations of 1,040 s, suggesting that cortical microtubules contribute to provide ER anchoring points. The anchoring points acted as the branching points of the ER tubules, resulting in the formation of multiway junctions. The density of the ER tubule junction positively correlated with the microtubule density in both elongating cells and mature cells of leaf epidermis, showing the requirement of microtubules for formation of the complex ER network. Taken together, our findings show that plants use microtubules for ER anchoring and ER tubule extension, which establish fine network structures of the ER within the cell. PMID:25367857

  2. 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

  3. Taxol Crystals Can Masquerade as Stabilized Microtubules

    PubMed Central

    Alsop, G. Bradley; Zhang, Dahong

    2008-01-01

    Taxol is a potent anti-mitotic drug used in chemotherapy, angioplastic stents, and cell biology research. By binding and stabilizing microtubules, Taxol inhibits their dynamics, crucial for cell division, motility, and survival. The drug has also been reported to induce formation of asters and bundles composed of stabilized microtubules. Surprisingly, at commonly used concentrations, Taxol forms crystals that rapidly bind fluorescent tubulin subunits, generating structures with an uncanny resemblance to microtubule asters and bundles. Kinetic and topological considerations suggest that tubulin subunits, rather than microtubules, bind the crystals. This sequestration of tubulin from the subunit pool would be expected to shift the equilibrium of free to polymerized tubulin to disfavor assembly. Our results imply that some previously reported Taxol-induced asters or bundles could include or be composed of tubulin-decorated Taxol crystals. Thus, reevaluation of certain morphological, chemical, and physical properties of Taxol-treated microtubules may be necessary. Moreover, our findings suggest a novel mechanism for chemotherapy-induced cytotoxicity in non-dividing cells, with far-reaching medical implications. PMID:18213384

  4. A study of microtubule dipole lattices

    NASA Astrophysics Data System (ADS)

    Nandi, Shubhendu

    Microtubules are cytoskeletal protein polymers orchestrating a host of important cellular functions including, but not limited to, cell support, cell division, cell motility and cell transport. In this thesis, we construct a toy-model of the microtubule lattice composed of vector Ising spins representing tubulin molecules, the building block of microtubules. Nearest-neighbor and next-to-nearest neighbor interactions are considered within an anisotropic dielectric medium. As a consequence of the helical topology, we observe that certain spin orientations render the lattice frustrated with nearest neighbor ferroelectric and next-to-nearest neighbor antiferroelectric bonds. Under these conditions, the lattice displays the remarkable property of stabilizing certain spin patterns that are robust to thermal fluctuations. We model this behavior in the framework of a generalized Ising model known as the J1 - J2 model and theoretically determine the set of stable patterns. Employing Monte-Carlo methods, we demonstrate the stability of such patterns in the microtubule lattice at human physiological temperatures. This suggests a novel biological mechanism for storing information in living organisms, whereby the tubulin spin (dipole moment) states become information bits and information gets stored in microtubules in a way that is robust to thermal fluctuations.

  5. Polyribosome targeting to microtubules: enrichment of specific mRNAs in a reconstituted microtubule preparation from sea urchin embryos

    PubMed Central

    1994-01-01

    A subset of mRNAs, polyribosomes, and poly(A)-binding proteins copurify with microtubules from sea urchin embryos. Several lines of evidence indicate that the interaction of microtubules with ribosomes is specific: a distinct stalk-like structure appears to mediate their association; ribosomes bind to microtubules with a constant stoichiometry through several purification cycles; and the presence of ribosomes in these preparations depends on the presence of intact microtubules. Five specific mRNAs are enriched with the microtubule- bound ribosomes, indicating that translation of specific proteins may occur on the microtubule scaffolding in vivo. PMID:7962079

  6. Binding of microtubule protein to DNA and chromatin: possibility of simultaneous linkage of microtubule to nucleic and assembly of the microtubule structure.

    PubMed Central

    Villasante, A; Corces, V G; Manso-Martínez, R; Avila, J

    1981-01-01

    Microtubule protein binds to DNA through microtubule associated polypeptides (MAPs). Among MAPs there is one high molecular weight polypeptide (MAP2) which interacts with DNA fundamentally through certain polynucleotide sequences. This interaction is not affected by the presence of histones and other chromosomal proteins. DNA can associate to assembled microtubules and when a determinate DNA/protein ratio is reached the nucleic acid behaves as a microtubule associated molecule. The nucleic acid fragments which preferentially bind to microtubules have been isolated and characterized. These fragments contain DNA regions enriched in repetitive sequences that hybridizes preferentially to the pericentromeric zone of metaphase chromosomes. These results give further support to the model of interaction microtubule-chromosome based upon the mediator function of the microtubule associated proteins. Images PMID:7232207

  7. A Barley ROP GTPase ACTIVATING PROTEIN Associates with Microtubules and Regulates Entry of the Barley Powdery Mildew Fungus into Leaf Epidermal Cells[C][W

    PubMed Central

    Hoefle, Caroline; Huesmann, Christina; Schultheiss, Holger; Börnke, Frederik; Hensel, Götz; Kumlehn, Jochen; Hückelhoven, Ralph

    2011-01-01

    Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry. PMID:21685259

  8. Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis[C][W][OA

    PubMed Central

    Liu, Xiaomin; Qin, Tao; Ma, Qianqian; Sun, Jingbo; Liu, Ziqiang; Yuan, Ming; Mao, Tonglin

    2013-01-01

    Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation. PMID:23653471

  9. Two Microtubule-associated Proteins of Arabidopsis MAP65s Promote Antiparallel Microtubule Bundling

    PubMed Central

    Gaillard, Jérémie; Neumann, Emmanuelle; Van Damme, Daniel; Stoppin-Mellet, Virginie; Ebel, Christine; Barbier, Elodie; Geelen, Danny

    2008-01-01

    The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that “zipper up” antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter. PMID:18667529

  10. 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.

  11. Mechanical model of kinesin moving on microtubule

    NASA Astrophysics Data System (ADS)

    To, Kiwing; Chou, Ya-Chang; Hsiao, Yi-Feng; Chen, Kuan-Hua

    Kinesins are biomolecules that serve as intercellular motors for carrying cellular cargos along microtubules. Although the mechanism of converting the chemical energy of ATP to mechanical work is not fully understood, the motion of a kinesin on a microtubule has been measured and two different mechanisms, namely the ``hand-over-hand'' and ``inchworm'', has been proposed. The particular shape of kinesin and microtubules suggest a possible mechanism for force generation similar to Brownian ratchet. Using a bead chain connected to two heads that are attracted to a vibrated ratchet plate as a scaled up analog of the kinesinmicrotubule system, we manage to simulate both ``handoverhand'' and ``inchworm'' motion [Chou, et. al., Physica A443, 66 (2015)]. In addition, we find that chain, which play the role of the stalk in a kinesin molecule, can also generate force by interacting with the ratchet plate [Chen, et. al. Phys. Rev. E87, 012711 (2013)].

  12. Orthotropic elastic shell model for buckling of microtubules.

    PubMed

    Wang, C Y; Ru, C Q; Mioduchowski, A

    2006-11-01

    In view of the fact that microtubules exhibit strong anisotropic elastic properties, an orthotropic elastic shell model for microtubules is developed to study buckling behavior of microtubules. The predicted critical pressure is found to agree well with recent unexplained experimental data on pressure-induced buckling of microtubules [Needleman, Phys. Rev. Lett. 93, 198104 (2004); Biophys. J. 89, 3410 (2005)] which are lower than that predicted by the isotropic shell model by four orders of magnitude. General buckling behavior of microtubules under axial compression or radial pressure is studied. The results show that the isotropic shell model greatly overestimates the bucking loads of microtubules, except columnlike axially compressed buckling of long microtubules (of length-to-diameter ratio larger than, say, 150). In particular, the present results also offer a plausible explanation for the length dependency of flexibility of microtubules reported in the literature.

  13. 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

  14. Micropattern-Guided Assembly of Overlapping Pairs of Dynamic Microtubules

    PubMed Central

    Fourniol, Franck J.; Li, Tai-De; Bieling, Peter; Mullins, R. Dyche; Fletcher, Daniel A.; Surrey, Thomas

    2014-01-01

    Interactions between antiparallel microtubules are essential for the organization of spindles in dividing cells. The ability to form immobilized antiparallel microtubule pairs in vitro, combined with the ability to image them via TIRF microscopy, permits detailed biochemical characterization of microtubule cross-linking proteins and their effects on microtubule dynamics. Here, we describe methods for chemical micropatterning of microtubule seeds on glass surfaces in configurations that specifically promote the formation of antiparallel microtubule overlaps in vitro. We demonstrate that this assay is especially well suited for reconstitution of minimal midzone overlaps stabilized by the antiparallel microtubule cross-linking protein PRC1 and its binding partners. The micropatterning method is suitable for use with a broad range of proteins, and the assay is generally applicable to any microtubule cross-linking protein. PMID:24630116

  15. Protofilament number in microtubules in cells of two parasitic nematodes.

    PubMed

    Davis, C; Gull, K

    1983-12-01

    The parasitic nematodes, Ascaridia galli and Trichostrongylus colubriformis, were prepared for electron microscopy with fixatives containing tannic acid, which allowed their microtubule protofilament number to be examined. In contrast to many mammalian tissues, the nematodes did not contain microtubules with 13 protofilaments. Ascaridia galli contained microtubules with 11 protofilaments in all tissues examined, including nerve, intestinal, pharyngeal, and hypodermal cells. Trichostrongylus colubriformis contained nerve cells, known as microtubule cells, with bundles of larger microtubules (approximately 30 nm in diameter) with 14 protofilaments. The microtubules in these cells did not appear to be continuous for the entire length of the axon. Other cells examined in T. colubriformis, including nerve, intestinal and pharyngeal cells, contained two distinct types of microtubules, one with 11 protofilaments and an approximate diameter of 25 nm, and one with 12 protofilaments and an approximate diameter of 27 nm. All cell types examined contained both types of microtubules.

  16. Dynamic microtubules and the texture of plant cell walls.

    PubMed

    Lloyd, Clive

    2011-01-01

    The relationship between microtubules and cell-wall texture has had a fitful history in which progress in one area has not been matched by progress in the other. For example, the idea that wall texture arises entirely from self-assembly, independently of microtubules, originated with electron microscopic analyses of fixed cells that gave no clue to the ability of microtubules to reorganize. Since then, live-cell studies have established the surprising dynamicity of plant microtubules involving collisions, changes in angle, parallelization, and rotation of microtubule tracks. Combined with proof that cellulose synthases do track along shifting microtubules, this offers more realistic models for the dynamic influence of microtubules on wall texture than could have been imagined in the electron microscopic era-the era from which most ideas on wall texture originate. This review revisits the classical literature on wall organization from the vantage point of current knowledge of microtubule dynamics.

