Asymmetries in kinesin-2 and cytoplasmic dynein contributions to melanosome transport.

PubMed

De Rossi, María Cecilia; De Rossi, María Emilia; Sued, Mariela; Rodríguez, Daniela; Bruno, Luciana; Levi, Valeria

2015-09-14

The mechanisms involved in bidirectional transport along microtubules remain largely unknown. We explored the collective action of kinesin-2 and dynein motors during transport of melanosomes in Xenopus laevis melanophores. These motors are attached to organelles through accessory proteins establishing a complex molecular linker. We determined both the stiffness of this linker and the organelles speed and observed that these parameters depended on the organelle size and cargo direction. Our results suggest that melanosome transport is driven by two dissimilar teams: whereas dynein motors compete with kinesin-2 affecting the properties of plus-end directed organelles, kinesin-2 does not seem to play a similar role during minus-end transport. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation*

PubMed Central

Neumayer, Gernot; Helfricht, Angela; Shim, Su Yeon; Le, Hoa Thi; Lundin, Cecilia; Belzil, Camille; Chansard, Mathieu; Yu, Yaping; Lees-Miller, Susan P.; Gruss, Oliver J.; van Attikum, Haico; Helleday, Thomas; Nguyen, Minh Dang

2012-01-01

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G0 and G1 phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage. PMID:23045526

MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus

PubMed Central

Vacca, Barbara; Sanchez-Heras, Elena; Steed, Emily; Balda, Maria S.; Ohnuma, Shin-Ichi; Sasai, Noriaki; Mayor, Roberto

2016-01-01

ABSTRACT Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus. MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis. PMID:27870636

MCM interference during licensing of DNA replication in Xenopus egg extracts-Possible Role of a C-terminal region of MCM3.

PubMed

Mimura, Satoru; Kubota, Yumiko; Takisawa, Haruhiko

2018-01-01

The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.

Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2.

PubMed

Ruane, Peter T; Gumy, Laura F; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J; Hoogenraad, Casper C; Allan, Victoria J

2016-06-07

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons.

Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2

PubMed Central

Ruane, Peter T.; Gumy, Laura F.; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J.; Hoogenraad, Casper C.; Allan, Victoria J.

2016-01-01

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons. PMID:27272132

A Chimeric Kinesin-1 Head/Kinesin-5 Tail Motor Switches between Diffusive and Processive Motility

PubMed Central

Thiede, Christina; Lakämper, Stefan; Wessel, Alok D.; Kramer, Stefanie; Schmidt, Christoph F.

2013-01-01

Homotetrameric kinesin-5 motors are essential for chromosome separation and assembly of the mitotic spindle. These kinesins bind between two microtubules (MTs) and slide them apart, toward the spindle poles. This process must be tightly regulated in mitosis. In in vitro assays, Eg5 moves diffusively on single MTs and switches to a directed mode between MTs. How allosteric communication between opposing motor domains works remains unclear, but kinesin-5 tail domains may be involved. Here we present a single-molecule fluorescence study of a tetrameric kinesin-1 head/kinesin-5 tail chimera, DK4mer. This motor exhibited fast processive motility on single MTs interrupted by pauses. Like Eg5, DK4mer diffused along MTs with ADP, and slid antiparallel MTs apart with ATP. In contrast to Eg5, diffusive and processive periods were clearly distinguishable. This allowed us to measure transition rates among states and for unbinding as a function of buffer ionic strength. These data, together with results from controls using tail-less dimers, indicate that there are two modes of interaction with MTs, separated by an energy barrier. This result suggests a scheme of motor regulation that involves switching between two bound states, possibly allosterically controlled by the opposing tetramer end. Such a scheme is likely to be relevant for the regulation of native kinesin-5 motors. PMID:23442865

Detection of the quantity of kinesin and microgravity-sensitive kinesin genes in rat bone marrow stromal cells grown in a simulated microgravity environment

NASA Astrophysics Data System (ADS)

Ni, Chengzhi; Wang, Chunyan; Li, Yuan; Li, Yinghui; Dai, Zhongquan; Zhao, Dongming; Sun, Hongyi; Wu, Bin

2011-06-01

Kinesin and kinesin-like proteins (KLPs) constitute a superfamily of microtubule motor proteins found in all eukaryotic organisms. Members of the kinesin superfamily are known to play important roles in many fundamental cellular and developmental processes. To date, few published studies have reported on the effects of microgravity on kinesin expression. In this paper, we describe the expression pattern and microgravity-sensitive genes of kinesin in rat bone marrow stromal cells cultured in a ground-based rotating bioreactor. The quantity of kinesin under the clinorotation condition was examined by immunoblot analysis with anti-kinesin. Furthermore, the distribution of kinesin at various times during clinorotation was determined by dual immunostaining, using anti-kinesin monoclonal antibody or anti-β-tubulin monoclonal antibody. In terms of kinesin quantity, we found that the ratios of the amounts of clinorotated/stationary KLPs decreased from clinorotation day 5 to day 10, although it increased on days 2 and 3. Immunofluorescence analysis revealed that kinesin in the nucleus was the first to be affected by simulated microgravity, following the kinesin at the periphery that was affected at various times during clinorotation. Real-time RT-PCR analysis of kinesin mRNA expression was performed and led to the identification of 3 microgravity-sensitive kinesin genes: KIF9, KIFC1, and KIF21A. Our results suggest that kinesin has a distinct expression pattern, and the identification of microgravity-sensitive kinesin genes offers insight into fundamental cell biology.

p120 catenin associates with kinesin and facilitates the transport of cadherin–catenin complexes to intercellular junctions

PubMed Central

Chen, Xinyu; Kojima, Shin-ichiro; Borisy, Gary G.; Green, Kathleen J.

2003-01-01

p120 catenin (p120) is a component of adherens junctions and has been implicated in regulating cadherin-based cell adhesion as well as the activity of Rho small GTPases, but its exact roles in cell–cell adhesion are unclear. Using time-lapse imaging, we show that p120-GFP associates with vesicles and exhibits unidirectional movements along microtubules. Furthermore, p120 forms a complex with kinesin heavy chain through the p120 NH2-terminal head domain. Overexpression of p120, but not an NH2-terminal deletion mutant deficient in kinesin binding, recruits endogenous kinesin to N-cadherin. Disruption of the interaction between N-cadherin and p120, or the interaction between p120 and kinesin, leads to a delayed accumulation of N-cadherin at cell–cell contacts during calcium-initiated junction reassembly. Our analyses identify a novel role of p120 in promoting cell surface trafficking of cadherins via association and recruitment of kinesin. PMID:14610057

Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

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

Morin, R D; Chang, E; Petrescu, A

2005-10-31

Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence betweenmore » the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.« less

Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites

PubMed Central

Babic, Milos; Russo, Gary J.; Wellington, Andrea J.; Sangston, Ryan M.; Gonzalez, Migdalia

2015-01-01

Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, we show that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, our analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state. PMID:25855186

Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesin-dependent microtubule motility

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

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

2007-01-01

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

Neck linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors.

PubMed

Shastry, Shankar; Hancock, William O

2010-05-25

Defining the mechanical and biochemical determinates of kinesin processivity is important for understanding how diverse kinesins are tuned for specific cellular functions. Because transmission of mechanical forces through the 14-18 amino acid neck linker domain underlies coordinated stepping, we investigated the role of neck linker length, charge, and structure in kinesin-1 and kinesin-2 motor behavior. For optimum comparison with kinesin-1, the KIF3A head and neck linker of kinesin-2 were fused to the kinesin-1 neck coil and rod. Extending the 14-residue kinesin-1 neck linker reduced processivity, and shortening the 17-residue kinesin-2 neck linker enhanced processivity. When a proline in the kinesin-2 neck linker was replaced, kinesin-1 and kinesin-2 run lengths scaled identically with neck linker length, despite moving at different speeds. In low-ionic-strength buffer, charge had a dominant effect on motor processivity, which resolves ongoing controversy regarding the effect of neck linker length on kinesin processivity. From stochastic simulations, the results are best explained by neck linker extension slowing strain-dependent detachment of the rear head along with diminishing strain-dependent inhibition of ATP binding. These results help delineate how interhead strain maximizes stepping and suggest that less processive kinesins are tuned to coordinate with other motors differently than the maximally processive kinesin-1. Copyright 2010 Elsevier Ltd. All rights reserved.

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

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

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

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 wasmore » 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.« less

Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms.

PubMed

Gao, William N D; Carpentier, David C J; Ewles, Helen A; Lee, Stacey-Ann; Smith, Geoffrey L

2017-08-01

Vaccinia virus (VACV) utilizes microtubule-mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin-1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin-1, yet in its absence VACV egress still occurs on microtubules. During a co-immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C-terminal tail of KLC2, to a region that overlaps the binding site of cellular 14-3-3 proteins. F12/E2 displaces 14-3-3 from KLC and, unlike 14-3-3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N-terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co-operatively enhance A36 association with KLC, particularly when using a KLC1-KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co-operatively associate with kinesin-1. © 2017 The Authors. Traffic published by John Wiley & Sons Ltd.

The beginning of kinesin's force-generating cycle visualized at 9-Å resolution

PubMed Central

Sindelar, Charles V.; Downing, Kenneth H.

2007-01-01

We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, in kinesin's poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in “ejecting” adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesin's putative force-delivering element (the “neck linker”). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself. PMID:17470637

Engineering of a novel Ca{sup 2+}-regulated kinesin molecular motor using a calmodulin dimer linker

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

Shishido, Hideki; Maruta, Shinsaku, E-mail: maruta@soka.ac.jp

Highlights: Black-Right-Pointing-Pointer Engineered kinesin-M13 and calmodulin involving single cysteine were prepared. Black-Right-Pointing-Pointer CaM mutant was cross-linked to dimer by bifunctional thiol reactive reagent. Black-Right-Pointing-Pointer Kinesin-M13 was dimerized via CaM dimer in the presence of calcium. Black-Right-Pointing-Pointer Function of the engineered kinesin was regulated by a Ca{sup 2+}-calmodulin dimer linker. -- Abstract: The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have 'on-off' control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesinmore » monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca{sup 2+}-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca{sup 2+}-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.« less

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization.

PubMed

Roux, Kyle J; Crisp, Melissa L; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L; Burke, Brian

2009-02-17

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin-Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1-3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning.

Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport

NASA Technical Reports Server (NTRS)

Tsai, M. Y.; Morfini, G.; Szebenyi, G.; Brady, S. T.

2000-01-01

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

Effective killing of the human pathogen Candida albicans by a specific inhibitor of non-essential mitotic kinesin Kip1p

PubMed Central

Chua, Penelope R; Roof, David M; Lee, Yan; Sakowicz, Roman; Clarke, David; Pierce, Dan; Stephens, Thoryn; Hamilton, Matthew; Morgan, Brad; Morgans, David; Nakai, Takashi; Tomasi, Adam; Maxon, Mary E

2007-01-01

Kinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in ‘rigor-like’, tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a non-essential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery. PMID:17573815

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization

PubMed Central

Roux, Kyle J.; Crisp, Melissa L.; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L.; Burke, Brian

2009-01-01

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin–Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1–3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning. PMID:19164528

The spindle kinesin-like protein HsEg5 is an autoantigen in systemic lupus erythematosus.

PubMed

Whitehead, C M; Winkfein, R J; Fritzler, M J; Rattner, J B

1996-10-01

Autoantibodies directed against the mitotic spindle apparatus (MSA) have been shown to target an antigen referred to as NuMA (nuclear mitotic apparatus). In this study, we identified a second MSA antigen as the spindle kinesin-like protein HsEg5. We studied the frequency of antibodies to HsEg5 in human sera that demonstrate the MSA pattern of staining, the frequency of autoantibodies to HsEg5 in patients with systemic lupus erythematosus (SLE), and the clinical features of patients with antibodies to HsEg5. A prototype serum from an SLE patient was used to isolate a 4.8-kilobase complementary DNA (cDNA) from a HeLa cDNA library. Western blot, immunoprecipitation, and sequence analysis revealed that the antigen was an approximately 130-kd protein, HsEg5. The frequency of autoantibodies to recombinant HsEg5 in 51 sera that demonstrated an MSA pattern of staining on HEp-2 and HeLa cells was detected by immunoblotting 2 constructs of the cDNA. The clinical features of patients with antibodies directed against HsEg5 was obtained by retrospective chart review. The antigen responsible for the MSA-35 pattern was identified as the human kinesin-like protein HsEg5. Seven of 51 sera (14%) that demonstrated an MSA pattern of staining reacted with recombinant HsEg5. Six of 7 of the HsEg5-positive patients (86%) had SLE, and 1 had Sjögren's syndrome. The indirect immunofluorescent staining pattern of sera that reacted with HsEg5 could be distinguished from the other sera that reacted with NuMA. In an unselected cohort of 52 SLE patients, 3 (6%) had autoantibodies reactive with the recombinant HsEg5. Autoantibodies to MSA fall into 2 major classes: those reactive with NuMA and those reactive with HsEg5. Autoantibodies to HsEg5 are found in a lower frequency than NuMA in sera that demonstrate the MSA pattern of staining and appear to be specifically associated with SLE. HsEg5 can be distinguished from NuMA by indirect immunofluorescence and Western blotting.

Structure of the SANT domain from the Xenopus chromatin remodeling factor ISWI

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

Horton, John R.; Elgar, Stuart J.; Khan, Seema I.

2008-09-17

The SANT (Swi3, Ada2, N-Cor, and TFIIIB) module was first described as a putative DNA-binding domain with strong similarity to the helix-turn-helix DNA binding domain of Myb-related proteins. The X-ray structure of the C-terminal one third portion of the ATPase ISWI of Drosophila melangoaster, containing both SANT and SLIDE (SANT-Like ISWI Domain), confirmed the overall helix-turn-helix structural architecture of SANT as well as SLIDE. However, the DNA-contacting residues in Myb are not conserved in SANT and the structurally corresponding residues in the ISWI SANT domain are acidic, and therefore incompatible with DNA interaction. Recent studies suggested that SANT domains mightmore » be a histone-tail-binding module, including the DNA binding SANT domain of c-Myb. Here they present the X-ray structure of Xenopus laevis ISWI SANT domain, derived from limited proteolysis of a C-terminal fragment of ISWI protein.« less

A Unique Role for Endothelial Cell Kinesin Light Chain 1, Variant 1 in Leukocyte Transendothelial Migration

PubMed Central

Cyrus, Bita F.; Muller, William A.

2017-01-01

A reservoir of parajunctional membrane in endothelial cells, the lateral border recycling compartment (LBRC), is critical for transendothelial migration (TEM). We have previously shown that targeted recycling of the LBRC to the site of TEM requires microtubules and a kinesin molecular motor. However, the identity of the kinesin and mechanism of cargo binding were not known. We show that microinjection of endothelial cells with a monoclonal antibody specific for kinesin-1 significantly blocked LBRC-targeted recycling and TEM. In complementary experiments, knocking down KIF5B, a ubiquitous kinesin-1 isoform, in endothelial cells significantly decreased targeted recycling of the LBRC and leukocyte TEM. Kinesin heavy chains move cargo along microtubules by one of many kinesin light chains (KLCs), which directly bind the cargo. Knocking down KLC 1 isoform variant 1 (KLC1C) significantly decreased LBRC-targeted recycling and TEM, whereas knocking down other isoforms of KLC1 had no effect. Re-expression of KLC1C resistant to the knockdown shRNA restored targeted recycling and TEM. Thus kinesin-1 and KLC1C are specifically required for targeted recycling and TEM. These data suggest that of the many potential combinations of the 45 kinesin family members and multiple associated light chains, KLC1C links the LBRC to kinesin-1 (KIF5B) during targeted recycling and TEM. Thus, KLC1C can potentially be used as a target for anti-inflammatory therapy. PMID:26994343

Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

NASA Technical Reports Server (NTRS)

Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

2002-01-01

Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

Deletion of the Tail Domain of the Kinesin-5 Cin8 Affects Its Directionality*

PubMed Central

Düselder, André; Fridman, Vladimir; Thiede, Christina; Wiesbaum, Alice; Goldstein, Alina; Klopfenstein, Dieter R.; Zaitseva, Olga; Janson, Marcel E.; Gheber, Larisa; Schmidt, Christoph F.

2015-01-01

The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end- to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8Δtail). In contrast to wild-type Cin8, the motility of single molecules of Cin8Δtail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8Δtail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8Δtail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus- and minus-end-directed motility. PMID:25991727

Nonlinear dynamics of C-terminal tails in cellular microtubules

NASA Astrophysics Data System (ADS)

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

2016-07-01

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

Motion of kinesin in a viscoelastic medium

NASA Astrophysics Data System (ADS)

Knoops, Gert; Vanderzande, Carlo

2018-05-01

Kinesin is a molecular motor that transports cargo along microtubules. The results of many in vitro experiments on kinesin-1 are described by kinetic models in which one transition corresponds to the forward motion and subsequent binding of the tethered motor head. We argue that in a viscoelastic medium like the cytosol of a cell this step is not Markov and has to be described by a nonexponential waiting time distribution. We introduce a semi-Markov kinetic model for kinesin that takes this effect into account. We calculate, for arbitrary waiting time distributions, the moment generating function of the number of steps made, and determine from this the average velocity and the diffusion constant of the motor. We illustrate our results for the case of a waiting time distribution that is Weibull. We find that for realistic parameter values, viscoelasticity decreases the velocity and the diffusion constant, but increases the randomness (or Fano factor).

Kinesin-dependent mechanism for controlling triglyceride secretion from the liver.

PubMed

Rai, Priyanka; Kumar, Mukesh; Sharma, Geetika; Barak, Pradeep; Das, Saumitra; Kamat, Siddhesh S; Mallik, Roop

2017-12-05

Despite massive fluctuations in its internal triglyceride content, the liver secretes triglyceride under tight homeostatic control. This buffering function is most visible after fasting, when liver triglyceride increases manyfold but circulating serum triglyceride barely fluctuates. How the liver controls triglyceride secretion is unknown, but is fundamentally important for lipid and energy homeostasis in animals. Here we find an unexpected cellular and molecular mechanism behind such control. We show that kinesin motors are recruited to triglyceride-rich lipid droplets (LDs) in the liver by the GTPase ARF1, which is a key activator of lipolysis. This recruitment is activated by an insulin-dependent pathway and therefore responds to fed/fasted states of the animal. In fed state, ARF1 and kinesin appear on LDs, consequently transporting LDs to the periphery of hepatocytes where the smooth endoplasmic reticulum (sER) is present. Because the lipases that catabolize LDs in hepatocytes reside on the sER, LDs can now be catabolized efficiently to provide triglyceride for lipoprotein assembly and secretion from the sER. Upon fasting, insulin is lowered to remove ARF1 and kinesin from LDs, thus down-regulating LD transport and sER-LD contacts. This tempers triglyceride availabiity for very low density lipoprotein assembly and allows homeostatic control of serum triglyceride in a fasted state. We further show that kinesin knockdown inhibits hepatitis-C virus replication in hepatocytes, likely because translated viral proteins are unable to transfer from the ER to LDs. Copyright © 2017 the Author(s). Published by PNAS.

Electrostatically Biased Binding of Kinesin to Microtubules

PubMed Central

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

2011-01-01

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

Acrylamide effects on kinesin-related proteins of the mitotic/meiotic spindle

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

Sickles, Dale W.; Sperry, Ann O.; Testino, Angie

The microtubule (MT) motor protein kinesin is a vital component of cells and organs expressing acrylamide (ACR) toxicity. As a mechanism of its potential carcinogenicity, we determined whether kinesins involved in cell division are inhibited by ACR similar to neuronal kinesin [Sickles, D.W., Brady, S.T., Testino, A.R., Friedman, M.A., and Wrenn, R.A. (1996). Direct effect of the neurotoxicant acrylamide on kinesin-based microtubule motility. Journal of Neuroscience Research 46, 7-17.] Kinesin-related genes were isolated from rat testes [Navolanic, P.M., and Sperry, A.O. (2000). Identification of isoforms of a mitotic motor in mammalian spermatogenesis. Biology of Reproduction 62, 1360-1369.], their kinesin-like proteinsmore » expressed in bacteria using recombinant DNA techniques and the effects of ACR, glycidamide (GLY) and propionamide (a non-neurotoxic metabolite) on the function of two of the identified kinesin motors were tested. KIFC5A MT bundling activity, required for mitotic spindle formation, was measured in an MT-binding assay. Both ACR and GLY caused a similar concentration-dependent reduction in the binding of MT; concentrations of 100 {mu}M ACR or GLY reduced its activity by 60%. KRP2 MT disassembling activity was assayed using the quantity of tubulin disassembled from taxol-stabilized MT. Both ACR and GLY inhibited KRP2-induced MT disassembly. GLY was substantially more potent; significant reductions of 60% were achieved by 500 {mu}M, a comparable inhibition by ACR required a 5 mM concentration. Propionamide had no significant effect on either kinesin, except KRP2 at 10 mM. This is the first report of ACR inhibition of a mitotic/meiotic motor protein. ACR (or GLY) inhibition of kinesin may be an alternative mechanism to DNA adduction in the production of cell division defects and potential carcinogenicity. We conclude that ACR may act on multiple kinesin family members and produce toxicities in organs highly dependent on microtubule-based functions.« less

Functional Analysis of Human Microtubule-based Motor Proteins, the Kinesins and Dyneins, in Mitosis/Cytokinesis Using RNA InterferenceD⃞V⃞

PubMed Central

Zhu, Changjun; Zhao, Jian; Bibikova, Marina; Leverson, Joel D.; Bossy-Wetzel, Ella; Fan, Jian-Bing; Abraham, Robert T.; Jiang, Wei

2005-01-01

Microtubule (MT)-based motor proteins, kinesins and dyneins, play important roles in multiple cellular processes including cell division. In this study, we describe the generation and use of an Escherichia coli RNase III-prepared human kinesin/dynein esiRNA library to systematically analyze the functions of all human kinesin/dynein MT motor proteins. Our results indicate that at least 12 kinesins are involved in mitosis and cytokinesis. Eg5 (a member of the kinesin-5 family), Kif2A (a member of the kinesin-13 family), and KifC1 (a member of the kinesin-14 family) are crucial for spindle formation; KifC1, MCAK (a member of the kinesin-13 family), CENP-E (a member of the kinesin-7 family), Kif14 (a member of the kinesin-3 family), Kif18 (a member of the kinesin-8 family), and Kid (a member of the kinesin-10 family) are required for chromosome congression and alignment; Kif4A and Kif4B (members of the kinesin-4 family) have roles in anaphase spindle dynamics; and Kif4A, Kif4B, MKLP1, and MKLP2 (members of the kinesin-6 family) are essential for cytokinesis. Using immunofluorescence analysis, time-lapse microscopy, and rescue experiments, we investigate the roles of these 12 kinesins in detail. PMID:15843429

SH3-like motif-containing C-terminal domain of staphylococcal teichoic acid transporter suggests possible function.

PubMed

Ko, Tzu-Ping; Tseng, Shih-Ting; Lai, Shu-Jung; Chen, Sheng-Chia; Guan, Hong-Hsiang; Shin Yang, Chia; Jung Chen, Chun; Chen, Yeh

2016-09-01

The negatively charged bacterial polysaccharides-wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising a transmembrane subunit TagG and an ATPase subunit TagH. We determined the crystal structure of the C-terminal domain of TagH (TagH-C) to investigate its function. The structure shows an N-terminal SH3-like subdomain wrapped by a C-terminal subdomain with an anti-parallel β-sheet and an outer shell of α-helices. A stretch of positively charged surface across the subdomain interface is flanked by two negatively charged regions, suggesting a potential binding site for negatively charged polymers, such as WTAs or acidic peptide chains. Proteins 2016; 84:1328-1332. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains

PubMed Central

1988-01-01

We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116- kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP- sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility. PMID:2974459

Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

PubMed Central

Signor, Dawn; Wedaman, Karen P.; Rose, Lesilee S.; Scholey, Jonathan M.

1999-01-01

Chemosensation in the nervous system of the nematode Caenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia. PMID:9950681

Unique players in the BMP pathway: Small C-terminal domain phosphatases dephosphorylate Smad1 to attenuate BMP signaling

PubMed Central

Knockaert, Marie; Sapkota, Gopal; Alarcón, Claudio; Massagué, Joan; Brivanlou, Ali H.

2006-01-01

Smad transcription factors are key signal transducers for the TGF-β/bone morphogenetic protein (BMP) family of cytokines and morphogens. C-terminal serine phosphorylation by TGF-β and BMP membrane receptors drives Smads into the nucleus as transcriptional regulators. Dephosphorylation and recycling of activated Smads is an integral part of this process, which is critical for agonist sensing by the cell. However, the nuclear phosphatases involved have remained unknown. Here we provide functional, biochemical, and embryological evidence identifying the SCP (small C-terminal domain phosphatase) family of nuclear phosphatases as mediators of Smad1 dephosphorylation in the BMP signaling pathway in vertebrates. Xenopus SCP2/Os4 inhibits BMP activity in the presumptive ectoderm and leads to neuralization. In Xenopus embryos, SCP2/Os4 and human SCP1, 2, and 3 cause selective dephosphorylation of Smad1 compared with Smad2, inhibiting BMP- and Smad1-dependent transcription and leading to the induction of the secondary dorsal axis. In human cells, RNAi-mediated depletion of SCP1 and SCP2 increases the extent and duration of Smad1 phosphorylation in response to BMP, the transcriptional action of Smad1, and the strength of endogenous BMP gene responses. The present identification of the SCP family as Smad C-terminal phosphatases sheds light on the events that attenuate Smad signaling and reveals unexpected links to the essential phosphatases that control RNA polymerase II in eukaryotes. PMID:16882717

The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm.

PubMed

Henson, J H; Cole, D G; Roesener, C D; Capuano, S; Mendola, R J; Scholey, J M

1997-01-01

We have utilized immunoblotting and light microscopic immunofluorescent staining methods to examine the expression and localization of sea urchin kinesin-II, a heterotrimeric plus end-directed microtubule motor protein (previously referred to as KRP(85/95)), in sea urchin and sand dollar sperm. We demonstrate the presence of the 85 K and 115 K subunits of kinesin-II in sperm and localize these proteins to the sperm flagella and midpiece. The kinesin-II localization pattern is punctate and discontinuous, and in the flagella it is quite distinct from the continuous labeling present in sperm labeled with anti-flagellar dynein. The kinesin-II staining is largely insensitive to prefixation detergent extraction, suggesting that it is not associated with membranous elements in the sperm. In the midpiece the kinesin-II staining is similar to the pattern present in sperm labeled with an anti-centrosomal antibody. To our knowledge, this is the first localization of kinesin-like proteins in mature sperm and corroborates the recent identification and localization of kinesin-like proteins in the flagella and basal body of the unicellular green alga Chlamydomonas. We hypothesize that kinesin-II in the sperm may play functional roles in intraflagellar transport and/or the formation of flagella during spermatogenesis.

The kinesin KIF9 and reggie/flotillin proteins regulate matrix degradation by macrophage podosomes

PubMed Central

Cornfine, Susanne; Himmel, Mirko; Kopp, Petra; el Azzouzi, Karim; Wiesner, Christiane; Krüger, Marcus; Rudel, Thomas; Linder, Stefan

2011-01-01

Podosomes are actin-based matrix contacts in a variety of cell types, most notably monocytic cells, and are characterized by their ability to lyse extracellular matrix material. Besides their dependence on actin regulation, podosomes are also influenced by microtubules and microtubule-dependent transport processes. Here we describe a novel role for KIF9, a previously little-characterized member of the kinesin motor family, in the regulation of podosomes in primary human macrophages. We find that small interfering RNA (siRNA)/short-hairpin RNA–induced knockdown of KIF9 significantly affects both numbers and matrix degradation of podosomes. Overexpression and microinjection experiments reveal that the unique C-terminal region of KIF9 is crucial for these effects, presumably through binding of specific interactors. Indeed, we further identify reggie-1/flotillin-2, a signaling mediator between intracellular vesicles and the cell periphery, as an interactor of the KIF9 C-terminus. Reggie-1 dynamically colocalizes with KIF9 in living cells, and, consistent with KIF9-mediated effects, siRNA-induced knockdown of reggies/flotillins significantly impairs matrix degradation by podosomes. In sum, we identify the kinesin KIF9 and reggie/flotillin proteins as novel regulators of macrophage podosomes and show that their interaction is critical for the matrix-degrading ability of these structures. PMID:21119006

Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

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

Faravelli, Alessandro; Dimasi, Nazzareno, E-mail: ndimasi@gmail.com

Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli,more » refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.« less

Kinesin motor protein as an electrostatic ratchet machine

NASA Astrophysics Data System (ADS)

Tsironis, George; Ciudad, Aleix; Sancho, Jose Maria

2008-03-01

Kinesin and related motor proteins utilize ATP fuel to propel themselves along the external surface of microtubules in a processive and directional fashion. We show that the observed step-like motion is possible through time varying charge distributions furnished by the ATP hydrolysis circle while the static charge configuration on the microtuble provides the guide for motion. Thus, while the chemical hydrolysis energy induces appropriate local conformational changes, the motor translational energy is fundamentally electrostatic. Numerical simulations of the mechanical equations of motion show that processivity and directionality are direct consequences of the ATP-dependent electrostatic interaction between the different charge distributions of kinesin and microtubule. Treating proterins as continuous dielectric media and using a Green's function formalism we find analytical expressions for the electrostatic energy in the vicinity of the protein surfaces. We calculate the Bjerrum length in the interior of the protein and analyze its dependence on the charge proximity to the protein interface. We apply these results to kinesin and estimate the pure electrostatic ATP-ADP interaction to be larger than 2k T.

Kinesin expands and stabilizes the GDP-microtubule lattice

NASA Astrophysics Data System (ADS)

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

2018-05-01

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

Two spatially distinct kinesin-14 proteins, Pkl1 and Klp2, generate collaborative inward forces against kinesin-5 Cut7 in S. pombe.

PubMed

Yukawa, Masashi; Yamada, Yusuke; Yamauchi, Tomoaki; Toda, Takashi

2018-01-04

Kinesin motors play central roles in bipolar spindle assembly. In many eukaryotes, spindle pole separation is driven by kinesin-5, which generates outward force. This outward force is balanced by antagonistic inward force elicited by kinesin-14 and/or dynein. In fission yeast, two kinesin-14 proteins, Pkl1 and Klp2, play an opposing role against the kinesin-5 motor protein Cut7. However, how the two kinesin-14 proteins coordinate individual activities remains elusive. Here, we show that although deletion of either pkl1 or klp2 rescues temperature-sensitive cut7 mutants, deletion of only pkl1 can bypass the lethality caused by cut7 deletion. Pkl1 is tethered to the spindle pole body, whereas Klp2 is localized along the spindle microtubule. Forced targeting of Klp2 to the spindle pole body, however, compensates for Pkl1 functions, indicating that cellular localizations, rather than individual motor specificities, differentiate between the two kinesin-14 proteins. Interestingly, human kinesin-14 (KIFC1 or HSET) can replace either Pkl1 or Klp2. Moreover, overproduction of HSET induces monopolar spindles, reminiscent of the phenotype of Cut7 inactivation. Taken together, this study has uncovered the biological mechanism whereby two different Kinesin-14 motor proteins exert their antagonistic roles against kinesin-5 in a spatially distinct manner. © 2018. Published by The Company of Biologists Ltd.

Nucleotide-induced conformations in the neck region of dimeric kinesin

PubMed Central

Skiniotis, Georgios; Surrey, Thomas; Altmann, Stephan; Gross, Heinz; Song, Young-Hwa; Mandelkow, Eckhard; Hoenger, Andreas

2003-01-01

The neck region of kinesin constitutes a key component in the enzyme’s walking mechanism. Here we applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin neck in dimeric and monomeric constructs complexed to microtubules. To this end we enhanced the visibility of this region by engineering an SH3 domain into the transition between neck linker and neck coiled coil. The resulting chimeric kinesin constructs remained functional as verified by physiology assays. In the presence of AMP–PNP the SH3 domains allowed us to identify the position of the neck in a well defined conformation and revealed its high flexibility in the absence of nucleotide. We show here the double-headed binding of dimeric kinesin along the same protofilament, which is characterized by the opposite directionality of neck linkers. In this configuration the neck coiled coil appears fully zipped. The position of the neck region in dimeric constructs is not affected by the presence of the tubulin C-termini as confirmed by subtilisin treatment of microtubules prior to motor decoration. PMID:12660159

[Motor protein Kinesin-6 and ischemic heart disease].

PubMed

Koroleva, O S; Zateĭshchikov, D A

2010-01-01

The review describes possible role of kinesins in development of coronary heart disease and efficacy of treatment with statins. Fourty five kinesins are represented in human body making up a superfamily of universal and simplest motor proteins which are expressed almost in all tissues. Level of kinesin 6 is 5% higher than expression of other kinesins in some segments of coronary arteries and it is relatively low in organs playing unknown role in susceptibility to atherosclerosis. As a result of several genoms wide association studies the role of polymorphic marker Thr719Arg of kinesin 6 gene in development of ischemic heart disease, myocardial infarction, and in efficacy of therapy with statins was revealed.

Molecular origin of the weak susceptibility of kinesin velocity to loads and its relation to the collective behavior of kinesins

PubMed Central

Wang, Qian; Diehl, Michael R.; Jana, Biman; Cheung, Margaret S.; Kolomeisky, Anatoly B.; Onuchic, José N.

2017-01-01

Motor proteins are active enzymatic molecules that support important cellular processes by transforming chemical energy into mechanical work. Although the structures and chemomechanical cycles of motor proteins have been extensively investigated, the sensitivity of a motor’s velocity in response to a force is not well-understood. For kinesin, velocity is weakly influenced by a small to midrange external force (weak susceptibility) but is steeply reduced by a large force. Here, we utilize a structure-based molecular dynamic simulation to study the molecular origin of the weak susceptibility for a single kinesin. We show that the key step in controlling the velocity of a single kinesin under an external force is the ATP release from the microtubule-bound head. Only under large loading forces can the motor head release ATP at a fast rate, which significantly reduces the velocity of kinesin. It underpins the weak susceptibility that the velocity will not change at small to midrange forces. The molecular origin of this velocity reduction is that the neck linker of a kinesin only detaches from the motor head when pulled by a large force. This prompts the ATP binding site to adopt an open state, favoring ATP release and reducing the velocity. Furthermore, we show that two load-bearing kinesins are incapable of equally sharing the load unless they are very close to each other. As a consequence of the weak susceptibility, the trailing kinesin faces the challenge of catching up to the leading one, which accounts for experimentally observed weak cooperativity of kinesins motors. PMID:28973894

Dynamic model of the force driving kinesin to move along microtubule-Simulation with a model system

NASA Astrophysics Data System (ADS)

Chou, Y. C.; Hsiao, Yi-Feng; To, Kiwing

2015-09-01

A dynamic model for the motility of kinesin, including stochastic-force generation and step formation is proposed. The force driving the motion of kinesin motor is generated by the impulse from the collision between the randomly moving long-chain stalk and the ratchet-shaped outer surface of microtubule. Most of the dynamical and statistical features of the motility of kinesin are reproduced in a simulation system, with (a) ratchet structures similar to the outer surface of microtubule, (b) a bead chain connected to two heads, similarly to the stalk of the real kinesin motor, and (c) the interaction between the heads of the simulated kinesin and microtubule. We also propose an experiment to discriminate between the conventional hand-over-hand model and the dynamic model.

Kinesin Steps Do Not Alternate in Size☆

PubMed Central

Fehr, Adrian N.; Asbury, Charles L.; Block, Steven M.

2008-01-01

Abstract Kinesin is a two-headed motor protein that transports cargo inside cells by moving stepwise on microtubules. Its exact trajectory along the microtubule is unknown: alternative pathway models predict either uniform 8-nm steps or alternating 7- and 9-nm steps. By analyzing single-molecule stepping traces from “limping” kinesin molecules, we were able to distinguish alternate fast- and slow-phase steps and thereby to calculate the step sizes associated with the motions of each of the two heads. We also compiled step distances from nonlimping kinesin molecules and compared these distributions against models predicting uniform or alternating step sizes. In both cases, we find that kinesin takes uniform 8-nm steps, a result that strongly constrains the allowed models. PMID:18083906

Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1

NASA Astrophysics Data System (ADS)

Xu, Jing

2013-03-01

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signalling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (approximately fourfold) without altering kinesin's single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results (Nat. Commun., 3:754, 2012) provide the first direct evidence of a protein kinase upregulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin. Work supported by NIGMS grants GM64624 to SPG, GM74830-06A1 to LH, GM76516 to LB, NS048501 to SJK, and AHA grant 825278F to JX.

Class I HDACs control a JIP1-dependent pathway for kinesin-microtubule binding in cardiomyocytes

PubMed Central

Blakeslee, Weston W.; Lin, Ying-Hsi; Stratton, Matthew S.; Tatman, Philip D.; Hu, Tianjing; Ferguson, Bradley S.; McKinsey, Timothy A.

2018-01-01

Class I histone deacetylase (HDAC) inhibitors block hypertrophy and fibrosis of the heart by suppressing pathological signaling and gene expression programs in cardiac myocytes and fibroblasts. The impact of HDAC inhibition in unstressed cardiac cells remains poorly understood. Here, we demonstrate that treatment of cultured cardiomyocytes with small molecule HDAC inhibitors leads to dramatic induction of c-Jun amino-terminal kinase (JNK)-interacting protein-1 (JIP1) mRNA and protein expression. In contrast to prior findings, elevated levels of endogenous JIP1 in cardiomyocytes failed to significantly alter JNK signaling or cardiomyocyte hypertrophy. Instead, HDAC inhibitor-mediated induction of JIP1 was required to stimulate expression of the kinesin heavy chain family member, KIF5A. We provide evidence for an HDAC-dependent regulatory circuit that promotes formation of JIP1:KIF5A:microtubule complexes that regulate intracellular transport of cargo such as autophagosomes. These findings define a novel role for class I HDACs in the control of the JIP1/kinesin axis in cardiomyocytes, and suggest that HDAC inhibitors could be used to alter microtubule transport in the heart. PMID:28886967

Engineered kinesin motor proteins amenable to small-molecule inhibition

PubMed Central

Engelke, Martin F.; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay; Blasius, T. Lynne; Soppina, Pushpanjali; Hancock, William O.; Gelfand, Vladimir I.; Verhey, Kristen J.