  17. Structural investigations into microtubule-MAP complexes.

    PubMed

    Hoenger, Andreas; Gross, Heinz

    2008-01-01

    Microtubules interact with a large variety of factors commonly referred to as either molecular motors (kinesins, dyneins) or structural microtubule-associated proteins (MAPs). MAPs do not exhibit motor activity, but regulate microtubule dynamics and their interactions with molecular motors, and organelles such as kinetochores or centrosomes. Structural investigations into microtubule-kinesin motor complexes are quite advanced today and by helical three-dimensional (3-D) analysis reveal a resolution of the motor-tubulin interface at <1.0 nm. However, due to their flexible structure MAPs like tau or MAP2C cannot be visualized in the same straightforward manner. Helical averaging usually reveals only the location of strong binding sites while the overall structure of the MAP remains unsolved. Other MAPs such as EB1 bind very selectively only to some parts of the microtubule lattice such as the lattice seam. Thus, they do not reveal a stoichiometric tubulin:MAP-binding ratio that would allow for a quantitative helical 3-D analysis. Therefore, to get a better view on the structure of microtubule-MAP complexes we often used a strategy that combined cryo-electron microscopy and helical or tomographic 3-D analysis with freeze-drying and high-resolution unidirectional surface shadowing. 3-D analysis of ice-embedded specimens reveals their full 3-D volume. This relies either on a repetitive structure following a helical symmetry that can be used for averaging or suffers from the limited resolution that is currently achievable with cryotomography. Surface metal shadowing exclusively images surface-exposed features at very high contrast, adding highly valuable information to 2-D or 3-D data of vitrified structures.

  18. Evolving tip structures can explain age-dependent microtubule catastrophe.

    PubMed

    Coombes, Courtney E; Yamamoto, Ami; Kenzie, Madeline R; Odde, David J; Gardner, Melissa K

    2013-07-22

    Microtubules are key structural and transport elements in cells. The dynamics at microtubule ends are characterized by periods of slow growth, followed by stochastic switching events termed "catastrophes," in which microtubules suddenly undergo rapid shortening. Growing microtubules are thought to be protected from catastrophe by a GTP-tubulin "cap": GTP-tubulin subunits add to the tips of growing microtubules but are subsequently hydrolyzed to GDP-tubulin subunits once they are incorporated into the microtubule lattice. Loss of the GTP-tubulin cap exposes GDP-tubulin subunits at the microtubule tip, resulting in a catastrophe event. However, the mechanistic basis for sudden loss of the GTP cap, leading to catastrophe, is not known. To investigate microtubule catastrophe events, we performed 3D mechanochemical simulations that account for interactions between neighboring protofilaments. We found that there are two separate factors that contribute to catastrophe events in the 3D simulation: the GTP-tubulin cap size, which settles into a steady-state value that depends on the free tubulin concentration during microtubule growth, and the structure of the microtubule tip. Importantly, 3D simulations predict, and both fluorescence and electron microscopy experiments confirm, that microtubule tips become more tapered as the microtubule grows. This effect destabilizes the tip and ultimately contributes to microtubule catastrophe. Thus, the likelihood of a catastrophe event may be intimately linked to the aging physical structure of the growing microtubule tip. These results have important consequences for catastrophe regulation in cells, as microtubule-associated proteins could promote catastrophe events in part by modifying microtubule tip structures.

  19. Polewards chromosome movement driven by microtubule depolymerization in vitro

    NASA Astrophysics Data System (ADS)

    Koshland, Douglas E.; Mitchison, T. J.; Kirschner, Marc W.

    1988-02-01

    We constructed complexes between isolated chromosomes and microtubules made from purified tubulin to study the movement of chromosomes towards the 'minus' end of microtubules in vitro, a process analogous to the movement of chromosomes towards the pole of the spindle at anaphase of mitosis. Our results show that the energy for this movement is derived solely from microtubule depolymerization, and indicate that anaphase movement of chromosomes is both powered and regulated by microtubule depolymerization at the kinetochore.

  20. Fluorescent microtubules break up under illumination

    PubMed Central

    1988-01-01

    We have synthesized three new fluorescent analogues of tubulin, using fluorescein or rhodamine groups attached to N-hydroxy-succinimidyl esters, and have partially characterized the properties of these analogues. We have also further characterized the tubulin derivatized with dichlorotriazinyl-aminofluorescein that has previously been used in this and other laboratories. Our results show that all four analogues assemble into microtubules which break up when exposed to light of the wavelengths that excite fluorescence. This sensitivity places severe constraints on the use of these analogues in studies of microtubule dynamics. PMID:3417772

  1. Colloidal Stabilization of Neurofilaments and Microtubules

    DTIC Science & Technology

    2002-06-01

    Alzheimer’s disease . To address this problem we used a set of biophysical methods, including atomic force microscopy, to investigate interfilament potentials. There are several main conclusions from the work under this award. First, microtubule associated proteins behave as though they are largely unstructured and can give rise to a long range repulsive force that is predominantly entropic in origin. This is an important finding that provides a biophysical mechanism that explains how microtubule spacing is maintained. Second, treating the unstructured proteins domains

  2. Cortical dynamics during cell motility are regulated by CRL3KLHL21 E3 ubiquitin ligase

    PubMed Central

    Courtheoux, Thibault; Enchev, Radoslav I.; Lampert, Fabienne; Gerez, Juan; Beck, Jochen; Picotti, Paola; Sumara, Izabela; Peter, Matthias

    2016-01-01

    Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3KLHL21 E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3KLHL21 activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions. PMID:27641145

  3. Arabidopsis homeodomain-leucine zipper IV proteins promote stomatal development and ectopically induce stomata beyond the epidermis.

    PubMed

    Peterson, Kylee M; Shyu, Christine; Burr, Christian A; Horst, Robin J; Kanaoka, Masahiro M; Omae, Minami; Sato, Yutaka; Torii, Keiko U

    2013-05-01

    The shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration. Advances have been made in our understanding of stomatal patterning and differentiation, and a set of 'master regulatory' transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis. Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages. Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata. A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2. Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta. Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.

  4. Controls on the emission of plant volatiles through stomata: Differential sensitivity of emission rates to stomatal closure explained

    NASA Astrophysics Data System (ADS)

    Niinemets, ÜLo; Reichstein, Markus

    2003-04-01

    Volatile (VOC) flux from leaves may be expressed as GSΔP, where GS is stomatal conductance to specific compound and ΔP partial pressure gradient between the atmosphere and substomatal cavities. It has been suggested that decreases in GS are balanced by increases in ΔP such that stomata cannot control VOC emission. Yet, responses of emission rates of various volatiles to experimental manipulations of stomatal aperture are contrasting. To explain these controversies, a dynamic emission model was developed considering VOC distribution between gas and liquid phases using Henry's law constant (H, Pa m3 mol-1). Our analysis demonstrates that highly volatile compounds such as isoprene and monoterpenes with H values on the order of 103 have gas and liquid pool half-times of a few seconds, and thus cannot be controlled by stomata. More soluble compounds such as alcohols and carboxylic acids with H values of 10-2-101 are controlled by stomata with the degree of stomatal sensitivity varying with H. Inability of compounds with high solubility to support a high partial pressure, and thus to balance ΔP in response to a decrease in GS is the primary explanation for different stomatal sensitivities. For compounds with low H, the analysis predicts bursts of emission after stomatal opening that accord with experimental observations, but that cannot be currently explained. Large within-leaf VOC pool sizes in compounds with low H also increase the system inertia to environmental fluctuations. In conclusion, dynamic models are necessary to simulate diurnal variability of the emissions of compounds that preferably partition to aqueous phase.

  5. Open or Close the Gate – Stomata Action Under the Control of Phytohormones in Drought Stress Conditions

    PubMed Central

    Daszkowska-Golec, Agata; Szarejko, Iwona

    2013-01-01

    Two highly specialized cells, the guard cells that surround the stomatal pore, are able to integrate environmental and endogenous signals in order to control the stomatal aperture and thereby the gas exchange. The uptake of CO2 is associated with a loss of water by leaves. Control of the size of the stomatal aperture optimizes the efficiency of water use through dynamic changes in the turgor of the guard cells. The opening and closing of stomata is regulated by the integration of environmental signals and endogenous hormonal stimuli. The various different factors to which the guard cells respond translates into the complexity of the network of signaling pathways that control stomatal movements. The perception of an abiotic stress triggers the activation of signal transduction cascades that interact with or are activated by phytohormones. Among these, abscisic acid (ABA), is the best-known stress hormone that closes the stomata, although other phytohormones, such as jasmonic acid, brassinosteroids, cytokinins, or ethylene are also involved in the stomatal response to stresses. As a part of the drought response, ABA may interact with jasmonic acid and nitric oxide in order to stimulate stomatal closure. In addition, the regulation of gene expression in response to ABA involves genes that are related to ethylene, cytokinins, and auxin signaling. In this paper, recent findings on phytohormone crosstalk, changes in signaling pathways including the expression of specific genes and their impact on modulating stress response through the closing or opening of stomata, together with the highlights of gaps that need to be elucidated in the signaling network of stomatal regulation, are reviewed. PMID:23717320

  6. Microtubules and the endoplasmic reticulum are highly interdependent structures

    PubMed Central

    1986-01-01

    The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system. PMID:3533956

  7. 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."

  8. Mmb1p binds mitochondria to dynamic microtubules

    PubMed Central

    Fu, Chuanhai; Jain, Deeptee; Costa, Judite; Velve-Casquillas, Guilhem; Tran, Phong T.

    2015-01-01

    Summary Background Mitochondria form a dynamics tubular network within the cell. Proper mitochondria movement and distribution are critical for their localized function in cell metabolism, growth, and survival. In mammalian cells, mechanisms of mitochondria positioning appear dependent on the microtubule cytoskeleton, with kinesin or dynein motors carrying mitochondria as cargos and distributing them throughout the microtubule network. Interestingly, the timescale of microtubule dynamics occurs in seconds, and the timescale of mitochondria distribution occurs in minutes. How does the cell couple these two time constants? Results Fission yeast also relies on microtubules for mitochondria distribution. We report here a new microtubule-dependent but motor-independent mechanism for proper mitochondria positioning in fission yeast. We identify the protein mmb1p, which binds to mitochondria and microtubules. Mmb1p attaches the tubular mitochondria to the microtubule lattice at multiple discrete interaction sites. Mmb1 deletion causes mitochondria to aggregate, with the long-term consequence of defective mitochondria distribution and cell death. Mmb1p decreases microtubule dynamicity. Conclusion Mmb1p is a new microtubule-mitochondria binding protein. We propose that mmb1p act to couple long-term mitochondria distribution to short-term microtubule dynamics by attenuating microtubule dynamics, thus enhancing the mitochondria-microtubule interaction time. PMID:21856157

  9. GDP-Tubulin Incorporation into Growing Microtubules Modulates Polymer Stability*

    PubMed Central

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

    2010-01-01

    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.” PMID:20371874

  10. Motors and MAPs collaborate to size up microtubules.

    PubMed

    Bechstedt, Susanne; Brouhard, Gary J

    2013-07-29

    Midzone microtubules keep chromosomes apart after segregation and provide a platform for cytokinesis factors. Reporting recently in Cell, Subramanian et al. (2013) describe how the motor protein kinesin-4 and the microtubule-associated protein PRC1 work together to mark microtubule ends for incorporation into the midzone in a length-dependent manner.

  11. The effects of enhanced UV-B radiation on growth, stomata, flavonoid, and ABA content in cucumber leaves

    NASA Astrophysics Data System (ADS)

    An, Lizhe; Wang, Jianhui; Liu, Yanhong; Chen, Tuo; Xu, Shijian; Feng, Huyuan; Wang, Xunling

    2003-06-01

    Cucumber plants (Cucumis sativus L. cv. Jinchun No 3) grown in a greenhouse were treated with three different biologically effective ultraviolet-B (UV-B) radiation levels: 1.28 kJ. m-2 (CK), 8.82kJ.m-2 (T1) and 12.6 kJ. m-2 (T2). Irradiances corresponded to 8% and 21% reduction in stratospheric ozone in Lanzhou. Plants at three-leaf stage were irradiated 7 h daily for 25 days. The growth, stomata, flavonoid and ABA content in cucumber leaves exposed to 3 levels of UV-B radiation were determined in this paper. The results indicated that, compared with the control after 25 days UV-B radiation, RI of cucumber under T1 treatment is -18.0% and RI under T2 treatment is -48% mostly because of the reduce of leave area and dry weight accompanying with the increase of SLW; the rate of stomata closure under the treatments of T1 and T2 on the 6th day was up to respectively 70% and 89%, and amounted to 90% and 100% on the 18th day, and the guard cells in some stomata apparatus became permanent pores and lost their function at the same time; with the duration of UV-B radiation, the rise of the absorbance to ultraviolet light (305nm) showed the content increase of flavonoid; Abscisic acid (ABA) was determined by means of ELISA which showed that under the T1 treatment, the content of ABA was up to maximum to 510% higher than that of the control on the 21st day, meanwhile, under the treatment of T2, it was the highest on the 18th day to 680% of the control, and then had a decrease tendency on 21st day. The result still indicated that ABA accumulation could be induced by enhanced UV-B the radiation. The bigger was the dose of radiation, the higher was the accumulation of ABA. When intensity of UV-B radiation went beyond the degree of endurance of cucumber plants, ABA content descended then. Cucumber plants resist enhanced UV-B radiation by means of improving the contents of ABA and flavonoid. The increase of ABA content in cucumber leaves could lead to the stomata closure. Therefore

  12. ABA induces H2O2 production in guard cells, but does not close the stomata on Vicia faba leaves developed at high air humidity.

    PubMed

    Arve, Louise E; Carvalho, Dália R A; Olsen, Jorunn E; Torre, Sissel

    2014-01-01

    Plants developed under constant high (> 85%) relative air humidity (RH) have larger stomata that are unable to close completely. One of the hypotheses for the less responsive stomata is that the plants have reduced sensitivity to abscisic acid (ABA). Both ABA and darkness are signals for stomatal closure and induce the production of the secondary messenger hydrogen peroxide (H2O2). In this study, the ability of Vicia faba plants developed in moderate or high RH to close the stomata in response to darkness, ABA and H2O2 was investigated. Moreover, the ability of the plants to produce H2O2 when treated with ABA or transferred to darkness was also assessed. Our results show that the ABA concentration in moderate RH is not increased during darkness even though the stomata are closing. This indicates that stomatal closure in V. faba during darkness is independent of ABA production. ABA induced both H2O2 production and stomatal closure in stomata formed at moderate RH. H2O2 production, as a result of treatment with ABA, was also observed in stomata formed at high RH, though the closing response was considerably smaller as compared with moderate RH. In either RH, leaf ABA concentration was not affected by darkness. Similarly to ABA treatment, darkness elicited both H2O2 production and stomatal closure following plant cultivation at moderate RH. Contrary to this, neither H2O2 production nor stomatal closure took place when stomata were formed at high RH. These results suggest that the reduced stomatal response in plants developed in continuous high RH is caused by one or more factors downstream of H2O2 in the signaling pathway toward stomatal closure.

  13. ABA induces H2O2 production in guard cells, but does not close the stomata on Vicia faba leaves developed at high air humidity

    PubMed Central

    Arve, Louise E; Carvalho, Dália RA; Olsen, Jorunn E; Torre, Sissel

    2014-01-01

    Plants developed under constant high (> 85%) relative air humidity (RH) have larger stomata that are unable to close completely. One of the hypotheses for the less responsive stomata is that the plants have reduced sensitivity to abscisic acid (ABA). Both ABA and darkness are signals for stomatal closure and induce the production of the secondary messenger hydrogen peroxide (H2O2). In this study, the ability of Vicia faba plants developed in moderate or high RH to close the stomata in response to darkness, ABA and H2O2 was investigated. Moreover, the ability of the plants to produce H2O2 when treated with ABA or transferred to darkness was also assessed. Our results show that the ABA concentration in moderate RH is not increased during darkness even though the stomata are closing. This indicates that stomatal closure in V. faba during darkness is independent of ABA production. ABA induced both H2O2 production and stomatal closure in stomata formed at moderate RH. H2O2 production, as a result of treatment with ABA, was also observed in stomata formed at high RH, though the closing response was considerably smaller as compared with moderate RH. In either RH, leaf ABA concentration was not affected by darkness. Similarly to ABA treatment, darkness elicited both H2O2 production and stomatal closure following plant cultivation at moderate RH. Contrary to this, neither H2O2 production nor stomatal closure took place when stomata were formed at high RH. These results suggest that the reduced stomatal response in plants developed in continuous high RH is caused by one or more factors downstream of H2O2 in the signaling pathway toward stomatal closure. PMID:25763494

  14. Effect of Aluminum, Iron, and Zinc Ions on the Assembly of Microtubules from Brain Microtubule Proteins.

    PubMed

    Shevtsov, P N; Shevtsova, E F; Burbaeva, G Sh

    2016-08-01

    Al(3+), Fe(3+), and Zn(2+) ions can disturb microtubule assembly from tubulin and microtubuleassociated proteins in rat brain. The main structural forms of these microtubules are rings and tangled bundles. These structures are formed only in the presence of Al(3+) and Fe(3+) ions. Therefore, Zn(2+) ions can be excluded from possible causes of structural abnormalities in microtubules during Alzheimer's disease. Al(3+) ions are the most probable etiological cause of Alzheimer's disease. The concentration of Al(3+) ions affecting the structure of microtubules is one order of magnitude lower than that of Fe(3+) ions (10 and 100 μM, respectively), which corresponds to their brain concentration reported in Alzheimer's disease.

  15. Novel insights into mammalian embryonic neural stem cell division: focus on microtubules

    PubMed Central

    Mora-Bermúdez, Felipe; Huttner, Wieland B.

    2015-01-01

    During stem cell divisions, mitotic microtubules do more than just segregate the chromosomes. They also determine whether a cell divides virtually symmetrically or asymmetrically by establishing spindle orientation and the plane of cell division. This can be decisive for the fate of the stem cell progeny. Spindle defects have been linked to neurodevelopmental disorders, yet the role of spindle orientation for mammalian neurogenesis has remained controversial. Here we explore recent advances in understanding how the microtubule cytoskeleton influences mammalian neural stem cell division. Our focus is primarily on the role of spindle microtubules in the development of the cerebral cortex. We also highlight unique characteristics in the architecture and dynamics of cortical stem cells that are tightly linked to their mode of division. These features contribute to setting these cells apart as mitotic “rule breakers,” control how asymmetric a division is, and, we argue, are sufficient to determine the fate of the neural stem cell progeny in mammals. PMID:26628750

  16. 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

  17. 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.

  18. 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.

  19. Sliding of STOP proteins on microtubules: a model system for diffusion-dependent microtubule motility.

    PubMed

    Margolis, R L; Job, D; Pabion, M; Rauch, C T

    1986-01-01

    STOP proteins, of 145 kD, act substoichiometrically to block end-wise disassembly of microtubules. STOPs bind to microtubules either during microtubule assembly or when added at steady state, and when binding to the polymers is apparently irreversible. They are not measurably lost from polymers under competition conditions, and there is no measurable exchange between polymers. Nonetheless, STOP proteins exhibit an extraordinary behavior: they "slide" laterally on the surface of the microtubule. Displacement is assayed by forming hybrid microtubules in which cold stable or cold labile region subunits are labeled. Displacement of STOPs on the polymer with time will cause labeled subunits of cold-stable regions to become increasingly cold labile in a manner reciprocal to cold stabilization of previously cold-labile subunits. Because equilibrium exchange of STOP proteins onto and off the polymers can be ruled out, the displacement of STOPs relative to subunits can only be explained by lateral diffusion or "sliding." Axonal transport and mitotic mechanisms were discussed as implications of such a lateral translocation mechanism for microtubule-dependent motility.

  20. Discodermolide interferes with the binding of tau protein to microtubules.

    PubMed

    Kar, Santwana; Florence, Gordon J; Paterson, Ian; Amos, Linda A

    2003-03-27

    We investigated whether discodermolide, a novel antimitotic agent, affects the binding to microtubules of tau protein repeat motifs. Like taxol, the new drug reduces the proportion of tau that pellets with microtubules. Despite their differing structures, discodermolide, taxol and tau repeats all bind to a site on beta-tubulin that lies within the microtubule lumen and is crucial in controlling microtubule assembly. Low concentrations of tau still bind strongly to the outer surfaces of preformed microtubules when the acidic C-terminal regions of at least six tubulin dimers are available for interaction with each tau molecule; otherwise binding is very weak.

  1. Analysis of microtubule polymerization dynamics in live cells

    PubMed Central

    Gierke, Sarah; Kumar, Praveen; Wittmann, Torsten

    2012-01-01

    Intracellular microtubule polymerization dynamics are spatiotemporally controlled by numerous microtubule-associated proteins and other mechanisms, and this regulation is central to many cell processes. Here, we give an overview and practical guide on how to acquire and analyze time-lapse sequences of dynamic microtubules in live cells by either fluorescently labeling entire microtubules or by utilizing proteins that specifically associate only with growing microtubule ends, and summarize the strengths and weaknesses of different approaches. We give practical recommendations for imaging conditions, and we also discuss important limitations of such analysis that are dictated by the maximal achievable spatial and temporal sampling frequencies. PMID:20719263

  2. Plant water use efficiency over geological time--evolution of leaf stomata configurations affecting plant gas exchange.