2016-01-01

The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest. PMID:27045608

Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling

PubMed Central

Morin, Ryan D.; Chang, Elbert; Petrescu, Anca; Liao, Nancy; Griffith, Malachi; Kirkpatrick, Robert; Butterfield, Yaron S.; Young, Alice C.; Stott, Jeffrey; Barber, Sarah; Babakaiff, Ryan; Dickson, Mark C.; Matsuo, Corey; Wong, David; Yang, George S.; Smailus, Duane E.; Wetherby, Keith D.; Kwong, Peggy N.; Grimwood, Jane; Brinkley, Charles P.; Brown-John, Mabel; Reddix-Dugue, Natalie D.; Mayo, Michael; Schmutz, Jeremy; Beland, Jaclyn; Park, Morgan; Gibson, Susan; Olson, Teika; Bouffard, Gerard G.; Tsai, Miranda; Featherstone, Ruth; Chand, Steve; Siddiqui, Asim S.; Jang, Wonhee; Lee, Ed; Klein, Steven L.; Blakesley, Robert W.; Zeeberg, Barry R.; Narasimhan, Sudarshan; Weinstein, John N.; Pennacchio, Christa Prange; Myers, Richard M.; Green, Eric D.; Wagner, Lukas; Gerhard, Daniela S.; Marra, Marco A.; Jones, Steven J.M.; Holt, Robert A.

2006-01-01

Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization. PMID:16672307

The expression pattern of the C-terminal kinesin gene kifc1 during the spermatogenesis of Sepiella maindroni.

PubMed

Tan, Fu-Qing; Ma, Xiao-Xin; Zhu, Jun-Quan; Yang, Wan-Xi

2013-12-10

In this study, we investigated the gene sequence and characteristic of kifc1 in Sepiella maindroni through PCR and RACE technology. Our research aimed particularly at the spatio-temporal expression pattern of kifc1 in the developmental testis through in situ hybridization. The particular role of kifc1 in the spermatogenesis of S. maindroni was our particular interest. Based on multiple protein sequence alignments of KIFC1 homologues, kifc1 gene from the testis of S. maindroni was identified, which consisted of 2432bp including a 2109 in-frame ORF corresponding to 703 continuous amino acids. The encoded polypeptide shared highest similarity with Octopus tankahkeei. Through the prediction of the secondary and tertiary structures, the motor domain of KIFC1 was conserved at the C-terminal, having putative ATP-binding and microtubule-binding motifs, while the N-terminal was more specific to bind various cargoes for cellular events. The stalk domain connecting between the C-terminal and N-terminal determined the direction of movement. According to RT-PCR results, the kifc1 gene is not tissue-specific, commonly detected in different tissues, for example, the testis, liver, stomach, muscle, caecum and gills. Through an in situ hybridization method, the expression pattern of KIFC1 protein mimics in the spermatogenesis of S. maindroni. During the primary stage of the spermatogenesis, the kifc1 mRNA signal was barely detectable. At the early spermatids, the signal started to be present. With the elongation of spermatids, the signals increased substantially. It peaked and gathered around the acrosome area when the spermatids began to transform to spindle shape. As the spermatids developed into mature sperm, the signal vanished. In summary, the expression of kfic1 at specific stages during spermiogenesis and its distribution shed light on the potential functions of this motor in major cytological transformations. The KIFC1 homologue may provide a direct shaping force to the

A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

PubMed Central

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

2017-01-01

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly. PMID:29021344

Importin-β Directly Regulates the Motor Activity and Turnover of a Kinesin-4.

PubMed

Ganguly, Anindya; DeMott, Logan; Zhu, Chuanmei; McClosky, Daniel D; Anderson, Charles T; Dixit, Ram

2018-03-12

Spatiotemporal regulation of kinesins is essential for microtubule-dependent intracellular transport. In plants, cell wall deposition depends on the FRA1 kinesin, whose abundance and motility are tightly controlled to match cellular growth rate. Here, we show that an importin-β, IMB4, regulates FRA1 activity in a developmental manner. IMB4 physically interacts with a PY motif in the FRA1 motor domain and inhibits its motility by preventing microtubule binding, while also protecting FRA1 against proteasome-mediated degradation, thus providing a mechanism to couple the motility and stability of FRA1. This regulatory mechanism is likely to be broadly applicable, based on the conservation of the PY motif in the motor domains of plant and animal kinesins and the direct interaction of multiple plant kinesins with IMB4. Together, our data establish IMB4 as a multi-functional regulator of FRA1 and reveal a mechanism for how plants control the magnitude of cargo transport needed for cell wall assembly. Copyright © 2018 Elsevier Inc. All rights reserved.

Processive movement of single kinesins on crowded microtubules visualized using quantum dots

PubMed Central

Seitz, Arne; Surrey, Thomas

2006-01-01

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

Expression, Refolding and Crystallizations of the Grb2-like (GADS) C-Terminal SH3 Domain Complexed with a SLP-76 Motif Peptide

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

Faravelli,A.; Dimasi, N.

The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.

Kinesin-5, a mitotic microtubule-associated motor protein, modulates neuronal migration

PubMed Central

Falnikar, Aditi; Tole, Shubha; Baas, Peter W.

2011-01-01

Kinesin-5 (also called Eg5 or kif11) is a homotetrameric motor protein that functions by modulating microtubule (MT)–MT interactions. In the case of mitosis, kinesin-5 slows the rate of separation of the half-spindles. In the case of the axon, kinesin-5 limits the frequency of transport of short MTs, and also limits the rate of axonal growth. Here we show that experimental inhibition of kinesin-5 in cultured migratory neurons results in a faster but more randomly moving neuron with a shorter leading process. As is the case with axons of stationary neurons, short MT transport frequency is notably enhanced in the leading process of the migratory neuron when kinesin-5 is inhibited. Conversely, overexpression of kinesin-5, both in culture and in developing cerebral cortex, causes migration to slow and even cease. Regions of anti-parallel MT organization behind the centrosome were shown to be especially rich in kinesin-5, implicating these regions as potential sites where kinesin-5 forces may be especially relevant. We posit that kinesin-5 acts as a “brake” on MT–MT interactions that modulates the advance of the entire MT apparatus. In so doing, kinesin-5 regulates the rate and directionality of neuronal migration and possibly the cessation of migration when the neuron reaches its destination. PMID:21411631

Casein Kinase 2 Reverses Tail-Independent Inhibition of Kinesin-1

NASA Astrophysics Data System (ADS)

Xu, Jing; Shu, Zhanyong; Anand, Preetha; Reddy, Babu; Cermelli, Silvia; Whisenant, Thomas; King, Stephen; Bardwell, Lee; Huang, Lan; Gross, Steven

2011-03-01

Kinesin-1 is a plus-end microtubule-based molecular motor, and defects in kinesin transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a direct head-tail interaction, but is believed to be active otherwise. In contrast, this study uncovers a fast but reversible inhibition distinct from the canonical auto-inhibition pathway. The majority of the initially active kinesin (full-length or tail-less) loses its ability to bind/interact with microtubule, and Casein Kinase 2 (CK2) reverses this inactivation (up to 4-fold) without altering kinesin's single motor properties. Motor phosphorylation is not required for this CK2 -mediated kinesin activation. In cultured mammalian cells, knockdown of CK2 level, but not kinase activity, was sufficient to decrease the force required to stall lipid droplet transport, consistent with a reduction in the number of active motors. We propose that CK2 forms a positive regulating complex with the motor. This study provides the first direct evidence of a protein kinase positively regulating kinesin-transport, and uncovers a pathway whereby inactive cargo-bound kinesin can be activated. This work is supported by NIGMS grants GM64624 and GM079156 to SPG, GM-74830 to LH, NIH grants GM76516 and GM60366 to LB, and AHA grant 825278F to JX.

Mutation of Rice BC12/GDD1, Which Encodes a Kinesin-Like Protein That Binds to a GA Biosynthesis Gene Promoter, Leads to Dwarfism with Impaired Cell Elongation[W][OA

PubMed Central

Li, Juan; Jiang, Jiafu; Qian, Qian; Xu, Yunyuan; Zhang, Cui; Xiao, Jun; Du, Cheng; Luo, Wei; Zou, Guoxing; Chen, Mingluan; Huang, Yunqing; Feng, Yuqi; Cheng, Zhukuan; Yuan, Ming; Chong, Kang

2011-01-01

The kinesins are a family of microtubule-based motor proteins that move directionally along microtubules and are involved in many crucial cellular processes, including cell elongation in plants. Less is known about kinesins directly regulating gene transcription to affect cellular physiological processes. Here, we describe a rice (Oryza sativa) mutant, gibberellin-deficient dwarf1 (gdd1), that has a phenotype of greatly reduced length of root, stems, spikes, and seeds. This reduced length is due to decreased cell elongation and can be rescued by exogenous gibberellic acid (GA3) treatment. GDD1 was cloned by a map-based approach, was expressed constitutively, and was found to encode the kinesin-like protein BRITTLE CULM12 (BC12). Microtubule cosedimentation assays revealed that BC12/GDD1 bound to microtubules in an ATP-dependent manner. Whole-genome microarray analysis revealed the expression of ent-kaurene oxidase (KO2), which encodes an enzyme involved in GA biosynthesis, was downregulated in gdd1. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that GDD1 bound to the element ACCAACTTGAA in the KO2 promoter. In addition, GDD1 was shown to have transactivation activity. The level of endogenous GAs was reduced in gdd1, and the reorganization of cortical microtubules was altered. Therefore, BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice. PMID:21325138

Motoring through: the role of kinesin superfamily proteins in female meiosis.

PubMed

Camlin, Nicole J; McLaughlin, Eileen A; Holt, Janet E

2017-07-01

The kinesin motor protein family consists of 14 distinct subclasses and 45 kinesin proteins in humans. A large number of these proteins, or their orthologues, have been shown to possess essential function(s) in both the mitotic and the meiotic cell cycle. Kinesins have important roles in chromosome separation, microtubule dynamics, spindle formation, cytokinesis and cell cycle progression. This article contains a review of the literature with respect to the role of kinesin motor proteins in female meiosis in model species. Throughout, we discuss the function of each class of kinesin proteins during oocyte meiosis, and where such data are not available their role in mitosis is considered. Finally, the review highlights the potential clinical importance of this family of proteins for human oocyte quality. To examine the role of kinesin motor proteins in oocyte meiosis. A search was performed on the Pubmed database for journal articles published between January 1970 and February 2017. Search terms included 'oocyte kinesin' and 'meiosis kinesin' in addition to individual kinesin names with the terms oocyte or meiosis. Within human cells 45 kinesin motor proteins have been discovered, with the role of only 13 of these proteins, or their orthologues, investigated in female meiosis. Furthermore, of these kinesins only half have been examined in mammalian oocytes, despite alterations occurring in gene transcripts or protein expression with maternal ageing, cryopreservation or behavioral conditions, such as binge drinking, for many of them. Kinesin motor proteins have distinct and important roles throughout oocyte meiosis in many non-mammalian model species. However, the functions these proteins have in mammalian meiosis, particularly in humans, are less clear owing to lack of research. This review brings to light the need for more experimental investigation of kinesin motor proteins, particularly those associated with maternal ageing, cryopreservation or exposure to

Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5*

PubMed Central

Liu, Liqiong; Richard, Jessica; Kim, Sunyoung; Wojcik, Edward J.

2014-01-01

Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable “next generation” target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are “druggable.” One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease. PMID:24737313

Detection of prosecretory mitogen lacritin in nonprimate tears primarily as a C-terminal-like fragment.

PubMed

Laurie, Diane E; Splan, Rebecca K; Green, Kari; Still, Katherine M; McKown, Robert L; Laurie, Gordon W

2012-09-12

Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal-reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.

Kinesin-microtubule interactions during gliding assays under magnetic force

NASA Astrophysics Data System (ADS)

Fallesen, Todd L.

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

A novel PKD2L1 C-terminal domain critical for trimerization and channel function.

PubMed

Zheng, Wang; Hussein, Shaimaa; Yang, JungWoo; Huang, Jun; Zhang, Fan; Hernandez-Anzaldo, Samuel; Fernandez-Patron, Carlos; Cao, Ying; Zeng, Hongbo; Tang, Jingfeng; Chen, Xing-Zhen

2015-03-30

As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is also called TRPP3 and has similar membrane topology as voltage-gated cation channels. PKD2L1 is involved in hedgehog signaling, intestinal development, and sour tasting. PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does not affect PKD2L1 channel function. It thus remains unclear how PKD2L1 proteins oligomerize into a functional channel. By SDS-PAGE, blue native PAGE and mutagenesis we here identified a novel C-terminal domain called C1 (K575-T622) involved in stronger homotrimerization than the non-overlapping CC2, and found that the PKD2L1 N-terminus is critical for dimerization. By electrophysiology and Xenopus oocyte expression, we found that C1, but not CC2, is critical for PKD2L1 channel function. Our co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits PKD2L1 trimerization as well as PKD2L1 and PKD2L1/PKD1L3 channel function. Thus, our study identified C1 as the first PKD2L1 domain essential for both PKD2L1 trimerization and channel function, and suggest that PKD2L1 and PKD2L1/PKD1L3 channels share the PKD2L1 trimerization process.

Downregulation of Ras C-terminal processing by JNK inhibition

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

Mouri, Wataru; Department of Neurosurgery, Yamagata University School of Medicine, Yamagata 990-9585; Biology Division, National Cancer Center Research Institute, Tokyo 104-0045

2008-06-27

After translation, Ras proteins undergo a series of modifications at their C-termini. This post-translational C-terminal processing is essential for Ras to become functional, but it remains unknown whether and how Ras C-terminal processing is regulated. Here we show that the C-terminal processing and subsequent plasma membrane localization of H-Ras as well as the activation of the downstream signaling pathways by H-Ras are prevented by JNK inhibition. Conversely, JNK activation by ultraviolet irradiation resulted in promotion of C-terminal processing of H-Ras. Furthermore, increased cell density promoted C-terminal processing of H-Ras most likely through an autocrine/paracrine mechanism, which was also blocked undermore » JNK-inhibited condition. Ras C-terminal processing was sensitive to JNK inhibition in the case of H- and N-Ras but not K-Ras, and in a variety of cell types. Thus, our results suggest for the first time that Ras C-terminal processing is a regulated mechanism in which JNK is involved.« less

A Structural Perspective on the Dynamics of Kinesin Motors

PubMed Central

Hyeon, Changbong; Onuchic, José N.

2011-01-01

Despite significant fluctuation under thermal noise, biological machines in cells perform their tasks with exquisite precision. Using molecular simulation of a coarse-grained model and theoretical arguments, we envisaged how kinesin, a prototype of biological machines, generates force and regulates its dynamics to sustain persistent motor action. A structure-based model, which can be versatile in adapting its structure to external stresses while maintaining its native fold, was employed to account for several features of kinesin dynamics along the biochemical cycle. This analysis complements our current understandings of kinesin dynamics and connections to experiments. We propose a thermodynamic cycle for kinesin that emphasizes the mechanical and regulatory role of the neck linker and clarify issues related to the motor directionality, and the difference between the external stalling force and the internal tension responsible for the head-head coordination. The comparison between the thermodynamic cycle of kinesin and macroscopic heat engines highlights the importance of structural change as the source of work production in biomolecular machines. PMID:22261064

Localization of the kinesin adaptor proteins trafficking kinesin proteins 1 and 2 in primary cultures of hippocampal pyramidal and cortical neurons.

PubMed

Loss, Omar; Stephenson, F Anne

2015-07-01

Neuronal function requires regulated anterograde and retrograde trafficking of mitochondria along microtubules by using the molecular motors kinesin and dynein. Previous work has established that trafficking kinesin proteins (TRAKs),TRAK1 and TRAK2, are kinesin adaptor proteins that link mitochondria to kinesin motor proteins via an acceptor protein in the mitochondrial outer membrane, etc. the Rho GTPase Miro. Recent studies have shown that TRAK1 preferentially controls mitochondrial transport in axons of hippocampal neurons by virtue of its binding to both kinesin and dynein motor proteins, whereas TRAK2 controls mitochondrial transport in dendrites resulting from its binding to dynein. This study further investigates the subcellular localization of TRAK1 and TRAK2 in primary cultures of hippocampal and cortical neurons by using both commercial antibodies and anti-TRAK1 and anti-TRAK2 antibodies raised in our own laboratory (in-house). Whereas TRAK1 was prevalently localized in axons of hippocampal and cortical neurons, TRAK2 was more prevalent in dendrites of hippocampal neurons. In cortical neurons, TRAK2 was equally distributed between axons and dendrites. Some qualitative differences were observed between commercial and in-house-generated antibody immunostaining. © 2015 Wiley Periodicals, Inc.

A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast.

PubMed

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

2017-12-01

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

Kinesin Mutations Cause Motor Neuron Disease Phenotypes by Disrupting Fast Axonal Transport in Drosophila

PubMed Central

Hurd, D. D.; Saxton, W. M.

1996-01-01

Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in those diseases. PMID:8913751

Detection of Prosecretory Mitogen Lacritin in Nonprimate Tears Primarily as a C-Terminal-Like Fragment

PubMed Central

Laurie, Diane E.; Splan, Rebecca K.; Green, Kari; Still, Katherine M.; McKown, Robert L.; Laurie, Gordon W.

2012-01-01

Purpose. Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. Methods. Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. Results. Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal–reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. Conclusions. Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair. PMID:22871838

Xenopus-derived glucagon-like peptide-1 and polyethylene-glycosylated glucagon-like peptide-1 receptor agonists: long-acting hypoglycaemic and insulinotropic activities with potential therapeutic utilities.

PubMed

Han, Jing; Fei, Yingying; Zhou, Feng; Chen, Xinyu; Zhang, Ying; Liu, Lin; Fu, Junjie

2018-02-01

Incretin-based therapies based on glucagon-like peptide-1 (GLP-1) receptor agonists are effective treatments of type 2 diabetes. Abundant research has focused on the development of long-acting GLP-1 receptor agonists. However, all GLP-1 receptor agonists in clinical use or development are based on human or Gila GLP-1. We have identified a potent GLP-1 receptor agonist, xGLP-1B, based on Xenopus GLP-1. To further modify the structure of xGLP-1B, alanine scanning was performed to study the structure -activity relationship of xGLP-1B. Two strategies were then employed to improve bioactivity. First, the C-terminal tail of lixisenatide was appended to cysteine-altered xGLP-1B analogues. Second, polyethylene glycol (PEG) chains with different molecular weights were conjugated with the peptides, giving a series of PEGylated conjugates. Comprehensive bioactivity studies of these conjugates were performed in vitro and in vivo. From the in vitro receptor activation potency and in vivo acute hypoglycaemic activities of conjugates 25 -36, 33 was identified as the best candidate for further biological assessments. Conjugate 33 exhibited prominent hypoglycaemic and insulinotropic activities, as well as improved pharmacokinetic profiles in vivo. The prolonged antidiabetic duration of 33 was further confirmed by pre-oral glucose tolerance tests (OGTT) and multiple OGTT. Furthermore, chronic treatment of db/db mice with 33 ameliorated non-fasting blood glucose and insulin levels, reduced HbA1c values and normalized their impaired glucose tolerance. Importantly, no in vivo toxicity was observed in mice treated with 33. Peptide 33 is a promising long-acting type 2 diabetes therapeutic deserving further investigation. © 2017 The British Pharmacological Society.

A universal pathway for kinesin stepping.

PubMed

Clancy, Bason E; Behnke-Parks, William M; Andreasson, Johan O L; Rosenfeld, Steven S; Block, Steven M

2011-08-14

Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin.

Expression, purification, and C-terminal site-specific PEGylation of cysteine-mutated glucagon-like peptide-1.

PubMed

Gao, Mingming; Tian, Hong; Ma, Chen; Gao, Xiangdong; Guo, Wei; Yao, Wenbing

2010-09-01

Glucagon-like peptide-1 (GLP-1) is attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical application is limited because of short biological half-life. This study was designed to produce a C-terminal site-specific PEGylated analog of cysteine-mutated GLP-1 (cGLP-1) to prolong its action. The gene of cGLP-1 was inserted into pET32a to construct a thioredoxinA fusion protein. After expression in BL21 (DE3) strain, the fusion protein was purified with Ni-affinity chromatography and then was PEGylated with methoxy-polyethylene glycol-maleimide (mPEG(10K)-MAL). The PEGylated fusion protein was purified with anion exchange chromatography and then was cleaved by enterokinase. The digested product was further purified with reverse-phase chromatography. Finally, 8.7 mg mPEG(10K)-cGLP-1 with a purity of up to 98% was obtained from the original 500 ml culture. The circular dichroism spectra indicated that mPEG(10K)-cGLP-1 maintained the secondary structure of native GLP-1. As compared with that of native GLP-1, the plasma glucose lowering activity of mPEG(10K)-cGLP-1 was significantly extended. These results suggest that our method will be useful in obtaining a large quantity of mPEG(10K)-cGLP-1 for further study and mPEG(10K)-cGLP-1 might find a role in the therapy of type 2 diabetes through C-terminal site-specific PEGylation.

Loading direction regulates the affinity of ADP for kinesin.

PubMed

Uemura, Sotaro; Ishiwata, Shin'ichi

2003-04-01

Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.

Kinesin regulation dynamics through cargo delivery, a single molecule investigation

NASA Astrophysics Data System (ADS)

Kovacs, Anthony; Kessler, Jonathan; Lin, Huawen; Dutcher, Susan; Wang, Yan Mei

2015-03-01

Kinesins are microtubule-based motors that deliver cargo to their destinations in a highly regulated manner. Although in recent years numerous regulators of cargo delivery have been identified, the regulation mechanism of kinesin through the cargo delivery and recycling process is not known. By performing single molecule fluorescence imaging measurements in Chlamydomonas flagella, which are 200 nm in diameter, 10 microns in length, and contain 9 sets of microtubule doublets, we tracked the intraflagellar transport (IFT) trains, BBSome cargo, and kinesin-2 motors through the cargo delivery process and determined the aforementioned dynamics. Upon arrival at the microtubule plus end at the flagellar tip, (1) IFT trains and BBSome cargo remain intact, dissociate together from kinesins and microtubules, and diffuse along flagellar membrane for a mean of 2.3 sec before commencing retrograde travel. (2) Kinesin motors remain bound to and diffuse along microtubules for 1.3 sec before dissociating into the flagellar lumen for recycling.

Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity

PubMed Central

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

2001-01-01

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

Evidence for involvement of NK₃ receptors in the anxiogenic-like effect of SP6-11(C-terminal), a metabolite of substance P, in rats evaluated in the elevated plus-maze.

PubMed

Duarte, Filipe Silveira; Duzzioni, Marcelo; Leme, Leandro Rinaldi; Smith, Saulo de Paiva; De Lima, Thereza C M

2016-04-15

Substance P (SP) is a neuropeptide widely expressed throughout the fear-processing pathways of the brain. SP is cleaved by several proteolytic enzymes in amino (N-) and carboxy (C-) terminal sequences, which can have biological activities per se. We have previously shown that the anxiogenic-like effects elicited by SP6-11(C-terminal), a specific metabolite of SP, are mediated via NK1 and NK2 receptors. Nevertheless, there are evidences that C-terminal fragments may have a greater affinity for NK3 receptors. The aim of the present study was to further investigate the possible involvement of NK3 receptors in the anxiogenic-like effects induced by SP6-11(C-terminal). Adult male Wistar rats were intracerebroventricularly (i.c.v.) treated with SR142801 (NK3 receptors antagonist) or vehicle one minute to prior SP6-11(C-terminal) or vehicle. Other experimental groups received SP6-11(C-terminal) or vehicle i.c.v. one minute prior to senktide (NK3 receptors agonist) or vehicle. After five minutes, the animals were behaviorally evaluated in the elevated plus-maze test (EPM). SR142801 (100 pmol) or SP6-11(C-terminal) (10 pmol) reduced all the parameters of open-arms exploration and increased the number of protected stretch-attend postures in the EPM, indicating an anxiogenic-like effect. Senktide (10 pmol) promoted an opposite effect on these behavioral parameters, characterizing an anxiolytic-like profile. Pretreatment with SR142801, in an ineffective dose, potentiated the SP6-11-induced anxiety, especially in the unprotected head-dipping and protected stretch-attend postures behaviors. Moreover, the anxiolytic-like effect induced by senktide (1 pmol) was prevented by SP6-11. Our results give support to the involvement of NK3 receptors in the anxiogenic-like actions of SP6-11(C-terminal), where this metabolite seems to behave as an antagonist, in a way similar to SR142801. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of a developmentally regulated nuclear localization signal in Xenopus

PubMed Central

1992-01-01

The 289 residue nuclear oncoprotein encoded by the adenovirus 5 Ela gene contains two peptide sequences that behave as nuclear localization signals (NLS). One signal, located at the carboxy terminus, is like many other known NLSs in that it consists of a short stretch of basic residues (KRPRP) and is constitutively active in cells. The second signal resides within an internal 45 residue region of E1a that contains few basic residues or sequences that resemble other known NLSs. Moreover, this internal signal functions in injected Xenopus oocytes, but not in transfected Xenopus A6 cells, suggesting that it could be regulated developmentally (Slavicek et al. 1989. J. Virol. 63:4047). In this study, we show that the activity of this signal is sensitive to ATP depletion in vivo, efficiently directs the import of a 50 kD fusion protein and can compete with the E1a carboxy-terminal NLS for nuclear import. In addition, we have delineated the precise amino acid residues that comprise the second E1a NLS, and have assessed its utilization during Xenopus embryogenesis. Using amino acid deletion and substitution analyses, we show that the signal consists of the sequence FV(X)7-20MXSLXYM(X)4MF. By expressing in Xenopus embryos a truncated E1a protein that contains only the second NLS and by monitoring its cytoplasmic/nuclear distribution during development with indirect immunofluorescence, we find that the second NLS is utilized up to the early neurula stage. In addition, there appears to be a hierarchy among the embryonic germ layers as to when the second NLS becomes nonfunctional. For this reason, we refer to this NLS as the developmentally regulated nuclear localization signal (drNLS). The implications of these findings for early development are discussed. PMID:1387407

Tumor immunology viewed from alternative animal models—the Xenopus story

PubMed Central

Banach, Maureen; Robert, Jacques

2017-01-01

a) Purpose of review Nonmammalian comparative animal models are important not only to gain fundamental evolutionary understanding of the complex interactions of tumors with the immune system, but also to better predict the applicability of novel immunotherapeutic approaches to humans. After reviewing recent advances in developing alternative models, we focus on the amphibian Xenopus laevis and its usefulness in deciphering the perplexing roles of MHC class I-like molecules and innate (i)T cells in tumor immunity. b) Recent findings Experiments using MHC-defined inbred and cloned animals, tumor cell lines, effective reagents, sequenced genomes, and adapted gene editing techniques in Xenopus, have revealed that the critical involvement of class I-like molecules and iT cells in tumor immunity has been conserved during evolution. c) Summary Comparative studies with the X. laevis tumor immunity model can contribute to the development of better and more efficient cancer immunotherapies. PMID:28944105

Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways.

PubMed

Sapkota, Gopal; Knockaert, Marie; Alarcón, Claudio; Montalvo, Ermelinda; Brivanlou, Ali H; Massagué, Joan

2006-12-29

Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state.

Potential involvement of kinesin-1 in the regulation of subcellular localization of Girdin

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

Muramatsu, Aya; Enomoto, Atsushi, E-mail: enomoto@iar.nagoya-u.ac.jp; Kato, Takuya

Girdin is an actin-binding protein that has multiple functions in postnatal neural development and cancer progression. We previously showed that Girdin is a regulator of migration for neuroblasts born from neural stem cells in the subventricular zone (SVZ) and the dentate gyrus of the hippocampus in the postnatal brain. Despite a growing list of Girdin-interacting proteins, the mechanism of Girdin-mediated migration has not been fully elucidated. Girdin interacts with Disrupted-In-Schizophrenia 1 and partitioning-defective 3, both of which have been shown to interact with the kinesin microtubule motor proteins. Based on this, we have identified that Girdin also interacts with kinesin-1,more » a member of neuronal kinesin proteins. Although a direct interaction of Girdin and kinesin-1 has not been determined, it is of interest to find that Girdin loss-of-function mutant mice with the mutation of a basic amino acid residue-rich region (Basic mut mice) exhibit limited interaction with kinesin-1. Furthermore, expression of a kinesin-1 mutant with motor defects, leads to Girdin mislocalization. Finally, consistent with previous studies on the role of kinesin proteins in trafficking a cell–cell adhesion molecule N-cadherin, Basic mut mice showed an aberrant expression pattern of N-cadherin in migrating SVZ neuroblasts. These findings suggest a potential role of Girdin/kinesin-1 interaction in the regulation of neuroblast migration in the postnatal brain. - Highlights: • Girdin is a regulator of migration for neuroblasts in the postnatal brain. • Girdin interacts with kinesin-1, a member of neuronal kinesin proteins. • Girdin mutant mice showed an aberrant expression of N-cadherin in neuroblasts.« less

Pressure-Induced Changes in the Structure and Function of the Kinesin-Microtubule Complex

PubMed Central

Nishiyama, Masayoshi; Kimura, Yoshifumi; Nishiyama, Yoshio; Terazima, Masahide

2009-01-01

Kinesin-1 is an ATP-driven molecular motor that “walks” along a microtubule by working two heads in a “hand-over-hand” fashion. The stepping motion is well-coordinated by intermolecular interactions between the kinesin head and microtubule, and is sensitively changed by applied forces. We demonstrate that hydrostatic pressure works as an inhibitory action on kinesin motility. We developed a high-pressure microscope that enables the application of hydrostatic pressures of up to 200 MPa (2000 bar). Under high-pressure conditions, taxol-stabilized microtubules were shortened from both ends at the same speed. The sliding velocity of kinesin motors was reversibly changed by pressure, and reached half-maximal value at ∼100 MPa. The pressure-velocity relationship was very close to the force-velocity relationship of single kinesin molecules, suggesting a similar inhibitory mechanism on kinesin motility. Further analysis showed that the pressure mainly affects the stepping motion, but not the ATP binding reaction. The application of pressure is thought to enhance the structural fluctuation and/or association of water molecules with the exposed regions of the kinesin head and microtubule. These pressure-induced effects could prevent kinesin motors from completing the stepping motion. PMID:19186149

A fluid membrane enhances the velocity of cargo transport by small teams of kinesin-1

NASA Astrophysics Data System (ADS)

Li, Qiaochu; Tseng, Kuo-Fu; King, Stephen J.; Qiu, Weihong; Xu, Jing

2018-03-01

Kinesin-1 (hereafter referred to as kinesin) is a major microtubule-based motor protein for plus-end-directed intracellular transport in live cells. While the single-molecule functions of kinesin are well characterized, the physiologically relevant transport of membranous cargos by small teams of kinesins remains poorly understood. A key experimental challenge remains in the quantitative control of the number of motors driving transport. Here we utilized "motile fraction" to overcome this challenge and experimentally accessed transport by a single kinesin through the physiologically relevant transport by a small team of kinesins. We used a fluid lipid bilayer to model the cellular membrane in vitro and employed optical trapping to quantify the transport of membrane-enclosed cargos versus traditional membrane-free cargos under identical conditions. We found that coupling motors via a fluid membrane significantly enhances the velocity of cargo transport by small teams of kinesins. Importantly, enclosing a cargo in a fluid lipid membrane did not impact single-kinesin transport, indicating that membrane-dependent velocity enhancement for team-based transport arises from altered interactions between kinesins. Our study demonstrates that membrane-based coupling between motors is a key determinant of kinesin-based transport. Enhanced velocity may be critical for fast delivery of cargos in live cells.

Depletion force induced collective motion of microtubules driven by kinesin

NASA Astrophysics Data System (ADS)

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

2015-10-01

considered a model system to experimentally investigate various aspects of group behavior and pattern formation by self-propelled objects. In the in vitro gliding assay, cytoskeletal filaments F-actin or microtubules are driven by the surface immobilized associated biomolecular motors myosin or dynein respectively. Although the F-actin/myosin or microtubule/dynein system was found to be promising in understanding the collective motion and pattern formation by self-propelled objects, the most widely used biomolecular motor system microtubule/kinesin could not be successfully employed so far in this regard. Failure in exhibiting collective motion by kinesin driven microtubules is attributed to the intrinsic properties of kinesin, which was speculated to affect the behavior of individual gliding microtubules and mutual interactions among them. In this work, for the first time, we have demonstrated the collective motion of kinesin driven microtubules by regulating the mutual interaction among the gliding microtubules, by employing a depletion force among them. Proper regulation of the mutual interaction among the gliding microtubules through the employment of the depletion force was found to allow the exhibition of collective motion and stream pattern formation by the microtubules. This work offers a universal means for demonstrating the collective motion using the in vitro gliding assay of biomolecular motor systems and will help obtain a meticulous understanding of the fascinating coordinated behavior and pattern formation by self-propelled objects. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr02213d

Modelling of internal architecture of kinesin nanomotor as a machine language.

PubMed

Khataee, H R; Ibrahim, M Y

2012-09-01

Kinesin is a protein-based natural nanomotor that transports molecular cargoes within cells by walking along microtubules. Kinesin nanomotor is considered as a bio-nanoagent which is able to sense the cell through its sensors (i.e. its heads and tail), make the decision internally and perform actions on the cell through its actuator (i.e. its motor domain). The study maps the agent-based architectural model of internal decision-making process of kinesin nanomotor to a machine language using an automata algorithm. The applied automata algorithm receives the internal agent-based architectural model of kinesin nanomotor as a deterministic finite automaton (DFA) model and generates a regular machine language. The generated regular machine language was acceptable by the architectural DFA model of the nanomotor and also in good agreement with its natural behaviour. The internal agent-based architectural model of kinesin nanomotor indicates the degree of autonomy and intelligence of the nanomotor interactions with its cell. Thus, our developed regular machine language can model the degree of autonomy and intelligence of kinesin nanomotor interactions with its cell as a language. Modelling of internal architectures of autonomous and intelligent bio-nanosystems as machine languages can lay the foundation towards the concept of bio-nanoswarms and next phases of the bio-nanorobotic systems development.

Single molecule FRET observation of kinesin-1’s head-tail interaction on microtubule

PubMed Central

Aoki, Takahiro; Tomishige, Michio; Ariga, Takayuki

2013-01-01

Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the micro-tubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility. PMID:27493553

RNA polymerase III mutants in TFIIFα-like C37 that cause terminator readthrough with no decrease in transcription output.

PubMed

Rijal, Keshab; Maraia, Richard J

2013-01-07

How eukaryotic RNA polymerases switch from elongation to termination is unknown. Pol III subunits Rpc53 and Rpc37 (C53/37) form a heterodimer homologous to TFIIFβ/α. C53/37 promotes efficient termination and together with C11 also mediates pol III recycling in vitro. We previously developed Schizosaccharomyces pombe strains that report on two pol III termination activities: RNA oligo(U) 3'-end cleavage, and terminator readthrough. We randomly mutagenized C53 and C37 and isolated many C37 mutants with terminator readthrough but no comparable C53 mutants. The majority of C37 mutants have strong phenotypes with up to 40% readthrough and map to a C-terminal tract previously localized near Rpc2p in the pol III active center while a minority represent a distinct class with weaker phenotype, less readthrough and 3'-oligo(U) lengthening. Nascent pre-tRNAs released from a terminator by C37 mutants have shorter 3'-oligo(U) tracts than in cleavage-deficient C11 double mutants indicating RNA 3'-end cleavage during termination. We asked whether termination deficiency affects transcription output in the mutants in vivo both by monitoring intron-containing nascent transcript levels and (14)C-uridine incorporation. Surprisingly, multiple termination mutants have no decrease in transcript output relative to controls. These data are discussed in context of current models of pol III transcription.

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry.

PubMed

Martin, Douglas S; Fathi, Reza; Mitchison, Timothy J; Gelles, Jeff

2010-03-23

As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."

A self-powered kinesin-microtubule system for smart cargo delivery

NASA Astrophysics Data System (ADS)

Jia, Yi; Dong, Weiguang; Feng, Xiyun; Li, Jieling; Li, Junbai

2014-11-01

A smart self-powered cargo delivery system that is composed of creatine phosphate kinase (CPK) microspheres, kinesins and microtubules is demonstrated. The CPK microsphere not only acts as an ATP generation and buffering system, but also as a carrier for cargo transport, thus realizing the easy loading and self-powered delivery of cargos at the same time.A smart self-powered cargo delivery system that is composed of creatine phosphate kinase (CPK) microspheres, kinesins and microtubules is demonstrated. The CPK microsphere not only acts as an ATP generation and buffering system, but also as a carrier for cargo transport, thus realizing the easy loading and self-powered delivery of cargos at the same time. Electronic supplementary information (ESI) available: Experimental details, Fig. S1-S4, and Mov. S1-S6. See DOI: 10.1039/c4nr04454a

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization*

PubMed Central

Wang, Weiyi; Jiang, Qiyang; Argentini, Manuela; Cornu, David; Gigant, Benoît; Knossow, Marcel; Wang, Chunguang

2012-01-01

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension. PMID:22403406

RNA polymerase III mutants in TFIIFα-like C37 that cause terminator readthrough with no decrease in transcription output

PubMed Central

Rijal, Keshab; Maraia, Richard J.

2013-01-01

How eukaryotic RNA polymerases switch from elongation to termination is unknown. Pol III subunits Rpc53 and Rpc37 (C53/37) form a heterodimer homologous to TFIIFβ/α. C53/37 promotes efficient termination and together with C11 also mediates pol III recycling in vitro. We previously developed Schizosaccharomyces pombe strains that report on two pol III termination activities: RNA oligo(U) 3′-end cleavage, and terminator readthrough. We randomly mutagenized C53 and C37 and isolated many C37 mutants with terminator readthrough but no comparable C53 mutants. The majority of C37 mutants have strong phenotypes with up to 40% readthrough and map to a C-terminal tract previously localized near Rpc2p in the pol III active center while a minority represent a distinct class with weaker phenotype, less readthrough and 3′-oligo(U) lengthening. Nascent pre-tRNAs released from a terminator by C37 mutants have shorter 3′-oligo(U) tracts than in cleavage-deficient C11 double mutants indicating RNA 3′-end cleavage during termination. We asked whether termination deficiency affects transcription output in the mutants in vivo both by monitoring intron-containing nascent transcript levels and 14C-uridine incorporation. Surprisingly, multiple termination mutants have no decrease in transcript output relative to controls. These data are discussed in context of current models of pol III transcription. PMID:23093604

The bipolar assembly domain of the mitotic motor kinesin-5

PubMed Central

Acar, Seyda; Carlson, David B.; Budamagunta, Madhu S.; Yarov-Yarovoy, Vladimir; Correia, John J.; Niñonuevo, Milady R.; Jia, Weitao; Tao, Li; Leary, Julie A.; Voss, John C.; Evans, James E.; Scholey, Jonathan M.

2013-01-01

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5’s bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil ‘BASS’ (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments. PMID:23299893

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

PubMed Central

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

2016-01-01

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

Functional diversification of the kinesin-14 family in land plants.