    PubMed

    Assouline, Shmuel; Or, Dani

    2013-01-01

    Plant gas exchange is a key process shaping global hydrological and carbon cycles and is often characterized by plant water use efficiency (WUE - the ratio of CO2 gain to water vapor loss). Plant fossil record suggests that plant adaptation to changing atmospheric CO2 involved correlated evolution of stomata density (d) and size (s), and related maximal aperture, amax . We interpreted the fossil record of s and d correlated evolution during the Phanerozoic to quantify impacts on gas conductance affecting plant transpiration, E, and CO2 uptake, A, independently, and consequently, on plant WUE. A shift in stomata configuration from large s-low d to small s-high d in response to decreasing atmospheric CO2 resulted in large changes in plant gas exchange characteristics. The relationships between gas conductance, gws , A and E and maximal relative transpiring leaf area, (amax ⋅d), exhibited hysteretic-like behavior. The new WUE trend derived from independent estimates of A and E differs from established WUE-CO2 trends for atmospheric CO2 concentrations exceeding 1,200 ppm. In contrast with a nearly-linear decrease in WUE with decreasing CO2 obtained by standard methods, the newly estimated WUE trend exhibits remarkably stable values for an extended geologic period during which atmospheric CO2 dropped from 3,500 to 1,200 ppm. Pending additional tests, the findings may affect projected impacts of increased atmospheric CO2 on components of the global hydrological cycle.

  3. Stomata open at night in pole-sized and mature ponderosa pine: implications for O3 exposure metrics.

    PubMed

    Grulke, N E; Alonso, R; Nguyen, T; Cascio, C; Dobrowolski, W

    2004-09-01

    Ponderosa pine (Pinus ponderosa Dougl. ex Laws.) is widely distributed in the western USA. We report the lack of stomatal closure at night in early summer for ponderosa pine at two of three sites investigated. Trees at a third site with lower nitrogen dioxide and nitric acid exposure, but greater drought stress, had slightly open stomata at night in early summer but closed stomata at night for the rest of the summer. The three sites had similar background ozone exposure during the summer of measurement (2001). Nighttime stomatal conductance (gs) ranged from one tenth to one fifth that of maximum daytime values. In general, pole-sized trees (< 40 years old) had greater nighttime gs than mature trees (> 250 years old). In late summer, nighttime gs was low (< 3.0 mmol H2O m(-2) s(-1)) for both tree size classes at all sites. Measurable nighttime gs has also been reported in other conifers, but the values we observed were higher. In June, nighttime ozone (O3) uptake accounted for 9, 5 and 3% of the total daily O3 uptake of pole-sized trees from west to east across the San Bernardino Mountains. In late summer, O3 uptake at night was < 2% of diel uptake at all sites. Nocturnal O3 uptake may contribute to greater oxidant injury development, especially in pole-sized trees in early summer.

  4. Prechilling of Xanthium strumarium L. Reduces Net Photosynthesis and, Independently, Stomatal Conductance, While Sensitizing the Stomata to CO21

    PubMed Central

    Drake, B.; Raschke, K.

    1974-01-01

    Greenhouse-grown plants of Xanthium strumarium L. were exposed in a growth cabinet to 10 C during days and 5 C during nights for periods of up to 120 hours. Subsequently, CO2 exchange, transpiration, and leaf temperature were measured on attached leaves and in leaf sections at 25 or 30 C, 19 C dew point of the air, 61 milliwatts per square centimeter irradiance, and CO2 concentrations between 0 and 1000 microliters per liter ambient air. Net photosynthesis and stomatal conductance decreased and dark respiration increased with increasing duration of prechilling. The reduction in net photosynthesis was not a consequence of decreased stomatal conductance because the intercellular CO2 concentration in prechilled leaves was equal to or greater than that in greenhouse-grown controls. The intercellular CO2 concentration at which one-half maximum net photosynthesis occurred remained the same in prechilled leaves and controls (175 to 190 microliters per liter). Stomata of the control plants responded to changes in the CO2 concentration of the air only slightly. Prechilling for 24 hours or more sensitized stomata to CO2; they responded to changes in CO2 concentration in the range from 100 to 1000 microliters per liter. PMID:16658795

  5. Target molecules of calmodulin on microtubules of Tetrahymena cilia

    SciTech Connect

    Hirano-Ohnishi, Junko; Watanabe, Yoshio )

    1988-09-01

    In the course of an attempt to isolate the calmodulin-binding proteins (CaMBPs) from cilia of Tetrahymena, it was found that some CaMBPs tend to interact with axonemal microtubules. The present study demonstrates this interaction by cosedimentation experiments using in vitro polymerized Tetrahymena axonemal microtubules and Tetrahymena CaMBPs purified from axonemes by calmodulin affinity column chromatography. Analysis by the ({sup 125}I)calmodulin overlay method showed that at least three CaMBPs (M{sub r} 69, 45, and 37 kDa) cosediment with microtubules. Furthermore, without any addition of exogenous CaMBPs, microtubules purified after three cycles of temperature-dependent polymerization and depolymerization included the above CaMBPs and additional CaMBPs which could not cosediment with microtubules. From the results, the authors have classified these microtubule-associated CaMBPs into two groups: (i) CaMBPs which interact with microtubules only during polymerization, and (ii) CaMBPs which interact not only with microtubules during polymerization, but also with polymerized microtubules. These results suggest that the microtubule-associated CaMBPs, especially those of the latter group, are located on the surface of ciliary microtubules, and may become the target molecules of calmodulin at Ca{sup 2+}-triggered ciliary reversal.

  6. Three-dimensional structure of cytoplasmic dynein bound to microtubules

    PubMed Central

    Mizuno, Naoko; Narita, Akihiro; Kon, Takahide; Sutoh, Kazuo; Kikkawa, Masahide

    2007-01-01

    Cytoplasmic dynein is a large, microtubule-dependent molecular motor (1.2 MDa). Although the structure of dynein by itself has been characterized, its conformation in complex with microtubules is still unknown. Here, we used cryoelectron microscopy (cryo-EM) to visualize the interaction between dynein and microtubules. Most dynein molecules in the nucleotide-free state are bound to the microtubule in a defined conformation and orientation. A 3D image reconstruction revealed that dynein's head domain, formed by a ring-like arrangement of AAA+ domains, is located ≈280 Å away from the center of the microtubule. The order of the AAA+ domains in the ring was determined by using recombinant markers. Furthermore, a 3D helical image reconstruction of microtubules with a dynein's microtubule binding domain [dynein stalk (DS)] revealed that the stalk extends perpendicular to the microtubule. By combining the 3D maps of the dynein-microtubule and DS-microtubule complexes, we present a model for how dynein in the nucleotide-free state binds to microtubules and discuss models for dynein's power stroke. PMID:18093913

  7. Organelles are transported on sliding microtubules in Reticulomyxa.

    PubMed

    Orokos, D D; Cole, R W; Travis, J L

    2000-12-01

    Organelles and plasma membrane domains appear to be transported along Reticulomyxa's microtubule cytoskeleton. Previously we demonstrated that organelle and cell surface transport share the same enzymatic properties and suggested that both are powered by the same cytoplasmic dynein. Motility analysis in Reticulomyxa is complicated by the fact that the microtubules also are motile and appear to "slide" bidirectionally throughout the network. We have utilized laser ablation to address this frame-of-reference problem as to how each transport component (microtubule sliding vs. organelle translocations) contributes to reactivated bidirectional translocation of organelles along the microtubule cytoskeleton. Laser ablation was used to cut microtubule bundles from lysed networks into 4-15-microm segments. After examining these reactivated cut fragments, it appears that the majority of organelles did not move relative to microtubule fragments, but remained attached to microtubules and moved as the microtubules slid. Microtubule sliding stops after 1-2 min and cannot be reactivated even when perfused with fresh ATP. Furthermore, once sliding stops, organelle transport also stops. Our findings indicate that the majority of Reticulomyxa pseudopodial organelles do not move along the surface of the microtubules, rather it is the sliding of the microtubules to which they are attached that moves them.

  8. Microtubule Dynamics Control Tail Retraction in Migrating Vascular Endothelial Cells†

    PubMed Central

    Ganguly, Anutosh; Yang, Hailing; Zhang, Hong; Cabral, Fernando; Patel, Kamala D.

    2014-01-01

    Drugs that target microtubules are potent inhibitors of angiogenesis but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; but, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish rather than elongated cells, rapid but non-directional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells while more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay. PMID:24107446

  9. 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.

  10. Photosynthesis-dependent and -independent responses of stomata to blue, red and green monochromatic light: differences between the normally oriented and inverted leaves of sunflower.

    PubMed

    Wang, Yin; Noguchi, Ko; Terashima, Ichiro

    2011-03-01

    The effects of growth light environment on stomatal light responses were analyzed. We inverted leaves of sunflower (Helianthus annuus) for 2 weeks until their full expansion, and measured gas exchange properties of the adaxial and abaxial sides separately. The sensitivity to light assessed as the increase in stomatal conductance was generally higher in the abaxial stomata than in the adaxial stomata, and these differences could not be completely changed by the inversion treatment. We also treated the leaves with DCMU to inhibit photosynthesis and evaluated the photosynthesis-dependent and -independent components of stomatal light responses. The red light response of stomata in both normally oriented and inverted leaves relied only on the photosynthesis-dependent component. The blue light response involved both the photosynthesis-dependent and photosynthesis-independent components, and the relative contributions of the two components differed between the normally oriented and inverted leaves. A green light response was observed only in the abaxial stomata, which also involved the photosynthesis-dependent and photosynthesis-independent components, strongly suggesting the existence of a green light receptor in sunflower leaves. Moreover, acclimation of the abaxial stomata to strong direct light eliminated the photosynthesis-independent component in the green light response. The results showed that stomatal responses to monochromatic light change considerably in response to growth light environment, although some of these responses appear to be determined inherently. © The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  11. What is the influence of ordinary epidermal cells and stomata on the leaf plasticity of coffee plants grown under full-sun and shady conditions?

    PubMed

    Pompelli, M F; Martins, S C V; Celin, E F; Ventrella, M C; Damatta, F M

    2010-11-01

    Stomata are crucial in land plant productivity and survival. In general, with lower irradiance, stomatal and epidermal cell frequency per unit leaf area decreases, whereas guard-cell length or width increases. Nevertheless, the stomatal index is accepted as remaining constant. The aim of this paper to study the influence of ordinary epidermal cells and stomata on leaf plasticity and the influence of these characteristics on stomata density, index, and sizes, in the total number of stomata, as well as the detailed distribution of stomata on a leaf blade. As a result, a highly significant positive correlation (R²(a) = 0.767 p ≤ 0.001) between stomatal index and stomatal density, and with ordinary epidermal cell density (R²(a) = 0.500 p ≤ 0.05), and a highly negative correlation between stomatal index and ordinary epidermal cell area (R²(a) = -0.571 p ≤ 0.001), were obtained. However in no instance was the correlation between stomatal index or stomatal density and stomatal dimensions taken into consideration. The study also indicated that in coffee, the stomatal index was 19.09% in shaded leaves and 20.08% in full-sun leaves. In this sense, variations in the stomatal index by irradiance, its causes and the consequences on plant physiology were discussed.