PubMed

Gicking, Allison M; Swentowsky, Kyle W; Dawe, R Kelly; Qiu, Weihong

2018-05-12

In most eukaryotes, cytoplasmic dynein serves as the primary cytoskeletal motor for minus-end-directed processes along microtubules. However, land plants lack dynein, having instead a large number of kinesin-14s, which suggests that kinesin-14s may have evolved to fill the cellular niche left by dynein. In addition, land plants do not have centrosomes, but contain specialized microtubule-based structures called phragmoplasts that facilitate the formation of new cell walls following cell division. This Review aims to compile the evidence for functional diversification of kinesin-14s in land plants. Known functions include spindle morphogenesis, microtubule-based trafficking, nuclear migration, chloroplast distribution, and phragmoplast expansion. Plant kinesin-14s have also evolved direct roles in chromosome segregation in maize and novel biochemical features such as actin transport and processive motility in the homodimeric state. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Amyloid-like Self-Assembly of a Cellular Compartment.

PubMed

Boke, Elvan; Ruer, Martine; Wühr, Martin; Coughlin, Margaret; Lemaitre, Regis; Gygi, Steven P; Alberti, Simon; Drechsel, David; Hyman, Anthony A; Mitchison, Timothy J

2016-07-28

Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Bidirectional motility of kinesin-5 motor proteins: structural determinants, cumulative functions and physiological roles.

PubMed

Singh, Sudhir Kumar; Pandey, Himanshu; Al-Bassam, Jawdat; Gheber, Larisa

2018-05-01

Mitotic kinesin-5 bipolar motor proteins perform essential functions in mitotic spindle dynamics by crosslinking and sliding antiparallel microtubules (MTs) apart within the mitotic spindle. Two recent studies have indicated that single molecules of Cin8, the Saccharomyces cerevisiae kinesin-5 homolog, are minus end-directed when moving on single MTs, yet switch directionality under certain experimental conditions (Gerson-Gurwitz et al., EMBO J 30:4942-4954, 2011; Roostalu et al., Science 332:94-99, 2011). This finding was unexpected since the Cin8 catalytic motor domain is located at the N-terminus of the protein, and such kinesins have been previously thought to be exclusively plus end-directed. In addition, the essential intracellular functions of kinesin-5 motors in separating spindle poles during mitosis can only be accomplished by plus end-directed motility during antiparallel sliding of the spindle MTs. Thus, the mechanism and possible physiological role of the minus end-directed motility of kinesin-5 motors remain unclear. Experimental and theoretical studies from several laboratories in recent years have identified additional kinesin-5 motors that are bidirectional, revealed structural determinants that regulate directionality, examined the possible mechanisms involved and have proposed physiological roles for the minus end-directed motility of kinesin-5 motors. Here, we summarize our current understanding of the remarkable ability of certain kinesin-5 motors to switch directionality when moving along MTs.

A Mutation in γ-Tubulin Alters Microtubule Dynamics and Organization and Is Synthetically Lethal with the Kinesin-like Protein Pkl1pV⃞

PubMed Central

Paluh, Janet L.; Nogales, Eva; Oakley, Berl R.; McDonald, Kent; Pidoux, Alison L.; Cande, W. Z.

2000-01-01

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component. PMID:10749926

Surface-Bound Casein Modulates the Adsorption and Activity of Kinesin on SiO2 Surfaces

PubMed Central

Ozeki, Tomomitsu; Verma, Vivek; Uppalapati, Maruti; Suzuki, Yukiko; Nakamura, Mikihiko; Catchmark, Jeffrey M.; Hancock, William O.

2009-01-01

Abstract Conventional kinesin is routinely adsorbed to hydrophilic surfaces such as SiO2. Pretreatment of surfaces with casein has become the standard protocol for achieving optimal kinesin activity, but the mechanism by which casein enhances kinesin surface adsorption and function is poorly understood. We used quartz crystal microbalance measurements and microtubule gliding assays to uncover the role that casein plays in enhancing the activity of surface-adsorbed kinesin. On SiO2 surfaces, casein adsorbs as both a tightly bound monolayer and a reversibly bound second layer that has a dissociation constant of 500 nM and can be desorbed by washing with casein-free buffer. Experiments using truncated kinesins demonstrate that in the presence of soluble casein, kinesin tails bind well to the surface, whereas kinesin head binding is blocked. Removing soluble casein reverses these binding profiles. Surprisingly, reversibly bound casein plays only a moderate role during kinesin adsorption, but it significantly enhances kinesin activity when surface-adsorbed motors are interacting with microtubules. These results point to a model in which a dynamic casein bilayer prevents reversible association of the heads with the surface and enhances association of the kinesin tail with the surface. Understanding protein-surface interactions in this model system should provide a framework for engineering surfaces for functional adsorption of other motor proteins and surface-active enzymes. PMID:19383474

Structure and expression of the Xenopus retinoblastoma gene.

PubMed

Destrée, O H; Lam, K T; Peterson-Maduro, L J; Eizema, K; Diller, L; Gryka, M A; Frebourg, T; Shibuya, E; Friend, S H

1992-09-01

We have cloned a Xenopus homology (XRb1) of the human retinoblastoma susceptibility gene. DNA sequence analysis shows that the XRb1 gene product is highly conserved in many regions. The leucine repeat motif and many of the potential cdc2 phosphorylation sites, as well as potential sites for other kinases, are retained. The region of the protein homologous to the SV40 T antigen binding site and the basic region directly C-terminal to the E1A binding site are all conserved. XRb1 gene expression at the RNA level was studied by Northern blot analysis. Transcripts of 4.2 and 10-kb are present as maternal RNA stores in the oocyte. While the 4.2-kb product is stable until at least the mid-blastula stage, the 10-kb transcript is selectively degraded. Between stages 11 and 13 the 10-kb transcript reappears and also a minor product of approximately 11 kb becomes apparent. Both the 4.2- and the 10-kb transcripts remain present until later stages of development and are also present in all adult tissues examined, although at differing levels. Antibodies raised against human p105Rb which recognize the protein product of the XRb1 gene, pXRb1, detect the Xenopus 99-kDa protein prior to the mid-blastula stage, but at lower levels than at later stages in development.

Coupling of kinesin ATP turnover to translocation and microtubule regulation: one engine, many machines.

PubMed

Friel, Claire T; Howard, Jonathon

2012-12-01

The cycle of ATP turnover is integral to the action of motor proteins. Here we discuss how variation in this cycle leads to variation of function observed amongst members of the kinesin superfamily of microtubule associated motor proteins. Variation in the ATP turnover cycle among superfamily members can tune the characteristic kinesin motor to one of the range of microtubule-based functions performed by kinesins. The speed at which ATP is hydrolysed affects the speed of translocation. The ratio of rate constants of ATP turnover in relation to association and dissociation from the microtubule influence the processivity of translocation. Variation in the rate-limiting step of the cycle can reverse the way in which the motor domain interacts with the microtubule producing non-motile kinesins. Because the ATP turnover cycle is not fully understood for the majority of kinesins, much work remains to show how the kinesin engine functions in such a wide variety of molecular machines.

A novel kinesin-like protein, KIF1Bbeta3 is involved in the movement of lysosomes to the cell periphery in non-neuronal cells.

PubMed

Matsushita, Masafumi; Tanaka, Shingo; Nakamura, Norihiro; Inoue, Hiroki; Kanazawa, Hiroshi

2004-03-01

The kinesin superfamily protein, KIF1Bbeta, a splice variant of KIF1B, is involved in the transport of synaptic vesicles in neuronal cells, and is also expressed in various non-neuronal tissues. To elucidate the functions of KIF1Bbeta in non-neuronal cells, we analyzed the intracellular localization of KIF1Bbeta and characterized its isoform expression profile. In COS-7 cells, KIF1B colocalized with lysosomal markers and expression of a mutant form of KIF1Bbeta, lacking the motor domain, impaired the intracellular distribution of lysosomes. A novel isoform of the kinesin-like protein, KIF1Bbeta3, was identified in rat and simian kidney. It lacks the 5th exon of the KIF1Bbeta-specific tail region. Overexpression of KIF1Bbeta3 induced the translocation of lysosomes to the cell periphery. However, overexpression of KIF1Bbeta3-Q98L, which harbors a pathogenic mutation associated with a familial neuropathy, Charcot-Marie-Tooth disease type 2 A, resulted in the abnormal perinuclear clustering of lysosomes. These results indicate that KIF1Bbeta3 is involved in the translocation of lysosomes from perinuclear regions to the cell periphery.

Microscale transport and sorting by kinesin molecular motors.

PubMed

Jia, Lili; Moorjani, Samira G; Jackson, Thomas N; Hancock, William O

2004-03-01

As biomolecular detection systems shrink in size, there is an increasing demand for systems that transport and position materials at micron- and nanoscale dimensions. Our goal is to combine cellular transport machinery-kinesin molecular motors and microtubules-with integrated optoelectronics into a hybrid biological/engineered microdevice that will bind, transport, and detect specific proteins, DNA/RNA molecules, viruses, or cells. For microscale transport, 1.5 microm deep channels were created with SU-8 photoresist on glass, kinesin motors adsorbed to the bottom of the channels, and the channel walls used to bend and redirect microtubules moving over the immobilized motors. Novel channel geometries were investigated as a means to redirect and sort microtubules moving in these channels. We show that DC and AC electric fields are sufficient to transport microtubules in solution, establishing an approach for redirecting microtubules moving in channels. Finally, we inverted the geometry to demonstrate that kinesins can transport gold nanowires along surface immobilized microtubules, providing a model for nanoscale directed assembly.

Expression patterns of Xenopus FGF receptor-like 1/nou-darake in early Xenopus development resemble those of planarian nou-darake and Xenopus FGF8.

PubMed

Hayashi, Shuichi; Itoh, Mari; Taira, Sumiko; Agata, Kiyokazu; Taira, Masanori

2004-08-01

Fibroblast growth factors (FGFs) mediate many cell-to-cell signaling events during early development. Nou-darake (ndk), a gene encoding an FGF receptor (FGFR)-like molecule, was found to be highly and specifically expressed in the head region of the planarian Dugesia japonica, and its functional analyses provided strong molecular evidence for the existence of a brain-inducing circuit based on the FGF signaling pathway. To analyze the role of ndk during vertebrate development, we isolated the Xenopus ortholog of ndk, the vertebrate FGFR-like 1 gene (XFGFRL1). Expression of XFGFRL1/Xndk was first detected in the anterior region at the late gastrula stage and dramatically increased at the early neurula stage in an overall anterior mesendodermal region, including the prechordal plate, paraxial mesoderm, anterior endoderm, and archenteron roof. This anterior expression pattern resembles that of ndk in planarians, suggesting that the expression of FGFRL1/ndk is conserved in evolution between these two distantly diverged organisms. During the tail bud stages, XFGFRL1/Xndk expression was detected in multiple regions, including the forebrain, eyes, midbrain-hindbrain boundary, otic vesicles, visceral arches, and somites. In many of these regions, XFGFRL1/Xndk was coexpressed with XFGF8, indicating that XFGFRL1/Xndk is a member of the XFGF8 synexpression group, which includes sprouty, sef, and isthmin. Copyright 2004 Wiley-Liss, Inc.

Effect of fuel concentration on cargo transport by a team of Kinesin motors

NASA Astrophysics Data System (ADS)

Takshak, Anjneya; Mishra, Nirvantosh; Kulkarni, Aditi; Kunwar, Ambarish

2017-02-01

Eukaryotic cells employ specialized proteins called molecular motors for transporting organelles and vesicles from one location to another in a regulated and directed manner. These molecular motors often work collectively in a team while transporting cargos. Molecular motors use cytoplasmic ATP as fuel, which is hydrolyzed to generate mechanical force. While the effect of ATP concentration on cargo transport by single Kinesin motor function is well understood, it is still unexplored, both theoretically and experimentally, how ATP concentration would affect cargo transport by a team of Kinesin motors. For instance, how does fuel concentration affect the travel distances and travel velocities of cargo? How cooperativity of Kinesin motors engaged on a cargo is affected by ATP concentration? To answer these questions, here we develop mechano-chemical models of cargo transport by a team of Kinesin motors. To develop these models we use experimentally-constrained mechano-chemical model of a single Kinesin motor as well as earlier developed mean-field and stochastic models of load sharing for cargo transport. Thus, our new models for cargo transport by a team of Kinesin motors include fuel concentration explicitly, which was not considered in earlier models. We make several interesting predictions which can be tested experimentally. For instance, the travel distances of cargos are very large at limited ATP concentrations in spite of very small travel velocity. Velocities of cargos driven by multiple Kinesin have a Michaelis-Menten dependence on ATP concentration. Similarly, cooperativity among the engaged Kinesin motors on the cargo shows a Michaelis-Menten type dependence, which attains a maximum value near physiological ATP concentrations. Our new results can be potentially useful in controlling artificial nano-molecular shuttles precisely for targeted delivery in various nano-technological applications.

Correlation Imaging Reveals Specific Crowding Dynamics of Kinesin Motor Proteins

NASA Astrophysics Data System (ADS)

Miedema, Daniël M.; Kushwaha, Vandana S.; Denisov, Dmitry V.; Acar, Seyda; Nienhuis, Bernard; Peterman, Erwin J. G.; Schall, Peter

2017-10-01

Molecular motor proteins fulfill the critical function of transporting organelles and other building blocks along the biopolymer network of the cell's cytoskeleton, but crowding effects are believed to crucially affect this motor-driven transport due to motor interactions. Physical transport models, like the paradigmatic, totally asymmetric simple exclusion process (TASEP), have been used to predict these crowding effects based on simple exclusion interactions, but verifying them in experiments remains challenging. Here, we introduce a correlation imaging technique to precisely measure the motor density, velocity, and run length along filaments under crowding conditions, enabling us to elucidate the physical nature of crowding and test TASEP model predictions. Using the kinesin motor proteins kinesin-1 and OSM-3, we identify crowding effects in qualitative agreement with TASEP predictions, and we achieve excellent quantitative agreement by extending the model with motor-specific interaction ranges and crowding-dependent detachment probabilities. These results confirm the applicability of basic nonequilibrium models to the intracellular transport and highlight motor-specific strategies to deal with crowding.

Kinesins and Myosins: Molecular Motors that Coordinate Cellular Functions in Plants.

PubMed

Nebenführ, Andreas; Dixit, Ram

2018-04-29

Kinesins and myosins are motor proteins that can move actively along microtubules and actin filaments, respectively. Plants have evolved a unique set of motors that function as regulators and organizers of the cytoskeleton and as drivers of long-distance transport of various cellular components. Recent progress has established the full complement of motors encoded in plant genomes and has revealed valuable insights into the cellular functions of many kinesin and myosin isoforms. Interestingly, several of the motors were found to functionally connect the two cytoskeletal systems and thereby to coordinate their activities. In this review, we discuss the available genetic, cell biological, and biochemical data for each of the plant kinesin and myosin families from the context of their subcellular mechanism of action as well as their physiological function in the whole plant. We particularly emphasize work that illustrates mechanisms by which kinesins and myosins coordinate the activities of the cytoskeletal system.

Construction of artificial cilia from microtubules and kinesins through a well-designed bottom-up approach.

PubMed

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

2018-04-05

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

Physical limits on kinesin-5–mediated chromosome congression in the smallest mitotic spindles

PubMed Central

McCoy, Kelsey M.; Tubman, Emily S.; Claas, Allison; Tank, Damien; Clancy, Shelly Applen; O’Toole, Eileen T.; Berman, Judith; Odde, David J.

2015-01-01

A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro­tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end–tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5–mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to

Role of the C-terminal extensions of alpha-crystallins. Swapping the C-terminal extension of alpha-crystallin to alphaB-crystallin results in enhanced chaperone activity.

PubMed

Pasta, Saloni Yatin; Raman, Bakthisaran; Ramakrishna, Tangirala; Rao, Ch Mohan

2002-11-29

Several small heat shock proteins contain a well conserved alpha-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of alphaA- and alphaB-crystallins, is not well understood. We have swapped the C-terminal extensions between alphaA- and alphaB-crystallins and generated two novel chimeric proteins, alphaABc and alphaBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric alphaB with the C-terminal extension of alphaA-crystallin, alphaBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric alphaA with the C-terminal extension of alphaB-crystallin, alphaABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that alphaBAc exhibits more solvent-exposed hydrophobic pockets than alphaA, alphaB, or alphaABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of alphaBAc differs from that of alphaB-crystallin whereas that of alphaABc overlaps with that of alphaA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.

The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner

PubMed Central

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

2017-01-01

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

Maternal syntabulin is required for dorsal axis formation and is a germ plasm component in Xenopus.

PubMed

Colozza, Gabriele; De Robertis, Edward M

2014-07-01

In amphibians and teleosts, early embryonic axial development is driven by maternally deposited mRNAs and proteins, called dorsal determinants, which migrate to the presumptive dorsal side of the embryo in a microtubule-dependent manner after fertilization. Syntabulin is an adapter protein that binds to kinesin KIF5B and to the transmembrane protein Syntaxin1. In zebrafish, a mutation in Syntabulin causes complete embryo ventralization. It is unknown whether Syntabulin plays an analogous role during early development of other species, a question addressed here in Xenopus laevis. in situ hybridization of syntabulin mRNA was carried out at different stages of Xenopus development. In oocytes, syntabulin transcripts were localized to the vegetal cortex of large oocytes and the mitochondrial cloud of very young oocytes. We extended the zebrafish data by finding that during cleavage Xenopus syntabulin mRNA localized to the germ plasm and was later expressed in primordial germ cells (PGCs). This new finding suggested a role for Syntabulin during germ cell differentiation. The functional role of maternal syntabulin mRNA was investigated by knock-down with phosphorothioate DNA antisense oligos followed by oocyte transfer. The results showed that syntabulin mRNA depletion caused the complete loss of dorso-anterior axis formation in frog embryos. Consistent with the ventralized phenotype, syntabulin-depleted embryos displayed severe reduction of dorsal markers and ubiquitous transcription of the ventral marker sizzled. Syntabulin was required for the maternal Wnt/β-Catenin signal, since ventralization could be completely rescued by injection of β-catenin (or syntabulin) mRNA. The data suggest an evolutionarily conserved role for Syntabulin, a protein that bridges microtubule motors and membrane vesicles, during dorso-ventral axis formation in the vertebrates. Copyright © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Transport of fungal RAB11 secretory vesicles involves myosin-5, dynein/dynactin/p25, and kinesin-1 and is independent of kinesin-3

PubMed Central

Peñalva, Miguel A.; Zhang, Jun; Xiang, Xin; Pantazopoulou, Areti

2017-01-01

Hyphal tip cells of the fungus Aspergillus nidulans are useful for studying long-range intracellular traffic. Post-Golgi secretory vesicles (SVs) containing the RAB11 orthologue RabE engage myosin-5 as well as plus end– and minus end–directed microtubule motors, providing an experimental system with which to investigate the interplay between microtubule and actin motors acting on the same cargo. By exploiting the fact that depolymerization of F-actin unleashes SVs focused at the apex by myosin-5 to microtubule-dependent motors, we establish that the minus end–directed transport of SVs requires the dynein/dynactin supercomplex. This minus end–directed transport is largely unaffected by genetic ablation of the Hook complex adapting early endosomes (EEs) to dynein but absolutely requires p25 in dynactin. Thus dynein recruitment to two different membranous cargoes, namely EEs and SVs, requires p25, highlighting the importance of the dynactin pointed-end complex to scaffold cargoes. Finally, by studying the behavior of SVs and EEs in null and rigor mutants of kinesin-3 and kinesin-1 (UncA and KinA, respectively), we demonstrate that KinA is the major kinesin mediating the anterograde transport of SVs. Therefore SVs arrive at the apex of A. nidulans by anterograde transport involving cooperation of kinesin-1 with myosin-5 and can move away from the apex powered by dynein. PMID:28209731

Discovery of a novel inhibitor of kinesin-like protein KIFC1.

PubMed

Zhang, Wei; Zhai, Ling; Wang, Yimin; Boohaker, Rebecca J; Lu, Wenyan; Gupta, Vandana V; Padmalayam, Indira; Bostwick, Robert J; White, E Lucile; Ross, Larry J; Maddry, Joseph; Ananthan, Subramaniam; Augelli-Szafran, Corinne E; Suto, Mark J; Xu, Bo; Li, Rongbao; Li, Yonghe

2016-04-15

Historically, drugs used in the treatment of cancers also tend to cause damage to healthy cells while affecting cancer cells. Therefore, the identification of novel agents that act specifically against cancer cells remains a high priority in the search for new therapies. In contrast with normal cells, most cancer cells contain multiple centrosomes which are associated with genome instability and tumorigenesis. Cancer cells can avoid multipolar mitosis, which can cause cell death, by clustering the extra centrosomes into two spindle poles, thereby enabling bipolar division. Kinesin-like protein KIFC1 plays a critical role in centrosome clustering in cancer cells, but is not essential for normal cells. Therefore, targeting KIFC1 may provide novel insight into selective killing of cancer cells. In the present study, we identified a small-molecule KIFC1 inhibitor, SR31527, which inhibited microtubule (MT)-stimulated KIFC1 ATPase activity with an IC50 value of 6.6 μM. By using bio layer interferometry technology, we further demonstrated that SR31527 bound directly to KIFC1 with high affinity (Kd=25.4 nM). Our results from computational modelling and saturation-transfer difference (STD)-NMR experiments suggest that SR31527 bound to a novel allosteric site of KIFC1 that appears suitable for developing selective inhibitors of KIFC1. Importantly, SR31527 prevented bipolar clustering of extra centrosomes in triple negative breast cancer (TNBC) cells and significantly reduced TNBC cell colony formation and viability, but was less toxic to normal fibroblasts. Therefore, SR31527 provides a valuable tool for studying the biological function of KIFC1 and serves as a potential lead for the development of novel therapeutic agents for breast cancer treatment. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Involvement of the kinesin family members KIF4A and KIF5C in intellectual disability and synaptic function.

PubMed

Willemsen, Marjolein H; Ba, Wei; Wissink-Lindhout, Willemijn M; de Brouwer, Arjan P M; Haas, Stefan A; Bienek, Melanie; Hu, Hao; Vissers, Lisenka E L M; van Bokhoven, Hans; Kalscheuer, Vera; Nadif Kasri, Nael; Kleefstra, Tjitske

2014-07-01

Kinesin superfamily (KIF) genes encode motor proteins that have fundamental roles in brain functioning, development, survival and plasticity by regulating the transport of cargo along microtubules within axons, dendrites and synapses. Mouse knockout studies support these important functions in the nervous system. The role of KIF genes in intellectual disability (ID) has so far received limited attention, although previous studies have suggested that many ID genes impinge on synaptic function. By applying next-generation sequencing (NGS) in ID patients, we identified likely pathogenic mutations in KIF4A and KIF5C. To further confirm the pathogenicity of these mutations, we performed functional studies at the level of synaptic function in primary rat hippocampal neurons. Four males from a single family with a disruptive mutation in the X-linked KIF4A (c.1489-8_1490delins10; p.?- exon skipping) showed mild to moderate ID and epilepsy. A female patient with a de novo missense mutation in KIF5C (c.11465A>C; p.(Glu237Lys)) presented with severe ID, epilepsy, microcephaly and cortical malformation. Knock-down of Kif4a in rat primary hippocampal neurons altered the balance between excitatory and inhibitory synaptic transmission, whereas the mutation in Kif5c affected its protein function at excitatory synapses. Our results suggest that mutations in KIF4A and KIF5C cause ID by tipping the balance between excitatory and inhibitory synaptic excitability. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

A method for multiprotein assembly in cells reveals independent action of kinesins in complex

PubMed Central

Norris, Stephen R.; Soppina, Virupakshi; Dizaji, Aslan S.; Schimert, Kristin I.; Sept, David; Cai, Dawen; Sivaramakrishnan, Sivaraj

2014-01-01

Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track. PMID:25365993

Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1

DOE PAGES

VanDelinder, Virginia; Adams, Peter G.; Bachand, George D.

2016-12-21

The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continuemore » to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.« less

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

PubMed

Semino, C E; Specht, C A; Raimondi, A; Robbins, P W

1996-05-14

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome

PubMed Central

Dodding, Mark P; Mitter, Richard; Humphries, Ashley C; Way, Michael

2011-01-01

Transport of cargoes by kinesin-1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit kinesin-1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define kinesin-1 binding. We now show that a bipartite tryptophan-based kinesin-1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for kinesin-1-dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan-based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases. PMID:21915095

Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic [published errata appear in J Cell Biol 1995 Mar;128(5):following 988 and 1995 May;129(3):893

PubMed Central

1995-01-01

The distribution and dynamics of both the ER and Golgi complex in animal cells are known to be dependent on microtubules; in many cell types the ER extends toward the plus ends of microtubules at the cell periphery and the Golgi clusters at the minus ends of microtubules near the centrosome. In this study we provide evidence that the microtubule motor, kinesin, is present on membranes cycling between the ER and Golgi and powers peripherally directed movements of membrane within this system. Immunolocalization of kinesin at both the light and electron microscopy levels in NRK cells using the H1 monoclonal antibody to kinesin heavy chain, revealed kinesin to be associated with all membranes of the ER/Golgi system. At steady-state at 37 degrees C, however, kinesin was most concentrated on peripherally distributed, pre- Golgi structures containing beta COP and vesicular stomatitis virus glycoprotein newly released from the ER. Upon temperature reduction or nocodazole treatment, kinesin's distribution shifted onto the Golgi, while with brefeldin A (BFA)-treatment, kinesin could be found in both Golgi-derived tubules and in the ER. This suggested that kinesin associates with membranes that constitutively cycle between the ER and Golgi. Kinesin's role on these membranes was examined by microinjecting kinesin antibody. Golgi-to-ER but not ER-to-Golgi membrane transport was found to be inhibited by the microinjected anti-kinesin, suggesting kinesin powers the microtubule plus end-directed recycling of membrane to the ER, and remains inactive on pre-Golgi intermediates that move toward the Golgi complex. PMID:7844144

Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast

PubMed Central

Rincon, Sergio A.; Lamson, Adam; Blackwell, Robert; Syrovatkina, Viktoriya; Fraisier, Vincent; Paoletti, Anne; Betterton, Meredith D.; Tran, Phong T.

2017-01-01

Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. Accordingly, Kinesin-5 inactivation results in force imbalance leading to monopolar spindle and chromosome segregation failure. In fission yeast, force balance is restored when both kinesin-5 Cut7 and kinesin-14 Pkl1 are deleted, restoring spindle bipolarity. Here we show that the cut7Δpkl1Δ spindle is fully competent for chromosome segregation independently of motor activity, except for kinesin-6 Klp9, which is required for anaphase spindle elongation. We demonstrate that cut7Δpkl1Δ spindle bipolarity requires the microtubule antiparallel bundler PRC1/Ase1 to recruit CLASP/Cls1 to stabilize microtubules. Brownian dynamics-kinetic Monte Carlo simulations show that Ase1 and Cls1 activity are sufficient for initial bipolar spindle formation. We conclude that pushing forces generated by microtubule polymerization are sufficient to promote spindle pole separation and the assembly of bipolar spindle in the absence of molecular motors. PMID:28513584

Kinesin Motor Enzymology: Chemistry, Structure, and Physics of Nanoscale Molecular Machines.

PubMed

Cochran, J C

2015-09-01

Molecular motors are enzymes that convert chemical potential energy into controlled kinetic energy for mechanical work inside cells. Understanding the biophysics of these motors is essential for appreciating life as well as apprehending diseases that arise from motor malfunction. This review focuses on kinesin motor enzymology with special emphasis on the literature that reports the chemistry, structure and physics of several different kinesin superfamily members.

A Kinesin-Related Protein Required for the Mitotic Spindle Assembly

DTIC Science & Technology

1999-05-01

8217 at 23 °C. The residual 20 ul can be frozen in liquid nitrogen and used to estimate total recovery if desired. 3. Remove supernatant as thoroughly...communicated to the XKCM1 protein, and what can these results tell us about how chromosomes are segregated? These are questions that are currently...METHODS 3.1 Preparation of Microtubule Substrates The effects of kinesins on MT dynamics can be assayed by using three different types of MT

The C-terminal region of the motor protein MCAK controls its structure and activity through a conformational switch

PubMed Central

Talapatra, Sandeep K; Harker, Bethany; Welburn, Julie PI

2015-01-01

The precise regulation of microtubule dynamics is essential during cell division. The kinesin-13 motor protein MCAK is a potent microtubule depolymerase. The divergent non-motor regions flanking the ATPase domain are critical in regulating its targeting and activity. However, the molecular basis for the function of the non-motor regions within the context of full-length MCAK is unknown. Here, we determine the structure of MCAK motor domain bound to its regulatory C-terminus. Our analysis reveals that the MCAK C-terminus binds to two motor domains in solution and is displaced allosterically upon microtubule binding, which allows its robust accumulation at microtubule ends. These results demonstrate that MCAK undergoes long-range conformational changes involving its C-terminus during the soluble to microtubule-bound transition and that the C-terminus-motor interaction represents a structural intermediate in the MCAK catalytic cycle. Together, our work reveals intrinsic molecular mechanisms underlying the regulation of kinesin-13 activity. DOI: http://dx.doi.org/10.7554/eLife.06421.001 PMID:25915621

The Mechanochemical Cycle of Mammalian Kinesin-2 KIF3A/B under Load.

PubMed

Andreasson, Johan O L; Shastry, Shankar; Hancock, William O; Block, Steven M

2015-05-04

The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length, and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains and not to the neck linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1. Copyright © 2015 Elsevier Ltd. All rights reserved.

The mechanochemical cycle of mammalian kinesin-2 KIF3A/B under load

PubMed Central

Andreasson, Johan O.L.; Shastry, Shankar; Hancock, William O.; Block, Steven M.

2015-01-01

Summary The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains, and not to the neck-linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1. PMID:25866395

Glider and Vision: two new families of miniature inverted-repeat transposable elements in Xenopus laevis genome.

PubMed

Lepetit, D; Pasquet, S; Olive, M; Thézé, N; Thiébaud, P

2000-01-01

We have characterised from Xenopus laevis two new short interspersed repetitive elements, we have named Glider and Vision, that belong to the family of miniature inverted-repeat transposable elements (MITEs). Glider was first characterised in an intronic region of the alpha-tropomyosin (alpha-TM) gene and database search has revealed the presence of this element in 10 other Xenopus laevis genes. Glider elements are about 150 bp long and for some of them, their terminal inverted repeats are flanked by potential target-site duplications. Evidence for the mobility of Glider element has been provided by the presence/absence of one element at corresponding location in duplicated alpha-TM genes. Vision element has been identified in the promoter region of the cyclin dependant kinase 2 gene (cdk2) where it is boxed in a Glider element. Vision is 284bp long and is framed by 14-bp terminal inverted repeats that are flanked by 7-bp direct repeats. We have estimated that there are about 20,000 and 300 copies of Glider and Vision respectively scattered throughout the Xenopus laevis genome. Every MITEs elements but two described in our study are found either in 5' or in 3' regulatory regions of genes suggesting a potential role in gene regulation.

Chromosome congression by kinesin-5 motor-mediated disassembly of longer kinetochore microtubules

PubMed Central

Gardner, Melissa K; Bouck, David C.; Paliulis, Leocadia V.; Meehl, Janet B.; O’Toole, Eileen T.; Haase, Julian; Soubry, Adelheid; Joglekar, Ajit P.; Winey, Mark; Salmon, Edward D.; Bloom, Kerry; Odde, David J.

2008-01-01

Summary During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus-ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely-conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus-end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression. PMID:19041752

Mutant analysis of Cdt1's function in suppressing nascent strand elongation during DNA replication in Xenopus egg extracts.

PubMed

Nakazaki, Yuta; Tsuyama, Takashi; Azuma, Yutaro; Takahashi, Mikiko; Tada, Shusuke

2017-09-02

The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity. Copyright © 2017 Elsevier Inc. All rights reserved.

A Dbf4p BRCA1 C-Terminal-Like Domain Required for the Response to Replication Fork Arrest in Budding Yeast

PubMed Central

Gabrielse, Carrie; Miller, Charles T.; McConnell, Kristopher H.; DeWard, Aaron; Fox, Catherine A.; Weinreich, Michael

2006-01-01

Dbf4p is an essential regulatory subunit of the Cdc7p kinase required for the initiation of DNA replication. Cdc7p and Dbf4p orthologs have also been shown to function in the response to DNA damage. A previous Dbf4p multiple sequence alignment identified a conserved ∼40-residue N-terminal region with similarity to the BRCA1 C-terminal (BRCT) motif called “motif N.” BRCT motifs encode ∼100-amino-acid domains involved in the DNA damage response. We have identified an expanded and conserved ∼100-residue N-terminal region of Dbf4p that includes motif N but is capable of encoding a single BRCT-like domain. Dbf4p orthologs diverge from the BRCT motif at the C terminus but may encode a similar secondary structure in this region. We have therefore called this the BRCT and DBF4 similarity (BRDF) motif. The principal role of this Dbf4p motif was in the response to replication fork (RF) arrest; however, it was not required for cell cycle progression, activation of Cdc7p kinase activity, or interaction with the origin recognition complex (ORC) postulated to recruit Cdc7p–Dbf4p to origins. Rad53p likely directly phosphorylated Dbf4p in response to RF arrest and Dbf4p was required for Rad53p abundance. Rad53p and Dbf4p therefore cooperated to coordinate a robust cellular response to RF arrest. PMID:16547092

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos.

PubMed

Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei

2010-02-01

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

[Crystallography of ATP hydrolysis mechanism in rat brain kinesin].

PubMed

Wan, Qun; Zhu, Pingting; Lü, Houning; Chen, Xinhong

2014-04-01

Rat brain kinesin is a conventional kinesin that uses the energy from ATP hydrolysis to walk along the microtubule progressively. Studying how the chemical energy in ATP is utilized for mechanical movement is important to understand this moving function. The monomeric motor domain, rK354, was crystallized. An ATP analog, AMPPNP, was soaked in the active site. Comparing the complex structure of rK354 x AMPPNP and that of rK354ADP, a hypothesis is proposed that Glu237 in the Switch II region sensors the presence of gamma-phosphate and transfers the signal to the microtubule binding region.

Structure-based molecular simulations reveal the enhancement of biased Brownian motions in single-headed kinesin.

PubMed

Kanada, Ryo; Kuwata, Takeshi; Kenzaki, Hiroo; Takada, Shoji

2013-01-01

Kinesin is a family of molecular motors that move unidirectionally along microtubules (MT) using ATP hydrolysis free energy. In the family, the conventional two-headed kinesin was experimentally characterized to move unidirectionally through "walking" in a hand-over-hand fashion by coordinated motions of the two heads. Interestingly a single-headed kinesin, a truncated KIF1A, still can generate a biased Brownian movement along MT, as observed by in vitro single molecule experiments. Thus, KIF1A must use a different mechanism from the conventional kinesin to achieve the unidirectional motions. Based on the energy landscape view of proteins, for the first time, we conducted a set of molecular simulations of the truncated KIF1A movements over an ATP hydrolysis cycle and found a mechanism exhibiting and enhancing stochastic forward-biased movements in a similar way to those in experiments. First, simulating stand-alone KIF1A, we did not find any biased movements, while we found that KIF1A with a large friction cargo-analog attached to the C-terminus can generate clearly biased Brownian movements upon an ATP hydrolysis cycle. The linked cargo-analog enhanced the detachment of the KIF1A from MT. Once detached, diffusion of the KIF1A head was restricted around the large cargo which was located in front of the head at the time of detachment, thus generating a forward bias of the diffusion. The cargo plays the role of a diffusional anchor, or cane, in KIF1A "walking."

A tethering mechanism controls the processivity and kinetochore-microtubule plus-end enrichment of the kinesin-8 Kif18A

PubMed Central

Stumpff, Jason; Du, Yaqing; English, Chauca A.; Maliga, Zoltan; Wagenbach, Michael; Asbury, Charles L.; Wordeman, Linda; Ohi, Ryoma

2011-01-01

Summary Metaphase chromosome positioning depends on Kif18A, a kinesin-8 that accumulates at and suppresses the dynamics of K-MT plus ends. By engineering Kif18A mutants that suppress MT dynamics but fail to concentrate at K-MT plus-ends, we identify a mechanism that allows Kif18A to accumulate at K-MT plus ends to a level required to suppress chromosome movements. Enrichment of Kif18A at K-MT plus-ends depends on its C-terminal tail domain, while the ability of Kif18A to suppress MT growth is conferred by the N-terminal motor domain. The Kif18A tail contains a second MT-binding domain that diffuses along the MT lattice, suggesting that it tethers the motor to the MT track. Consistently, the tail enhances Kif18A processivity and is crucial for it to accumulate at K-MT plus-ends. The heightened processivity of Kif18A, conferred by its tail domain, thus promotes concentration of Kif18A at K-MT plus-ends, where it suppresses their dynamics to control chromosome movements. PMID:21884977

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

PubMed Central

Nika, Heinz; Hawke, David H.; Angeletti, Ruth Hogue

2014-01-01

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures. PMID:24688319

Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis.

PubMed

Dagle, John M; Sabel, Jaime L; Littig, Jennifer L; Sutherland, Lillian B; Kolker, Sandra J; Weeks, Daniel L

2003-10-15

The experimental manipulation of early embryologic events, resulting in the misexpression of the homeobox transcription factor pitx2, is associated with subsequent defects of laterality in a number of vertebrate systems. To clarify the role of one pitx2 isoform, pitx2c, in determining the left-right axis of amphibian embryos, we examined the heart and gut morphology of Xenopus laevis embryos after attenuating pitx2c mRNA levels using chemically modified antisense oligonucleotides. We demonstrate that the partial depletion of pitx2c mRNA in these embryos results in alteration of both cardiac morphology and intestinal coiling. The most common cardiac abnormality seen was a failure of rightward migration of the outflow tract, while the most common intestinal laterality phenotype seen was a full reversal in the direction of coiling, each present in 23% of embryos injected with the pitx2c antisense oligonucleotide. An abnormality in either the heart or gut further predisposed to a malformation in the other. In addition, a number of other cardiac anomalies were observed after pitx2c mRNA attenuation, including abnormalities of atrial septation, extracellular matrix restriction, relative atrial-ventricular chamber positioning, and restriction of ventricular development. Many of these findings correlate with cardiac defects previously reported in pitx2 null and hypomorphic mice, but can now be assigned specifically to attenuation of the pitx2c isoform in Xenopus.

SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity.

PubMed

Muramatsu, Tomonari; Takemoto, Chie; Kim, Yong-Tae; Wang, Hongfei; Nishii, Wataru; Terada, Takaho; Shirouzu, Mikako; Yokoyama, Shigeyuki

2016-11-15

The 3C-like protease (3CL pro ) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N- and C-terminal autoprocessing sites, by recognizing P4-P1 and P1'. In this study, we determined the crystal structure of 3CL pro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3' position [Phe(P3')] is snugly accommodated in the S3' pocket. Mutations of Phe(P3') impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C- and N-terminal sites, as follows. The S3' subsite is formed by Phe(P2)-induced conformational changes of 3CL pro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3' subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3'). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4-P1 with Leu(P2) inhibits protease activity, whereas that with Phe(P2) exhibits a much smaller inhibitory effect, because Phe(P3') is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2).

Decrypting the structural, dynamic, and energetic basis of a monomeric kinesin interacting with a tubulin dimer in three ATPase states by all-atom molecular dynamics simulation.

PubMed

Chakraborty, Srirupa; Zheng, Wenjun

2015-01-27

We have employed molecular dynamics (MD) simulation to investigate, with atomic details, the structural dynamics and energetics of three major ATPase states (ADP, APO, and ATP state) of a human kinesin-1 monomer in complex with a tubulin dimer. Starting from a recently solved crystal structure of ATP-like kinesin-tubulin complex by the Knossow lab, we have used flexible fitting of cryo-electron-microscopy maps to construct new structural models of the kinesin-tubulin complex in APO and ATP state, and then conducted extensive MD simulations (total 400 ns for each state), followed by flexibility analysis, principal component analysis, hydrogen bond analysis, and binding free energy analysis. Our modeling and simulation have revealed key nucleotide-dependent changes in the structure and flexibility of the nucleotide-binding pocket (featuring a highly flexible and open switch I in APO state) and the tubulin-binding site, and allosterically coupled motions driving the APO to ATP transition. In addition, our binding free energy analysis has identified a set of key residues involved in kinesin-tubulin binding. On the basis of our simulation, we have attempted to address several outstanding issues in kinesin study, including the possible roles of β-sheet twist and neck linker docking in regulating nucleotide release and binding, the structural mechanism of ADP release, and possible extension and shortening of α4 helix during the ATPase cycle. This study has provided a comprehensive structural and dynamic picture of kinesin's major ATPase states, and offered promising targets for future mutational and functional studies to investigate the molecular mechanism of kinesin motors.

Identification of Carboxypeptidase Substrates by C-Terminal COFRADIC.

PubMed

Tanco, Sebastian; Aviles, Francesc Xavier; Gevaert, Kris; Lorenzo, Julia; Van Damme, Petra

2017-01-01

We here present a detailed procedure for studying protein C-termini and their posttranslational modifications by C-terminal COFRADIC. In fact, this procedure can enrich for both C-terminal and N-terminal peptides through a combination of a strong cation exchange fractionation step at low pH, which removes the majority of nonterminal peptides in whole-proteome digests, while the actual COFRADIC step segregates C-terminal peptides from N-terminal peptides. When used in a differential mode, C-terminal COFRADIC allows for the identification of neo-C-termini generated by the action of proteases, which in turn leads to the identification of protease substrates. More specifically, this technology can be applied to determine the natural substrate repertoire of carboxypeptidases on a proteome-wide scale.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

PubMed

Reifenberger, Matthew S; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Alli, Ahmed A; Eaton, Douglas C; Alli, Abdel A

2014-07-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. Copyright © 2014 the American Physiological Society.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells

PubMed Central

Reifenberger, Matthew S.; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Eaton, Douglas C.; Alli, Abdel A.

2014-01-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na+ channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. PMID:24829507

Regulation of microtubule-based transport by MAP4

PubMed Central

Semenova, Irina; Ikeda, Kazuho; Resaul, Karim; Kraikivski, Pavel; Aguiar, Mike; Gygi, Steven; Zaliapin, Ilya; Cowan, Ann; Rodionov, Vladimir

2014-01-01

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect. PMID:25143402

Light responses in rods of vitamin A-deprived Xenopus.

PubMed

Solessio, Eduardo; Umino, Yumiko; Cameron, David A; Loew, Ellis; Engbretson, Gustav A; Knox, Barry E; Barlow, Robert B

2009-09-01

Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A-deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration. The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry. The vitamin A-deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A-deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds(-1)) was twofold greater than the maximum levels attainable by control rods ( approximately 7 seconds(-1)). Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.

Isolation and Expression Profile of the Ca2+-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis

PubMed Central

Lee, Ra Mi; Ryu, Rae Hyung; Jeong, Seong Won; Oh, Soo Jin; Huang, Hue; Han, Jin Soo; Lee, Chi Ho; Lee, C. Justin; Jan, Lily Yeh

2011-01-01

To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA. PMID:21826170

Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors.

PubMed

Furuta, Ken'ya; Furuta, Akane; Toyoshima, Yoko Y; Amino, Misako; Oiwa, Kazuhiro; Kojima, Hiroaki

2013-01-08

Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

Gene structure, transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chicken.

PubMed

Pinheiro, Pedro L C; Cardoso, João C R; Gomes, Ana S; Fuentes, Juan; Power, Deborah M; Canário, Adelino V M

2010-12-01

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken. The PTH-L gene is present throughout the vertebrates with the exception of placental mammals. Gene structure of PTH and PTH-L seems to be conserved in vertebrates while PTHrP gene structure is divergent and has acquired new exons and alternative promoters. Splice variants of PTHrP and PTH-L are common in Xenopus and chicken and transcripts of the former have a widespread tissue distribution, although PTH-L is more restricted. PTH is widely expressed in fish tissue but from Xenopus to mammals becomes largely restricted to the parathyroid gland. The N-terminal (1-34) region of PTH, PTHrP and PTH-L in Xenopus and chicken share high sequence conservation and the capacity to modify calcium fluxes across epithelia suggesting a conserved role in calcium metabolism possibly via similar receptors. The parathyroid hormone family contains 3 principal members, PTH, PTHrP and the recently identified PTH-L. In teleosts there are 5 genes which encode PTHrP (2), PTH (2) and PTH-L and in tetrapods there are 3 genes (PTHrP, PTH and PTH-L), the exception is placental mammals which have 2 genes and lack PTH-L. It is hypothesized that

Updating the profile of C-terminal MECP2 deletions in Rett syndrome

PubMed Central

Bebbington, A; Percy, A; Christodoulou, J; Ravine, D; Ho, G; Jacoby, P; Anderson, A; Pineda, M; Ben Zeev, B; Bahi-Buisson, N; Smeets, E; Leonard, H

2014-01-01

Objectives This study aimed to compare the phenotype of Rett syndrome cases with C-terminal deletions to that of cases with different MECP2 mutations and to examine the phenotypic variation within C-terminal deletions. Methods Cases were selected from InterRett, an international database and from the population-based Australian Rett Syndrome Database. Cases (n=832) were included if they had a pathogenic MECP2 mutation in which the nature of the amino acid change was known. Three severity scale systems were used, and individual aspects of the phenotype were also compared. Results Lower severity was associated with C-terminal deletions (n=79) compared to all other MECP2 mutations (e.g. Pineda scale C-terminals mean 15.0 (95% CI 14.0–16.0) vs 16.2 (15.9–16.5). Cases with C-terminal deletions were more likely to have a normal head circumference (odds ratio 3.22, 95% CI 1.53 – 6.79) and weight (odds ratio 2.97, 95% CI 1.25–5.76). Onset of stereotypies tended to be later (median age 2.5 years vs 2 years, p<0.001 from survival analysis), and age of learning to walk tended to be earlier (median age 1.6 years vs 2 years, p=0.002 from survival analysis). Those with C-terminal deletions occurring later in the region had lower average severity scores than those occurring earlier in the region. Conclusion In terms of overall severity C-terminal deletion cases would appear to be in the middle of the range. In terms of individual aspects of phenotype growth and ability to ambulate appear to be particular strengths. By pooling data internationally this study has achieved the case numbers to provide a phenotypic profile of C-terminal deletions in Rett syndrome. PMID:19914908

An evolutionarily conserved motif in the TAB1 C-terminal region is necessary for interaction with and activation of TAK1 MAPKKK.

PubMed

Ono, K; Ohtomo, T; Sato, S; Sugamata, Y; Suzuki, M; Hisamoto, N; Ninomiya-Tsuji, J; Tsuchiya, M; Matsumoto, K

2001-06-29

TAK1, a member of the MAPKKK family, is involved in the intracellular signaling pathways mediated by transforming growth factor beta, interleukin 1, and Wnt. TAK1 kinase activity is specifically activated by the TAK1-binding protein TAB1. The C-terminal 68-amino acid sequence of TAB1 (TAB1-C68) is sufficient for TAK1 interaction and activation. Analysis of various truncated versions of TAB1-C68 defined a C-terminal 30-amino acid sequence (TAB1-C30) necessary for TAK1 binding and activation. NMR studies revealed that the TAB1-C30 region has a unique alpha-helical structure. We identified a conserved sequence motif, PYVDXA/TXF, in the C-terminal domain of mammalian TAB1, Xenopus TAB1, and its Caenorhabditis elegans homolog TAP-1, suggesting that this motif constitutes a specific TAK1 docking site. Alanine substitution mutagenesis showed that TAB1 Phe-484, located in the conserved motif, is crucial for TAK1 binding and activation. The C. elegans homolog of TAB1, TAP-1, was able to interact with and activate the C. elegans homolog of TAK1, MOM-4. However, the site in TAP-1 corresponding to Phe-484 of TAB1 is an alanine residue (Ala-364), and changing this residue to Phe abrogates the ability of TAP-1 to interact with and activate MOM-4. These results suggest that the Phe or Ala residue within the conserved motif of the TAB1-related proteins is important for interaction with and activation of specific TAK1 MAPKKK family members in vivo.

Microtubule defects influence kinesin-based transport in vitro.

NASA Astrophysics Data System (ADS)

Xu, Jing

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

Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas.

PubMed

Martinez, J M; Elfarissi, H; De Velasco, B; Ochoa, G H; Miller, A M; Clark, Y M; Matsumoto, B; Robles, L J

2000-01-01

Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos

PubMed Central

Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li

2013-01-01

. We did not observe a significant change of mesodermal and endodermal marker gene expression, while after stage 40, about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil, but other structures outside the gastrointestinal tract were relatively normal. To test if the phenotypes were specifically caused by the knockdown of IRE1α, a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1α was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC, to investigate the functional domain of IRE1α. Injection of IRE1αΔC-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α. In order to investigate if IRE1α/XBP1 pathway was involved in gut development, 50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage. CONCLUSION: IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway. PMID:23345945

Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos.

PubMed

Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li

2013-01-14

significant change of mesodermal and endodermal marker gene expression, while after stage 40, about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil, but other structures outside the gastrointestinal tract were relatively normal. To test if the phenotypes were specifically caused by the knockdown of IRE1α, a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1α was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC, to investigate the functional domain of IRE1α. Injection of IRE1αΔC-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α. In order to investigate if IRE1α/XBP1 pathway was involved in gut development, 50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage. IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.

BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes along Different Microtubule Tracks.

PubMed

Guardia, Carlos M; Farías, Ginny G; Jia, Rui; Pu, Jing; Bonifacino, Juan S

2016-11-15

The multiple functions of lysosomes are critically dependent on their ability to undergo bidirectional movement along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes is mediated by kinesin and dynein motors, respectively. We recently described a multi-subunit complex named BORC that recruits the small GTPase Arl8 to lysosomes to promote their kinesin-dependent movement toward the cell periphery. Here, we show that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1Bβ and KIF1A). Remarkably, KIF5B preferentially moves lysosomes on perinuclear tracks enriched in acetylated α-tubulin, whereas KIF1Bβ and KIF1A drive lysosome movement on more rectilinear, peripheral tracks enriched in tyrosinated α-tubulin. These findings establish BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Common regulation by BORC enables coordinate control of lysosome movement in different regions of the cell. Published by Elsevier Inc.

BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes Along Different Microtubule Tracks

PubMed Central

Guardia, Carlos M.; Farías, Ginny G.; Jia, Rui; Pu, Jing; Bonifacino, Juan S.

2016-01-01

Summary The multiple functions of lysosomes are critically dependent on their ability to undergo bidirectional movement along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes is mediated by kinesin and dynein motors, respectively. We recently described a multisubunit complex named BORC that recruits the small GTPase Arl8 to lysosomes to promote their kinesin-dependent movement toward the cell periphery. Here we show that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1Bβ and KIF1A). Remarkably, KIF5B preferentially moves lysosomes on perinuclear tracks enriched in acetylated α-tubulin, whereas KIF1Bβ and KIF1A drive lysosome movement on more rectilinear, peripheral tracks enriched in tyrosinated α-tubulin. These findings establish BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Common regulation by BORC enables coordinate control of lysosome movement in different regions of the cell. PMID:27851960

Chlamydomonas Kinesin-II–dependent Intraflagellar Transport (IFT): IFT Particles Contain Proteins Required for Ciliary Assembly in Caenorhabditis elegans Sensory Neurons

PubMed Central

Cole, Douglas G.; Diener, Dennis R.; Himelblau, Amy L.; Beech, Peter L.; Fuster, Jason C.; Rosenbaum, Joel L.

1998-01-01

We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519–5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979–992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia. PMID:9585417

Synthesis, purification and crystallographic studies of the C-terminal sterol carrier protein type 2 (SCP-2) domain of human hydroxysteroid dehydrogenase-like protein 2.

PubMed

Cheng, Zhong; Li, Yao; Sui, Chun; Sun, Xiaobo; Xie, Yong

2015-07-01

Human hydroxysteroid dehydrogenase-like protein 2 (HSDL2) is a member of the short-chain dehydrogenase/reductase (SDR) subfamily of oxidoreductases and contains an N-terminal catalytic domain and a C-termianl sterol carrier protein type 2 (SCP-2) domain. In this study, the C-terminal SCP-2 domain of human HSDL2, including residues Lys318-Arg416, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystal belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 70.4, c = 60.6 Å, α = β = 90, γ = 120°. Two protein molecules are present in the asymmetric unit, resulting in a Matthews coefficient of 2.16 Å(3) Da(-1) and an approximate solvent content of 43%.

Kinesin-8 Motors Improve Nuclear Centering by Promoting Microtubule Catastrophe

NASA Astrophysics Data System (ADS)

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

2015-02-01

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

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments

PubMed Central

Kozłowska, Małgorzata; Tarczewska, Aneta; Jakób, Michał; Bystranowska, Dominika; Taube, Michał; Kozak, Maciej; Czarnocki-Cieciura, Mariusz; Dziembowski, Andrzej; Orłowski, Marek; Tkocz, Katarzyna; Ożyhar, Andrzej

2017-01-01

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a β-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins. PMID:28074868

CENP-E Kinesin Interacts with SKAP Protein to Orchestrate Accurate Chromosome Segregation in Mitosis*

PubMed Central

Huang, Yuejia; Wang, Wenwen; Yao, Phil; Wang, Xiwei; Liu, Xing; Zhuang, Xiaoxuan; Yan, Feng; Zhou, Jinhua; Du, Jian; Ward, Tarsha; Zou, Hanfa; Zhang, Jiancun; Fang, Guowei; Ding, Xia; Dou, Zhen; Yao, Xuebiao

2012-01-01

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochore. Although previous studies show that the mitotic kinesin CENP-E forms a link between attachment of the spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying dynamic kinetochore-microtubule interactions in mammalian cells remains elusive. Here, we identify a novel interaction between CENP-E and SKAP that functions synergistically in governing dynamic kinetochore-microtubule interactions. SKAP binds to the C-terminal tail of CENP-E in vitro and is essential for an accurate kinetochore-microtubule attachment in vivo. Immunoelectron microscopic analysis indicates that SKAP is a constituent of the kinetochore corona fibers of mammalian centromeres. Depletion of SKAP or CENP-E by RNA interference results in a dramatic reduction of inter-kinetochore tension, which causes chromosome mis-segregation with a prolonged delay in achieving metaphase alignment. Importantly, SKAP binds to microtubules in vitro, and this interaction is synergized by CENP-E. Based on these findings, we propose that SKAP cooperates with CENP-E to orchestrate dynamic kinetochore-microtubule interaction for faithful chromosome segregation. PMID:22110139

Regulation of early Xenopus development by ErbB signaling

PubMed Central

Nie, Shuyi; Chang, Chenbei

2008-01-01

ErbB signaling has long been implicated in cancer formation and progression and is shown to regulate cell division, migration and death during tumorigenesis. The functions of the ErbB pathway during early vertebrate embryogenesis, however, are not well understood. Here we report characterization of ErbB activities during early frog development. Gain-of-function analyses show that EGFR, ErbB2 and ErbB4 induce ectopic tumor-like cell mass that contains increased numbers of mitotic cells. Both the muscle and the neural markers are expressed in these ectopic protrusions. ErbBs also induce mesodermal markers in ectodermal explants. Loss-of-function studies using carboxyl terminal-truncated dominant-negative ErbB receptors demonstrate that blocking ErbB signals leads to defective gastrulation movements and malformation of the embryonic axis with a reduction in the head structures in early frog embryos. These data, together with the observation that ErbBs are expressed early during frog embryogenesis, suggest that ErbBs regulate cell proliferation, movements and embryonic patterning during early Xenopus development. PMID:16258939

Controlling self-assembly of microtubule spools via kinesin motor density

PubMed Central

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

2014-01-01

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

Controlling self-assembly of microtubule spools via kinesin motor density.

PubMed

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

2014-11-21

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

Isolation of Xenopus frizzled-10A and frizzled-10B genomic clones and their expression in adult tissues and embryos.

PubMed

Moriwaki, J; Kajita, E; Kirikoshi, H; Koike, J; Sagara, N; Yasuhiko, Y; Saitoh, T; Hirai, M; Katoh, M; Shiokawa, K

2000-11-19

Frizzled genes, encoding WNT receptors, play key roles in cell fate determination. Here, we isolated two Xenopus frizzled genes (Xfz10A and Xfz10B), probably reflecting pseudotetraploidy in Xenopus. Xfz10A (586 amino acids) and Xfz10B (580 amino acids) both encoded by a single exon, consisted of the N-terminal cysteine-rich domain, seven transmembrane domains, and the C-terminal Ser/Thr-X-Val motif. Xfz10A and Xfz10B were 97.0% identical at the amino acid level, and Xfz10B was 100% identical to previously reported Xfz9, yet Xfz10A was 85.3% and 62.4% identical to FZD10 and FZD9, respectively. Xfz10 mRNA appeared as 3.4 kb in adult tissues and embryos. RT-PCR analyses revealed the expression of more Xfz10A mRNA in stomach, kidney, eye, skeletal muscle, and skin, and more Xfz10B mRNA in heart and ovary, but in embryos, two mRNAs were equally expressed from the blastula stage with their peak expression at the late gastrula stage. The main site of Xfz10 mRNA expression was neural fold at the neurula stage and the dorsal region of midbrain, hindbrain, and spinal cord at the tadpole stage. These results suggest that Xfz10 has important roles in neural tissue formation. Copyright 2000 Academic Press.

Islet-1 is required for ventral neuron survival in Xenopus

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

Shi, Yu; Zhao, Shuhua; Li, Jiejing

Islet-1 is a LIM domain transcription factor involved in several processes of embryonic development. Xenopus Islet-1 (Xisl-1) has been shown to be crucial for proper heart development. Here we show that Xisl-1 and Xisl-2 are differentially expressed in the nervous system in Xenopus embryos. Knock-down of Xisl-1 by specific morpholino leads to severe developmental defects, including eye and heart failure. Staining with the neuronal markers N-tubulin and Xisl-1 itself reveals that the motor neurons and a group of ventral interneurons are lost in the Xisl-1 morphants. Terminal dUTP nick-end labeling (TUNEL) analysis shows that Xisl-1 morpholino injection induces extensive apoptosismore » in the ventral neural plate, which can be largely inhibited by the apoptosis inhibitor M50054. We also find that over-expression of Xisl-1 is able to promote cell proliferation and induce Xstat3 expression in the injected side, suggesting a potential role for Xisl-1 in the regulation of cell proliferation in co-operation with the Jak-Stat pathway.« less

The nervus terminalis in larval and adult Xenopus laevis.

PubMed

Hofmann, M H; Meyer, D L

1989-09-25

Nervus terminalis (nt) projections were studied by HRP injections into one nostril in adult Xenopus and in Xenopus tadpoles. Central nt targets are: medial septum, preoptic nucleus, nucleus of the anterior commissure, and hypothalamus (mainly ipsilaterally). In Xenopus tadpoles, additional fibers reach the ipsilateral dorsal thalamus and the mesencephalic tegmentum, bilaterally; furthermore, hypothalamic projections are bilateral. Xenopus tadpole nt connections resemble those of adult urodeles more closely than the projections of frogs. However, Xenopus tadpoles lack nt innervation of the medial septum.

Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

PubMed Central

Stith, Bradley J.

2015-01-01

This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal*

PubMed Central

Login, Frédéric H.; Wolf-Watz, Hans

2015-01-01

All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca2+-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15). U15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the “classical” N-terminal secretion signal, U15 interacted with the ATPase YscN. Although U15 is critical for YscUCC secretion, deletion of the C-terminal secretion signal of YscUCC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion. PMID:26338709

C8orf46 homolog encodes a novel protein Vexin that is required for neurogenesis in Xenopus laevis.

PubMed

Moore, Kathryn B; Logan, Mary A; Aldiri, Issam; Roberts, Jacqueline M; Steele, Michael; Vetter, Monica L

2018-05-01

Neural basic helix-loop helix (bHLH) transcription factors promote progenitor cell differentiation by activation of downstream target genes that coordinate neuronal differentiation. Here we characterize a neural bHLH target gene in Xenopus laevis, vexin (vxn; previously sbt1), that is homologous to human c8orf46 and is conserved across vertebrate species. C8orf46 has been implicated in cancer progression, but its function is unknown. Vxn is transiently expressed in differentiating progenitors in the developing central nervous system (CNS), and is required for neurogenesis in the neural plate and retina. Its function is conserved, since overexpression of either Xenopus or mouse vxn expands primary neurogenesis and promotes early retinal cell differentiation in cooperation with neural bHLH factors. Vxn protein is localized to the cell membrane and the nucleus, but functions in the nucleus to promote neural differentiation. Vxn inhibits cell proliferation, and works with the cyclin-dependent kinase inhibitor p27Xic1 (cdkn1b) to enhance neurogenesis and increase levels of the proneural protein Neurog2. We propose that vxn provides a key link between neural bHLH activity and execution of the neurogenic program. Copyright © 2018 Elsevier Inc. All rights reserved.

An Improved Optical Tweezers Assay for Measuring the Force Generation of Single Kinesin Molecules

PubMed Central

Nicholas, Matthew P.; Rao, Lu; Gennerich, Arne

2014-01-01

Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments. PMID:24633799

Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B.

PubMed

Novas, Rossina; Cardenas-Rodriguez, Magdalena; Lepanto, Paola; Fabregat, Matías; Rodao, Magela; Fariello, María Inés; Ramos, Mauricio; Davison, Camila; Casanova, Gabriela; Alfaya, Lucía; Lecumberry, Federico; González-Sapienza, Gualberto; Irigoín, Florencia; Badano, Jose L

2018-02-14

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, polydactyly, renal disease and mental retardation. CCDC28B is a BBS-associated protein that we have previously shown plays a role in cilia length regulation whereby its depletion results in shortened cilia both in cells and Danio rerio (zebrafish). At least part of that role is achieved by its interaction with the mTORC2 component SIN1, but the mechanistic details of this interaction and/or additional functions that CCDC28B might play in the context of cilia remain poorly understood. Here we uncover a novel interaction between CCDC28B and the kinesin 1 molecular motor that is relevant to cilia. CCDC28B interacts with kinesin light chain 1 (KLC1) and the heavy chain KIF5B. Notably, depletion of these kinesin 1 components results in abnormally elongated cilia. Furthermore, through genetic interaction studies we demonstrate that kinesin 1 regulates ciliogenesis through CCDC28B. We show that kinesin 1 regulates the subcellular distribution of CCDC28B, unexpectedly, inhibiting its nuclear accumulation, and a ccdc28b mutant missing a nuclear localization motif fails to rescue the phenotype in zebrafish morphant embryos. Therefore, we uncover a previously unknown role of kinesin 1 in cilia length regulation that relies on the BBS related protein CCDC28B.

A Molecular atlas of Xenopus respiratory system development.

PubMed

Rankin, Scott A; Thi Tran, Hong; Wlizla, Marcin; Mancini, Pamela; Shifley, Emily T; Bloor, Sean D; Han, Lu; Vleminckx, Kris; Wert, Susan E; Zorn, Aaron M

2015-01-01

Respiratory system development is regulated by a complex series of endoderm-mesoderm interactions that are not fully understood. Recently Xenopus has emerged as an alternative model to investigate early respiratory system development, but the extent to which the morphogenesis and molecular pathways involved are conserved between Xenopus and mammals has not been systematically documented. In this study, we provide a histological and molecular atlas of Xenopus respiratory system development, focusing on Nkx2.1+ respiratory cell fate specification in the developing foregut. We document the expression patterns of Wnt/β-catenin, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) signaling components in the foregut and show that the molecular mechanisms of respiratory lineage induction are remarkably conserved between Xenopus and mice. Finally, using several functional experiments we refine the epistatic relationships among FGF, Wnt, and BMP signaling in early Xenopus respiratory system development. We demonstrate that Xenopus trachea and lung development, before metamorphosis, is comparable at the cellular and molecular levels to embryonic stages of mouse respiratory system development between embryonic days 8.5 and 10.5. This molecular atlas provides a fundamental starting point for further studies using Xenopus as a model to define the conserved genetic programs controlling early respiratory system development. © 2014 Wiley Periodicals, Inc.

In Vitro Motility of Liver Connexin Vesicles along Microtubules Utilizes Kinesin Motors*

PubMed Central

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

2011-01-01

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

C-Terminal residues in small potassium channel blockers OdK1 and OSK3 from scorpion venom fine-tune the selectivity.

PubMed

Kuzmenkov, Alexey I; Peigneur, Steve; Chugunov, Anton O; Tabakmakher, Valentin M; Efremov, Roman G; Tytgat, Jan; Grishin, Eugene V; Vassilevski, Alexander A

2017-05-01

We report isolation, sequencing, and electrophysiological characterization of OSK3 (α-KTx 8.8 in Kalium and Uniprot databases), a potassium channel blocker from the scorpion Orthochirus scrobiculosus venom. Using the voltage clamp technique, OSK3 was tested on a wide panel of 11 voltage-gated potassium channels expressed in Xenopus oocytes, and was found to potently inhibit Kv1.2 and Kv1.3 with IC 50 values of ~331nM and ~503nM, respectively. OdK1 produced by the scorpion Odontobuthus doriae differs by just two C-terminal residues from OSK3, but shows marked preference to Kv1.2. Based on the charybdotoxin-potassium channel complex crystal structure, a model was built to explain the role of the variable residues in OdK1 and OSK3 selectivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Synergistic effect with Phe-Gly-Leu-Met-NH2 of the C-terminal of substance P and insulin-like growth factor-1 on epithelial wound healing of rabbit cornea

PubMed Central

Nakamura, Masatsugu; Chikama, Tai-ichiro; Nishida, Teruo

1999-01-01

We previously reported that substance P and insulin-like growth factor-1 (IGF-1) synergistically stimulate corneal epithelial wound healing in vitro and in vivo. We wished to identify which portion of the amino acid sequence of substance P might be responsible for this synergism.Corneal epithelial migration was not affected by the addition of any one of the following factors: substance P; Phe-Gly-Leu-Met-NH2 (C-terminal of substance P); Val-Gly-Leu-Met-NH2 (C-terminal of neurokinin A, neurokinin B, and kassinin); Tyr-Gly-Leu-Met-NH2 (C-terminal of physalaemin); Ile-Gly-Leu-Met-NH2 (C-terminal of eledoisin); or Gly-Leu-Met-NH2 (common C-terminal of tachykinins).In the presence of IGF-1, only substance P and Phe-Gly-Leu-Met-NH2 were synergistic in stimulating corneal epithelial migration in a dose-dependent fashion.The combination of Phe-Gly-Leu-Met-NH2 and IGF-1 did not affect the incorporation of [3H]-thymidine into corneal epithelial cells.Treatment with Phe-Gly-Leu-Met-NH2 and IGF-1, but not with Phe-Gly-Leu-Met-NH2 or IGF-1 alone, increased attachment of corneal epithelial cells to a fibronectin matrix.The levels of α5 and β1 integrin were not affected by Phe-Gly-Leu-Met-NH2 or IGF-1 alone, but they were significantly increased by the combination of Phe-Gly-Leu-Met-NH2 and IGF-1.Topical application of the same combination facilitated corneal epithelial wound closure in vivo.These results demonstrated that Phe-Gly-Leu-Met-NH2, a sequence of 4 amino-acids of the C-terminal of substance P, is the minimum sequence necessary to produce the synergistic effects of substance P and IGF-1 on corneal epithelial wound healing. PMID:10385250

Establishment and characterization of Xenopus oviduct cells in primary culture

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

Marsh, J.; Tata, J.R.

1987-11-01

Based on previously established procedure of Xenopus hepatocytes, the authors describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response, from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the samemore » characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes. The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfill the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.« less

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal.

PubMed

Login, Frédéric H; Wolf-Watz, Hans

2015-10-23

All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca(2+)-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15). U15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the "classical" N-terminal secretion signal, U15 interacted with the ATPase YscN. Although U15 is critical for YscUCC secretion, deletion of the C-terminal secretion signal of YscUCC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The C-terminal extension of human RTEL1, mutated in Hoyeraal-Hreidarsson syndrome, contains harmonin-N-like domains.

PubMed

Faure, Guilhem; Revy, Patrick; Schertzer, Michael; Londono-Vallejo, Arturo; Callebaut, Isabelle

2014-06-01

Several studies have recently shown that germline mutations in RTEL1, an essential DNA helicase involved in telomere regulation and DNA repair, cause Hoyeraal-Hreidarsson syndrome (HHS), a severe form of dyskeratosis congenita. Using original new softwares, facilitating the delineation of the different domains of the protein and the identification of remote relationships for orphan domains, we outline here that the C-terminal extension of RTEL1, downstream of its catalytic domain and including several HHS-associated mutations, contains a yet unidentified tandem of harmonin-N-like domains, which may serve as a hub for partner interaction. This finding highlights the potential critical role of this region for the function of RTEL1 and gives insights into the impact that the identified mutations would have on the structure and function of these domains. © 2013 Wiley Periodicals, Inc.

Detectable states, cycle fluxes, and motility scaling of molecular motor kinesin: An integrative kinetic graph theory analysis

NASA Astrophysics Data System (ADS)

Ren, Jie

2017-12-01

The process by which a kinesin motor couples its ATPase activity with concerted mechanical hand-over-hand steps is a foremost topic of molecular motor physics. Two major routes toward elucidating kinesin mechanisms are the motility performance characterization of velocity and run length, and single-molecular state detection experiments. However, these two sets of experimental approaches are largely uncoupled to date. Here, we introduce an integrative motility state analysis based on a theorized kinetic graph theory for kinesin, which, on one hand, is validated by a wealth of accumulated motility data, and, on the other hand, allows for rigorous quantification of state occurrences and chemomechanical cycling probabilities. An interesting linear scaling for kinesin motility performance across species is discussed as well. An integrative kinetic graph theory analysis provides a powerful tool to bridge motility and state characterization experiments, so as to forge a unified effort for the elucidation of the working mechanisms of molecular motors.

The Genome of the Western Clawed Frog Xenopus tropicalis

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

Hellsten, Uffe; Harland, Richard M.; Gilchrist, Michael J.

2009-10-01

The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes over 20,000 protein-coding genes, including orthologs of at least 1,700 human disease genes. Over a million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like other tetrapods, the genome contains gene deserts enriched for conserved non-coding elements. The genome exhibits remarkable shared synteny with humanmore » and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.« less

Molecular sorting by electrical steering of microtubules in kinesin-coated channels.

PubMed

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

2006-05-12

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

Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

Scythe regulates apoptosis through modulating ubiquitin-mediated proteolysis of the Xenopus elongation factor XEF1AO

PubMed Central

Minami, Ryosuke; Shimada, Masumi; Yokosawa, Hideyoshi; Kawahara, Hiroyuki

2007-01-01

Scythe was originally identified as a novel Reaper-binding anti-apoptotic protein, although the mechanisms of its functions remain largely obscure. Our previous analysis revealed that Scythe can bind to a proteasomal subunit via N-terminal domains and that the domains are required for appropriate development of Xenopus embryos. In the present study, we show evidence that the N-terminus of Scythe interacts with XEF1AO, a maternal form of Xenopus laevis EF1A that was suggested to be a potential inducer of apoptosis in vertebrates, and that the binding enhances the poly-ubiquitin modification and subsequent degradation of XEF1AO. Scythe is required for degradation of XEF1AO, since immunodepletion of Scythe from embryonic extracts stabilized XEF1AO significantly. Furthermore, we show that apoptosis induced by accumulation of XEF1AO can be suppressed by co-expression of the full-length form of Scythe. These observations indicate that the proteolytic regulation of XEF1AO, mediated through Scythe, is essential to prevent inappropriate accumulation of XEF1AO and resulting apoptotic events during the course of Xenopus development. PMID:17428197

Single olfactory organ associated with prosencephalic malformation and cyclopia in a Xenopus laevis tadpole.

PubMed

Magrassi, L; Graziadei, P P

1987-06-02

A cyclops Xenopus laevis tadpole with a single olfactory organ is described. At a stage comparable to 48, the telencephalon was severely atrophic and only the region where the olfactory fibres terminated appeared to have the cytoarchitecture of the olfactory bulb. In this animal the central nervous system (CNS) appeared normally developed only posterior to the preoptic area. The hypothesis of a diencephalic origin of the region where the olfactory fibres terminated is discussed in the light of our previous results of olfactory placode transplantation. By analogy between this case and other malformations (cyclopia, holoprosencephaly) in higher vertebrates and humans, the need is emphasized for a more precise anatomical description of the olfactory input in related malformations.

Examining kinesin processivity within a general gating framework

PubMed Central

Andreasson, Johan OL; Milic, Bojan; Chen, Geng-Yuan; Guydosh, Nicholas R; Hancock, William O; Block, Steven M

2015-01-01

Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity. DOI: http://dx.doi.org/10.7554/eLife.07403.001 PMID:25902401

EBF proteins participate in transcriptional regulation of Xenopus muscle development.

PubMed

Green, Yangsook Song; Vetter, Monica L

2011-10-01

EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells. Copyright © 2010 Elsevier Inc. All rights reserved.

Genes encoding Xenopus laevis Ig L chains: Implications for the evolution of [kappa] and [lambda] chains

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

Zezza, D.J.; Stewart, S.E.; Steiner, L.A.

1992-12-15

Xenopus laevis Ig contain two distinct types of L chains, designated [rho] or L1 and [sigma] or L2. The authors have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and Cmore » gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian [kappa] chains than to those of mammalian [lambda] chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J[kappa]. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V[kappa]. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5[prime]-flanking region, but diverges in sequence 5[prime] to position [minus]95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3[prime]-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3[prime]-ends, as in V[kappa]. Taken together, the data suggest that Xenopus L1 L chain genes are members of the [kappa] gene family. 80 refs., 9 figs.« less

Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide.

PubMed

Schumacher, Jens; Bacic, Tijana; Staritzbichler, René; Daneschdar, Matin; Klamp, Thorsten; Arnold, Philipp; Jägle, Sabrina; Türeci, Özlem; Markl, Jürgen; Sahin, Ugur

2018-04-13

Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element

Three new members of the RNP protein family in Xenopus.

PubMed Central

Good, P J; Rebbert, M L; Dawid, I B

1993-01-01

Many RNP proteins contain one or more copies of the RNA recognition motif (RRM) and are thought to be involved in cellular RNA metabolism. We have previously characterized in Xenopus a nervous system specific gene, nrp1, that is more similar to the hnRNP A/B proteins than to other known proteins (K. Richter, P. J. Good, and I. B. Dawid (1990), New Biol. 2, 556-565). PCR amplification with degenerate primers was used to identify additional cDNAs encoding two RRMs in Xenopus. Three previously uncharacterized genes were identified. Two genes encode hnRNP A/B proteins with two RRMs and a glycine-rich domain. One of these is the Xenopus homolog of the human A2/B1 gene; the other, named hnRNP A3, is similar to both the A1 and A2 hnRNP genes. The Xenopus hnRNP A1, A2 and A3 genes are expressed throughout development and in all adult tissues. Multiple protein isoforms for the hnRNP A2 gene are predicted that differ by the insertion of short peptide sequences in the glycine-rich domain. The third newly isolated gene, named xrp1, encodes a protein that is related by sequence to the nrp1 protein but is expressed ubiquitously. Despite the similarity to nuclear RNP proteins, both the nrp1 and xrp1 proteins are localized to the cytoplasm in the Xenopus oocyte. The xrp1 gene may have a function in all cells that is similar to that executed by nrp1 specifically within the nervous system. Images PMID:8451200

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

PubMed Central

Schäper, Simon; Steinchen, Wieland; Krol, Elizaveta; Altegoer, Florian; Skotnicka, Dorota; Bange, Gert; Becker, Anke

2017-01-01

Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal “RxxxR” motif and a β-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP–responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP–responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins. PMID:28559336

Host-defense peptides from skin secretions of the octoploid frogs Xenopus vestitus and Xenopus wittei (Pipidae): insights into evolutionary relationships.

PubMed

Mechkarska, Milena; Coquet, Laurent; Leprince, Jérôme; Jouenne, Thierry; Vaudry, Hubert; Michalak, Katarzyna; Michalak, Pawel; Conlon, J Michael

2014-09-01

The primary structures of host-defense peptides have proved useful in elucidating the evolution history of frogs. Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from the octoploid frogs, Xenopus vestitus (Kivu clawed frog) and Xenopus wittei (De Witte's clawed frog) in the family Pipidae. Structural characterization demonstrated that the X. vestitus peptides belong to the magainin (3 peptides), peptide glycine-leucine-amide (PGLa; 4 peptides), xenopsin-precursor fragment (XPF; 1 peptide), and caerulein-precursor fragment (CPF; 5 peptides) families. The X. wittei peptides comprise magainin (4 peptides), PGLa (1 peptide), XPF (2 peptides), and CPF (7 peptides). In addition, secretions from both species contain caerulein, identical to the peptide from Xenopus laevis, but X. wittei secretions contains the novel peptide [R4K]xenopsin. The variability in the numbers of paralogs in each peptide family indicates a selective silencing of the host-defense peptide genes following the polyploidization events. The primary structures of the peptides provide insight into phylogenetic relationships among the octoploid Xenopus frogs. The data support a sister-group relationship between X. vestitus and Xenopus lenduensis, suggestive of bifurcating speciation after allopolyploidization, whereas X. wittei is more closely related to the Xenopus amieti-Xenopus andrei group suggesting a common tetraploid ancestor. Consistent with previous data, the CPF peptides showed the highest growth inhibitory activity against bacteria with CPF-W6 (GIGSLLAKAAKLAAGLV.NH2) combining high antimicrobial potency against Staphylococcus aureus (MIC=4 μM) with relatively low hemolytic activity (LC50=190 μM). Copyright © 2014 Elsevier Inc. All rights reserved.

Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C-terminal sequence.