  12. Brassinosteroid-regulated GSK3/Shaggy-like Kinases Phosphorylate Mitogen-activated Protein (MAP) Kinase Kinases, Which Control Stomata Development in Arabidopsis thaliana*

    PubMed Central

    Khan, Mamoona; Rozhon, Wilfried; Bigeard, Jean; Pflieger, Delphine; Husar, Sigrid; Pitzschke, Andrea; Teige, Markus; Jonak, Claudia; Hirt, Heribert; Poppenberger, Brigitte

    2013-01-01

    Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module. PMID:23341468

  13. A 14,000 year vegetation history of a hypermaritime island on the outer Pacific coast of Canada based on fossil pollen, spores and conifer stomata

    NASA Astrophysics Data System (ADS)

    Lacourse, Terri; Delepine, J. Michelle; Hoffman, Elizabeth H.; Mathewes, Rolf W.

    2012-11-01

    Pollen and conifer stomata analyses of lake sediments from Hippa Island on the north coast of British Columbia were used to reconstruct the vegetation history of this small hypermaritime island. Between 14,000 and 13,230 cal yr BP, the island supported diverse herb-shrub communities dominated by Cyperaceae, Artemisia and Salix. Pinus contorta and Picea sitchensis stomata indicate that these conifers were present among the herb-shrub communities, likely as scattered individuals. Transition to open P. contorta woodland by 13,000 cal yr BP was followed by increases in Alnus viridis, Alnus rubra and P. sitchensis. After 12,000 cal yr BP, Pinus-dominated communities were replaced by dense P. sitchensis and Tsuga heterophylla forest with Lysichiton americanus and fern understory. Thuja plicata stomata indicate that this species was present by 8700 cal yr BP, but the pollen record suggests that its populations did not expand to dominate regional rainforests, along with Tsuga and Picea, until after 6600 cal yr BP. Conifer stomata indicate that species may be locally present for hundreds to thousands of years before pollen exceed thresholds routinely used to infer local species arrival. When combined, pollen and conifer stomata can provide a more accurate record of paleovegetation than either when used alone.

  14. Expansion and Polarity Sorting in Microtubule-Dynein Bundles

    NASA Astrophysics Data System (ADS)

    Zemel, A.; Mogilner, A.

    Interactions of multiple molecular motors with dynamicpolymers, such as actin and microtubules, form the basis for many processes in the cell cytoskeleton. One example is the active `sorting' of microtubule bundles by dynein molecular motors into aster-like arrays of microtubules; in these bundles dynein motors cross-link and slide neighboring microtubules apart. A number of models have been suggested to quantify the active dynamics of cross-linked bundles of polar filaments. In the case of densely packed bundles, however, a major complication arises from the fact that each microtubule interacts with multiple neighboring filaments. To explicitly take these interactions into account we performed detailed computer simulations in which the equations of motion for all microtubules in the bundle were iteratively solved. Our simulations demonstrate the phenomenon of polarity sorting and reveal the variable-rate of the concurrent bundle expansion and its dependence on the nature of the microtubule-motor interactions.

  15. Mathematical modeling of microtubule dynamics: insights into physiology and disease.

    PubMed

    Buxton, Gavin A; Siedlak, Sandra L; Perry, George; Smith, Mark A

    2010-12-01

    Computer models of microtubule dynamics have provided the basis for many of the theories on the cellular mechanics of the microtubules, their polymerization kinetics, and the diffusion of tubulin and tau. In the three-dimensional model presented here, we include the effects of tau concentration and the hydrolysis of GTP-tubulin to GDP-tubulin and observe the emergence of microtubule dynamic instability. This integrated approach simulates the essential physics of microtubule dynamics in a cellular environment. The model captures the structure of the microtubules as they undergo steady state dynamic instabilities in this simplified geometry, and also yields the average number, length, and cap size of the microtubules. The model achieves realistic geometries and simulates cellular structures found in degenerating neurons in disease states such as Alzheimer disease. Further, this model can be used to simulate microtubule changes following the addition of antimitotic drugs which have recently attracted attention as chemotherapeutic agents.

  16. Effects of Tau on Flow-Aligned Microtubule Bundles

    NASA Astrophysics Data System (ADS)

    Ross, Jennifer L.; Kuchnir Fygenson, D.

    2003-03-01

    Microtubules are cylindrical crystals of the protein tubulin with 17nm inner diameter and 25nm outer diameter. Recent structural studies suggest that the microtubule wall may be porous to small molecules. We have investigated the mobility of molecules in bundles of flow aligned microtubules. We find the spacing between the microtubules in the bundle is increased by the addition of tau, a microtubule associated protein. In the absence of tau, flow can be used to make tightly packed bundles of microtubules. Adding tau causes the tight bundles to swell and separate. We use fluorescence recovery after photobleaching (FRAP) to quantify the mobility of a taxol, a small drug that binds to the microtubule interior.

  17. Microtubules search for chromosomes by pivoting around the spindle pole

    NASA Astrophysics Data System (ADS)

    Tolic-Norrelykke, Iva

    2014-03-01

    During cell division, proper segregation of genetic material between the two daughter cells requires that the spindle microtubules attach to the chromosomes via kinetochores, protein complexes on the chromosome. The central question, how microtubules find kinetochores, is still under debate. We observed in fission yeast that kinetochores are captured by microtubules pivoting around the spindle pole body, instead of growing towards the kinetochores. By introducing a theoretical model, we show that the observed angular movement of microtubules is sufficient to explain the process of kinetochore capture. Our theory predicts that the speed of the capture process depends mainly on how fast microtubules pivot. We confirmed this prediction experimentally by speeding up and slowing down microtubule pivoting. Thus, microtubules explore space by pivoting, as they search for intracellular targets such as kinetochores.

  18. Centrosome centering and decentering by microtubule network rearrangement

    PubMed Central

    Letort, Gaëlle; Nedelec, Francois; Blanchoin, Laurent; Théry, Manuel

    2016-01-01

    The centrosome is positioned at the cell center by pushing and pulling forces transmitted by microtubules (MTs). Centrosome decentering is often considered to result from asymmetric, cortical pulling forces exerted in particular by molecular motors on MTs and controlled by external cues affecting the cell cortex locally. Here we used numerical simulations to investigate the possibility that it could equally result from the redistribution of pushing forces due to a reorientation of MTs. We first showed that MT gliding along cell edges and pivoting around the centrosome regulate MT rearrangement and thereby direct the spatial distribution of pushing forces, whereas the number, dynamics, and stiffness of MTs determine the magnitude of these forces. By modulating these parameters, we identified different regimes, involving both pushing and pulling forces, characterized by robust centrosome centering, robust off-centering, or “reactive” positioning. In the last-named conditions, weak asymmetric cues can induce a misbalance of pushing and pulling forces, resulting in an abrupt transition from a centered to an off-centered position. Taken together, these results point to the central role played by the configuration of the MTs on the distribution of pushing forces that position the centrosome. We suggest that asymmetric external cues should not be seen as direct driver of centrosome decentering and cell polarization but instead as inducers of an effective reorganization of the MT network, fostering centrosome motion to the cell periphery. PMID:27440925

  19. Halogenated auxins affect microtubules and root elongation in Lactuca sativa

    NASA Technical Reports Server (NTRS)

    Zhang, N.; Hasenstein, K. H.

    2000-01-01

    We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.

  20. Halogenated auxins affect microtubules and root elongation in Lactuca sativa

    NASA Technical Reports Server (NTRS)

    Zhang, N.; Hasenstein, K. H.

    2000-01-01

    We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.

  1. Centrosome centering and decentering by microtubule network rearrangement.

    PubMed

    Letort, Gaëlle; Nedelec, Francois; Blanchoin, Laurent; Théry, Manuel

    2016-09-15

    The centrosome is positioned at the cell center by pushing and pulling forces transmitted by microtubules (MTs). Centrosome decentering is often considered to result from asymmetric, cortical pulling forces exerted in particular by molecular motors on MTs and controlled by external cues affecting the cell cortex locally. Here we used numerical simulations to investigate the possibility that it could equally result from the redistribution of pushing forces due to a reorientation of MTs. We first showed that MT gliding along cell edges and pivoting around the centrosome regulate MT rearrangement and thereby direct the spatial distribution of pushing forces, whereas the number, dynamics, and stiffness of MTs determine the magnitude of these forces. By modulating these parameters, we identified different regimes, involving both pushing and pulling forces, characterized by robust centrosome centering, robust off-centering, or "reactive" positioning. In the last-named conditions, weak asymmetric cues can induce a misbalance of pushing and pulling forces, resulting in an abrupt transition from a centered to an off-centered position. Taken together, these results point to the central role played by the configuration of the MTs on the distribution of pushing forces that position the centrosome. We suggest that asymmetric external cues should not be seen as direct driver of centrosome decentering and cell polarization but instead as inducers of an effective reorganization of the MT network, fostering centrosome motion to the cell periphery.

  2. Stochastic models for plant microtubule self-organization and structure.

    PubMed

    Eren, Ezgi C; Dixit, Ram; Gautam, Natarajan

    2015-12-01

    One of the key enablers of shape and growth in plant cells is the cortical microtubule (CMT) system, which is a polymer array that forms an appropriately-structured scaffolding in each cell. Plant biologists have shown that stochastic dynamics and simple rules of interactions between CMTs can lead to a coaligned CMT array structure. However, the mechanisms and conditions that cause CMT arrays to become organized are not well understood. It is prohibitively time-consuming to use actual plants to study the effect of various genetic mutations and environmental conditions on CMT self-organization. In fact, even computer simulations with multiple replications are not fast enough due to the spatio-temporal complexity of the system. To redress this shortcoming, we develop analytical models and methods for expeditiously computing CMT system metrics that are related to self-organization and array structure. In particular, we formulate a mean-field model to derive sufficient conditions for the organization to occur. We show that growth-prone dynamics itself is sufficient to lead to organization in presence of interactions in the system. In addition, for such systems, we develop predictive methods for estimation of system metrics such as expected average length and number of CMTs over time, using a stochastic fluid-flow model, transient analysis, and approximation algorithms tailored to our problem. We illustrate the effectiveness of our approach through numerical test instances and discuss biological insights.

  3. Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

    PubMed

    Kenney, Justin W; Genheden, Maja; Moon, Kyung-Mee; Wang, Xuemin; Foster, Leonard J; Proud, Christopher G

    2016-01-01

    Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons.