PubMed

Sommer, J M; Nguyen, T T; Wang, C C

1994-08-15

Import of proteins into the glycosomes of T. brucei resembles the peroxisomal protein import in that C-terminal SKL-like tripeptide sequences can function as targeting signals. Many of the glycosomal proteins do not, however, possess such C-terminal tripeptide signals. Among these, phosphoenolpyruvate carboxykinase (PEPCK (ATP)) was thought to be targeted to the glycosomes by an N-terminal or an internal targeting signal. A limited similarity to the N-terminal targeting signal of rat peroxisomal thiolase exists at the N-terminus of T. brucei PEPCK. However, we found that this peroxisomal targeting signal does not function for glycosomal protein import in T. brucei. Further studies of the PEPCK gene revealed that the C-terminus of the predicted protein does not correspond to the previously deduced protein sequence of 472 amino acids due to a -1 frame shift error in the original DNA sequence. Readjusting the reading frame of the sequence results in a predicted protein of 525 amino acids in length ending in a tripeptide serine-arginine-leucine (SRL), which is a potential targeting signal for import into the glycosomes. A fusion protein of firefly luciferase, without its own C-terminal SKL targeting signal, and T. brucei PEPCK is efficiently imported into the glycosomes when expressed in procyclic trypanosomes. Deletion of the C-terminal SRL tripeptide or the last 29 amino acids of PEPCK reduced the import only by about 50%, while a deletion of the last 47 amino acids completely abolished the import. These results suggest that T. brucei PEPCK may contain a second, internal glycosomal targeting signal upstream of the C-terminal SRL sequence.

Evolution of Heat Sensors Drove Shifts in Thermosensation between Xenopus Species Adapted to Different Thermal Niches*

PubMed Central

Saito, Shigeru; Ohkita, Masashi; Saito, Claire T.; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

2016-01-01

Temperature is one of the most critical environmental factors affecting survival, and thus species that inhabit different thermal niches have evolved thermal sensitivities suitable for their respective habitats. During the process of shifting thermal niches, various types of genes expressed in diverse tissues, including those of the peripheral to central nervous systems, are potentially involved in the evolutionary changes in thermosensation. To elucidate the molecular mechanisms behind the evolution of thermosensation, thermal responses were compared between two species of clawed frogs (Xenopus laevis and Xenopus tropicalis) adapted to different thermal environments. X. laevis was much more sensitive to heat stimulation than X. tropicalis at the behavioral and neural levels. The activity and sensitivity of the heat-sensing TRPA1 channel were higher in X. laevis compared with those of X. tropicalis. The thermal responses of another heat-sensing channel, TRPV1, also differed between the two Xenopus species. The species differences in Xenopus TRPV1 heat responses were largely determined by three amino acid substitutions located in the first three ankyrin repeat domains, known to be involved in the regulation of rat TRPV1 activity. In addition, Xenopus TRPV1 exhibited drastic species differences in sensitivity to capsaicin, contained in chili peppers, between the two Xenopus species. Another single amino acid substitution within Xenopus TRPV1 is responsible for this species difference, which likely alters the neural and behavioral responses to capsaicin. These combined subtle amino acid substitutions in peripheral thermal sensors potentially serve as a driving force for the evolution of thermal and chemical sensation. PMID:27022021

Kinesin spindle protein (KSP) inhibitors. Part 4: Structure-based design of 5-alkylamino-3,5-diaryl-4,5-dihydropyrazoles as potent, water-soluble inhibitors of the mitotic kinesin KSP.

PubMed

Cox, Christopher D; Torrent, Maricel; Breslin, Michael J; Mariano, Brenda J; Whitman, David B; Coleman, Paul J; Buser, Carolyn A; Walsh, Eileen S; Hamilton, Kelly; Schaber, Michael D; Lobell, Robert B; Tao, Weikang; South, Vicki J; Kohl, Nancy E; Yan, Youwei; Kuo, Lawrence C; Prueksaritanont, Thomayant; Slaughter, Donald E; Li, Chunze; Mahan, Elizabeth; Lu, Bing; Hartman, George D

2006-06-15

Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.

Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: implications for chemotherapy-induced peripheral neuropathy.

PubMed

LaPointe, Nichole E; Morfini, Gerardo; Brady, Scott T; Feinstein, Stuart C; Wilson, Leslie; Jordan, Mary Ann

2013-07-01

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious, painful and dose-limiting side effect of cancer drugs that target microtubules. The mechanisms underlying the neuronal damage are unknown, but may include disruption of fast axonal transport, an essential microtubule-based process that moves cellular components over long distances between neuronal cell bodies and nerve terminals. This idea is supported by the "dying back" pattern of degeneration observed in CIPN, and by the selective vulnerability of sensory neurons bearing the longest axonal projections. In this study, we test the hypothesis that microtubule-targeting drugs disrupt fast axonal transport using vesicle motility assays in isolated squid axoplasm and a cell-free microtubule gliding assay with defined components. We compare four clinically-used drugs, eribulin, vincristine, paclitaxel and ixabepilone. Of these, eribulin is associated with a relatively low incidence of severe neuropathy, while vincristine has a relatively high incidence. In vesicle motility assays, we found that all four drugs inhibited anterograde (conventional kinesin-dependent) fast axonal transport, with the potency being vincristine=ixabepilone>paclitaxel=eribulin. Interestingly, eribulin and paclitaxel did not inhibit retrograde (cytoplasmic dynein-dependent) fast axonal transport, in contrast to vincristine and ixabepilone. Similarly, vincristine and ixabepilone both exerted significant inhibitory effects in an in vitro microtubule gliding assay consisting of recombinant kinesin (kinesin-1) and microtubules composed of purified bovine brain tubulin, whereas paclitaxel and eribulin had negligible effects. Our results suggest that (i) inhibition of microtubule-based fast axonal transport may be a significant contributor to neurotoxicity induced by microtubule-targeting drugs, and (ii) that individual microtubule-targeting drugs affect fast axonal transport through different mechanisms. Copyright © 2013 Elsevier Inc

Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.

PubMed

Heikkila, John J; Kaldis, Angelo; Abdulle, Rashid

2006-01-01

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

Amphibian (Xenopus sp.) iodothyronine deiodinase ...

EPA Pesticide Factsheets

The U.S. EPA-MED amphibian thyroid group is currently screening chemicals for inhibition of human iodothyronine deiodinase activity as components of the thyroid system important in human development. Amphibians are a bellwether taxonomic group to gauge toxicity of chemicals in the environment. Amphibian thyroid function is not only important in development but also metamorphosis. Xenopus sp. have been used extensively as model organisms and are well characterized genetically. We propose to screen a list of chemicals (selected from the human DIO screening results) to test for inhibition of Xenopus deiodinases. Large quantities of the enzymes will be produced using an adenovirus system. Our preliminary results show that there may be catalytic differences between human and Xenopus deiodinases. The Twin Ports Early Career Scientists is a new group formed within the Duluth-Superior scientific community. This presentation will provide a basic introduction to my research and our mission at EPA, and help to establish networking and collaboration relationships across disciplines and institutions.

Conservation and divergence of ADAM family proteins in the Xenopus genome

PubMed Central

2010-01-01

Background Members of the disintegrin metalloproteinase (ADAM) family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST) databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species. Results Based on the X. tropicalis genome sequence and EST databases, we identified Xenopus orthologues of mammalian ADAMs and obtained full-length cDNA clones for these genes. The deduced protein sequences, synteny and exon-intron boundaries are conserved between most human and X. tropicalis orthologues. The alternative splicing patterns of certain Xenopus ADAM genes, such as adams 22 and 28, are similar to those of their mammalian orthologues. However, we were unable to identify an orthologue for ADAM7 or 8. The Xenopus orthologue of ADAM15, an active metalloproteinase in mammals, does not contain the conserved zinc-binding motif and is hence considered proteolytically inactive. We also found evidence for gain of ADAM genes in Xenopus as compared to other species. There is a homologue of ADAM10 in Xenopus that is missing in most mammals. Furthermore, a single scaffold of X. tropicalis genome contains four genes encoding ADAM28 homologues, suggesting genome duplication in this region. Conclusions Our genome-wide analysis of ADAM genes in X. tropicalis revealed both conservation and evolutionary divergence of these genes in this amphibian species. On the one hand, all ADAMs implicated in normal development and health in other species are conserved in X. tropicalis. On the other hand, some ADAM genes and ADAM protease

Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat.

PubMed Central

Tsuji, S; Qureshi, M A; Hou, E W; Fitch, W M; Li, S S

1994-01-01

The nucleotide sequences of the cDNAs encoding LDH (EC 1.1.1.27) subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously. Images PMID:7937776

Characterization of X-OCRL, a Xenopus laevis homologue of OCRL-1, the Lowe oculocerebrorenal syndrome candidate gene

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

Reilly, D.S.; Nussbaum, R.L.

1994-09-01

The Lowe oculocerebrorenal syndrome (OCRL) is an X-linked disease characterized by congenital cataract, mental retardation, and renal tubular dysfunction. A candidate cDNA, OCRL-1, was identified by positional cloning and mutations in OCRL-1 have been detected in patients with Lowe syndrome. The OCRL-1 nucleotide sequence encodes a predicted protein of 968 amino acids and shares 51% amino acid identity with a human inositol polyphosphate-5-phosphatase. This suggests that the underlying defect in OCRL may be due to a defect in inositol phosphate metabolism. The isolation of OCRL-1 provides the opportunity to investigate its function through the use of animal model systems. Wemore » have isolated a partial cDNA clone encoding an OCRL-1 homologue, X-OCRL, from the South African clawed frog, Xenopus laevis. We used a portion of the human cDNA to screen a Xenopus laevis embryo cDNA library and isolated four positive clones. One clone, 42-5A, is a 650 bp insert with over 75% amino acid identity to the corresponding region of the human OCRL-1 sequence. 42-5A detects messenger RNA in adult Xenopus brain, stomach, small intestine, skin, muscle, lung, blood, and oviduct. X-OCRL messenger RNA is first detected during late gastrula and continues to be expressed throughout Xenopus development. In situ hybridization studies are underway to identify the cellular localization of X-OCRL expression in Xenopus embryos and adult tissues. We are especially interested in characterizing X-OCRL expression during formation of the amphibian lens since congenital cataracts are a constant feature of the human disease.« less

Morphology of the caudal spinal cord in Rana (Ranidae) and Xenopus (Pipidae) tadpoles.

PubMed

Nishikawa, K; Wassersug, R

1988-03-08

Using a variety of neuroanatomical and histological techniques, we compare the spinal cord and peripheral nerve distribution in the tails of larvae from Xenopus laevis and three species of Rana. The relatively large, postsacral spinal cord of Xenopus contains abundant motoneurons and their axons. Spinal nerves exit from the spinal cord in a regular array, one nerve per myotome, from the cervical region to near the end of the tail. Somata of motoneurons innervating caudal myotomes are found along the entire length of the tail. In contrast, the caudal cord of Rana is reduced to a filum terminale consisting of little more than an ependymal tube; spinal nerves to all caudal myotomes leave the cord in the sacral region and reach their motor targets via a cauda equina and caudal plexus. Motoneuron cell bodies innervating caudal myotomes are found only in the sacral region. The Rana larval pattern is similar to that of adult frogs and mammals, whereas the Xenopus larval pattern is more like that of salamanders and reptiles. These gross neuroanatomical differences are not due to differences in the size or developmental stage of the tadpoles, but instead are associated with differences in the swimming behavior of the larvae. The presence of motoneurons in the caudal spinal cord of Xenopus may provide local intermyotomal control within the tail; the elongated topography of the cord appears to permit finer, rostral-to-caudal regulation of neuromuscular activity. The Rana spinal cord, on the other hand--with motoneurons clustered anteriorly--may produce concurrent firing of adjacent ipsilateral myotomes, but at the expense of fine intermyotomal regulation. The fact that nerves in the tail of Xenopus enter and exit from the spinal cord locally, as opposed to far anteriorly as in Rana, means that for tadpoles of the same size, reflex arc lengths are many times shorter in Xenopus.

Genetic screens for mutations affecting development of Xenopus tropicalis.

PubMed

Goda, Tadahiro; Abu-Daya, Anita; Carruthers, Samantha; Clark, Matthew D; Stemple, Derek L; Zimmerman, Lyle B

2006-06-01

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

C-terminal domains of bacterial proteases: structure, function and the biotechnological applications.

PubMed

Huang, J; Wu, C; Liu, D; Yang, X; Wu, R; Zhang, J; Ma, C; He, H

2017-01-01

C-terminal domains widely exist in the C-terminal region of multidomain proteases. As a β-sandwich domain in multidomain protease, the C-terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C-terminal protease domains are described. Furthermore, the application prospects of C-terminal domains, including polycystic kidney disease, prepeptidase C-terminal and collagen-binding domain, in the area of medicine and biological artificial materials are also discussed. © 2016 The Society for Applied Microbiology.

Localization of a microtubule organizing center by kinesin motors

NASA Astrophysics Data System (ADS)

Arita, Chikashi; Bosche, Jonas; Lück, Alexander; Santen, Ludger

2017-12-01

Molecular motors are proteins which bind to a polarized cytoskeletal filament and move steadily along it. Molecular motors of the kinesin family move along microtubules (MTs), which are a component of the cytoskeleton. A very processive kinesin motor Kip3p, is known to promote catastrophes and pausing of MT, in particular on cortical contact. These properties play an important role in positioning the mitotic spindle in budding yeast. We present a theoretical approach to positioning of MT networks under confinement. In order to explore a localization mechanism of a microtubule organizing center (MTOC), we introduce an idealized system of two MTs connected by a MTOC. The dynamics of Kip3p is modeled by interacting stochastic particles, which allows us to study the effects of motor-induced depolymerization in a finite volume. We find that localization in the middle of the cavity is realized in a parameter regime where the motor densities on the MTs are increasing with the distance from the MTOC. Localization at an asymmetric position is also possible by tuning model parameters.

Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

PubMed Central

Grover, Rahul; Fischer, Janine; Schwarz, Friedrich W.; Walter, Wilhelm J.; Schwille, Petra; Diez, Stefan

2016-01-01

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. PMID:27803325

1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain.

PubMed

Hörnig, Christina; Albert, Dana; Fischer, Lutz; Hörnig, Michael; Rådmark, Olof; Steinhilber, Dieter; Werz, Oliver

2005-07-22

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.

Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.

PubMed

Tao, Y; Strelkov, S V; Mesyanzhinov, V V; Rossmann, M G

1997-06-15

Oligomeric coiled-coil motifs are found in numerous protein structures; among them is fibritin, a structural protein of bacteriophage T4, which belongs to a class of chaperones that catalyze a specific phage-assembly process. Fibritin promotes the assembly of the long tail fibers and their subsequent attachment to the tail baseplate; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and, thus, prevents infection. The structure of fibritin had been predicted from sequence and biochemical analyses to be mainly a triple-helical coiled coil. The determination of its structure at atomic resolution was expected to give insights into the assembly process and biological function of fibritin, and the properties of modified coiled-coil structures in general. The three-dimensional structure of fibritin E, a deletion mutant of wild-type fibritin, was determined to 2.2 A resolution by X-ray crystallography. Three identical subunits of 119 amino acid residues form a trimeric parallel coiled-coil domain and a small globular C-terminal domain about a crystallographic threefold axis. The coiled-coil domain is divided into three segments that are separated by insertion loops. The C-terminal domain, which consists of 30 residues from each subunit, contains a beta-propeller-like structure with a hydrophobic interior. The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C-terminal

Predicting the stochastic guiding of kinesin-driven microtubules in microfabricated tracks: a statistical-mechanics-based modeling approach.

PubMed

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

2010-01-01

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

Crystallographic analysis of the conserved C-terminal domain of transcription factor Cdc73 from Saccharomyces cerevisiae reveals a GTPase-like fold.

PubMed

Chen, Hongkai; Shi, Nuo; Gao, Yongxiang; Li, Xu; Teng, Maikun; Niu, Liwen

2012-08-01

The yeast Paf1 complex (Paf1C), which is composed of the proteins Paf1, Cdc73, Ctr9, Leo1 and Rtf1, accompanies RNA polymerase II from the promoter to the 3'-end formation site of mRNA- and snoRNA-encoding genes. As one of the first identified subunits of Paf1C, yeast Cdc73 (yCdc73) takes part in many transcription-related processes, including binding to RNA polymerase II, recruitment and activation of histone-modification factors and communication with other transcriptional activators. The human homologue of yCdc73, parafibromin, has been identified as a tumour suppressor linked to breast, renal and gastric cancers. However, the functional mechanism of yCdc73 has until recently been unclear. Here, a 2.2 Å resolution crystal structure of the highly conserved C-terminal region of yCdc73 is reported. It revealed that yCdc73 appears to have a GTPase-like fold. However, no GTPase activity was observed. The crystal structure of yCdc73 will shed new light on the modes of function of Cdc73 and Paf1C.

Chromosome and mitotic spindle dynamics in fission yeast kinesin-8 mutants

NASA Astrophysics Data System (ADS)

Crapo, Ammon M.; Gergley, Zachary R.; McIntosh, J. Richard; Betterton, M. D.

2014-03-01

Fission yeast proteins Klp5p and Klp6p are plus-end directed motors of the kinesin-8 family which promote microtubule (MT) depolymerization and also affect chromosome segregation, but the mechanism of these activities is not well understood. Using live-cell time-lapse fluorescence microscopy of fission yeast wild-type (WT) and klp5/6 mutant strains, we quantify and compare the dynamics of kinetochore motion and mitotic spindle length in 3D. In WT cells, the spindle, once formed, remains a consistent size and chromosomes are correctly organized and segregated. In kinesin-8 mutants, spindles undergo large length fluctuations of several microns. Kinetochore motions are also highly fluctuating, with kinetochores frequently moving away from the spindle rather than toward it. We observe transient pushing of chromosomes away from the spindle by as much as 10 microns in distance.

Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

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

Delorme, Caroline; Joshi, Monika; Allingham, John S., E-mail: allinghj@queensu.ca

2012-11-30

Highlights: Black-Right-Pointing-Pointer The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. Black-Right-Pointing-Pointer The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. Black-Right-Pointing-Pointer The MBP-Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance,more » we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the 'ATP state' of the mechanochemical cycle. This site differs from the Kar3 neck-core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.« less

47 CFR 25.134 - Licensing provisions of Very Small Aperture Terminal (VSAT) and C-band Small Aperture Terminal...

Code of Federal Regulations, 2011 CFR

2011-10-01

... Terminal (VSAT) and C-band Small Aperture Terminal (CSAT) networks. 25.134 Section 25.134 Telecommunication...) and C-band Small Aperture Terminal (CSAT) networks. (a)(1) VSAT networks operating in the 12/14 GHz bands. All applications for digital VSAT networks granted on or before September 15, 2005, with a...

47 CFR 25.134 - Licensing provisions of Very Small Aperture Terminal (VSAT) and C-band Small Aperture Terminal...

Code of Federal Regulations, 2010 CFR

2010-10-01

... Terminal (VSAT) and C-band Small Aperture Terminal (CSAT) networks. 25.134 Section 25.134 Telecommunication...) and C-band Small Aperture Terminal (CSAT) networks. (a)(1) VSAT networks operating in the 12/14 GHz bands. All applications for digital VSAT networks granted on or before September 15, 2005, with a...

C-terminal interactions mediate the quaternary dynamics of αB-crystallin

PubMed Central

Hilton, Gillian R.; Hochberg, Georg K. A.; Laganowsky, Arthur; McGinnigle, Scott I.; Baldwin, Andrew J.; Benesch, Justin L. P.

2013-01-01

αB-crystallin is a highly dynamic, polydisperse small heat-shock protein that can form oligomers ranging in mass from 200 to 800 kDa. Here we use a multifaceted mass spectrometry approach to assess the role of the C-terminal tail in the self-assembly of αB-crystallin. Titration experiments allow us to monitor the binding of peptides representing the C-terminus to the αB-crystallin core domain, and observe individual affinities to both monomeric and dimeric forms. Notably, we find that binding the second peptide equivalent to the core domain dimer is considerably more difficult than the first, suggesting a role of the C-terminus in regulating assembly. This finding motivates us to examine the effect of point mutations in the C-terminus in the full-length protein, by quantifying the changes in oligomeric distribution and corresponding subunit exchange rates. Our results combine to demonstrate that alterations in the C-terminal tail have a significant impact on the thermodynamics and kinetics of αB-crystallin. Remarkably, we find that there is energy compensation between the inter- and intra-dimer interfaces: when one interaction is weakened, the other is strengthened. This allosteric communication between binding sites on αB-crystallin is likely important for its role in binding target proteins. PMID:23530258

Parsing the roles of neck-linker docking and tethered head diffusion in the stepping dynamics of kinesin.

PubMed

Zhang, Zhechun; Goldtzvik, Yonathan; Thirumalai, D

2017-11-14

Kinesin walks processively on microtubules (MTs) in an asymmetric hand-over-hand manner consuming one ATP molecule per 16-nm step. The individual contributions due to docking of the approximately 13-residue neck linker to the leading head (deemed to be the power stroke) and diffusion of the trailing head (TH) that contributes in propelling the motor by 16 nm have not been quantified. We use molecular simulations by creating a coarse-grained model of the MT-kinesin complex, which reproduces the measured stall force as well as the force required to dislodge the motor head from the MT, to show that nearly three-quarters of the step occurs by bidirectional stochastic motion of the TH. However, docking of the neck linker to the leading head constrains the extent of diffusion and minimizes the probability that kinesin takes side steps, implying that both the events are necessary in the motility of kinesin and for the maintenance of processivity. Surprisingly, we find that during a single step, the TH stochastically hops multiple times between the geometrically accessible neighboring sites on the MT before forming a stable interaction with the target binding site with correct orientation between the motor head and the [Formula: see text] tubulin dimer.

Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of Caenorhabditis elegans

PubMed Central

Burghoorn, Jan; Dekkers, Martijn P. J.; Rademakers, Suzanne; de Jong, Ton; Willemsen, Rob; Jansen, Gert

2007-01-01

In the cilia of the nematode Caenorhabditis elegans, anterograde intraflagellar transport (IFT) is mediated by two kinesin-2 complexes, kinesin II and OSM-3 kinesin. These complexes function together in the cilia middle segments, whereas OSM-3 alone mediates transport in the distal segments. Not much is known about the mechanisms that compartmentalize the kinesin-2 complexes or how transport by both kinesins is coordinated. Here, we identify DYF-5, a conserved MAP kinase that plays a role in these processes. Fluorescence microscopy and EM revealed that the cilia of dyf-5 loss-of-function (lf) animals are elongated and are not properly aligned into the amphid channel. Some cilia do enter the amphid channel, but the distal ends of these cilia show accumulation of proteins. Consistent with these observations, we found that six IFT proteins accumulate in the cilia of dyf-5(lf) mutants. In addition, using genetic analyses and live imaging to measure the motility of IFT proteins, we show that dyf-5 is required to restrict kinesin II to the cilia middle segments. Finally, we show that, in dyf-5(lf) mutants, OSM-3 moves at a reduced speed and is not attached to IFT particles. We propose that DYF-5 plays a role in the undocking of kinesin II from IFT particles and in the docking of OSM-3 onto IFT particles. PMID:17420466

Evolution of Courtship Songs in Xenopus : Vocal Pattern Generation and Sound Production.

PubMed

Leininger, Elizabeth C; Kelley, Darcy B

2015-01-01

The extant species of African clawed frogs (Xenopus and Silurana) provide an opportunity to link the evolution of vocal characters to changes in the responsible cellular and molecular mechanisms. In this review, we integrate several robust lines of research: evolutionary trajectories of Xenopus vocalizations, cellular and circuit-level mechanisms of vocalization in selected Xenopus model species, and Xenopus evolutionary history and speciation mechanisms. Integrating recent findings allows us to generate and test specific hypotheses about the evolution of Xenopus vocal circuits. We propose that reduced vocal sex differences in some Xenopus species result from species-specific losses of sexually differentiated neural and neuromuscular features. Modification of sex-hormone-regulated developmental mechanisms is a strong candidate mechanism for reduced vocal sex differences.

Characteristics of concatemeric GABAA receptors containing α4/δ subunits expressed in Xenopus oocytes

PubMed Central

Shu, Hong-Jin; Bracamontes, John; Taylor, Amanda; Wu, Kyle; Eaton, Megan M; Akk, Gustav; Manion, Brad; Evers, Alex S; Krishnan, Kathiresan; Covey, Douglas F; Zorumski, Charles F; Steinbach, Joe Henry; Mennerick, Steven

2012-01-01

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. KEY RESULTS δ-β2 −α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors. PMID:21950777

Tug-of-war of microtubule filaments at the boundary of a kinesin- and dynein-patterned surface

NASA Astrophysics Data System (ADS)

Ikuta, Junya; Kamisetty, Nagendra K.; Shintaku, Hirofumi; Kotera, Hidetoshi; Kon, Takahide; Yokokawa, Ryuji

2014-06-01

Intracellular cargo is transported by multiple motor proteins. Because of the force balance of motors with mixed polarities, cargo moves bidirectionally to achieve biological functions. Here, we propose a microtubule gliding assay for a tug-of-war study of kinesin and dynein. A boundary of the two motor groups is created by photolithographically patterning gold to selectively attach kinesin to the glass and dynein to the gold surface using a self-assembled monolayer. The relationship between the ratio of two antagonistic motor numbers and the velocity is derived from a force-velocity relationship for each motor to calculate the detachment force and motor backward velocity. Although the tug-of-war involves >100 motors, values are calculated for a single molecule and reflect the collective dynein and non-collective kinesin functions when they work as a team. This assay would be useful for detailed in vitro analysis of intracellular motility, e.g., mitosis, where a large number of motors with mixed polarities are involved.

Tug-of-war of microtubule filaments at the boundary of a kinesin- and dynein-patterned surface

PubMed Central

Ikuta, Junya; Kamisetty, Nagendra K.; Shintaku, Hirofumi; Kotera, Hidetoshi; Kon, Takahide; Yokokawa, Ryuji

2014-01-01

Intracellular cargo is transported by multiple motor proteins. Because of the force balance of motors with mixed polarities, cargo moves bidirectionally to achieve biological functions. Here, we propose a microtubule gliding assay for a tug-of-war study of kinesin and dynein. A boundary of the two motor groups is created by photolithographically patterning gold to selectively attach kinesin to the glass and dynein to the gold surface using a self-assembled monolayer. The relationship between the ratio of two antagonistic motor numbers and the velocity is derived from a force-velocity relationship for each motor to calculate the detachment force and motor backward velocity. Although the tug-of-war involves >100 motors, values are calculated for a single molecule and reflect the collective dynein and non-collective kinesin functions when they work as a team. This assay would be useful for detailed in vitro analysis of intracellular motility, e.g., mitosis, where a large number of motors with mixed polarities are involved. PMID:24923426

Hyperinnervation improves Xenopus laevis limb regeneration.

PubMed

Mitogawa, Kazumasa; Makanae, Aki; Satoh, Akira

2018-01-15

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis

PubMed Central

Wen, Luan; Fu, Liezhen; Guo, Xiaogang; Chen, Yonglong; Shi, Yun-Bo

2015-01-01

Histone methylations have been implicated to play important roles in diverse cellular processes. Of particular interest is the methylation of histone H3K79, which is catalyzed by an evolutionarily conserved methyltransferase, disruptor of telomeric silencing (Dot1)-like (Dot1L). To investigate the role of Dot1L during vertebrate development, we have generated a Dot1L-specific transcription activator-like effector nuclease (TALEN) nuclease to knockdown endogenous Dot1L in Xenopus tropicalis, a diploid species highly related to the well-known developmental model Xenopus laevis, a pseudotetraploid amphibian. We show that the TALEN was extremely efficient in mutating Dot1L when expressed in fertilized eggs, creating essentially Dot1L knockout embryos with little H3K79 methylation. Importantly, we observed that Dot1L knockdown had no apparent effect on embryogenesis because normally feeding tadpoles were formed, consistent with the lack of maternal Dot1L expression. On the other hand, Dot1L knockdown severely retarded the growth of the tadpoles and led to tadpole lethality prior to metamorphosis. These findings suggest that Dot1L and H3K79 methylation play an important role for tadpole growth and development prior to metamorphosis into a frog. Our findings further reveal interesting similarities and differences between Xenopus and mouse development and suggest the existence of 2 separate phases of vertebrate development with distinct requirements for epigenetic modifications.—Wen, L., Fu, L., Guo, X., Chen, Y., Shi, Y.-B. Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis. PMID:25366346

C-terminal oligomerization of podocin mediates interallelic interactions.

PubMed

Stráner, Pál; Balogh, Eszter; Schay, Gusztáv; Arrondel, Christelle; Mikó, Ágnes; L'Auné, Gerda; Benmerah, Alexandre; Perczel, András; K Menyhárd, Dóra; Antignac, Corinne; Mollet, Géraldine; Tory, Kálmán

2018-07-01

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3' mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283-382): principally through the first C-terminal helical region (H1, 283-313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332-348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r 2  = 0.68, P = 9.2 × 10 -32 ). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals. Copyright © 2018 Elsevier B.V. All rights reserved.

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

PubMed Central

Yang, Yinshan; Labesse, Gilles; Carrère-Kremer, Séverine; Esteves, Kevin; Kremer, Laurent

2012-01-01

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg2+ deprivation, but previous work suggested that MgtC is not a Mg2+ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg2+ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg2+ concentration, indicating that it does not bind Mg2+. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg2+ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain. PMID:22984256

Xenopus egg cytoplasm with intact actin.

PubMed

Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

2014-01-01

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy.

PubMed

Myers, Stephanie M; Collins, Ian

2016-01-01

The kinesin class of microtubule-associated motor proteins present attractive anticancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms and explore the implications for future anticancer drug development against these targets.

Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy

PubMed Central

Myers, Stephanie M.; Collins, Ian

2016-01-01

The kinesin class of microtubule-associated motor proteins present attractive anti-cancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms, and explore the implications for future anticancer drug development against these targets. PMID:26976726

Multimotor Transport in a System of Active and Inactive Kinesin-1 Motors

PubMed Central

Scharrel, Lara; Ma, Rui; Schneider, René; Jülicher, Frank; Diez, Stefan

2014-01-01

Long-range directional transport in cells is facilitated by microtubule-based motor proteins. One example is transport in a nerve cell, where small groups of motor proteins, such as kinesins and cytoplasmic dynein, work together to ensure the supply and clearance of cellular material along the axon. Defects in axonal transport have been linked to Alzheimer’s and other neurodegenerative diseases. However, it is not known in detail how multimotor-based cargo transport is impaired if a fraction of the motors are defective. To mimic impaired multimotor transport in vitro, we performed gliding motility assays with varying fractions of active kinesin-1 motors and inactive kinesin-1 motor mutants. We found that impaired transport manifests in multiple motility regimes: 1), a fast-motility regime characterized by gliding at velocities close to the single-molecule velocity of the active motors; 2), a slow-motility regime characterized by gliding at close-to zero velocity or full stopping; and 3), a regime in which fast and slow motilities coexist. Notably, the transition from the fast to the slow regime occurred sharply at a threshold fraction of active motors. Based on single-motor parameters, we developed a stochastic model and a mean-field theoretical description that explain our experimental findings. Our results demonstrate that impaired multimotor transport mostly occurs in an either/or fashion: depending on the ratio of active to inactive motors, transport is either performed at close to full speed or is out of action. PMID:25028878

Antisense and sense poly(A)-RNAs from the Xenopus laevis pyruvate dehydrogenase gene loci are regulated with message production during embryogenesis.

PubMed

Islam, N; Poitras, L; Gagnon, F; Moss, T

1996-10-17

The structure and temporal expression of two Xenopus cDNAs encoding the beta subunit of pyruvate dehydrogenase (XPdhE1 beta) have been determined. XPdhE1 beta was 88% homologous to mature human PdhE1 beta, but the putative N-terminal mitochondrial signal peptide was poorly conserved. Zygotic expression of XPdhE1 beta mRNA was detected at neural tube closure and increased until stage 40. RT-PCR cloning identified a short homology to a protein kinase open reading frame within the 3' non-coding sequence of the XPdhE1 beta cDNAs. This homology, which occurred on the antisense cDNA strand, was shown by strand specific RT-PCR to be transcribed in vivo as part of an antisense RNA. Northern analysis showed that this RNA formed part of an abundant and heterogeneous population of antisense and sense poly(A)-RNAs transcribed from the XPdhE1 beta loci and coordinately regulated with message production.

Slowed Relaxation in Fatigued Skeletal Muscle Fibers of Xenopus and Mouse

PubMed Central

Westerblad, Håkan; Lännergren, Jan; Allen, David G.

1997-01-01

Slowing of relaxation is an important characteristic of skeletal muscle fatigue. The aim of the present study was to quantify the relative contribution of altered Ca2+ handling (calcium component) and factors down-stream to Ca2+ (cross-bridge component) to the slowing of relaxation in fatigued fibers of Xenopus and mouse. Two types of Xenopus fibers were used: easily fatigued, type 1 fibers and fatigue resistant, type 2 fibers. In these Xenopus fibers the free myoplasmic [Ca2+] ([Ca2+]i) was measured with indo-1, and the relaxation of Ca2+-derived force, constructed from tetanic [Ca2+]i records and in vivo [Ca2+]i-force curves, was analyzed. An alternative method was used in both Xenopus and mouse fibers: fibers were rapidly shortened during the initial phase of relaxation, and the time to the peak of force redevelopment was measured. These two methods gave similar results and showed proportional slowing of the calcium and cross-bridge components of relaxation in both fatigued type 1 and type 2 Xenopus fibers, whereas only the cross-bridge component was slowed in fatigued mouse fibers. Ca2+ removal from the myoplasm during relaxation was markedly less effective in Xenopus fibers as compared to mouse fibers. Fatigued Xenopus fibers displayed a reduced rate of sarcoplasmic reticulum Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Some fibers were stretched at various times during relaxation. The resistance to these stretches was increased during fatigue, especially in Xenopus fibers, which indicates that longitudinal movements during relaxation had become less pronounced and this might contribute to the increased cross-bridge component of relaxation in fatigue. In conclusion, slowing of relaxation in fatigued Xenopus fibers is caused by impaired Ca2+ handling and altered cross-bridge kinetics, whereas the slowing in mouse fibers is only due to altered cross-bridge kinetics. PMID:9089444

Cloning of noggin gene from hydra and analysis of its functional conservation using Xenopus laevis embryos.

PubMed

Chandramore, Kalpana; Ito, Yuzuro; Takahashi, Shuji; Asashima, Makoto; Ghaskadbi, Surendra

2010-01-01

Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin-like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)-signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV-induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.

Friend of GATA (FOG) Interacts with the Nucleosome Remodeling and Deacetylase Complex (NuRD) to Support Primitive Erythropoiesis in Xenopus laevis

PubMed Central

Mimoto, Mizuho S.; Christian, Jan L.

2012-01-01

Friend of GATA (FOG) plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC) development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD), but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect. PMID:22235346

Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8.

PubMed

Shin, Yongdae; Du, Yaqing; Collier, Scott E; Ohi, Melanie D; Lang, Matthew J; Ohi, Ryoma

2015-07-21

Kinesin-8s are plus-end-directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are "ultraprocessive," a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end-directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.

Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8

PubMed Central

Shin, Yongdae; Du, Yaqing; Collier, Scott E.; Ohi, Melanie D.; Lang, Matthew J.; Ohi, Ryoma

2015-01-01

Kinesin-8s are plus-end–directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are “ultraprocessive,” a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end–directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle. PMID:26150501

Isthmin is a novel secreted protein expressed as part of the Fgf-8 synexpression group in the Xenopus midbrain-hindbrain organizer.

PubMed

Pera, Edgar M; Kim, James I; Martinez, Sarah L; Brechner, Mariel; Li, Su Yu; Wessely, Oliver; De Robertis, E M

2002-08-01

Patterning of the central nervous system is regulated by a signaling center located at the midbrain-hindbrain boundary (MHB), or isthmus organizer. Fibroblast growth factors secreted from the MHB are required and sufficient to direct the ordered growth and regionalization of the midbrain and anterior hindbrain. In an unbiased secretion cloning screen of Xenopus gastrula embryos we identified a novel gene, which we designated as Isthmin (xIsm) due to its prominent expression at the MHB. xIsm encodes a secreted protein of 449 amino acids containing one copy of the thrombospondin type 1 repeat (TSR). We also found orthologous Isthmin genes in human (hIsm) and mouse (mIsm), as well as a gene encoding an Isthmin-like human unknown protein (hIsm-l). The conservation of a unique carboxy-terminal region between hIsm and hIsm-l suggests that Isthmin is the founding member of a new family of secreted proteins. xIsm was strongly expressed maternally in the Xenopus egg and showed zygotic expression in the ventral blastopore lip, notochord, and MHB. Additional expression domains were detected in neural crest, ear vesicle, and developing blood islands. Interestingly, xIsm was co-expressed with Fibroblast growth factor-8 (xFgf-8) at multiple sites including the MHB, indicating that these two genes are part of a synexpression group which also includes sprouty and sef homologs.

C-terminal motifs in promyelocytic leukemia protein isoforms critically regulate PML nuclear body formation.

PubMed

Li, Chuang; Peng, Qiongfang; Wan, Xiao; Sun, Haili; Tang, Jun

2017-10-15

Promyelocytic leukemia protein (PML) nuclear bodies (NBs), which are sub-nuclear protein structures, are involved in a variety of important cellular functions. PML-NBs are assembled by PML isoforms, and contact between small ubiquitin-like modifiers (SUMOs) with the SUMO interaction motif (SIM) are critically involved in this process. PML isoforms contain a common N-terminal region and a variable C-terminus. However, the contribution of the C-terminal regions to PML-NB formation remains poorly defined. Here, using high-resolution microscopy, we show that mutation of the SIM distinctively influences the structure of NBs formed by each individual PML isoform, with that of PML-III and PML-V minimally changed, and PML-I and PML-IV dramatically impaired. We further identify several C-terminal elements that are important in regulating NB structure and provide strong evidence to suggest that the 8b element in PML-IV possesses a strong ability to interact with SUMO-1 and SUMO-2, and critically participates in NB formation. Our findings highlight the importance of PML C-termini in NB assembly and function, and provide molecular insight into the PML-NB assembly of each distinctive isoform. © 2017. Published by The Company of Biologists Ltd.

Identification of a candidate CD5 homologue in the amphibian Xenopus laevis.

PubMed

Jürgens, J B; Gartland, L A; Du Pasquier, L; Horton, J D; Göbel, T W; Cooper, M D

1995-11-01

We identified a novel T cell Ag in the South African clawed toad (Xenopus laevis) by a mAb designated 2B1. This Ag is present in relatively high levels on most thymocytes, approximately 65% of splenocytes, 55% of PBL, and 65% of intestinal lymphocytes, but is rarely seen on IgM+ B cells in any of these tissues. Lymphocytes bearing the 2B1 Ag proliferate in response to stimulation with Con A or PHA, whereas the 2B1- lymphocytes are reactive to LPS. Biochemical analysis indicates that this Ag is a differentially phosphorylated glycoprotein of 71 to 82 kDa. The protein core of 64 kDa bears both N- and O-linked carbohydrate side chains. The amino-terminal protein sequence of the 2B1 Ag shares significant homology with both the macrophage scavenger receptor type 1 motif and the mammalian CD5/CD6 family. The biochemical characteristics and cellular distribution of the 2B1 Ag suggest that it represents the CD5 homologue in X. laevis. While T cells constitutively express this highly conserved molecule, Xenopus B cells acquire the CD5 homologue only when they are stimulated in the presence of T cells.

Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis

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

Aranguren, Xabier L., E-mail: xabier.lopezaranguren@med.kuleuven.be; Beerens, Manu, E-mail: manu.beerens@med.kuleuven.be; Vandevelde, Wouter, E-mail: woutervandevelde@gmail.com

Highlights: {yields} COUP-TFII deficiency in zebrafish affects arterio-venous EC specification. {yields} COUP-TFII is indispensable for lymphatic development in zebrafish. {yields} COUP-TFII knockdown in Xenopus disrupts lymphatic EC differentiation and migration. {yields} COUP-TFII's role in EC fate decisions is evolutionary conserved. -- Abstract: Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment ofmore » venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.« less

Dusty plasma sheath-like structure in the region of lunar terminator

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

Popel, S. I.; Zelenyi, L. M.; Atamaniuk, B.

2015-12-15

The main properties of the dusty plasma layer near the surface over the illuminated and dark parts of the Moon are described. They are used to realize dusty plasma behaviour and to determine electric fields over the terminator region. Possibility of the existence of a dusty plasma sheath-like structure in the region of lunar terminator is shown. The electric fields excited in the terminator region are demonstrated to be on the order of 300 V/m. These electric fields can result in rise of dust particles of the size of 2–3 μm up to an altitude of about 30 cm over the lunar surfacemore » that explains the effect of “horizon glow” observed at the terminator by Surveyor lunar lander.« less

Comparison of Glutamate Turnover in Nerve Terminals and Brain Tissue During [1,6-13C2]Glucose Metabolism in Anesthetized Rats.

PubMed

Patel, Anant B; Lai, James C K; Chowdhury, Golam I M; Rothman, Douglas L; Behar, Kevin L

2017-01-01

The 13 C turnover of neurotransmitter amino acids (glutamate, GABA and aspartate) were determined from extracts of forebrain nerve terminals and brain homogenate, and fronto-parietal cortex from anesthetized rats undergoing timed infusions of [1,6- 13 C 2 ]glucose or [2- 13 C]acetate. Nerve terminal 13 C fractional labeling of glutamate and aspartate was lower than those in whole cortical tissue at all times measured (up to 120 min), suggesting either the presence of a constant dilution flux from an unlabeled substrate or an unlabeled (effectively non-communicating on the measurement timescale) glutamate pool in the nerve terminals. Half times of 13 C labeling from [1,6- 13 C 2 ]glucose, as estimated by least squares exponential fitting to the time course data, were longer for nerve terminals (Glu C4 , 21.8 min; GABA C2 21.0 min) compared to cortical tissue (Glu C4 , 12.4 min; GABA C2 , 14.5 min), except for Asp C3 , which was similar (26.5 vs. 27.0 min). The slower turnover of glutamate in the nerve terminals (but not GABA) compared to the cortex may reflect selective effects of anesthesia on activity-dependent glucose use, which might be more pronounced in the terminals. The 13 C labeling ratio for glutamate-C4 from [2- 13 C]acetate over that of 13 C-glucose was twice as large in nerve terminals compared to cortex, suggesting that astroglial glutamine under the 13 C glucose infusion was the likely source of much of the nerve terminal dilution. The net replenishment of most of the nerve terminal amino acid pools occurs directly via trafficking of astroglial glutamine.

Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

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

Mertz, L.M.; Catt, K.J.

1991-10-01

The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNAmore » in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.« less

Family-specific Kinesin Structures Reveal Neck-linker Length Based on Initiation of the Coiled-coil*

PubMed Central

Phillips, Rebecca K.; Peter, Logan G.; Gilbert, Susan P.

2016-01-01

Kinesin-1, -2, -5, and -7 generate processive hand-over-hand 8-nm steps to transport intracellular cargoes toward the microtubule plus end. This processive motility requires gating mechanisms to coordinate the mechanochemical cycles of the two motor heads to sustain the processive run. A key structural element believed to regulate the degree of processivity is the neck-linker, a short peptide of 12–18 residues, which connects the motor domain to its coiled-coil stalk. Although a shorter neck-linker has been correlated with longer run lengths, the structural data to support this hypothesis have been lacking. To test this hypothesis, seven kinesin structures were determined by x-ray crystallography. Each included the neck-linker motif, followed by helix α7 that constitutes the start of the coiled-coil stalk. In the majority of the structures, the neck-linker length differed from predictions because helix α7, which initiates the coiled-coil, started earlier in the sequence than predicted. A further examination of structures in the Protein Data Bank reveals that there is a great disparity between the predicted and observed starting residues. This suggests that an accurate prediction of the start of a coiled-coil is currently difficult to achieve. These results are significant because they now exclude simple comparisons between members of the kinesin superfamily and add a further layer of complexity when interpreting the results of mutagenesis or protein fusion. They also re-emphasize the need to consider factors beyond the kinesin neck-linker motif when attempting to understand how inter-head communication is tuned to achieve the degree of processivity required for cellular function. PMID:27462072

Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix

PubMed Central

Macías, María J.; Teijido, Oscar; Zifarelli, Giovanni; Martin, Pau; Ramirez-Espain, Ximena; Zorzano, Antonio; Palacín, Manuel; Pusch, Michael; Estévez, Raúl

2006-01-01

Myotonia is a state of hyperexcitability of skeletal-muscle fibres. Mutations in the ClC-1 Cl− channel cause recessive and dominant forms of this disease. Mutations have been described throughout the protein-coding region, including three sequence variations (A885P, R894X and P932L) in a distal C-terminal stretch of residues [CTD (C-terminal domain) region] that are not conserved between CLC proteins. We show that surface expression of these mutants is reduced in Xenopus oocytes compared with wild-type ClC-1. Functional, biochemical and NMR spectroscopy studies revealed that the CTD region encompasses a segment conserved in most voltage-dependent CLC channels that folds with a secondary structure containing a short type II poly-proline helix. We found that the myotonia-causing mutation A885P disturbs this structure by extending the poly-proline helix. We hypothesize that this structural modification results in the observed alteration of the common gate that acts on both pores of the channel. We provide the first experimental investigation of structural changes resulting from myotonia-causing mutations. PMID:17107341

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1.

PubMed

Real Hernandez, Luis M; Gonzalez de Mejia, Elvira

2017-04-01

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption. Copyright © 2017 Elsevier Inc. All rights reserved.

Metallic Glass Wire Based Localization of Kinesin/Microtubule Bio-molecular Motility System

NASA Astrophysics Data System (ADS)

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

2014-03-01

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

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating*

PubMed Central

Chen, Geng-Yuan; Arginteanu, David F. J.; Hancock, William O.

2015-01-01

The kinesin-2 family motor KIF3A/B works together with dynein to bidirectionally transport intraflagellar particles, melanosomes, and neuronal vesicles. Compared with kinesin-1, kinesin-2 is less processive, and its processivity is more sensitive to load, suggesting that processivity may be controlled by different gating mechanisms. We used stopped-flow and steady-state kinetics experiments, along with single-molecule and multimotor assays to characterize the entire kinetic cycle of a KIF3A homodimer that exhibits motility similar to that of full-length KIF3A/B. Upon first encounter with a microtubule, the motor rapidly exchanges both mADP and mATP. When adenosine 5′-[(β,γ)-imido]triphosphate was used to entrap the motor in a two-head-bound state, exchange kinetics were unchanged, indicating that rearward strain in the two-head-bound state does not alter nucleotide binding to the front head. A similar lack of front head gating was found when intramolecular strain was enhanced by shortening the neck linker domain from 17 to 14 residues. In single-molecule assays in ADP, the motor dissociates at 2.1 s−1, 20-fold slower than the stepping rate, demonstrating the presence of rear head gating. In microtubule pelleting assays, the KDMt is similar in ADP and ATP. The data and accompanying simulations suggest that, rather than KIF3A processivity resulting from strain-dependent regulation of nucleotide binding (front head gating), the motor spends a significant fraction of its hydrolysis cycle in a low affinity state but dissociates only slowly from this state. This work provides a mechanism to explain differences in the load-dependent properties of kinesin-1 and kinesin-2. PMID:25657001

Insights into amyloid-like aggregation of H2 region of the C-terminal domain of nucleophosmin.

PubMed

Russo, Anna; Diaferia, Carlo; La Manna, Sara; Giannini, Cinzia; Sibillano, Teresa; Accardo, Antonella; Morelli, Giancarlo; Novellino, Ettore; Marasco, Daniela

2017-02-01

Nucleophosmin (NPM1) is a multifunctional protein involved in a variety of biological processes including the pathogenesis of several human malignancies and is the most frequently mutated gene in Acute Myeloid Leukemia (AML). To deepen the role of protein regions in its biological activities, lately we reported on the structural behavior of dissected C-terminal domain (CTD) helical fragments. Unexpectedly the H2 (residues 264-277) and H3 AML-mutated regions showed a remarkable tendency to form amyloid-like assemblies with fibrillar morphology and β-sheet structure that resulted as toxic when exposed to human neuroblastoma cells. More recently NPM1 was found to be highly expressed and toxic in neurons of mouse models of Huntington's disease (HD). Here we investigate the role of each residue in the β-strand aggregation process of H2 region of NPM1 by performing a systematic alanine scan of its sequence and structural and kinetic analyses of aggregation of derived peptides by means of Circular Dichorism (CD) and Thioflavin T (Th-T) assay. These solution state investigations pointed out the crucial role exerted by the basic amyloidogenic stretch of H2 (264-271) and to shed light on the initial and main interactions involved in fibril formation we performed studies on fibrils deriving from the related Ala peptides through the analysis of fibrils with birefringence of polarized optical microscopy and wide-angle X-ray scattering (WAXS). This analysis suggested that the presence of branched Ile 269 conferred preferential packing patterns that, instead, appeared geometrically hampered by the aromatic side-chain of Phe 268 . Present investigations could be useful to deepen the knowledge of AML molecular mechanisms and the role of cytoplasmatic aggregates of NPM1c+. Copyright © 2016 Elsevier B.V. All rights reserved.

Temporal control of bidirectional lipid-droplet motion in Drosophila depends on the ratio of kinesin-1 and its co-factor Halo

PubMed Central

Arora, Gurpreet K.; Tran, Susan L.; Rizzo, Nicholas; Jain, Ankit; Welte, Michael A.

2016-01-01

ABSTRACT During bidirectional transport, individual cargoes move continuously back and forth along microtubule tracks, yet the cargo population overall displays directed net transport. How such transport is controlled temporally is not well understood. We analyzed this issue for bidirectionally moving lipid droplets in Drosophila embryos, a system in which net transport direction is developmentally controlled. By quantifying how the droplet distribution changes as embryos develop, we characterize temporal transitions in net droplet transport and identify the crucial contribution of the previously identified, but poorly characterized, transacting regulator Halo. In particular, we find that Halo is transiently expressed; rising and falling Halo levels control the switches in global distribution. Rising Halo levels have to pass a threshold before net plus-end transport is initiated. This threshold level depends on the amount of the motor kinesin-1: the more kinesin-1 is present, the more Halo is needed before net plus-end transport commences. Because Halo and kinesin-1 are present in common protein complexes, we propose that Halo acts as a rate-limiting co-factor of kinesin-1. PMID:26906417

Seeing the future: using Xenopus to understand eye regeneration.

PubMed

Tseng, Ai-Sun

2017-01-01

Studies of Xenopus eye development have contributed considerably to the understanding of vertebrate neurogenesis, including eye field specification, cell fate determination and identification of genes critical for eye formation. This knowledge has served as a solid foundation for cellular and molecular examinations of the robust regenerative capacity of the Xenopus eye. The retina, lens, and the optic nerve are capable of regeneration after injury in both larval and adult stages. Here, we discuss the current models for studying eye regeneration in Xenopus and their potential applications for providing insights into human eye diseases. As Xenopus has many of the same tools that are available for other regeneration models, we thus highlight the distinct strengths and versatility of this organism that make it especially suited for extrapolating and testing strategies aimed at promoting regeneration and repair in eye tissues. Furthermore, we outline a promising future for the use of new techniques and approaches to address outstanding questions in understanding eye regeneration. © 2017 Wiley Periodicals, Inc.

KCH kinesin drives nuclear transport and cytoskeletal coalescence for tip cell growth.

PubMed

Yamada, Moé; Goshima, Gohta

2018-06-07

Long-distance transport along microtubules (MTs) is critical for intracellular organisation. In animals, antagonistic motor proteins kinesin (plus end-directed) and dynein (minus end-directed) drive cargo transport. In land plants, however, the identity of motors responsible for transport is poorly understood, as genes encoding cytoplasmic dynein are absent in plant genomes. How other functions of dynein are brought about in plants also remains unknown. Here, we show that a subclass of the kinesin-14 family, KCH (kinesin with calponin homology domain)-which can also bind actin-drives MT minus end-directed nuclear transport in the moss Physcomitrella patens. When all four KCH genes were deleted, the nucleus was not maintained in the cell centre, but was translocated to the apical end of protonemal cells. In the knockout (KO) line, apical cell tip growth was also severely suppressed. KCH was localized to MTs, including at the MT focal point near the tip of protonemal cells, where MT plus ends coalesced with actin filaments. MT focus was not stably maintained in KCH KO lines, whereas actin destabilisation also disrupted the MT focus in wild-type lines despite KCH remaining on unfocused MTs. KCH had distinct functions in nuclear transport and tip growth, as a truncated KCH construct restored nuclear transport activity, but not tip growth retardation of the KO line. Thus, our study identified KCH as a long-distance retrograde transporter as well as a MT crosslinker, reminiscent of the versatile animal dynein. © 2018 American Society of Plant Biologists. All rights reserved.

KinG Is a Plant-Specific Kinesin That Regulates Both Intra- and Intercellular Movement of SHORT-ROOT.

PubMed

Spiegelman, Ziv; Lee, Chin-Mei; Gallagher, Kimberly L

2018-01-01

Both endogenous plant proteins and viral movement proteins associate with microtubules to promote their movement through plasmodesmata. The association of viral movement proteins with microtubules facilitates the formation of virus-associated replication complexes, which are required for the amplification and subsequent spread of the virus. However, the role of microtubules in the intercellular movement of plant proteins is less clear. Here we show that the SHORT-ROOT (SHR) protein, which moves between cells in the root to regulate root radial patterning, interacts with a type-14 kinesin, KINESIN G (KinG). KinG is a calponin homology domain kinesin that directly interacts with the SHR-binding protein SIEL (SHR-INTERACING EMBRYONIC LETHAL) and localizes to both microtubules and actin. Since SIEL and SHR associate with endosomes, we suggest that KinG serves as a linker between SIEL, SHR, and the plant cytoskeleton. Loss of KinG function results in a decrease in the intercellular movement of SHR and an increase in the sensitivity of SHR movement to treatment with oryzalin. Examination of SHR and KinG localization and dynamics in live cells suggests that KinG is a nonmotile kinesin that promotes the pausing of SHR-associated endosomes. We suggest a model in which interaction of KinG with SHR allows for the formation of stable movement complexes that facilitate the cell-to-cell transport of SHR. © 2018 American Society of Plant Biologists. All Rights Reserved.

NtKRP, a kinesin-12 protein, regulates embryo/seed size and seed germination via involving in cell cycle progression at the G2/M transition.

PubMed

Tian, Shujuan; Wu, Jingjing; Li, Fen; Zou, Jianwei; Liu, Yuwen; Zhou, Bing; Bai, Yang; Sun, Meng-Xiang

2016-10-25

Kinesins comprise a superfamily of microtubule-based motor proteins involved in essential processes in plant development, but few kinesins have been functionally identified during seed development. Especially, few kinesins that regulate cell division during embryogenesis have been identified. Here we report the functional characterization of NtKRP, a motor protein of the kinesin-12 family. NtKRP is predominantly expressed in embryos and embryonic roots. NtKRP RNAi lines displayed reductions in cell numbers in the meristematic zone, in embryonic root length, and in mature embryo and seed sizes. Furthermore, we also show that CDKA;1 binds to NtKRP at the consensus phosphorylation sites and that the decreased cell numbers in NtKRP-silenced embryos are due to a delay in cell division cycle at the G2/M transition. In addition, binding between the cargo-binding tail domain of NtKRP and CDKA; 1 was also determined. Our results reveal a novel molecular pathway that regulates embryo/seed development and critical role of kinesin in temporal and spatial regulation of a specific issue of embryo developmental.

Expression and functional analyses of a Kinesin gene GhKIS13A1 from cotton (Gossypium hirsutum) fiber.

PubMed

Li, Yan-Jun; Zhu, Shou-Hong; Zhang, Xin-Yu; Liu, Yong-Chang; Xue, Fei; Zhao, Lan-Jie; Sun, Jie

2017-06-12

Cotton fiber, a natural fiber widely used in the textile industry, is differentiated from single cell of ovule epidermis. A large number of genes are believed to be involved in fiber formation, but so far only a few fiber genes have been isolated and functionally characterized in this developmental process. The Kinesin13 subfamily was found to play key roles during cell division and cell elongation, and was considered to be involved in the regulation of cotton fiber development. The full length of coding sequence of GhKIS13A1 was cloned using cDNA from cotton fiber for functional characterization. Expression pattern analysis showed that GhKIS13A1 maintained a lower expression level during cotton fiber development. Biochemical assay showed that GhKIS13A1 has microtubule binding activity and basal ATPase activity that can be activated significantly by the presence of microtubules. Overexpression of GhKIS13A1 in Arabidopsis reduced leaf trichomes and the percentage of three-branch trichomes, and increased two-branch and shriveled trichomes compared to wild-type. Additionally, the expression of GhKIS13A1 in the Arabidopsis Kinesin-13a-1 mutant rescued the defective trichome branching pattern of the mutant, making its overall trichome branching pattern back to normal. Our results suggested that GhKIS13A1 is functionally compatible with AtKinesin-13A regarding their role in regulating the number and branching pattern of leaf trichomes. Given the developmental similarities between cotton fibers and Arabidopsis trichomes, it is speculated that GhKIS13A1 may also be involved in the regulation of cotton fiber development.

The unique N-terminal zinc finger of synaptotagmin-like protein 4 reveals FYVE structure.

PubMed

Miyamoto, Kazuhide; Nakatani, Arisa; Saito, Kazuki

2017-12-01

Synaptotagmin-like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N-terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N-terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross-brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C 4 C 4 -type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin-conjugating enzyme (E2)-binding capability, cross-brace structures with eight zinc-ligating residues are needed as the scaffold. The cross-brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs. © 2017 The Protein Society.

Kinesin-1 and mitochondrial motility control by discrimination of structurally equivalent but distinct subdomains in Ran-GTP-binding domains of Ran-binding protein 2.

PubMed

Patil, Hemangi; Cho, Kyoung-in; Lee, James; Yang, Yi; Orry, Andrew; Ferreira, Paulo A

2013-03-27

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein-protein and protein-phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD(n = 1-4)) with close structural homology to the PH domain of Bruton's tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure-function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260,000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.

Xenopus laevis ribosomal protein genes: isolation of recombinant cDNA clones and study of the genomic organization.

PubMed Central

Bozzoni, I; Beccari, E; Luo, Z X; Amaldi, F

1981-01-01

Poly-A+ mRNA from Xenopus laevis oocytes, partially enriched for r-protein coding capacity has been used as starting material for preparing a cDNA bank in plasmid pBR322. The clones containing sequences specific for r-proteins have been selected by translation of the complementary mRNAs. Clones for six different r-proteins have been identified and utilized as probes for studying their genomic organization. Two gene copies per haploid genome were found for r-proteins L1, L14, S19, and four-five for protein S1, S8 and L32. Moreover a population polymorphism has been observed for the genomic regions containing sequences for r-protein S1, S8 and L14. Images PMID:6112733

Assembly of viral particles in Xenopus oocytes: pre-surface-antigens regulate secretion of the hepatitis B viral surface envelope particle.

PubMed Central

Standring, D N; Ou, J H; Rutter, W J

1986-01-01

Infection with hepatitis B virus (HBV) is associated with the production of a viral envelope particle that contains membrane lipids, surface antigen (S), and two presurface-antigens (pre-S) comprised of the entire S moiety with approximately 55 (pre-S2) and 174 (pre-S1) additional NH2-terminal amino acids. We show here that Xenopus oocytes injected with synthetic S mRNA assemble and secrete characteristic 22-nm viral envelope particles. In contrast, pre-S1 and pre-S2 antigens are synthesized but not secreted. By coinjecting mRNAs, we found that synthesis of high levels of pre-S proteins specifically inhibits S antigen secretion. On the other hand, high levels of S synthesis can drive the secretion of small amounts of either pre-S antigen. These observations are consistent with a model for viral envelope assembly in which both S and pre-S proteins are incorporated into a multimeric particle, presumably via interactions between the S protein domains, while the pre-S amino-terminal moieties regulate the secretion of this structure. Our results indicate that Xenopus oocytes will provide a powerful system for studying the morphogenesis of simple structures of viral or cellular origin. Images PMID:3467308

Alpha-A crystallin: quantitation of C-terminal modification during lens aging

NASA Technical Reports Server (NTRS)

Takemoto, L.; Gopalakrishnan, S.; Spooner, B. S. (Principal Investigator)

1994-01-01

Previous studies have demonstrated that the C-terminal region of alpha-A crystallin is susceptible to age-dependent, posttranslational modification. To quantitate the amount of modification, alpha-A crystallin was purified from total proteins of the aging bovine lens, then digested with lys-C endoproteinase. Reverse phase, high pressure liquid chromatography was used to resolve and quantitate the resulting peptides, to determine the amount of C-terminal peptide relative to peptides from other regions of the protein that have not been reported to undergo modification. The results indicate that relative to alpha-A crystallin from newborn lens, posttranslational modification has occurred in approximately 45-55% of the C-terminal region from mature lens. These results demonstrate extensive modification of the C-terminal region of alpha-A crystallin from the mature lens, indicating that during the aging process, posttranslational modifications in this region may make significant contributions to the aggregated state and/or molecular chaperone properties of the molecule.

The KAC family of kinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants.

PubMed

Suetsugu, Noriyuki; Sato, Yoshikatsu; Tsuboi, Hidenori; Kasahara, Masahiro; Imaizumi, Takato; Kagawa, Takatoshi; Hiwatashi, Yuji; Hasebe, Mitsuyasu; Wada, Masamitsu

2012-11-01

Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.

Engineered tug-of-war between kinesin and dynein controls direction of microtubule transport in vivo

PubMed Central

Rezaul, Karim; Gupta, Dipika; Semenova, Irina; Ikeda, Kazuho; Kraikivski, Pavel; Yu, Ji; Cowan, Ann; Zaliapin, Ilya; Rodionov, Vladimir

2017-01-01

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus-end directed kinesins and minus-end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT-based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug-of-war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug-of-war between opposing MT motors alone, by attaching large number of kinesin-1 motors to organelles transported by dynein to minus-ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus-end-directed dynein-dependent MT runs, leading to a reversal of the overall direction of dynein-driven organelles in vivo. Therefore in the absence of external regulators tug-of-war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo. PMID:26843027

How does the Xenopus laevis embryonic cell cycle avoid spatial chaos?

PubMed Central

Gelens, Lendert; Huang, Kerwyn Casey; Ferrell, James E.

2015-01-01

Summary Theoretical studies have shown that a deterministic biochemical oscillator can become chaotic when operating over a sufficiently large volume, and have suggested that the Xenopus laevis cell cycle oscillator operates close to such a chaotic regime. To experimentally test this hypothesis, we decreased the speed of the post-fertilization calcium wave, which had been predicted to generate chaos. However, cell divisions were found to develop normally and eggs developed into normal tadpoles. Motivated by these experiments, we carried out modeling studies to understand the prerequisites for the predicted spatial chaos. We showed that this type of spatial chaos requires oscillatory reaction dynamics with short pulse duration, and postulated that the mitotic exit in Xenopus laevis is likely slow enough to avoid chaos. In systems with shorter pulses, chaos may be an important hazard, as in cardiac arrhythmias, or a useful feature, as in the pigmentation of certain mollusk shells. PMID:26212326

Electroclinic effect in a chiral carbosilane-terminated 5-phenylpyrimidine liquid crystal with 'de Vries-like' properties.

PubMed

Schubert, Christopher P J; Müller, Carsten; Wand, Michael D; Giesselmann, Frank; Lemieux, Robert P

2015-08-14

The chiral carbosilane-terminated liquid crystal 2-[(2S,3S)-2,3-difluorohexyloxy]-5-[4-(12,12,14,14,16,16-hexamethyl-12,14,16-trisilaheptadecyloxy)phenyl]pyrimidine () undergoes a smectic A*-smectic C* phase transition with a maximum layer contraction of only 0.2%. It exhibits an electroclinic effect (ECE) comparable to that reported for the 'de Vries-like' liquid crystal and shows no appreciable optical stripe defects due to horizontal chevron formation.

CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes

PubMed Central

Nagel, G; Barbry, P; Chabot, H; Brochiero, E; Hartung, K; Grygorczyk, R

2005-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR–ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain. PMID:15746174

Ciliary targeting of olfactory CNG channels requires the CNGB1b subunit and the kinesin-2 motor protein, KIF17.

PubMed

Jenkins, Paul M; Hurd, Toby W; Zhang, Lian; McEwen, Dyke P; Brown, R Lane; Margolis, Ben; Verhey, Kristen J; Martens, Jeffrey R

2006-06-20

Nonmotile cilia on olfactory sensory neurons (OSNs) compartmentalize signaling molecules, including odorant receptors and cyclic nucleotide-gated (CNG) channels, allowing for efficient, spatially confined responses to sensory stimuli . Little is known about the mechanisms of the ciliary targeting of olfactory CNG channels, composed of three subunits: CNGA2, CNGA4, and CNGB1b . Recent reports suggest that subunit composition of the retinal CNG channel influences localization, leading to disease . However, the mechanistic role of subunits in properly targeting native olfactory CNG channels remains unclear. Here, we show that heteromeric assembly with CNGB1b, containing a critical carboxy-terminal motif (RVxP), is required for ciliary trafficking of olfactory CNG channels. Movement of proteins within the cilia is governed by intraflagellar transport (IFT), a process that facilitates bidirectional movement of cargo along microtubules. Work in C. elegans has established that heterotrimeric and homodimeric kinesin-2 family members play a critical role in anterograde transport . In mammalian systems, the heterotrimeric KIF3a/KIF3b/KAP-3 complex plays a clear role in IFT; however, no role has been established for KIF17, the mammalian homolog of OSM-3 . Here, we demonstrate that KIF17 is required for olfactory CNG channel targeting, providing novel insights into mechanisms of mammalian ciliary transport.

Roles of the N- and C-terminal sequences in Hsp27 self-association and chaperone activity

PubMed Central

Lelj-Garolla, Barbara; Mauk, A Grant

2012-01-01

The small heat shock protein 27 (Hsp27 or HSPB1) is an oligomeric molecular chaperone in vitro that is associated with several neuromuscular, neurological, and neoplastic diseases. Although aspects of Hsp27 biology are increasingly well known, understanding of the structural basis for these involvements or of the functional properties of the protein remains limited. As all 11 human small heat shock proteins (sHsps) possess an α-crystallin domain, their varied functional and physiological characteristics must arise from contributions of their nonconserved sequences. To evaluate the role of two such sequences in Hsp27, we have studied three Hsp27 truncation variants to assess the functional contributions of the nonconserved N- and C-terminal sequences. The N-terminal variants Δ1–14 and Δ1–24 exhibit little chaperone activity, somewhat slower but temperature-dependent subunit exchange kinetics, and temperature-independent self-association with formation of smaller oligomers than wild-type Hsp27. The C-terminal truncation variants exhibit chaperone activity at 40 °C but none at 20 °C, limited subunit exchange, and temperature-independent self-association with an oligomer distribution at 40 °C that is very similar to that of wild-type Hsp27. We conclude that more of the N-terminal sequence than simply the WPDF domain is essential in the formation of larger, native-like oligomers after binding of substrate and/or in binding of Hsp27 to unfolding peptides. On the other hand, the intrinsically flexible C-terminal region drives subunit exchange and thermally-induced unfolding, both of which are essential to chaperone activity at low temperature and are linked to the temperature dependence of Hsp27 self-association. PMID:22057845

Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

PubMed Central

Wilson, Brenda A.; Ponferrada, Virgilio G.; Vallance, Jefferson E.; Ho, Mengfei

1999-01-01

We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells. PMID:9864199

Dusty plasma sheath-like structure in the lunar terminator region

NASA Astrophysics Data System (ADS)

Popel, Sergey; Zelenyi, Lev; Atamaniuk, Barbara

2016-07-01

The main properties of the dusty plasma layer near the surface over the illuminated and dark parts of the Moon are described. They are used to realize dusty plasma behaviour and to determine electric fields over the terminator region. Possibility of the existence of a dusty plasma sheath-like structure [1] in the region of lunar terminator is shown. The electric fields excited in the terminator region are demonstrated to be on the order of 300 V/m. These electric fields can result in rise of dust particles of the size of a few micrometers up to an altitude of about 30 cm over the lunar surface that explains the effect of ``horizon glow" observed at the terminator by Surveyor lunar lander. This work was supported in part by the Presidium of the Russian Academy of Sciences (under Fundamental Research Program No. 7, ``Experimental and Theoretical Study of the Solar System Objects and Stellar Planet Systems. Transient Explosion Processes in Astrophysics" and the Russian Foundation for Basic Research (Project No. 15-02-05627-a). [1] S. I. Popel, L. M. Zelenyi, and B. Atamaniuk, Phys. Plasmas 22, 123701 (2015); doi: 10.1063/1.4937368.

Mitotic Chromosome Biorientation in Fission Yeast Is Enhanced by Dynein and a Minus-end–directed, Kinesin-like Protein

PubMed Central

Spiridonov, Ilia S.; McIntosh, J. Richard

2007-01-01

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end–directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole. PMID:17409356

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

PubMed Central

Sardar, Harjinder S.; Luczak, Vincent G.; Lopez, Maria M.; Lister, Bradford C.; Gilbert, Susan P.

2010-01-01

Summary Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis including establishment of microtubule (MT)-chromosome linkage and movement of monooriented chromosomes on kinetochore microtubules (kMTs) for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus-ends. A human CENP-E construct was engineered, expressed, and purified which yielded the CENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity marked MTs exhibited CENP-E promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end elongated MTs showed CENP-E at the MT plus-end. The time dependence of CENP-E promoted MT elongation in solution best fit a single exponential function (kobs = 5.1 s−1), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E as part of its function in chromosome kinetochore-MT linkage plays a direct role in MT elongation. PMID:20797864

C-terminals in the mouse branchiomotor nuclei originate from the magnocellular reticular formation.

PubMed

Matsui, Toshiyasu; Hongo, Yu; Haizuka, Yoshinori; Kaida, Kenichi; Matsumura, George; Martin, Donna M; Kobayashi, Yasushi

2013-08-26

Large cholinergic synaptic boutons called "C-terminals" contact motoneurons and regulate their excitability. C-terminals in the spinal somatic motor nuclei originate from cholinergic interneurons in laminae VII and X that express a transcription factor Pitx2. Cranial motor nuclei contain another type of motoneuron: branchiomotor neurons. Although branchiomotor neurons receive abundant C-terminal projections, the neural source of these C-terminals remains unknown. In the present study, we first examined whether cholinergic neurons express Pitx2 in the reticular formation of the adult mouse brainstem, as in the spinal cord. Although Pitx2-positive cholinergic neurons were observed in the magnocellular reticular formation and region around the central canal in the caudal medulla, none was present more rostrally in the brainstem tegmentum. We next explored the origin of C-terminals in the branchiomotor nuclei by using biotinylated dextran amine (BDA). BDA injections into the magnocellular reticular formation of the medulla and pons resulted in the labeling of numerous C-terminals in the branchiomotor nuclei: the ambiguous, facial, and trigeminal motor nuclei. Our results revealed that the origins of C-terminals in the branchiomotor nuclei are cholinergic neurons in the magnocellular reticular formation not only in the caudal medulla, but also at more rostral levels of the brainstem, which lacks Pitx2-positive neurons. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

C-terminals in the mouse branchiomotor nuclei originate from the magnocellular reticular formation

PubMed Central

Matsui, Toshiyasu; Hongo, Yu; Haizuka, Yoshinori; Kaida, Kenichi; Matsumura, George; Martin, Donna M.; Kobayashi, Yasushi

2013-01-01

Large cholinergic synaptic boutons called "C-terminals" contact motoneurons and regulate their excitability. C-terminals in the spinal somatic motor nuclei originate from cholinergic interneurons in laminae VII and X that express a transcription factor Pitx2. Cranial motor nuclei contain another type of motoneuron: branchiomotor neurons. Although branchiomotor neurons receive abundant C-terminal projections, the neural source of these C-terminals remains unknown. In the present study, we first examined whether cholinergic neurons express Pitx2 in the reticular formation of the adult mouse brainstem, as in the spinal cord. Although Pitx2-positive cholinergic neurons were observed in the magnocellular reticular formation and region around the central canal in the caudal medulla, none was present more rostrally in the brainstem tegmentum. We next explored the origin of C-terminals in the branchiomotor nuclei by using biotinylated dextran amine (BDA). BDA injections into the magnocellular reticular formation of the medulla and pons resulted in the labeling of numerous C-terminals in the branchiomotor nuclei: the ambiguous, facial, and trigeminal motor nuclei. Our results revealed that the origins of C-terminals in the branchiomotor nuclei are cholinergic neurons in the magnocellular reticular formation not only in the caudal medulla, but also at more rostral levels of the brainstem, which lacks Pitx2-positive neurons. PMID:23756176

Elucidation of the Hsp90 C-terminal Inhibitor Binding Site

PubMed Central

Matts, Robert L.; Dixit, Anshuman; Peterson, Laura B.; Sun, Liang; Voruganti, Sudhakar; Kalyanaraman, Palgunan; Hartson, Steve D.; Verkhivker, Gennady M.; Blagg, Brian S. J.

2011-01-01

The Hsp90 chaperone machine is required for the folding, activation and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains, however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein. PMID:21548602

Insulin-like growth factor binding protein-2: contributions of the C-terminal domain to insulin-like growth factor-1 binding.

PubMed

Kibbey, Megan M; Jameson, Mark J; Eaton, Erin M; Rosenzweig, Steven A

2006-03-01

Signaling by the insulin-like growth factor (IGF)-1 receptor (IGF-1R) has been implicated in the promotion and aggressiveness of breast, prostate, colorectal, and lung cancers. The IGF binding proteins (IGFBPs) represent a class of natural IGF antagonists that bind to and sequester IGF-1/2 from the IGF-1R, making them attractive candidates as therapeutics for cancer prevention and control. Recombinant human IGFBP-2 significantly attenuated IGF-1-stimulated MCF-7 cell proliferation with coaddition of 20 or 100 nM IGFBP-2 (50 or 80% inhibition, respectively). We previously identified IGF-1 contact sites both upstream and downstream of the CWCV motif (residues 247-250) in human IGFBP-2 (J Biol Chem 276:2880-2889, 2001). To further test their contributions to IGFBP-2 function, the single tryptophan in human IGFBP-2, Trp-248, was selectively cleaved with 2-(2'nitrophenylsulfenyl)-3-methyl-3 bromoindolenine (BNPS-skatole) and the BNPS-skatole products IGFBP-2(1-248) and IGFBP-2(249-289) as well as IGFBP-2(1-190) were expressed as glutathione S-transferase-fusion proteins and purified. Based on competition binding analysis, deletion of residues 249 to 289 caused an approximately 20-fold decrease in IGF-1 binding affinity (IGFBP-2 EC50 = 0.35 nM and IGFBP-2(1-248) = 7 nM). Removal of the remainder of the C-terminal domain had no further effect on affinity (IGFBP-2(1-190) EC50 = 9.2 nM). In kinetic assays, IGFBP-2(1-248) and IGFBP-2(1-190) exhibited more rapid association and dissociation rates than full-length IGFBP-2. These results confirm that regions upstream and downstream of the CWCV motif participate in IGF-1 binding. They further support the development of full-length IGFBP-2 as a cancer therapeutic.

Age-dependent loss of the C-terminal amino acid from alpha crystallin

NASA Technical Reports Server (NTRS)

Emmons, T.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1992-01-01

Antiserum made against the C-terminal region of alpha-A crystallin was used to monitor the purification of a tryptic peptide containing the C-terminus of the molecule from fetal versus adult bovine lenses. Mass spectral analysis of the peptide preparations obtained from these lenses demonstrated the presence of a peptide (T20) containing an intact C-terminus from fetal lenses and the presence of an additional peptide (T20') from older lenses that contained a cleaved C-terminal serine. These results demonstrate an age-dependent processing of alpha-A crystallin in the bovine lens, resulting in removal of the C-terminal amino acid residue.

Detection and initial characterization of protein entities consisting of the HIV glycoprotein cytoplasmic C-terminal domain alone.

PubMed

Pfeiffer, Tanya; Ruppert, Thomas; Schaal, Heiner; Bosch, Valerie

2013-06-20

Employing antibodies against the cytoplasmic tail of the HIV-1 glycoprotein (Env-CT), in addition to gp160/gp41, we have identified several novel small Env proteins (<25kD) in HIV-1 transfected and infected cells. Mass spectrometric and mutational analyses show that two mechanisms contribute to their generation. Thus the protein, designated Tr-Env-CT (for truncated Env-CT), consists of the C-terminal 139 amino acids (aa) of Env (aa 718-856) with the N-terminal Q718 modified to pyroglutamic acid. It is likely derived from full-length Env protein by proteolytic processing. A further heterogeneous set of slightly larger proteins, termed Env-CT* species, are rather derived from spliced mRNAs containing only those Env C-terminal residues (aa 719-856) which overlap with the second tat and rev coding exons. They are N-terminally extended in the same reading frame. It is conceivable that essential Env-CT functions may be fulfilled by these novel species rather than by the full-length glycoprotein itself. Copyright © 2013 Elsevier Inc. All rights reserved.

Xenopus: An Emerging Model for Studying Congenital Heart Disease

PubMed Central

Kaltenbrun, Erin; Tandon, Panna; Amin, Nirav M.; Waldron, Lauren; Showell, Chris; Conlon, Frank L.