  4. 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

  5. [Coordination effect between vapor water loss through plant stomata and liquid water supply in soil-plant-atmosphere continuum (SPAC): a review].

    PubMed

    Liu, Li-Min; Qi, Hua; Luo, Xin-Lan; Zhang, Xuan

    2008-09-01

    Some important phenomena and behaviors concerned with the coordination effect between vapor water loss through plant stomata and liquid water supply in SPAC were discussed in this paper. A large amount of research results showed that plants show isohydric behavior when the plant hydraulic and chemical signals cooperate to promote the stomatal regulation of leaf water potential. The feedback response of stomata to the change of environmental humidity could be used to explain the midday depression of stomatal conductance and photosynthesis under drought condition, and also, to interpret the correlation between stomatal conductance and hydraulic conductance. The feed-forward response of stomata to the change of environmental humidity could be used to explain the hysteresis response of stomatal conductance to leaf-atmosphere vapor pressure deficit. The strategy for getting the most of xylem transport requires the rapid stomatal responses to avoid excess cavitation and the corresponding mechanisms for reversal of cavitation in short time.

  6. Saccharin enhances neurite extension by regulating organization of the microtubules.

    PubMed

    Yamashita, Hiroo; Muroi, Yoshikage; Ishii, Toshiaki

    2013-11-06

    In the present study, we found that saccharin, an artificial calorie-free sweetener, promotes neurite extension in the cultured neuronal cells. The purposes of this study are to characterize the effect of saccharine on neurite extension and to determine how saccharin enhances neurite extension. The analyses were performed using mouse neuroblastoma N1E-115 cells and rat pheochromocytoma PC12 cells. Neurite extension was evaluated by counting the cells bearing neurites and measuring the length of neurites. Formation, severing and transportation of the microtubules were evaluated by immunostaining and western blotting analysis. Deprivation of glucose increased the number of N1E-115 cells bearing long processes. And the effect was inhibited by addition of glucose. Saccharin increased the number of these cells bearing long processes in a dose-dependent manner and total neurite length and longest neurite length in each cell. Saccharin also had a similar effect on NGF-treated PC12 cells. Saccharin increased the amount of the microtubules reconstructed after treatment with nocodazole, a disruptor of microtubules. The effect of saccharin on microtubule reconstruction was not influenced by dihydrocytochalasin B, an inhibitor of actin polymerization, indicating that saccharin enhances microtubule formation without requiring actin dynamics. In the cells treated with vinblastine, an inhibitor of microtubule polymerization, after microtubule reorganization, filamentous microtubules were observed more distantly from the centrosome in saccharin-treated cells, indicating that saccharin enhances microtubule severing and/or transportation. These results suggest that saccharin enhances neurite extension by promoting microtubule organization. © 2013.

  7. Molecular architecture of the Dam1 complex–microtubule interaction

    PubMed Central

    Legal, Thibault; Zou, Juan; Sochaj, Alicja; Rappsilber, Juri

    2016-01-01

    Mitosis is a highly regulated process that allows the equal distribution of the genetic material to the daughter cells. Chromosome segregation requires the formation of a bipolar mitotic spindle and assembly of a multi-protein structure termed the kinetochore to mediate attachments between condensed chromosomes and spindle microtubules. In budding yeast, a single microtubule attaches to each kinetochore, necessitating robustness and processivity of this kinetochore–microtubule attachment. The yeast kinetochore-localized Dam1 complex forms a direct interaction with the spindle microtubule. In vitro, the Dam1 complex assembles as a ring around microtubules and couples microtubule depolymerization with cargo movement. However, the subunit organization within the Dam1 complex, its higher-order oligomerization and how it interacts with microtubules remain under debate. Here, we used chemical cross-linking and mass spectrometry to define the architecture and subunit organization of the Dam1 complex. This work reveals that both the C termini of Duo1 and Dam1 subunits interact with the microtubule and are critical for microtubule binding of the Dam1 complex, placing Duo1 and Dam1 on the inside of the ring structure. Integrating this information with available structural data, we provide a coherent model for how the Dam1 complex self-assembles around microtubules. PMID:26962051

  8. Neurodegeneration and microtubule dynamics: death by a thousand cuts

    PubMed Central

    Dubey, Jyoti; Ratnakaran, Neena; Koushika, Sandhya P.

    2015-01-01

    Microtubules form important cytoskeletal structures that play a role in establishing and maintaining neuronal polarity, regulating neuronal morphology, transporting cargo, and scaffolding signaling molecules to form signaling hubs. Within a neuronal cell, microtubules are found to have variable lengths and can be both stable and dynamic. Microtubule associated proteins, post-translational modifications of tubulin subunits, microtubule severing enzymes, and signaling molecules are all known to influence both stable and dynamic pools of microtubules. Microtubule dynamics, the process of interconversion between stable and dynamic pools, and the proportions of these two pools have the potential to influence a wide variety of cellular processes. Reduced microtubule stability has been observed in several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), and tauopathies like Progressive Supranuclear Palsy. Hyperstable microtubules, as seen in Hereditary Spastic Paraplegia (HSP), also lead to neurodegeneration. Therefore, the ratio of stable and dynamic microtubules is likely to be important for neuronal function and perturbation in microtubule dynamics might contribute to disease progression. PMID:26441521

  9. Microtubule Stabilization Leads to Growth Reorientation in Arabidopsis Trichomes

    PubMed Central

    Mathur, Jaideep; Chua, Nam-Hai

    2000-01-01

    The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein–microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions. PMID:10760237

  10. Microtubules Growth Rate Alteration in Human Endothelial Cells

    PubMed Central

    Alieva, Irina B.; Zemskov, Evgeny A.; Kireev, Igor I.; Gorshkov, Boris A.; Wiseman, Dean A.; Black, Stephen M.; Verin, Alexander D.

    2010-01-01

    To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC) and “fast” (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules. PMID:20445745

  11. Microtubules growth rate alteration in human endothelial cells.

    PubMed

    Alieva, Irina B; Zemskov, Evgeny A; Kireev, Igor I; Gorshkov, Boris A; Wiseman, Dean A; Black, Stephen M; Verin, Alexander D

    2010-01-01

    To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with "normal" (similar to those in monolayer EC) and "fast" (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.

  12. Tubulin acetylation protects long-lived microtubules against mechanical ageing.

    PubMed

    Portran, Didier; Schaedel, Laura; Xu, Zhenjie; Théry, Manuel; Nachury, Maxence V

    2017-04-01

    Long-lived microtubules endow the eukaryotic cell with long-range transport abilities. While long-lived microtubules are acetylated on Lys40 of α-tubulin (αK40), acetylation takes place after stabilization and does not protect against depolymerization. Instead, αK40 acetylation has been proposed to mechanically stabilize microtubules. Yet how modification of αK40, a residue exposed to the microtubule lumen and inaccessible to microtubule-associated proteins and motors, could affect microtubule mechanics remains an open question. Here we develop FRET-based assays that report on the lateral interactions between protofilaments and find that αK40 acetylation directly weakens inter-protofilament interactions. Congruently, αK40 acetylation affects two processes largely governed by inter-protofilament interactions, reducing the nucleation frequency and accelerating the shrinkage rate. Most relevant to the biological function of acetylation, microfluidics manipulations demonstrate that αK40 acetylation enhances flexibility and confers resilience against repeated mechanical stresses. Thus, unlike deacetylated microtubules that accumulate damage when subjected to repeated stresses, long-lived microtubules are protected from mechanical ageing through their acquisition of αK40 acetylation. In contrast to other tubulin post-translational modifications that act through microtubule-associated proteins, motors and severing enzymes, intraluminal acetylation directly tunes the compliance and resilience of microtubules.

  13. 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

  14. CSPP and CSPP-L associate with centrosomes and microtubules and differently affect microtubule organization.

    PubMed

    Patzke, Sebastian; Stokke, Trond; Aasheim, Hans-Christian

    2006-10-01

    We recently described the identification of a centrosome/spindle pole associated protein, CSPP, involved in cell cycle progression. Here we report a CSPP isoform denoted CSPP-L, with a 294 amino acids longer N-terminus and a 51 amino acids insertion located in the coiled-coil mid-domain. Expression analysis indicates an inverse cell cycle dependent regulation. CSPP mRNA expression is highest in G1 whereas CSPP-L expression is highest in G2/M. Ectopic expression of CSPP-L impairs cell cycle progression weaker in G1 than CSPP. Furthermore, normal mitotic phenotypes were observed in CSPP-L but not in CSPP transfectants. CSPP-L relocates from spindle microtubules and poles in metaphase to the mid-spindle in anaphase and concentrates at the mid-body in telophase/cytokinesis. CSPP-L high-expressing mitotic cells were predominantly characterized by lagging chromosomes or monopolar spindles, in contrast to the predominant multipolar spindles observed with CSPP expression. The different effects of CSPP and CSPP-L on microtubule organization in mitosis depend on the coiled-coil mid-domain insertion. The common C-terminal domain is required to repress that activity until mitosis. Notably, this C-terminal domain alone can associate with centrosomes in a microtubule independent manner. Taken together, CSPP and CSPP-L interact with centrosomes and microtubules and can differently affect microtubule organization. Copyright 2006 Wiley-Liss, Inc.

  15. Dynamic Concentration of Motors in Microtubule Arrays

    NASA Astrophysics Data System (ADS)

    Nédélec, François; Surrey, Thomas; Maggs, A. C.

    2001-04-01

    We present experimental and theoretical studies of the dynamics of molecular motors in microtubule arrays and asters. By solving a convection-diffusion equation we find that the density profile of motors in a two-dimensional aster is characterized by continuously varying exponents. Simulations are used to verify the assumptions of the continuum model. We observe the concentration profiles of kinesin moving in quasi-two-dimensional artificial asters by fluorescent microscopy and compare with our theoretical results.

  16. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2012-02-15

    radio wave to the proteins in a heat bath. Experiment: We felt three years back in 2008, that it was not sufficient just to try to build the...solution individually or as a combination, produced microtubules were isolated for single nanowire measurement of resonance levels. Ultrasound power...varied between pico-watt to femto-watt, if Mg2+ is not added, effect of ultrasound pumping is not observed, so Mg2+ is essential, GTP is not. Phase

  17. Microtubule Actin Cross-Linking Factor (Macf)

    PubMed Central

    Leung, Conrad L.; Sun, Dongming; Zheng, Min; Knowles, David R.; Liem, Ronald K.H.