2011-01-01

Congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death. Clinical studies have identified a number of congenital heart syndromes associated with mutations in genes that are involved in the complex process of cardiogenesis. The African clawed frog, Xenopus, has been instrumental in studies of vertebrate heart development and provides a valuable tool to investigate the molecular mechanisms underlying human congenital heart diseases. In this review, we discuss the methodologies that make Xenopus an ideal model system to investigate heart development and disease. We also outline congenital heart conditions linked to cardiac genes that have been well-studied in Xenopus and describe some emerging technologies that will further aid in the study of these complex syndromes. PMID:21538812

Crystallization and X-ray diffraction analysis of the CH domain of the cotton kinesin GhKCH2

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

Qin, Xinghua; The Fourth Military Medical University, No. 169 Changlexi Road, Xincheng District, Xi’an 710032, People’s Republic of; Chen, Ziwei

The cloning, expression, purification and crystallization of the CH domain of the plant-specific kinesin GhKCH2 is reported. GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin (K{sub d} = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30–166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000 as a precipitant. The crystalsmore » diffracted to a resolution of 2.5 Å and belonged to space group P2{sub 1}, with unit-cell parameters a = 41.57, b = 81.92, c = 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å{sup 3} Da{sup −1}, corresponding to a solvent content of 44.8%.« less

Structural Basis for Toughness and Flexibility in the C-terminal Passenger Domain of an Acinetobacter Trimeric Autotransporter Adhesin*

PubMed Central

Koiwai, Kotaro; Hartmann, Marcus D.; Linke, Dirk; Lupas, Andrei N.; Hori, Katsutoshi

2016-01-01

Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. PMID:26698633

Graphics Software For VT Terminals

NASA Technical Reports Server (NTRS)

Wang, Caroline

1991-01-01

VTGRAPH graphics software tool for DEC/VT computer terminal or terminals compatible with it, widely used by government and industry. Callable in FORTRAN or C language, library program enabling user to cope with many computer environments in which VT terminals used for window management and graphic systems. Provides PLOT10-like package plus color or shade capability for VT240, VT241, and VT300 terminals. User can easily design more-friendly user-interface programs and design PLOT10 programs on VT terminals with different computer systems. Requires ReGis graphics set terminal and FORTRAN compiler.

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: functional coupling of the sphingosine-1-phosphate receptor to PLC-xbeta in Xenopus oocytes.

PubMed

Noh, S J; Kim, M J; Shim, S; Han, J K

1998-08-01

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+-dependent oscillatory Cl- currents by acting through membrane-bound receptors. External application of 50 microM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-microM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca2+-dependent Cl- currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, because Cl- currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xbeta), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xbeta, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein alpha subunits that were identified in Xenopus laevis; G(q)alpha, G11alpha, G0alpha, and G(i1)alpha. Among AS-ODNs against the G alphas tested, AS-G(q)alpha and AS-G(i1)alpha to S1P and AS-G(q)alpha and AS-G11alpha to LPA specifically reduced current responses, respectively, to about 20-30% of controls. These results demonstrate that LPA and S1P, although they have similar structural

75 FR 998 - Terminate Long Range Aids to Navigation (Loran-C) Signal

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-07

... to Navigation (Loran-C) Signal AGENCY: U.S. Coast Guard, DHS. ACTION: Notice. SUMMARY: On October 28... Act allows for the termination of the Loran-C system subject to the Coast Guard certifying that termination of the Loran-C signal will not adversely impact the safety of maritime navigation and the...

Cyclic AMP-dependent regulation of P-type calcium channels expressed in Xenopus oocytes.

PubMed

Fournier, F; Bourinet, E; Nargeot, J; Charnet, P

1993-05-01

Xenopus oocytes injected with rat cerebellum mRNA, express voltage-dependent calcium channels (VDCC). These were identified as P-type Ca2+ channels by their insensitivity to dihydropyridines and omega-conotoxin and by their blockade by Agelenopsis aperta venom (containing the funnel-web spider toxins: FTX and omega-Aga-IV-A). Coinjection of cerebellar mRNA and antisense oligonucleotide complementary to the dihydropyridine-resistant brain Ca2+ channel, named BI [Mori Y. et al. (1991) Nature 350:398-402] or rbA [Starr T. V. B. et al. (1991) Proc Natl Acad Sci USA 88:5621-5625], strongly reduced the expressed Ba2+ current suggesting that these clones encode a P-type VDCC. The macroscopic Ca2+ channel activity was increased by direct intraoocyte injection of cAMP. This increase in current amplitude was concomitant with a slowing of current inactivation, and was attributed to activation of protein kinase A, since it could be antagonized by a peptidic inhibitor of this enzyme. Positive regulation of P-type VDCC could be of importance in Purkinje neurons and motor nerve terminals where this channel is predominant.

Charged and Hydrophobic Surfaces on the A Chain of Shiga-Like Toxin 1 Recognize the C-Terminal Domain of Ribosomal Stalk Proteins

PubMed Central

McCluskey, Andrew J.; Bolewska-Pedyczak, Eleonora; Jarvik, Nick; Chen, Gang; Sidhu, Sachdev S.; Gariépy, Jean

2012-01-01

Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A1 chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A1 chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A1 variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A1 chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A1 chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A1 chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs. PMID:22355345

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity.

PubMed

Tuli, Amit; Thiery, Jerome; James, Ashley M; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B; Orange, Jordan S; Lieberman, Judy; Brenner, Michael B

2013-12-01

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity

PubMed Central

Tuli, Amit; Thiery, Jerome; James, Ashley M.; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B.; Orange, Jordan S.; Lieberman, Judy; Brenner, Michael B.

2013-01-01

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity. PMID:24088571

The unexpected teratogenicity of RXR antagonist UVI3003 via activation of PPARγ in Xenopus tropicalis

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

Zhu, Jingmin

2017-01-01

The RXR agonist (triphenyltin, TPT) and the RXR antagonist (UVI3003) both show teratogenicity and, unexpectedly, induce similar malformations in Xenopus tropicalis embryos. In the present study, we exposed X. tropicalis embryos to UVI3003 in seven specific developmental windows and identified changes in gene expression. We further measured the ability of UVI3003 to activate Xenopus RXRα (xRXRα) and PPARγ (xPPARγ) in vitro and in vivo. We found that UVI3003 activated xPPARγ either in Cos7 cells (in vitro) or Xenopus embryos (in vivo). UVI3003 did not significantly activate human or mouse PPARγ in vitro; therefore, the activation of Xenopus PPARγ by UVI3003more » is novel. The ability of UVI3003 to activate xPPARγ explains why UVI3003 and TPT yield similar phenotypes in Xenopus embryos. Our results indicate that activating PPARγ leads to teratogenic effects in Xenopus embryos. More generally, we infer that chemicals known to specifically modulate mammalian nuclear hormone receptors cannot be assumed to have the same activity in non-mammalian species, such as Xenopus. Rather they must be tested for activity and specificity on receptors of the species in question to avoid making inappropriate conclusions. - Highlights: • UVI3003 is a RXRs antagonist and shows teratogenicity to Xenopus embryos. • UVI3003 activated xPPARγ either in Cos7 cells or Xenopus embryos. • UVI3003 did not activate human or mouse PPARγ in Cos7 cells. • Activating PPARγ leads to teratogenic effects in Xenopus embryos.« less

Alternative C-Terminal Helix Orientation Alters Chemokine Function

PubMed Central

Kuo, Je-Hung; Chen, Ya-Ping; Liu, Jai-Shin; Dubrac, Alexandre; Quemener, Cathy; Prats, Hervé; Bikfalvi, Andreas; Wu, Wen-guey; Sue, Shih-Che

2013-01-01

Chemokines, a subfamily of cytokines, are small, secreted proteins that mediate a variety of biological processes. Various chemokines adopt remarkable conserved tertiary structure comprising an anti-parallel β-sheet core domain followed by a C-terminal helix that packs onto the β-sheet. The conserved structural feature has been considered critical for chemokine function, including binding to cell surface receptor. The recently isolated variant, CXCL4L1, is a homologue of CXCL4 chemokine (or platelet factor 4) with potent anti-angiogenic activity and differed only in three amino acid residues of P58L, K66E, and L67H. In this study we show by x-ray structural determination that CXCL4L1 adopts a previously unrecognized structure at its C terminus. The orientation of the C-terminal helix protrudes into the aqueous space to expose the entire helix. The alternative helix orientation modifies the overall chemokine shape and surface properties. The L67H mutation is mainly responsible for the swing-out effect of the helix, whereas mutations of P58L and K66E only act secondarily. This is the first observation that reports an open conformation of the C-terminal helix in a chemokine. This change leads to a decrease of its glycosaminoglycan binding properties and to an enhancement of its anti-angiogenic and anti-tumor effects. This unique structure is recent in evolution and has allowed CXCL4L1 to gain novel functional properties. PMID:23536183

Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse

PubMed Central

Sadananda, Aparna; Hamid, Runa; Doodhi, Harinath; Ghosal, Debnath; Girotra, Mukul; Jana, Swadhin Chandra; Ray, Krishanu

2012-01-01

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin based movement of large protein-aggregates aids this process. Choline acetyl-transferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates towards synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution. PMID:22486887

Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor l-arginine

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

Garnett, James A.; Baumberg, Simon; Stockley, Peter G.

2007-11-01

The crystal structure of the C-terminal domain hexameric core of AhrC, with bound corepressor (l-arginine), has been solved at 1.95 Å resolution. Binding of l-arginine results in a rotation between the two trimers of the hexamer, leading to the activation of the DNA-binding state. The arginine repressor/activator protein (AhrC) from Bacillus subtilis belongs to a large family of multifunctional transcription factors that are involved in the regulation of bacterial arginine metabolism. AhrC interacts with operator sites in the promoters of arginine biosynthetic and catabolic operons, acting as a transcriptional repressor at biosynthetic sites and an activator of transcription at catabolicmore » sites. AhrC is a hexamer of identical subunits, each having two domains. The C-terminal domains form the core of the protein and are involved in oligomerization and l-arginine binding. The N-terminal domains lie on the outside of the compact core and play a role in binding to 18 bp DNA operators called ARG boxes. The C-terminal domain of AhrC has been expressed, purified and characterized, and also crystallized as a hexamer with the bound corepressor l-arginine. Here, the crystal structure refined to 1.95 Å is presented.« less

Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

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

Dehennaut, Vanessa; EA 4020, Laboratoire de Regulation des Signaux de Division, USTL, IFR147, Villeneuve d'Ascq; Hanoulle, Xavier

2008-05-02

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads tomore » a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.« less

Discovery of a vezatin-like protein for dynein-mediated early endosome transport

PubMed Central

Yao, Xuanli; Arst, Herbert N.; Wang, Xiangfeng; Xiang, Xin

2015-01-01

Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo. PMID:26378255

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth*

PubMed Central

Kelly, Geraldine M.; Buckley, Deirdre A.; Kiely, Patrick A.; Adams, David R.; O'Connor, Rosemary

2012-01-01

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the 1248SFYYS1252 motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling. PMID:22685298

C-terminal peptide extension via gas-phase ion/ion reactions

PubMed Central

Peng, Zhou; McLuckey, Scott A.

2015-01-01

The formation of peptide bonds is of great importance from both a biological standpoint and in routine organic synthesis. Recent work from our group demonstrated the synthesis of peptides in the gas-phase via ion/ion reactions with sulfo-NHS reagents, which resulted in conjugation of individual amino acids or small peptides to the N-terminus of an existing ‘anchor’ peptide. Here, we demonstrate a complementary approach resulting in the C-terminal extension of peptides. Individual amino acids or short peptides can be prepared as reagents by incorporating gas phase-labile protecting groups to the reactive C-terminus and then converting the N-terminal amino groups to the active ketenimine reagent. Gas-phase ion/ion reactions between the anionic reagents and doubly protonated “anchor” peptide cations results in extension of the “anchor” peptide with new amide bond formation at the C-terminus. We have demonstrated that ion/ion reactions can be used as a fast, controlled, and efficient means for C-terminal peptide extension in the gas phase. PMID:26640400

Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

PubMed Central

Moreira, Gustavo M. S. G.; Conceição, Fabricio R.; McBride, Alan J. A.; Pinto, Luciano da S.

2013-01-01

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins. PMID:24260572

Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

PubMed

Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

2013-01-01

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure.

PubMed

Matsui, Takeshi; Tabayashi, Ayako; Iwano, Megumi; Shinmyo, Atsuhiko; Kato, Ko; Nakayama, Hideki

2011-02-01

Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.

The relationship between the agonist-induced activation and desensitization of the human tachykinin NK2 receptor expressed in Xenopus oocytes

PubMed Central

Maudsley, S; Gent, J P; Findlay, J B C; Donnelly, D

1998-01-01

Repeated applications of neurokinin A (NKA) to oocytes injected with 25 ng wild-type hNK2 receptor cRNA caused complete attenuation of second and subsequent NKA-induced responses while analogous experiments using repeated applications of GR64349 and [Nle10]NKA(4–10) resulted in no such desensitization. This behaviour has been previously attributed to the ability of the different ligands to stabilize different active conformations of the receptor that have differing susceptibilities to receptor kinases (Nemeth & Chollet, 1995).However, for Xenopus oocytes injected (into the nucleus) with 10 ng wild-type hNK2 receptor cDNA, a single 100 nM concentration of any of the three ligands resulted in complete desensitization to further concentrations.On the other hand, none of the ligands caused any desensitization in oocytes injected with 0.25 ng wild-type hNK2 receptor cRNA, even at concentrations up to 10 μM.The two N-terminally truncated analogues of neurokinin A have a lower efficacy than NKA and it is likely that it is this property which causes the observed differences in desensitization, rather than the formation of alternative active states of the receptor.The peak calcium-dependent chloride current is not a reliable measure of maximal receptor stimulation and efficacy is better measured in this system by studying agonist-induced desensitization.The specific adenylyl cyclase inhibitor SQ22536 can enhance NKA and GR64349-mediated desensitization which suggests that agonist-induced desensitization involves the inhibition of adenylyl cyclase and the subsequent down-regulation of the cyclic AMP-dependent protein kinase, possibly by cross-talk to a second signalling pathway. PMID:9690859

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

PubMed

Shiokawa, K; Kai, M; Higo, T; Kaito, C; Yokoska, J; Yasuhiko, Y; Kajita, E; Nagano, M; Yamada, Y; Shibata, M; Muto, T; Shinga, J; Hara, H; Takayama, E; Fukamachi, H; Yaoita, Y; Igarashi, K

2000-06-01

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

ATP hydrolysis in Eg5 kinesin involves a catalytic two-water mechanism.

PubMed

Parke, Courtney L; Wojcik, Edward J; Kim, Sunyoung; Worthylake, David K

2010-02-19

Motor proteins couple steps in ATP binding and hydrolysis to conformational switching both in and remote from the active site. In our kinesin.AMPPPNP crystal structure, closure of the active site results in structural transformations appropriate for microtubule binding and organizes an orthosteric two-water cluster. We conclude that a proton is shared between the lytic water, positioned for gamma-phosphate attack, and a second water that serves as a general base. To our knowledge, this is the first experimental detection of the catalytic base for any ATPase. Deprotonation of the second water by switch residues likely triggers subsequent large scale structural rearrangements. Therefore, the catalytic base is responsible for initiating nucleophilic attack of ATP and for relaying the positive charge over long distances to initiate mechanotransduction. Coordination of switch movements via sequential proton transfer along paired water clusters may be universal for nucleotide triphosphatases with conserved active sites, such as myosins and G-proteins.

The C-terminal region of alpha-crystallin: involvement in protection against heat-induced denaturation

NASA Technical Reports Server (NTRS)

Takemoto, L.; Emmons, T.; Horwitz, J.; Spooner, B. S. (Principal Investigator)

1993-01-01

Recent studies have demonstrated that the alpha-crystallins can protect other proteins against heat-induced denaturation and aggregation. To determine the possible involvement of the C-terminal region in this activity, the alpha-crystallins were subjected to limited tryptic digestion, and the amount of cleavage from the N-terminal and C-terminal regions of the alpha-A and alpha-B crystallin chains was assessed using antisera specific for these regions. Limited tryptic digestion resulted in cleavage only from the C-terminal region of alpha-A crystallin. This trypsin-treated alpha-A crystallin preparation showed a decreased ability to protect proteins from heat-induced aggregation using an in vitro assay. Together, these results demonstrate that the C-terminal region of alpha-A crystallin is important for its ability to protect against heat-induced aggregation, which is consistent with the hypothesis that post-translational changes that are known to occur at the C-terminal region may have significant effects on the ability of alpha-A crystallin to protect against protein denaturation in vivo.

Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins

PubMed Central

Sitar, Tomasz; Popowicz, Grzegorz M.; Siwanowicz, Igor; Huber, Robert; Holak, Tad A.

2006-01-01

Insulin-like growth factor-binding proteins (IGFBPs) control bioavailability, activity, and distribution of insulin-like growth factor (IGF)1 and -2 through high-affinity IGFBP/IGF complexes. IGF-binding sites are found on N- and C-terminal fragments of IGFBPs, the two conserved domains of IGFBPs. The relative contributions of these domains to IGFBP/IGF complexation has been difficult to analyze, in part, because of the lack of appropriate three-dimensional structures. To analyze the effects of N- and C-terminal domain interactions, we determined several x-ray structures: first, of a ternary complex of N- and C-terminal domain fragments of IGFBP4 and IGF1 and second, of a “hybrid” ternary complex using the C-terminal domain fragment of IGFBP1 instead of IGFBP4. We also solved the binary complex of the N-terminal domains of IGFBP4 and IGF1, again to analyze C- and N-terminal domain interactions by comparison with the ternary complexes. The structures reveal the mechanisms of IGF signaling regulation via IGFBP binding. This finding supports research into the design of IGFBP variants as therapeutic IGF inhibitors for diseases of IGF disregulation. In IGFBP4, residues 1–38 form a rigid disulphide bond ladder-like structure, and the first five N-terminal residues bind to IGF and partially mask IGF residues responsible for the type 1 IGF receptor binding. A high-affinity IGF1-binding site is located in a globular structure between residues 39 and 82. Although the C-terminal domains do not form stable binary complexes with either IGF1 or the N-terminal domain of IGFBP4, in the ternary complex, the C-terminal domain contacts both and contributes to blocking of the IGF1 receptor-binding region of IGF1. PMID:16924115

The transport of Staufen2-containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen-activated protein kinase pathway.

PubMed

Jeong, Ji-Hye; Nam, Yeon-Ju; Kim, Seok-Yong; Kim, Eung-Gook; Jeong, Jooyoung; Kim, Hyong Kyu

2007-09-01

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.

77 FR 2127 - Birmingham Terminal Railway, L.L.C.-Acquisition and Operation Exemption-Birmingham Southern...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-13

... Terminal Railway, L.L.C.--Acquisition and Operation Exemption--Birmingham Southern Railroad Company Birmingham Terminal Railway, L.L.C. (BHRR), a noncarrier, has filed a verified notice of exemption under 49... Exemption--Birmingham Terminal Railway, L.L.C., wherein Watco Holdings, Inc., seeks Board approval to...

Evidence for new C-terminally truncated variants of α- and β-tubulins

PubMed Central

Aillaud, Chrystelle; Bosc, Christophe; Saoudi, Yasmina; Denarier, Eric; Peris, Leticia; Sago, Laila; Taulet, Nicolas; Cieren, Adeline; Tort, Olivia; Magiera, Maria M.; Janke, Carsten; Redeker, Virginie; Andrieux, Annie; Moutin, Marie-Jo

2016-01-01

Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the –EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same –EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with –EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development. PMID:26739754

Molecular characterization of myelin protein zero in Xenopus laevis peripheral nerve

NASA Astrophysics Data System (ADS)

Xie, Bo; Luo, Xiaoyang; Zhao, Cheng; Priest, Christina Marie; Chan, Shiu-Yung; O'Connor, Peter B.; Kirschner, Daniel A.; Costello, Catherine E.

2007-12-01

Myelin protein zero (P0), a glycosylated single-pass transmembrane protein, is essential in the formation and maintenance of peripheral nervous system (PNS) compact myelin. P0 in Xenopus (xP0) exists primarily as a dimeric form that remains stable after various physical and chemical treatments. In exploring the nature of the interactions underlying the dimer stability, we found that xP0 dimer dissociated into monomer during continuous elution gel electrophoresis and conventional SDS-PAGE, indicating that the dimer is stabilized by non-covalent interactions. Furthermore, as some of the gel-purified monomer re-associated into dimer on SDS-PAGE gels, there is likely a dynamic equilibrium between xP0 dimer and monomer in vivo. Because the carbohydrate and fatty acyl moieties may be crucial for the adhesion role of P0, we used sensitive mass spectrometry approaches to elucidate the detailed N-glycosylation and S-acylation profiles of xP0. Asn92 was determined to be the single, fully-occupied glycosylation site of xP0, and a total of 12 glycans was detected that exhibited new structural features compared with those observed from P0 in other species: (1) the neutral glycans were composed mainly of high mannose and hybrid types; (2) 5 of 12 were acidic glycans, among which three were sialylated and the other two were sulfated; (3) none of the glycans had core fucosylation; and (4) no glucuronic acid, hence no HNK-1 epitope, was detected. The drastically different carbohydrate structures observed here support the concept of the species-specific variation in N-glycosylation of P0. Cys152 was found to be acylated with stearoyl (C18:0), whereas palmitoyl (C16:0) is the corresponding predominant fatty acyl group on P0 from higher vertebrates. We propose that the unique glycosylation and acylation patterns of Xenopus P0 may underlie its unusual dimerization behavior. Our results should shed light on the understanding of the phylogenetic development of P0's adhesion role in PNS

Global gene expression during early differentiation of Xenopus (Silurana) tropicalis gonad tissues

EPA Science Inventory

African clawed frog Xenopus sp. has been used extensively for developmental biology and toxicology research. Xenopus (Silurana) tropicalis has been coveted more recently for genomics research because its diploid genome has been sequenced. Amid concerns of environmental pollutants...

Development and aminergic neuromodulation of a spinal locomotor network controlling swimming in Xenopus larvae.

PubMed

Sillar, K T; Reith, C A; McDearmid, J R

1998-11-16

In this article we review our research on the development and intrinsic neuromodulation of a spinal network controlling locomotion in a simple vertebrate. Swimming in hatchling Xenopus embryos is generated by a restricted network of well-characterized spinal neurons. This network produces a stereotyped motor pattern which, like real swimming, involves rhythmic activity that alternates across the body and progresses rostrocaudally with a brief delay between muscle segments. The stereotypy results from motoneurons discharging a single impulse in each cycle; because all motoneurons appear to behave similarly there is little scope for altering the output to the myotomes from one cycle to the next. Just one day later, however, Xenopus larvae generate a more complex and flexible motor pattern in which motoneurons can discharge a variable number of impulses which contribute to ventral root bursts in each cycle. This maturation of swimming is due, in part, to the influence of serotonin released from brain-stem raphespinal interneurons whose axonal projections innervate the cord early in larval life. Larval swimming is differentially modulated by both serotonin and by noradrenaline: serotonin leads to relatively fast, intense swimming whereas noradrenaline favors slower, weaker activity. Thus, these two biogenic amines select opposite extremes from the spectrum of possible output patterns that the swimming network can produce. Our studies on the cellular and synaptic effects of the amines indicate that they can control the strength of reciprocal glycinergic inhibition in the spinal cord. Serotonin and noradrenaline act presynaptically on the terminals of glycinergic commissural interneurons to weaken and strengthen, respectively, crossed glycinergic inhibition during swimming. As a result, serotonin reduces and noradrenaline increases interburst intervals. The membrane properties of spinal neurons are also affected by the amines. In particular, serotonin can induce

Inference of genetic network of Xenopus frog egg: improved genetic algorithm.

PubMed

Wu, Shinq-Jen; Chou, Chia-Hsien; Wu, Cheng-Tao; Lee, Tsu-Tian

2006-01-01

An improved genetic algorithm (IGA) is proposed to achieve S-system gene network modeling of Xenopus frog egg. Via the time-courses training datasets from Michaelis-Menten model, the optimal parameters are learned. The S-system can clearly describe activative and inhibitory interaction between genes as generating and consuming process. We concern the mitotic control in cell-cycle of Xenopus frog egg to realize cyclin-Cdc2 and Cdc25 for MPF activity. The proposed IGA can achieve global search with migration and keep the best chromosome with elitism operation. The generated gene regulatory networks can provide biological researchers for further experiments in Xenopus frog egg cell cycle control.

A Novel de novo CDH1 Germline Variant Aids in the Classification of C-terminal E-cadherin Alterations Predicted to Escape Nonsense-Mediated mRNA Decay.

PubMed

Krempely, Kate; Karam, Rachid

2018-05-24

Most truncating CDH1 pathogenic alterations confer an elevated lifetime risk of diffuse gastric cancer and lobular breast cancer. However, transcripts containing carboxyl-terminal (C-terminal) premature stop codons have been demonstrated to escape the nonsense-mediated mRNA decay (NMD) pathway, and gastric and breast cancer risks associated with these truncations should be carefully evaluated. A female patient underwent multigene panel testing due to a personal history of invasive lobular breast cancer diagnosed at age 54, which identified the germline CDH1 nonsense alteration, c.2506G>T (p.E836*), in the last exon of the gene. Subsequent parental testing for the alteration was negative and additional short tandem repeat analysis confirmed the familial relationships and the de novo occurrence in the proband. Based on the de novo occurrence, clinical history, and rarity in general population databases, this alteration was classified as a likely pathogenic variant. This is the most C-terminal pathogenic alteration reported to date. Additionally, this alteration contributed to the classification of six other upstream CDH1 C-terminal truncating variants as pathogenic or likely pathogenic. Identifying the most distal pathogenic alteration provides evidence to classify other C-terminal truncating variants as either pathogenic or benign, a fundamental step to offering pre-symptomatic screening and prophylactic procedures to the appropriate patients. Cold Spring Harbor Laboratory Press.

Transforming Growth Factor Beta (TGFβ) Is Produced by and Influences the Proliferative Response of Xenopus laevis Lymphocytes

PubMed Central

Haynes, Laura

1993-01-01

Both TGF/β2 and 5 have been described in the South African clawed frog Xenopus laevis and have been cloned from the tadpole-derived fibroblast cell line, XTC. Because TGFβ has such a profound inhibitory effect on the mammalian immune system, this study was performed to determine whether TGFβ: (a) has any in vitro effects on the growth of Xenopus lymphoblasts, and (b) is produced by mitogen-activated Xenopus lymphocytes. Following stimulation with mitogen or alloantigen, T lymphocytes from Xenopus secrete a T-cell growth factor (TCGF) that is functionally homologous to mammalian interleukin-2 (IL-2). Both recombinant human TGFβ1 and Xenopus TGFβ5 inhibit TCGF-induced proliferation of Xenopus splenic blasts and this inhibition can be reversed with anti-pan TGFβ antiserum. The Xenopus mitogen-induced saturated ammonium sulfate precipitated TCGF-containing supernatant (SAS TCGF SN) also contains latent TGFβ as assayed on mink lung fibroblasts and Xenopus splenic blasts, and experiments utilizing anti-TGFβ antiserum showed that only TGFβ5 is present in this supernatant. PMID:8281035

The Xenopus Tgfbi is required for embryogenesis through regulation of canonical Wnt signalling.

PubMed

Wang, Feng; Hu, Wanzhou; Xian, Jian; Ohnuma, Shin-ichi; Brenton, James D

2013-07-01

Tgfbi, a fasciclin family extracellular matrix protein, has various roles in human diseases from corneal dystrophies to cancer. However, the molecular mechanisms that underlie its functions are poorly understood. Here, we studied the role of Tgfbi during Xenopus embryogenesis. During gastrulation and immediately after, Xtgfbi is expressed at developmentally important signaling centers including the dorsal marginal zone, notochord and floorplate. Xtgfbi knockdown by anti-sense morpholinos causes defective organizer induction, patterning and differentiation of muscle, neuron and neural crests, similar to suppression of canonical Wnt signaling. In Xenopus embryos and animal caps as well as DLD-1 cells, we show that Tgfbi is strongly required for the full activation of the canonical Wnt pathway by promoting phosphorylation of GSK3β and consequently enhancing the stabilization and nuclear localization of β-catenin. Further analysis shows that Tgfbi is likely to promote GSK3β phosphorylation through integrin-linked kinase. Copyright © 2013 Elsevier Inc. All rights reserved.

Urate oxidase is imported into peroxisomes recognizing the C-terminal SKL motif of proteins.

PubMed

Miura, S; Oda, T; Funai, T; Ito, M; Okada, Y; Ichiyama, A

1994-07-01

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.

Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO).

PubMed

Alborghetti, Marcos Rodrigo; Furlan, Ariane da Silva; da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

2013-01-01

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

PubMed Central

da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L.; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

2013-01-01

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth. PMID:24116125

ACE-like hydrolysis of gastrin analogs and CCK-8 by fundic mucosal cells of different species with release of the amidated C-terminal dipeptide.

PubMed

Dubreuil, P; Fulcrand, P; Rodriguez, M; Laur, J; Bali, J P; Martinez, J

1990-06-19

Various gastrin analogues and CCK-8 (Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) are hydrolyzed in vitro by angiotensin-converting enzyme (ACE), the main and initial cleavage occurring at the Met-Asp (or Leu-Asp) bond, releasing the C-terminal dipeptide amide Asp-Phe-NH2. Tetragastrin analogues (e.g., Boc-Trp-Leu-Asp-Phe-NH2) are degraded by a vesicular membrane fraction from rat gastric mucosa, yielding the C-terminal dipeptide Asp-Phe-NH2. We report here on the degradation of gastrin analogues and CCK-8 by a gastric mucosal cell preparation containing specific gastrin receptors. We have shown that gastrin analogues were specifically degraded by gastric mucosal cells from different species (e.g., rabbit and dog) at 37 degrees C (pH 7.4), releasing the C-terminal dipeptide Asp-Phe-NH2, similarly to ACE. This cleavage was found to be temperature and pH sensitive, and was inhibited by metalloproteinase inhibitors and by captopril, strongly suggesting that this enzymatic system closely resembles ACE. We have also demonstrated that a close correlation seems to exist between the apparent affinity of the gastrin analogues for gastrin receptors on gastric mucosal cells, and their ability of being hydrolyzed by this cell preparation. Moreover, all gastrin analogues which have been demonstrated to act as gastrin antagonists remained unaffected in the incubation conditions.

Clustered Xenopus keratin genes: A genomic, transcriptomic, and proteomic analysis.

PubMed

Suzuki, Ken-Ichi T; Suzuki, Miyuki; Shigeta, Mitsuki; Fortriede, Joshua D; Takahashi, Shuji; Mawaribuchi, Shuuji; Yamamoto, Takashi; Taira, Masanori; Fukui, Akimasa

2017-06-15

Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations

PubMed Central

Bentley, Marvin; Decker, Helena; Luisi, Julie

2015-01-01

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin–binding domain–tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations. PMID:25624392

Structures of the Gasdermin D C-Terminal Domains Reveal Mechanisms of Autoinhibition.

PubMed

Liu, Zhonghua; Wang, Chuanping; Rathkey, Joseph K; Yang, Jie; Dubyak, George R; Abbott, Derek W; Xiao, Tsan Sam

2018-05-01

Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The molecular mechanisms of autoinhibition for GSDMD are poorly characterized. Here we report the crystal structures of the human and murine GSDMD C-terminal domains, which differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis. Our results suggest that GSDMDs may employ a distinct mode of intramolecular domain interaction and autoinhibition, which may be relevant to its unique role in pyroptosis downstream of inflammasome activation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Probing the Xenopus laevis inner ear transcriptome for biological function

PubMed Central

2012-01-01

Background The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis. Results Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip® Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization. Conclusions We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome, thereby addressing the

Kinesin and Dynein Mechanics: Measurement Methods and Research Applications.

PubMed

Abraham, Zachary; Hawley, Emma; Hayosh, Daniel; Webster-Wood, Victoria A; Akkus, Ozan

2018-02-01

Motor proteins play critical roles in the normal function of cells and proper development of organisms. Among motor proteins, failings in the normal function of two types of proteins, kinesin and dynein, have been shown to lead many pathologies, including neurodegenerative diseases and cancers. As such, it is critical to researchers to understand the underlying mechanics and behaviors of these proteins, not only to shed light on how failures may lead to disease, but also to guide research toward novel treatment and nano-engineering solutions. To this end, many experimental techniques have been developed to measure the force and motility capabilities of these proteins. This review will (a) discuss such techniques, specifically microscopy, atomic force microscopy (AFM), optical trapping, and magnetic tweezers, and (b) the resulting nanomechanical properties of motor protein functions such as stalling force, velocity, and dependence on adenosine triphosophate (ATP) concentrations will be comparatively discussed. Additionally, this review will highlight the clinical importance of these proteins. Furthermore, as the understanding of the structure and function of motor proteins improves, novel applications are emerging in the field. Specifically, researchers have begun to modify the structure of existing proteins, thereby engineering novel elements to alter and improve native motor protein function, or even allow the motor proteins to perform entirely new tasks as parts of nanomachines. Kinesin and dynein are vital elements for the proper function of cells. While many exciting experiments have shed light on their function, mechanics, and applications, additional research is needed to completely understand their behavior.

Triphenylbutanamines: Kinesin Spindle Protein Inhibitors with in Vivo Antitumor Activity†

PubMed Central

2012-01-01

The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent C-trityl analogues exhibit Kiapp ≤ 10 nM and GI50 ≈ 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies. PMID:22248262

Variants of the Xenopus laevis ribosomal transcription factor xUBF are developmentally regulated by differential splicing.

PubMed

Guimond, A; Moss, T

1992-07-11

XUBF is a Xenopus ribosomal transcription factor of the HMG-box family which contains five tandemly disposed homologies to the HMG1 & 2 DNA binding domains. XUBF has been isolated as a protein doublet and two cDNAs encoding the two molecular weight variants have been characterised. The major two forms of xUBF identified differ by the presence or absence of a 22 amino acid segment lying between HMG-boxes 3 and 4. Here we show that the mRNAs for these two forms of xUBF are regulated during development and differentiation over a range of nearly 20 fold. By isolating two of the xUBF genes, it was possible to show that both encoded the variable 22 amino acid segment in exon 12. Oocyte splicing assays and the sequencing of PCR-generated cDNA fragments, demonstrated that the transcripts from one of these genes were differentially spliced in a developmentally regulated manner. Transcripts from the second gene were found to be predominantly or exclusively spliced to produce the lower molecular weight form of xUBF. Expression of a high molecular weight form from yet a third gene was also detected. Although the intron-exon structures of the Xenopus and mouse UBF genes were found to be essentially identical, the differential splicing of exon 8 found in mammals, was not detected in Xenopus.

Calculating the Degradation Rate of Individual Proteins Using Xenopus Extract Systems.

PubMed

McDowell, Gary S; Philpott, Anna

2018-05-16

The Xenopus extract system has been used extensively as a simple, quick, and robust method for assessing the stability of proteins against proteasomal degradation. In this protocol, methods are provided for assessing the half-life of in vitro translated radiolabeled proteins using Xenopus egg or embryo extracts. © 2019 Cold Spring Harbor Laboratory Press.

Molecular characterization of the alpha subunit of complement component C8 (GcC8alpha) in the nurse shark (Ginglymostoma cirratum).

PubMed

Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L

2009-09-01

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of alpha, beta, and gamma subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8alpha and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8gamma-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8alpha respectively. Southern blot analysis showed GcC8alpha exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8alpha orthologs and as a sister taxa to the Xenopus. 2009 Elsevier Ltd.

Molecular characterization of the alpha subunit of complement component C8 (GcC8α) in the nurse shark (Ginglymostoma cirratum)

PubMed Central

Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L.

2009-01-01

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of α, β, and γ subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8α and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8γ-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4 % identity with human and Xenopus C8α respectively. Southern blot analysis showed GcC8α exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8α orthologs and as a sister taxa to the Xenopus. PMID:19524681

Survey of the vestibulum, and behavior of Xenopus laevis larvae developed during a 7-days space flight.

PubMed

Briegleb, W; Neubert, J; Schatz, A; Klein, T; Kruse, B

1986-01-01

Aquatic animals have almost no body weight related proprioception for spatial orientation. Xenopus larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; Xenopus larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity. For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D-1-Mission). The further evaluation of the "weightless-larvae" revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the l-g controls. An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown Xenopus eggs. The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.

Survey of the vestibulum, and behavior of xenopus laevis larvae developed during a 7-days space flight

NASA Astrophysics Data System (ADS)

Briegleb, W.; Neubert, J.; Schatz, A.; Klein, T.; Kruse, B.

Aquatic animals have almost no body weight related proprioception for spatial orientation. Xenopus larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; Xenopus larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity. For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D1-Mission). The further evaluation of the ``weightless-larvae'' revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the 1-g controls. An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown Xenopus eggs. The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.

Xenopus laevis - A success story in biological research in Space

NASA Astrophysics Data System (ADS)

Horn, E.