    1999-01-01

    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 NH2 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 NH2-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. PMID:10601340

  18. Molecular Communication: Simulation of Microtubule Topology

    NASA Astrophysics Data System (ADS)

    Moore, Michael J.; Enomoto, Akihiro; Nakano, Tadashi; Kayasuga, Atsushi; Kojima, Hiroaki; Sakakibara, Hitoshi; Oiwa, Kazuhiro; Suda, Tatsuya

    Molecular communication is one method for communication among biological nanomachines. Nanomachines are artificial or biological nano-scale devices that perform simple computation, sensing, or actuation. Future applications using nanomachines may require various communication mechanisms. For example, broadcast is one primitive communication for transmission from one sender to many receivers. In this paper, we discuss preliminary work on designing a molecular communication system that is adapted from the molecular motor transport mechanism existing in biological cells. In the proposed molecular motor mechanism, a sender releases information molecules, and molecular motors transport the information molecules along microtubule filaments to receiver nanomachines up to hundreds of micrometers away. This paper describes some possible arrangements for microtubule filaments and simulations to evaluate sending of one information molecule to many receivers. The simulation results indicate that the proposed molecular motor system transports simulated information molecules (100nm radius spheres) more quickly than a diffusion-only communication and that placement of receivers at the plus-end of microtubules results in lower propagation delay.

  19. Mechanics of microtubules and viral capsids

    NASA Astrophysics Data System (ADS)

    Schmidt, Christoph F.

    2004-03-01

    Polymeric macromolecular assemblies play crucial roles in biology, from DNA to the cytoskeleton or the cell membrane. I will report on recent measurements of the elastic properties of two types of 2D-crystalline protein shells which we have probed at the nanometer scale by indentation with a scanning force microscope (SFM) tip. Microtubules are cylindrical shells and we find a linear elastic regime that can be described by both thin-shell theory and finite element methods, in which microtubules are modeled as homogeneous hollow tubes. We also find a non-linear regime and catastrophic collapse of the microtubules under large loads. The main physics of protein shells at the nanometer scale shows simultaneously aspects of continuum elasticity in their linear response, as well as molecular graininess in their non-linear behavior. Bacteriophages use highly ordered proteinaceous shells to protect their genome from the environment and, interestingly, also to store elastic energy for the injection process. We have studied empty and filled bacteriophage Phi29 shells, again by SFM indentation. These shells are approximately ellipsoidal. We again find a regime of linear elastic response, followed by non-linear response and break-down. The linear regime can again be described by thin shell theory, assuming a homogeneous material, but we observe, already in the linear regime, signatures of the substructure of the shells.

  20. Self-organization of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Ndlec, F. J.; Surrey, T.; Maggs, A. C.; Leibler, S.

    1997-09-01

    Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly `pathways'.

  1. Microtubule self-organisation depends upon gravity

    NASA Astrophysics Data System (ADS)

    Tabony, J.; Pochon, N.; Papaseit, C.

    2001-01-01

    The molecular processes by which gravity is transduced into biological systems are poorly, if at all, understood. Under equilibrium conditions, chemical and biochemical structures do not depend upon gravity. It has been proposed that biological systems might show a gravity dependence by way of the bifurcation properties of certain types of non-linear chemical reactions that are far-from-equilibrium. We have found that in-vitro preparations of microtubules, an important element of the cellular cytoskeleton, show this type of behaviour. On earth, the solutions show macroscopic self-ordering, and the morphology of the structures that form depend upon the orientation of the sample with respect to gravity at a critical moment at an early stage in the development of the self-organised state. An experiment carried out in a sounding rocket, showed that as predicted by theories of this type, no self-organisation occurs when the microtubules are assembled under low gravity conditions. This is an experimental demonstration of how a very simple biochemical system, containing only two molecules, can be gravity sensitive. At a molecular level this behaviour results from an interaction of gravity with macroscopic concentration and density fluctuations that arise from the processes of microtubule contraction and elongation.

  2. Validation of microtubule-associated Tobacco mosaic virus RNA movement and involvement of microtubule-aligned particle trafficking.

    PubMed

    Boyko, Vitaly; Hu, Quanan; Seemanpillai, Mark; Ashby, Jamie; Heinlein, Manfred

    2007-08-01

    Functional studies of Tobacco mosaic virus (TMV) infection using virus derivatives expressing functional, dysfunctional, and temperature-sensitive movement protein (MP) mutants indicated that the cell-to-cell transport of TMV RNA is functionally correlated with the association of MP with microtubules. However, the role of microtubules in the movement process during early infection remains unclear, since MP accumulates on microtubules rather late in infection and treatment of plants with microtubule-disrupting agents fails to strongly interfere with cell-to-cell movement of TMV RNA. To further test the role of microtubules in TMV cell-to-cell movement, we investigated TMV strain Ni2519, which is temperature-sensitive for movement. We demonstrate that the temperature-sensitive defect in movement is correlated with temperature-sensitive changes in the localization of MP to microtubules. Furthermore, we show that during early phases of recovery from non-permissive conditions, the MP localizes to microtubule-associated particles. Similar particles are found in cells at the leading front of spreading TMV infection sites. Initially mobile, the particles become immobile when MP starts to accumulate along the length of the particle-associated microtubules. Our observations confirm a role for microtubules in the spread of TMV infection and associate this role with microtubule-associated trafficking of MP-containing particles in cells engaged in the cell-to-cell movement of the TMV genome.

  3. A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei

    PubMed Central

    1992-01-01

    The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule- associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins. PMID:1348252

  4. A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei.

    PubMed

    Hemphill, A; Affolter, M; Seebeck, T

    1992-04-01

    The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule-associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins.

  5. Stomata are less responsive to environmental stimuli in high background ozone in Dactylis glomerata and Ranunculus acris.

    PubMed

    Wagg, Serena; Mills, Gina; Hayes, Felicity; Wilkinson, Sally; Davies, William J

    2013-04-01

    Two mesotrophic grassland species, Ranunculus acris and Dactylis glomerata were exposed to a range of ozone treatments (16.2-89.5 ppb 24 h mean) and two watering regimes under naturally fluctuating photosynthetically active radiation (PAR), vapour pressure deficit (VPD) and temperature. Stomatal conductance was measured throughout the experiments, and the combined data set (>1000 measurements) was analysed for effects of low and high ozone on responses to environmental stimuli. We show that when D. glomerata and R. acris were grown in 72.6-89.5 ppb ozone the stomata consistently lose the ability to respond, or have reduced response, to naturally fluctuating environmental conditions in comparison to their response in low ozone. The maximum stomatal conductance (g(max)) was also significantly higher in the high ozone treatment for D. glomerata. We discuss the hypotheses for the reduced sensitivity of stomatal closure to a changing environment and the associated implications for ozone flux modelling.

  6. Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud

    PubMed Central

    Shaw, Sidney L.; Yeh, Elaine; Maddox, Paul; Salmon, E.D.; Bloom, Kerry

    1997-01-01

    Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at ≅0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud. PMID:9362516

  7. Co-ordination of physiological and morphological responses of stomata to elevated [CO2] in vascular plants.

    PubMed

    Haworth, Matthew; Elliott-Kingston, Caroline; McElwain, Jennifer C

    2013-01-01

    Plant stomata display a wide range of short-term behavioural and long-term morphological responses to atmospheric carbon dioxide concentration ([CO(2)]). The diversity of responses suggests that plants may have different strategies for controlling gas exchange, yet it is not known whether these strategies are co-ordinated in some way. Here, we test the hypothesis that there is co-ordination of physiological (via aperture change) and morphological (via stomatal density change) control of gas exchange by plants. We examined the response of stomatal conductance (G(s)) to instantaneous changes in external [CO(2)] (C(a)) in an evolutionary cross-section of vascular plants grown in atmospheres of elevated [CO(2)] (1,500 ppm) and sub-ambient [O(2)] (13.0 %) compared to control conditions (380 ppm CO(2), 20.9 % O(2)). We found that active control of stomatal aperture to [CO(2)] above current ambient levels was not restricted to angiosperms, occurring in the gymnosperms Lepidozamia peroffskyana and Nageia nagi. The angiosperm species analysed appeared to possess a greater respiratory demand for stomatal movement than gymnosperm species displaying active stomatal control. Those species with little or no control of stomatal aperture (termed passive) to C(a) were more likely to exhibit a reduction in stomatal density than species with active stomatal control when grown in atmospheres of elevated [CO(2)]. The relationship between the degree of stomatal aperture control to C(a) above ambient and the extent of any reduction in stomatal density may suggest the co-ordination of physiological and morphological responses of stomata to [CO(2)] in the optimisation of water use efficiency. This trade-off between stomatal control strategies may have developed due to selective pressures exerted by the costs associated with passive and active stomatal control.

  8. RabGAP22 Is Required for Defense to the Vascular Pathogen Verticillium longisporum and Contributes to Stomata Immunity

    PubMed Central

    Roos, Jonas; Bejai, Sarosh; Oide, Shinichi; Dixelius, Christina

    2014-01-01

    Verticillium longisporum is a soil-borne pathogen with a preference for plants within the family Brassicaceae. Following invasion of the roots, the fungus proliferates in the plant vascular system leading to stunted plant growth, chlorosis and premature senescence. RabGTPases have been demonstrated to play a crucial role in regulating multiple responses in plants. Here, we report on the identification and characterization of the Rab GTPase-activating protein RabGAP22 gene from Arabidopsis, as an activator of multiple components in the immune responses to V. longisporum. RabGAP22Pro:GUS transgenic lines showed GUS expression predominantly in root meristems, vascular tissues and stomata, whereas the RabGAP22 protein localized in the nucleus. Reduced RabGAP22 transcript levels in mutants of the brassinolide (BL) signaling gene BRI1-ASSOCIATED RECEPTOR KINASE 1, together with a reduction of fungal proliferation following BL pretreatment, suggested RabGAP22 to be involved in BL-mediated responses. Pull-down assays revealed SERINE:GLYOXYLATE AMINOTRANSFERASE (AGT1) as an interacting partner during V. longisporum infection and bimolecular fluorescence complementation (BiFC) showed the RabGAP22-AGT1 protein complex to be localized in the peroxisomes. Further, fungal-induced RabGAP22 expression was found to be associated with elevated endogenous levels of the plant hormones jasmonic acid (JA) and abscisic acid (ABA). An inadequate ABA response in rabgap22-1 mutants, coupled with a stomata-localized expression of RabGAP22 and impairment of guard cell closure in response to V. longisporum and Pseudomonas syringae, suggest that RabGAP22 has multiple roles in innate immunity. PMID:24505423

  9. Ecological distribution of leaf stomata and trichomes among tree species in a Malaysian lowland tropical rain forest.

    PubMed

    Ichie, Tomoaki; Inoue, Yuta; Takahashi, Narumi; Kamiya, Koichi; Kenzo, Tanaka

    2016-07-01

    The vertical structure of a tropical rain forest is complex and multilayered, with strong variation of micro-environment with height up to the canopy. We investigated the relation between morphological traits of leaf surfaces and tree ecological characteristics in a Malaysian tropical rain forest. The shapes and densities of stomata and trichomes on the abaxial leaf surfaces and their relation with leaf characteristics such as leaf area and leaf mass per area (LMA) were studied in 136 tree species in 35 families with different growth forms in the tropical moist forest. Leaf physiological properties were also measured in 50 canopy and emergent species. Most tree species had flat type (40.4 %) or mound type (39.7 %) stomata. In addition, 84 species (61.76 %) in 22 families had trichomes, including those with glandular (17.65 %) and non-glandular trichomes (44.11 %). Most leaf characteristics significantly varied among the growth form types: species in canopy and emergent layers and canopy gap conditions had higher stomatal density, stomatal pore index (SPI), trichome density and LMA than species in understory and subcanopy layers, though the relation of phylogenetically independent contrasts to each characteristic was not statistically significant, except for leaf stomatal density, SPI and LMA. Intrinsic water use efficiency in canopy and emergent tree species with higher trichome densities was greater than in species with lower trichome densities. These results suggest that tree species in tropical rain forests adapt to a spatial difference in their growth forms, which are considerably affected by phylogenetic context, by having different stomatal and trichome shapes and/or densities.

  10. Differences in the way potassium chloride and sucrose solutions effect osmotic potential of significance to stomata aperture modulation.

    PubMed

    Cochrane, Thomas T; Cochrane, Thomas A

    2009-03-01

    Guard cell solution osmotic potential changes resulting in the opening and closing of stomata apertures follow an initial influx of potassium ions, their substitution with sucrose molecules and the subsequent reduction of the latter. To provide an insight into the osmotic mechanism of the changes, the new equation for calculating osmotic pressure, which equates the difference between the energy of pure water across a semi-permeable membrane interface with that of solution water, was used to compare the osmotic properties of KCl and sucrose. For sucrose solutions, the effect of the sucrose molecules in increasing the spacing of the solution water was mainly responsible for osmotic potential; this contrasted with K+ + Cl(-) ions where their spacing effect was only a little higher to that of water held to those ions. At solute concentrations giving an osmotic potential level of -3.0 MPa near that of turgid guard cells, the spacing effect on the potential of the unattached solution water molecules caused by sucrose, but in its theoretical absence, was estimated as -2.203 MPa compared with -1.431 MPa for KCl. In contrast, the potential attributed to water molecules firmly held to the K+ + Cl(-) ions was -1.212 MPa versus zero for sucrose. The potential to keep the sucrose molecules in solution was -0.797 MPa compared with -0.357 MPa for KCl. The findings illustrate that the way KCl effects osmotic pressure is very different to that of sucrose. It is concluded that stomata aperture modulation is closely linked to the osmotic properties of its guard cell solution solutes.

  11. Stomata-controlled nighttime COS fluxes in a boreal forest: implications for the use of COS as a GPP tracer

    NASA Astrophysics Data System (ADS)

    Kooijmans, Linda M. J.; Maseyk, Kadmiel; Seibt, Ulli; Vesala, Timo; Mammarella, Ivan; Baker, Ian T.; Franchin, Alessandro; Kolari, Pasi; Sun, Wu; Keskinen, Helmi; Levula, Janne; Chen, Huilin

    2016-04-01

    Carbonyl Sulfide (COS) is a promising new tracer that can be used to partition the Net Ecosystem Exchange into gross primary production (GPP) and respiration. COS and CO2 vegetation fluxes are closely related as these gases share the same diffusion pathway into stomata. This close coupling is the fundamental principle for the use of COS as tracer for GPP. Nonetheless, in contrast to CO2 , the uptake of COS by vegetation is not light-dependent, and therefore the vegetative uptake of COS can continue during the night as long as stomata are open. Nighttime stomatal conductance is observed in a variety of studies, and also nighttime depletion of COS concentrations is reported several times but it is not confirmed with field measurements that the depletion of COS in the night is indeed driven by stomatal opening. In the summer of 2015 a campaign took place at the SMEAR II site in Hyytiälä, Finland to provide better constrained COS flux data for boreal forests using a combination of COS measurements, i.e. atmospheric profile concentrations up to 125 m, eddy-covariance fluxes and soil chamber fluxes, and collocated measurements of stomatal conductance and 222Radon. A high correlation between concentrations of 222Radon and COS implies that the radon-tracer method is a valuable tool to derive nighttime ecosystem COS fluxes. We find that soils contribute to 17% of the total ecosystem COS flux during nighttime in the peak growing season. Nighttime ecosystem COS fluxes show a correlation with stomatal conductance (R2 = 0.3), indicating that nighttime COS fluxes are primarily driven by vegetation. The COS vegetation fluxes will be compared with calculated fluxes from the Simple Biosphere model. Furthermore, the nighttime vegetative COS uptake covers a substantial fraction (25%) of the daily maximum COS uptake by vegetation. Accurate quantification of nighttime COS uptake is required to be able to use COS as a useful tracer for GPP.

  12. Cellular Samurai: katanin and the severing of microtubules.

    PubMed

    Quarmby, L

    2000-08-01

    Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

  13. 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

  14. 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.

  15. Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro

    SciTech Connect

    Walker, R.A.; Inoue, S.; Salmon, E.D.

    1989-03-01

    The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a GTP cap. A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of stabilizing cap, possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

  16. The cortical cytoskeletal network and cell-wall dynamics in the unicellular charophycean green alga Penium margaritaceum

    PubMed Central

    Ochs, Julie; LaRue, Therese; Tinaz, Berke; Yongue, Camille; Domozych, David S.

    2014-01-01

    Background and Aims Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division. Methods Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy. Key Results The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division. Conclusions The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre

  17. The cortical cytoskeletal network and cell-wall dynamics in the unicellular charophycean green alga Penium margaritaceum.

    PubMed

    Ochs, Julie; LaRue, Therese; Tinaz, Berke; Yongue, Camille; Domozych, David S

    2014-10-01

    Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division. Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy. The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division. The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre-division wall expansion and the zone where a cross

  18. Cortical granule translocation is microfilament mediated and linked to meiotic maturation in the sea urchin oocyte.

    PubMed

    Wessel, Gary M; Conner, Sean D; Berg, Linnea

    2002-09-01

    Cortical granules exocytose after the fusion of egg and sperm in most animals, and their contents function in the block to polyspermy by creating an impenetrable extracellular matrix. Cortical granules are synthesized throughout oogenesis and translocate en masse to the cell surface during meiosis where they remain until fertilization. As the mature oocyte is approximately 125 micro m in diameter (Lytechinus variegatus), many of the cortical granules translocate upwards of 60 micro m to reach the cortex within a 4 hour time window. We have investigated the mechanism of this coordinated vesicular translocation event. Although the stimulus to reinitiate meiosis in sea urchin oocytes is not known, we found many different ways to reversibly inhibit germinal vesicle breakdown, and used these findings to discover that meiotic maturation and cortical granule translocation are inseparable. We also learned that cortical granule translocation requires association with microfilaments but not microtubules. It is clear from endocytosis assays that microfilament motors are functional prior to meiosis, even though cortical granules do not use them. However, just after GVBD, cortical granules attach to microfilaments and translocate to the cell surface. This latter conclusion is based on organelle stratification within the oocyte followed by positional quantitation of the cortical granules. We conclude from these studies that maturation promoting factor (MPF) activation stimulates vesicle association with microfilaments, and is a key regulatory step in the coordinated translocation of cortical granules to the egg cortex.

  19. A genetic screen for suppressors and enhancers of the Drosophila cdk1-cyclin B identifies maternal factors that regulate microtubule and microfilament stability.

    PubMed Central

    Ji, Jun-Yuan; Haghnia, Marjan; Trusty, Cory; Goldstein, Lawrence S B; Schubiger, Gerold

    2002-01-01

    Coordination between cell-cycle progression and cytoskeletal dynamics is important for faithful transmission of genetic information. In early Drosophila embryos, increasing maternal cyclin B leads to higher Cdk1-CycB activity, shorter microtubules, and slower nuclear movement during cycles 5-7 and delays in nuclear migration to the cortex at cycle 10. Later during cycle 14 interphase of six cycB embryos, we observed patches of mitotic nuclei, chromosome bridges, abnormal nuclear distribution, and small and large nuclei. These phenotypes indicate disrupted coordination between the cell-cycle machinery and cytoskeletal function. Using these sensitized phenotypes, we performed a dosage-sensitive genetic screen to identify maternal proteins involved in this process. We identified 10 suppressors classified into three groups: (1) gene products regulating Cdk1 activities, cdk1 and cyclin A; (2) gene products interacting with both microtubules and microfilaments, Actin-related protein 87C; and (3) gene products interacting with microfilaments, chickadee, diaphanous, Cdc42, quail, spaghetti-squash, zipper, and scrambled. Interestingly, most of the suppressors that rescue the astral microtubule phenotype also reduce Cdk1-CycB activities and are microfilament-related genes. This suggests that the major mechanism of suppression relies on the interactions among Cdk1-CycB, microtubule, and microfilament networks. Our results indicate that the balance among these different components is vital for normal early cell cycles and for embryonic development. Our observations also indicate that microtubules and cortical microfilaments antagonize each other during the preblastoderm stage. PMID:12454065

  20. Theoretical Description of Microtubule Dynamics in Fission Yeast During Interphase

    NASA Astrophysics Data System (ADS)

    Oei, Yung-Chin; Jiménez-Dalmaroni, Andrea; Vilfan, Andrej; Duke, Thomas

    2009-03-01

    Fission yeast (S. pombe) is a unicellular organism with a characteristic cylindrical shape. Cell growth during interphase is strongly influenced by microtubule self-organization - a process that has been experimentally well characterised. The microtubules are organized in 3 to 4 bundles, called ``interphase microtubule assemblies'' (IMAs). Each IMA is composed of several microtubules, arranged with their dynamic ``plus'' ends facing the cell tips and their ``minus'' ends overlapping at the cell middle. Although the main protein factors involved in interphase microtubule organization have been identified, an understanding of how their collective interaction with microtubules leads to the organization and structures observed in vivo is lacking. We present a physical model of microtubule dynamics that aims to provide a quantitative description of the self-organization process. First, we solve equations for the microtubule length distribution in steady-state, taking into account the way that a limited tubulin pool affects the nucleation, growth and shrinkage of microtubules. Then we incorporate passive and active crosslinkers (the bundling factor Ase1 and molecular motor Klp2) and investigate the formation of IMA structures. Analytical results are complemented by a 3D stochastic simulation.

  1. Microtubules Modulate F-actin Dynamics during Neuronal Polarization.

    PubMed

    Zhao, Bing; Meka, Durga Praveen; Scharrenberg, Robin; König, Theresa; Schwanke, Birgit; Kobler, Oliver; Windhorst, Sabine; Kreutz, Michael R; Mi