A feature of sensory, neuronal and motor systems is the existence of a critical period during their development. Environmental modifications, in particular stimulus depri-vation, during this period of life affects development in a long-term manner. For gravity sensory systems, space flights offer the only opportunity for deprivation conditions. Studies in the amphibian Xenopus laevis presented the most complete picture. The presentation demonstrates the importance of Xenopus laevis as an ex-perimental model animal in the past and even for future research in Space. Studies are presented which range from fertilization in Space and anatomical studies during early development under weightlessness up to post-flight studies on the anatomy of the peripheral sense organ, the spinal motor activity and behavior. Gravity depriva-tion induces anatomical as well as behavioral and neurophysiological modifications, which are normalized either during flight (thickening of the blastocoel roof) or after reentry in 1g-conditions (swimming and reflex behavior, spinal motor activity). The physiological changes can be explained by mechanisms of physiological adaptation. However, the studies also revealed stages which were insensitive to gravity depriva-tion; they point to the existence of a critical period. Observations on morphological mal-formations are described which are reversible after termination of microgravity and which are linked to a depression of vestibular reflex behavior. They might be caused by a competition between dorsalization and ventralization inducing growth factors. This observation offers the possibility for a genetic approach in finding ba-sics for microgravity effects on the development of Xenopus, and in a general frame, on the development of vertebrates including men. At the present stage of research, it remains open whether adaptive processes during exposure to altered gravity or the existence of a critical period in vestibular development are responsible for

Participation of Xenopus Elr-type Proteins in Vegetal mRNA Localization during Oogenesis*

PubMed Central

Arthur, Patrick K.; Claussen, Maike; Koch, Susanne; Tarbashevich, Katsiaryna; Jahn, Olaf; Pieler, Tomas

2009-01-01

Directional transport of specific mRNAs is of primary biological relevance. In Xenopus oocytes, mRNA localization to the vegetal pole is important for germ layer formation and germ cell development. Using a biochemical approach, we identified Xenopus Elr-type proteins, homologs of the Hu/ELAV proteins, as novel components of the vegetal mRNA localization machinery. They bind specifically to the localization elements of several different vegetally localizing Xenopus mRNAs, and they are part of one RNP together with other localization proteins, such as Vg1RBP and XStaufen 1. Blocking Elr-type protein binding by either localization element mutagenesis or antisense morpholino oligonucleotide-mediated masking of their target RNA structures, as well as overexpression of wild type and mutant ElrB proteins, interferes with vegetal localization in Xenopus oocytes. PMID:19458392

Musashi and Plasticity of Xenopus and Axolotl Spinal Cord Ependymal Cells

PubMed Central

Chernoff, Ellen A. G.; Sato, Kazuna; Salfity, Hai V. N.; Sarria, Deborah A.; Belecky-Adams, Teri

2018-01-01

The differentiated state of spinal cord ependymal cells in regeneration-competent amphibians varies between a constitutively active state in what is essentially a developing organism, the tadpole of the frog Xenopus laevis, and a quiescent, activatable state in a slowly growing adult salamander Ambystoma mexicanum, the Axolotl. Ependymal cells are epithelial in intact spinal cord of all vertebrates. After transection, body region ependymal epithelium in both Xenopus and the Axolotl disorganizes for regenerative outgrowth (gap replacement). Injury-reactive ependymal cells serve as a stem/progenitor cell population in regeneration and reconstruct the central canal. Expression patterns of mRNA and protein for the stem/progenitor cell-maintenance Notch signaling pathway mRNA-binding protein Musashi (msi) change with life stage and regeneration competence. Msi-1 is missing (immunohistochemistry), or at very low levels (polymerase chain reaction, PCR), in both intact regeneration-competent adult Axolotl cord and intact non-regeneration-competent Xenopus tadpole (Nieuwkoop and Faber stage 62+, NF 62+). The critical correlation for successful regeneration is msi-1 expression/upregulation after injury in the ependymal outgrowth and stump-region ependymal cells. msi-1 and msi-2 isoforms were cloned for the Axolotl as well as previously unknown isoforms of Xenopus msi-2. Intact Xenopus spinal cord ependymal cells show a loss of msi-1 expression between regeneration-competent (NF 50–53) and non-regenerating stages (NF 62+) and in post-metamorphosis froglets, while msi-2 displays a lower molecular weight isoform in non-regenerating cord. In the Axolotl, embryos and juveniles maintain Msi-1 expression in the intact cord. In the adult Axolotl, Msi-1 is absent, but upregulates after injury. Msi-2 levels are more variable among Axolotl life stages: rising between late tailbud embryos and juveniles and decreasing in adult cord. Cultures of regeneration-competent Xenopus tadpole

Molecular characterization of a KIF3B-like kinesin gene in the testis of Octopus tankahkeei (Cephalopoda, Octopus).

PubMed

Dang, Ran; Zhu, Jun-Quan; Tan, Fu-Qing; Wang, Wei; Zhou, Hong; Yang, Wan-Xi

2012-05-01

KIF3B is known for maintaining and assembling cilia and flagellum. To date, the function of KIF3B and its relationship with KIF3A during spermiogenesis in the cephalopod Octopus tankahkeei remains unknown. In the present study, we characterized a gene encoding a homologue of rat KIF3B in the O. tankahkeei testis and examined its temporal and spatial expression pattern during spermiogenesis. The cDNA of KIF3B was obtained with degenerate and RACE PCR and the distribution pattern of ot-kif3b were observed with RT-PCR. The morphological development during spermiogenesis was illustrated by histological and transmission electron microscopy and mRNA expression of ot-kif3b was observed by in situ hybridization. The 2,365 nucleotides cDNA consisted of a 102 bp 5' untranslated region (UTR), a 2,208 bp open reading frame (ORF) encoding a protein of 736 amino acids, and a 55 bp 3' UTR. Multiple alignments revealed that the putative Ot-KIF3B shared 68, 68, 69, 68, and 67% identity with that of Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis, respectively, along with high identities with Ot-KIF3A in fundamental structures. Ot-kif3b transcripts appeared gradually in early spermatids, increased in intermediate spermatids and maximized in drastically remodeled and final spermatids. The kif3b gene is identified and its expression pattern is demonstrated for the first time in O. tankahkeei. Compared to ot-kif3a reported by our laboratory before, our data suggested that the putative heterodimeric motor proteins Ot-KIF3A/B may be involved in intraspermatic transport and might contribute to structural changes during spermiogenesis.

Evidence for new C-terminally truncated variants of α- and β-tubulins.

PubMed

Aillaud, Chrystelle; Bosc, Christophe; Saoudi, Yasmina; Denarier, Eric; Peris, Leticia; Sago, Laila; Taulet, Nicolas; Cieren, Adeline; Tort, Olivia; Magiera, Maria M; Janke, Carsten; Redeker, Virginie; Andrieux, Annie; Moutin, Marie-Jo

2016-02-15

Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the -EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same -EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with -EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development. © 2016 Aillaud 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).

ZWY Sex Determination in Xenopus tropicalis

EPA Science Inventory

Most vertebrate species with described genetic sex determination are either male (XY) or female (ZW) heterogametic. To date, studies with Xenopus species indicate that members of this genus operate under a ZW sex determination system. We used two different approaches and demonst...

Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse.

PubMed Central

Kominami, R; Mishima, Y; Urano, Y; Sakai, M; Muramatsu, M

1982-01-01

A Eco RI 6.6 kb DNA fragment containing the 3'-end of 28S ribosomal RNA gene of the mouse was detected by Southern blot hybridization, and cloned in a lambda-phage vector. The site of transcription termination and the processed 3'-end of 28S RNA were determined on the cloned fragment and the surrounding nucleotide sequence determined. The 3'-terminal nucleotides of mouse 28S RNA are similar to those of yeast, Drosophila and Xenopus although the homology was lost drastically beyond the 3'-end of 28S RNA. 45S precursor RNA terminated at 30 nucleotides downstream from the 3'-end of 28S RNA gene. A structure of a dyad symmetry with a loop was found immediately prior to the termination site of 45S RNA. The rDNA termination site thus shares some common features with termination sites recognized by other RNA polymerases. Images PMID:6281727

Mice that lack the C-terminal region of Reelin exhibit behavioral abnormalities related to neuropsychiatric disorders

PubMed Central

Sakai, Kaori; Shoji, Hirotaka; Kohno, Takao; Miyakawa, Tsuyoshi; Hattori, Mitsuharu

2016-01-01

The secreted glycoprotein Reelin is believed to play critical roles in the pathogenesis of several neuropsychiatric disorders. The highly basic C-terminal region (CTR) of Reelin is necessary for efficient activation of its downstream signaling, and the brain structure of knock-in mice that lack the CTR (ΔC-KI mice) is impaired. Here, we performed a comprehensive behavioral test battery on ΔC-KI mice, in order to evaluate the effects of partial loss-of-function of Reelin on brain functions. The ΔC-KI mice were hyperactive and exhibited reduced anxiety-like and social behaviors. The working memory in ΔC-KI mice was impaired in a T-maze test. There was little difference in spatial reference memory, depression-like behavior, prepulse inhibition, or fear memory between ΔC-KI and wild-type mice. These results suggest that CTR-dependent Reelin functions are required for some specific normal brain functions and that ΔC-KI mice recapitulate some aspects of neuropsychiatric disorders, such as schizophrenia, bipolar disorder, and autism spectrum disorder. PMID:27346785

Synaptic-like vesicles and candidate transduction channels in mechanosensory terminals

PubMed Central

Bewick, Guy S

2015-01-01

This article summarises progress to date over an exciting and very enjoyable first 15 years of collaboration with Bob Banks. Our collaboration began when I contacted him with (to me) an unexpected observation that a dye used to mark recycling synaptic vesicle membrane at efferent terminals also labelled muscle spindle afferent terminals. This observation led to the re-discovery of a system of small clear vesicles present in all vertebrate primary mechanosensory nerve terminals. These synaptic-like vesicles (SLVs) have been, and continue to be, the major focus of our work. This article describes our characterisation of the properties and functional significance of these SLVs, combining our complementary skills: Bob’s technical expertise and encyclopaedic knowledge of mechanosensation with my experience of synaptic vesicles and the development of the styryl pyridinium dyes, of which the most widely used is FM1-43. On the way we have found that SLVs seem to be part of a constitutive glutamate secretory system necessary to maintain the stretch-sensitivity of spindle endings. The glutamate activates a highly unusual glutamate receptor linked to phospholipase D activation, which we have termed the PLD-mGluR. It has a totally distinct pharmacology first described in the hippocampus nearly 20 years ago but, like the SLVs that were first described over 50 years ago, has since been little researched. Yet, our evidence and literature searches suggest this glutamate/SLV/PLD-mGluR system is a ubiquitous feature of mechanosensory endings and, at least for spindles, is essential for maintaining mechanosensory function. This article summarises how this system integrates with the classical model of mechanosensitive channels in spindles and other mechanosensory nerve terminals, including hair follicle afferents and baroreceptors controlling blood pressure. Finally, in this time when there is an imperative to show translational relevance, I describe how this fascinating system

Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis.

PubMed

Grimaldi, Annalisa; Tettamanti, Gianluca; Martin, Benjamin L; Gaffield, William; Pownall, Mary E; Hughes, Simon M

2004-07-01

In tetrapod phylogeny, the dramatic modifications of the trunk have received less attention than the more obvious evolution of limbs. In somites, several waves of muscle precursors are induced by signals from nearby tissues. In both amniotes and fish, the earliest myogenesis requires secreted signals from the ventral midline carried by Hedgehog (Hh) proteins. To determine if this similarity represents evolutionary homology, we have examined myogenesis in Xenopus laevis, the major species from which insight into vertebrate mesoderm patterning has been derived. Xenopus embryos form two distinct kinds of muscle cells analogous to the superficial slow and medial fast muscle fibres of zebrafish. As in zebrafish, Hh signalling is required for XMyf5 expression and generation of a first wave of early superficial slow muscle fibres in tail somites. Thus, Hh-dependent adaxial myogenesis is the likely ancestral condition of teleosts, amphibia and amniotes. Our evidence suggests that midline-derived cells migrate to the lateral somite surface and generate superficial slow muscle. This cell re-orientation contributes to the apparent rotation of Xenopus somites. Xenopus myogenesis in the trunk differs from that in the tail. In the trunk, the first wave of superficial slow fibres is missing, suggesting that significant adaptation of the ancestral myogenic programme occurred during tetrapod trunk evolution. Although notochord is required for early medial XMyf5 expression, Hh signalling fails to drive these cells to slow myogenesis. Later, both trunk and tail somites develop a second wave of Hh-independent slow fibres. These fibres probably derive from an outer cell layer expressing the myogenic determination genes XMyf5, XMyoD and Pax3 in a pattern reminiscent of amniote dermomyotome. Thus, Xenopus somites have characteristics in common with both fish and amniotes that shed light on the evolution of somite differentiation. We propose a model for the evolutionary adaptation of

cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

PubMed

Wahli, W; Martinez, E; Corthésy, B; Cardinaux, J R

1989-01-01

Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin

The Phragmoplast-Orienting Kinesin-12 Class Proteins Translate the Positional Information of the Preprophase Band to Establish the Cortical Division Zone in Arabidopsis thaliana[C][W

PubMed Central

Lipka, Elisabeth; Gadeyne, Astrid; Stöckle, Dorothee; Zimmermann, Steffi; De Jaeger, Geert; Ehrhardt, David W.; Kirik, Viktor; Van Damme, Daniel; Müller, Sabine

2014-01-01

The preprophase band (PPB) is a faithful but transient predictor of the division plane in somatic cell divisions. Throughout mitosis the PPBs positional information is preserved by factors that continuously mark the division plane at the cell cortex, the cortical division zone, by their distinct spatio-temporal localization patterns. However, the mechanism maintaining these identity factors at the plasma membrane after PPB disassembly remains obscure. The pair of kinesin-12 class proteins PHRAGMOPLAST ORIENTING KINESIN1 (POK1) and POK2 are key players in division plane maintenance. Here, we show that POK1 is continuously present at the cell cortex, providing a spatial reference for the site formerly occupied by the PPB. Fluorescence recovery after photobleaching analysis combined with microtubule destabilization revealed dynamic microtubule-dependent recruitment of POK1 to the PPB during prophase, while POK1 retention at the cortical division zone in the absence of cortical microtubules appeared static. POK function is strictly required to maintain the division plane identity factor TANGLED (TAN) after PPB disassembly, although POK1 and TAN recruitment to the PPB occur independently during prophase. Together, our data suggest that POKs represent fundamental early anchoring components of the cortical division zone, translating and preserving the positional information of the PPB by maintaining downstream identity markers. PMID:24972597

Induction of filopodia-like protrusions in N1E-115 neuroblastoma cells by diacylglycerol kinase γ independent of its enzymatic activity: potential novel function of the C-terminal region containing the catalytic domain of diacylglycerol kinase γ.

PubMed

Tanino, Fumihiko; Maeda, Yuki; Sakai, Hiromichi; Sakane, Fumio

2013-01-01

Type I diacylglycerol kinase (DGK) isozymes (α, β, and γ) contain recoverin homology domains and calcium-binding EF-hand motifs at their N-termini. The γ-isoform of DGK is abundantly expressed in retinal and Purkinje cells; however, its function in neuronal cells remains unknown. Here, we report that the mRNA and protein levels of DGKγ, but not DGKα or β, were markedly increased in N1E-115 neuroblastoma cells upon cellular differentiation by serum starvation. Interestingly, overexpression of wild-type DGKγ, which was partially located at the plasma membrane, considerably induced the formation of slender, filopodia-like cytoplasmic projections from N1E-115 cell bodies. Deletion of the recoverin homology domain and the EF-hand motifs, which potentiated the plasma membrane localization of the isozyme, significantly enhanced the formation of the filopodia-like protrusions. Intriguingly, the catalytic activity of the isozyme is not essential for the protrusion formation. The N-terminal half of the catalytic domain and a short stretch of amino acid residues at the C-terminus are responsible for plasma membrane localization and filopodia-like process formation. Taken together, we have described a potentially novel morphological function of the C-terminal DGKγ catalytic region that is independent of its enzymatic activity.

Cell lineage tracing during Xenopus tail regeneration.

PubMed

Gargioli, Cesare; Slack, Jonathan M W

2004-06-01

The tail of the Xenopus tadpole will regenerate following amputation, and all three of the main axial structures - the spinal cord, the notochord and the segmented myotomes - are found in the regenerated tail. We have investigated the cellular origin of each of these three tissue types during regeneration. We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method. The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

PubMed Central

Nebreda, A R; Hunt, T

1993-01-01

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified. Images PMID:8387916

Light regulation of the abundance of mRNA encoding a nucleolin-like protein localized in the nucleoli of pea nuclei.

PubMed Central

Tong, C G; Reichler, S; Blumenthal, S; Balk, J; Hsieh, H L; Roux, S J

1997-01-01

A cDNA encoding a nucleolar protein was selected from a pea (Pisum sativum) plumule library, cloned, and sequenced. The translated sequence of the cDNA has significant percent identity to Xenopus laevis nucleolin (31%), the alfalfa (Medicago sativa) nucleolin homolog (66%), and the yeast (Saccharomyces cerevisiae) nucleolin homolog (NSR1) (28%). It also has sequence patterns in its primary structure that are characteristic of all nucleolins, including an N-terminal acidic motif, RNA recognition motifs, and a C-terminal Gly- and Arg-rich domain. By immunoblot analysis, the polyclonal antibodies used to select the cDNA bind selectively to a 90-kD protein in purified pea nuclei and nucleoli and to an 88-kD protein in extracts of Escherichia coli expressing the cDNA. In immunolocalization assays of pea plumule cells, the antibodies stained primarily a region surrounding the fibrillar center of nucleoli, where animal nucleolins are typically found. Southern analysis indicated that the pea nucleolin-like protein is encoded by a single gene, and northern analysis showed that the labeled cDNA binds to a single band of RNA, approximately the same size and the cDNA. After irradiation of etiolated pea seedlings by red light, the mRNA level in plumules decreased during the 1st hour and then increased to a peak of six times the 0-h level at 12 h. Far-red light reversed this effect of red light, and the mRNA accumulation from red/far-red light irradiation was equal to that found in the dark control. This indicates that phytochrome may regulate the expression of this gene. PMID:9193096

Probing Mitotic CENP-E Kinesin with the Tethered Cargo Motion Assay and Laser Tweezers.

PubMed

Gudimchuk, Nikita; Tarasovetc, Ekaterina V; Mustyatsa, Vadim; Drobyshev, Alexei L; Vitre, Benjamin; Cleveland, Don W; Ataullakhanov, Fazly I; Grishchuk, Ekaterina L

2018-06-05

Coiled-coil stalks of various kinesins differ significantly in predicted length and structure; this is an adaption that helps these motors carry out their specialized functions. However, little is known about the dynamic stalk configuration in moving motors. To gain insight into the conformational properties of the transporting motors, we developed a theoretical model to predict Brownian motion of a microbead tethered to the tail of a single, freely walking molecule. This approach, which we call the tethered cargo motion (TCM) assay, provides an accurate measure of the mechanical properties of motor-cargo tethering, verified using kinesin-1 conjugated to a microbead via DNA links in vitro. Applying the TCM assay to the mitotic kinesin CENP-E unexpectedly revealed that when walking along a microtubule track, this highly elongated molecule with a contour length of 230 nm formed a 20-nm-long tether. The stalk of a walking CENP-E could not be extended fully by application of sideways force with optical tweezers (up to 4 pN), implying that CENP-E carries its cargo in a compact configuration. Assisting force applied along the microtubule track accelerates CENP-E walking, but this increase does not depend on the presence of the CENP-E stalk. Our results suggest that the unusually large stalk of CENP-E has little role in regulating its function as a transporter. The adjustable stalk configuration may represent a regulatory mechanism for controlling the physical reach between kinetochore-bound CENP-E and spindle microtubules, or it may assist localizing various kinetochore regulators in the immediate vicinity of the kinetochore-embedded microtubule ends. The TCM assay and underlying theoretical framework will provide a general guide for determining the dynamic configurations of various molecular motors moving along their tracks, freely or under force. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death.

PubMed

Bougé, Anne-Laure; Parmentier, Marie-Laure

2016-03-01

In neurodegenerative diseases such as Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown, in particular with respect to the microtubule (MT)-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of affected individuals. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for MTs, there is, to our knowledge, no indication that an excess of this protein could affect mitosis. Here, we studied the effect of an excess of human Tau (hTau) protein on cell mitosis in vivo. Using the Drosophila developing wing disc epithelium as a model, we show that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. We studied the mechanism of action of hTau and found that the MT-binding domain of hTau is responsible for these defects. We also demonstrate that the effects of hTau occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). We finally show that this deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc), as well as in human HeLa cells. By demonstrating that MT-bound Tau inhibits the Eg5 kinesin and cell mitosis, our work provides a new framework to consider the role of Tau in neurodegenerative diseases. © 2016. Published by The Company of Biologists Ltd.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

PubMed

Corthésy, B; Cardinaux, J R; Claret, F X; Wahli, W

1989-12-01

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A

PubMed Central

Mennella, Vito; Tan, Dong-Yan; Buster, Daniel W.; Asenjo, Ana B.; Rath, Uttama; Ma, Ao; Sosa, Hernando J.

2009-01-01

Microtubule (MT)-destabilizing kinesin 13s perform fundamental roles throughout the cell cycle. In this study, we show that the Drosophila melanogaster kinesin 13, KLP10A, is phosphorylated in vivo at a conserved serine (S573) positioned within the α-helix 5 of the motor domain. In vitro, a phosphomimic KLP10A S573E mutant displays a reduced capacity to depolymerize MTs but normal affinity for the MT lattice. In cells, replacement of endogenous KLP10A with KLP10A S573E dampens MT plus end dynamics throughout the cell cycle, whereas a nonphosphorylatable S573A mutant apparently enhances activity during mitosis. Electron microscopy suggests that KLP10A S573 phosphorylation alters its association with the MT lattice, whereas molecular dynamics simulations reveal how KLP10A phosphorylation can alter the kinesin–MT interface without changing important structural features within the motor’s core. Finally, we identify casein kinase 1α as a possible candidate for KLP10A phosphorylation. We propose a model in which phosphorylation of the KLP10A motor domain provides a regulatory switch controlling the time and place of MT depolymerization. PMID:19687256

Polycystin-1 C-terminal Cleavage Is Modulated by Polycystin-2 Expression*

PubMed Central

Bertuccio, Claudia A.; Chapin, Hannah C.; Cai, Yiqiang; Mistry, Kavita; Chauvet, Veronique; Somlo, Stefan; Caplan, Michael J.

2009-01-01

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway. PMID:19491093

The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis

PubMed Central

Zhou, Junsong; Wu, Yi; Wang, Lu; Rauova, Lubica; Hayes, Vincent M.; Poncz, Mortimer; Essex, David W.

2015-01-01

Protein disulfide isomerase (PDI) has two distinct CGHC redox-active sites; however, the contribution of these sites during different physiologic reactions, including thrombosis, is unknown. Here, we evaluated the role of PDI and redox-active sites of PDI in thrombosis by generating mice with blood cells and vessel wall cells lacking PDI (Mx1-Cre Pdifl/fl mice) and transgenic mice harboring PDI that lacks a functional C-terminal CGHC motif [PDI(ss-oo) mice]. Both mouse models showed decreased fibrin deposition and platelet accumulation in laser-induced cremaster arteriole injury, and PDI(ss-oo) mice had attenuated platelet accumulation in FeCl3-induced mesenteric arterial injury. These defects were rescued by infusion of recombinant PDI containing only a functional C-terminal CGHC motif [PDI(oo-ss)]. PDI infusion restored fibrin formation, but not platelet accumulation, in eptifibatide-treated wild-type mice, suggesting a direct role of PDI in coagulation. In vitro aggregation of platelets from PDI(ss-oo) mice and PDI-null platelets was reduced; however, this defect was rescued by recombinant PDI(oo-ss). In human platelets, recombinant PDI(ss-oo) inhibited aggregation, while recombinant PDI(oo-ss) potentiated aggregation. Platelet secretion assays demonstrated that the C-terminal CGHC motif of PDI is important for P-selectin expression and ATP secretion through a non-αIIbβ3 substrate. In summary, our results indicate that the C-terminal CGHC motif of PDI is important for platelet function and coagulation. PMID:26529254

Accumulation of Cytoplasmic Dynein and Dynactin at Microtubule Plus Ends in Aspergillus nidulans Is Kinesin DependentV⃞

PubMed Central

Zhang, Jun; Li, Shihe; Fischer, Reinhard; Xiang, Xin

2003-01-01

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end–directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF. PMID:12686603

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole.

PubMed

Taniguchi, Yuka; Watanabe, Kenji; Mochii, Makoto

2014-06-18

Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans.

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole

PubMed Central

2014-01-01

Background Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. Results In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. Conclusion As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans. PMID:24941877

Ski represses BMP signaling in Xenopus and mammalian cells

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

kluo@lbl.gov

2001-05-16

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells bymore » directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-{beta} family members.« less

The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

PubMed Central

Yankiwski, Victor; Noonan, James P; Neff, Norma F

2001-01-01

Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10) or PML (promyelocytic leukemia) nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies. PMID:11472631

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2014-10-01

AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL INVESTIGATOR...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH

Membrane transport of WAVE2 and lamellipodia formation require Pak1 that mediates phosphorylation and recruitment of stathmin/Op18 to Pak1-WAVE2-kinesin complex.

PubMed

Takahashi, Kazuhide; Suzuki, Katsuo

2009-05-01

Membrane transport of WAVE2 that leads to lamellipodia formation requires a small GTPase Rac1, the motor protein kinesin, and microtubules. Here we explore the possibility of whether the Rac1-dependent and kinesin-mediated WAVE2 transport along microtubules is regulated by a p21-activated kinase Pak as a downstream effector of Rac1. We find that Pak1 constitutively binds to WAVE2 and is transported with WAVE2 to the leading edge by stimulation with hepatocyte growth factor (HGF). Concomitantly, phosphorylation of tubulin-bound stathmin/Op18 at serine 25 (Ser25) and Ser38, microtubule growth, and stathmin/Op18 binding to kinesin-WAVE2 complex were induced. The HGF-induced WAVE2 transport, lamellipodia formation, stathmin/Op18 phosphorylation at Ser38 and binding to kinesin-WAVE2 complex, but not stathmin/Op18 phosphorylation at Ser25 and microtubule growth, were abrogated by Pak1 inhibitor IPA-3 and Pak1 depletion with small interfering RNA (siRNA). Moreover, stathmin/Op18 depletion with siRNA caused significant inhibition of HGF-induced WAVE2 transport and lamellipodia formation, with HGF-independent promotion of microtubule growth. Collectively, it is suggested that Pak1 plays a critical role in HGF-induced WAVE2 transport and lamellipodia formation by directing Pak1-WAVE2-kinesin complex toward the ends of growing microtubules through phosphorylation and recruitment of tubulin-bound stathmin/Op18 to the complex.

Functional mechanism of C-terminal tail in the enzymatic role of porcine testicular carbonyl reductase: a combined experiment and molecular dynamics simulation study of the C-terminal tail in the enzymatic role of PTCR.

PubMed

Son, Minky; Bang, Woo Young; Park, Chanin; Lee, Yuno; Kwon, Seul Gi; Kim, Sam Woong; Kim, Chul Wook; Lee, Keun Woo

2014-01-01

Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR) superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C-terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV). Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT) were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily.

Functional Mechanism of C-Terminal Tail in the Enzymatic Role of Porcine Testicular Carbonyl Reductase: A Combined Experiment and Molecular Dynamics Simulation Study of the C-Terminal Tail in the Enzymatic Role of PTCR

PubMed Central

Park, Chanin; Lee, Yuno; Kwon, Seul Gi; Kim, Sam Woong; Kim, Chul Wook; Lee, Keun Woo

2014-01-01

Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR) superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C- terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV). Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT) were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily. PMID:24646606

OCT imaging of craniofacial anatomy in xenopus embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Deniz, Engin; Jonas, Stephan M.; Griffin, John; Hooper, Michael C.; Choma, Michael A.; Khokha, Mustafa K.

2016-03-01

The etiology of craniofacial defects is incompletely understood. The ability to obtain large amounts of gene sequence data from families affected by craniofacial defects is opening up new ways to understand molecular genetic etiological factors. One important link between gene sequence data and clinical relevance is biological research into candidate genes and molecular pathways. We present our recent research using OCT as a nondestructive phenotyping modality of craniofacial morphology in Xenopus embryos, an important animal model for biological research in gene and pathway discovery. We define 2D and 3D scanning protocols for a standardized approach to craniofacial imaging in Xenopus embryos. We define standard views and planar reconstructions for visualizing normal anatomy and landmarks. We compare these views and reconstructions to traditional histopathology using alcian blue staining. In addition to being 3D, nondestructive, and having much faster throughout, OCT can identify craniofacial features that are lost during traditional histopathological preparation. We also identify quantitative morphometric parameters to define normative craniofacial anatomy. We also note that craniofacial and cardiac defects are not infrequently present in the same patient (e.g velocardiofacial syndrome). Given that OCT excels at certain aspects of cardiac imaging in Xenopus embryos, our work highlights the potential of using OCT and Xenopus to study molecular genetic factors that impact both cardiac and craniofacial development.

The small heat shock protein, HSP30, is associated with aggresome-like inclusion bodies in proteasomal inhibitor-, arsenite-, and cadmium-treated Xenopus kidney cells.

PubMed

Khan, Saad; Khamis, Imran; Heikkila, John J

2015-11-01

In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with the proteasomal inhibitor, MG132, or the environmental toxicants, sodium arsenite or cadmium chloride, induced the accumulation of the small heat shock protein, HSP30, in total and in both soluble and insoluble protein fractions. Immunocytochemical analysis revealed the presence of relatively large HSP30 structures primarily in the perinuclear region of the cytoplasm. All three of the stressors promoted the formation of aggresome-like inclusion bodies as determined by immunocytochemistry and laser scanning confocal microscopy using a ProteoStat aggresome dye and additional aggresomal markers, namely, anti-γ-tubulin and anti-vimentin antibodies. Further analysis revealed that HSP30 co-localized with these aggresome-like inclusion bodies. In most cells, HSP30 was found to envelope or occur within these structures. Finally, we show that treatment of cells with withaferin A, a steroidal lactone with anti-inflammatory, anti-tumor, and proteasomal inhibitor properties, also induced HSP30 accumulation that co-localized with aggresome-like inclusion bodies. It is possible that proteasomal inhibitor or metal/metalloid-induced formation of aggresome-like inclusion bodies may sequester toxic protein aggregates until they can be degraded. While the role of HSP30 in these aggresome-like structures is not known, it is possible that they may be involved in various aspects of aggresome-like inclusion body formation or transport. Copyright © 2015 Elsevier Inc. All rights reserved.

Parallel Allostery by cAMP and PDE Coordinates Activation and Termination Phases in cAMP Signaling.

PubMed

Krishnamurthy, Srinath; Tulsian, Nikhil Kumar; Chandramohan, Arun; Anand, Ganesh S

2015-09-15

The second messenger molecule cAMP regulates the activation phase of the cAMP signaling pathway through high-affinity interactions with the cytosolic cAMP receptor, the protein kinase A regulatory subunit (PKAR). Phosphodiesterases (PDEs) are enzymes responsible for catalyzing hydrolysis of cAMP to 5' AMP. It was recently shown that PDEs interact with PKAR to initiate the termination phase of the cAMP signaling pathway. While the steps in the activation phase are well understood, steps in the termination pathway are unknown. Specifically, the binding and allosteric networks that regulate the dynamic interplay between PKAR, PDE, and cAMP are unclear. In this study, PKAR and PDE from Dictyostelium discoideum (RD and RegA, respectively) were used as a model system to monitor complex formation in the presence and absence of cAMP. Amide hydrogen/deuterium exchange mass spectrometry was used to monitor slow conformational transitions in RD, using disordered regions as conformational probes. Our results reveal that RD regulates its interactions with cAMP and RegA at distinct loci by undergoing slow conformational transitions between two metastable states. In the presence of cAMP, RD and RegA form a stable ternary complex, while in the absence of cAMP they maintain transient interactions. RegA and cAMP each bind at orthogonal sites on RD with resultant contrasting effects on its dynamics through parallel allosteric relays at multiple important loci. RD thus serves as an integrative node in cAMP termination by coordinating multiple allosteric relays and governing the output signal response. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

7 CFR 1c.113 - Suspension or termination of IRB approval of research.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 1 2011-01-01 2011-01-01 false Suspension or termination of IRB approval of research... § 1c.113 Suspension or termination of IRB approval of research. An IRB shall have authority to suspend or terminate approval of research that is not being conducted in accordance with the IRB's...

Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

PubMed Central

Chen, Xianling; Chen, Xiaole; Li, Ding; Fan, Yingjuan; Xu, Jianhua; Chen, Yuanzhong; Wu, Lixian

2017-01-01

Heat shock protein 90 (Hsp90) contains amino (N)–terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states. PMID:28036294

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

PubMed

Huang, Chao; Zhang, Zhe; Chen, Lihan; Lee, Hank W; Ayrapetov, Marina K; Zhao, Ting C; Hao, Yimei; Gao, Jinsong; Yang, Chunzhang; Mehta, Gautam U; Zhuang, Zhengping; Zhang, Xiaoren; Hu, Guohong; Chin, Y Eugene

2018-06-01

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

PubMed Central

Guedon, G; Sovia, D; Ebel, J P; Befort, N; Remy, P

1985-01-01

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:4092696

Human Topoisomerase I C-Terminal Domain Fragment Containing the Active Site Tyrosine is a Molten Globule: Implication for the Formation of Competent Productive Complex

PubMed Central

Punchihewa, Chandanamali; Dai, Jixun; Carver, Megan; Yang, Danzhou

2007-01-01

Human topoisomerase I (topo I) is an essential cellular enzyme that relaxes DNA supercoiling. The 6.3 kDa C-terminal domain of topo I contains the active site tyrosine (Tyr723) but lacks enzymatic activity by itself. Activity can be fully reconstituted when the C-terminal is associated with the 56 kDa core domain. Even though several crystal structures of topo I/DNA complexes are available, crystal structures of the free topo I protein or its individual domain fragments have been difficult to obtain. In this report we analyze the human topo I C-terminal domain structure using a variety of biophysical methods. Our results indicate that this fragment protein (topo6.3) appears to be in a molten globule state. It appears to have a native-like tertiary fold that contains a large population of α-helix secondary structure and extensive surface hydrophobic regions. Topo6.3 is known to be readily activated with the association of the topo I core domain, and the molten globule state of topo6.3 is likely to be an energy-favorable conformation for the free topo I C-terminal domain protein. The structural fluctuation and plasticity may represent an efficient mechanism in the topo I functional pathway, where the flexibility aids in the complementary association with the core domain and in the formation of a fully productive topo I complex. PMID:17434318

The human estrogen receptor can regulate exogenous but not endogenous vitellogenin gene promoters in a Xenopus cell line

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

Seiler-Tuyns, A.; Merillat, A.M.; Haefliger, D.N.

Transfection of a human estrogen receptor cDNA expression vector (HEO) into cultured Xenopus kidney cells confers estrogen responsiveness to the recipient cells as demonstrated by the hormone dependent expression of co-transfected Xenopus vitellogenin-CAT chimeric genes. The estrogen stimulation of these vit-CAT genes is dependent upon the presence of the vitellogenin estrogen responsive element (ERE) in their 5{prime} flanking region. Thus, functional human estrogen receptor (hER) can be synthesized in heterologous lower vertebrate cells and can act as a trans-acting regulatory factor that is necessary, together with estradiol, for the induction of the vit-CAT constructs in these cells. In addition, vitellogeninmore » minigenes co-transfected with the HEO expression vector also respond to hormonal stimulation. Their induction is not higher than that of the vit-CAT chimeric genes. It suggests that in the Xenopus kidney cell line B 3.2, the structural parts of the vitellogenin minigenes do not play a role in the induction process. Furthermore, no stabilizing effect of estrogen on vitellogenin mRNA is observed in these cells.« less

Polystyrene nanoparticles affect Xenopus laevis development

NASA Astrophysics Data System (ADS)

Tussellino, Margherita; Ronca, Raffaele; Formiggini, Fabio; Marco, Nadia De; Fusco, Sabato; Netti, Paolo Antonio; Carotenuto, Rosa

2015-02-01

Exposing living organisms to nanoparticulates is potentially hazardous, in particular when it takes place during embryogenesis. In this investigation, we have studied the effects of 50-nm-uncoated polystyrene nanoparticles (PSNPs) as a model to investigate the suitability of their possible future employments. We have used the standardized Frog Embryo Teratogenesis Assay- Xenopus test during the early stages of larval development of Xenopus laevis, and we have employed either contact exposure or microinjections. We found that the embryos mortality rate is dose dependent and that the survived embryos showed high percentage of malformations. They display disorders in pigmentation distribution, malformations of the head, gut and tail, edema in the anterior ventral region, and a shorter body length compared with sibling untreated embryos. Moreover, these embryos grow more slowly than the untreated embryos. Expressions of the mesoderm markers, bra (T-box Brachyury gene), myod1 (myogenic differentiation1), and of neural crest marker sox9 (sex SRY (determining region Y-box 9) transcription factor sox9), are modified. Confocal microscopy showed that the nanoparticles are localized in the cytoplasm, in the nucleus, and in the periphery of the digestive gut cells. Our data suggest that PSNPs are toxic and show a potential teratogenic effect for Xenopus larvae. We hypothesize that these effects may be due either to the amount of NPs that penetrate into the cells and/or to the "corona" effect caused by the interaction of PSNPs with cytoplasm components. The three endpoints of our study, i.e., mortality, malformations, and growth inhibition, suggest that the tests we used may be a powerful and flexible bioassay in evaluating pollutants in aquatic embryos.

Inappropriate Expression of an NLP Effector in Colletotrichum orbiculare Impairs Infection on Cucurbitaceae Cultivars via Plant Recognition of the C-Terminal Region.

PubMed

Azmi, Nur Sabrina Ahmad; Singkaravanit-Ogawa, Suthitar; Ikeda, Kyoko; Kitakura, Saeko; Inoue, Yoshihiro; Narusaka, Yoshihiro; Shirasu, Ken; Kaido, Masanori; Mise, Kazuyuki; Takano, Yoshitaka

2018-01-01

The hemibiotrophic pathogen Colletotrichum orbiculare preferentially expresses a necrosis and ethylene-inducing peptide 1 (Nep1)-like protein named NLP1 during the switch to necrotrophy. Here, we report that the constitutive expression of NLP1 in C. orbiculare blocks pathogen infection in multiple Cucurbitaceae cultivars via their enhanced defense responses. NLP1 has a cytotoxic activity that induces cell death in Nicotiana benthamiana. However, C. orbiculare transgenic lines constitutively expressing a mutant NLP1 lacking the cytotoxic activity still failed to infect cucumber, indicating no clear relationship between cytotoxic activity and the NLP1-dependent enhanced defense. NLP1 also possesses the microbe-associated molecular pattern (MAMP) sequence called nlp24, recognized by Arabidopsis thaliana at its central region, similar to NLPs of other pathogens. Surprisingly, inappropriate expression of a mutant NLP1 lacking the MAMP signature is also effective for blocking pathogen infection, uncoupling the infection block from the corresponding MAMP. Notably, the deletion analyses of NLP1 suggested that the C-terminal region of NLP1 is critical to enhance defense in cucumber. The expression of mCherry fused with the C-terminal 32 amino acids of NLP1 was enough to trigger the defense of cucurbits, revealing that the C-terminal region of the NLP1 protein is recognized by cucurbits and, then, terminates C. orbiculare infection.

Insulin-like growth factor 1 regulation of proliferation and differentiation of Xenopus laevis myogenic cells in vitro.

PubMed

Miyata, Sairi; Yada, Tomotaka; Ishikawa, Natsuko; Taheruzzaman, Kazi; Hara, Ryohei; Matsuzaki, Takashi; Nishikawa, Akio

2017-03-01

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1's cell differentiation promotion, suggesting the possibility that IGF-1's differentiation-promotion effect is an indirect effect via IGF-1's cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100-500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-L-thyronine (T 3 ) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T 3 . In larval tail cells, cell count was 76% lower in the presence of T 3 , and IGF-1 did not promote proliferation and differentiation in T 3 -containing medium. In larval dorsal cells, cell count was also lower in the presence of T 3 , but IGF-1 enhanced proliferation and differentiation in T 3 -containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T 3 and helps accelerate dorsal muscle remodeling during metamorphosis.

C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

PubMed Central

Brieger, Angela; Plotz, Guido; Hinrichsen, Inga; Passmann, Sandra; Adam, Ronja; Zeuzem, Stefan

2012-01-01

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. PMID:22348133

Urea Dependent (15)N NMR-Relaxation Studies on PfP2 Multimers Reveal that the C-Terminal Behaves like an Independent Intrinsically Disordered Peptide.

PubMed

Mishra, Pushpa; Hosur, Ramakrishna V

2015-01-01

Intrinsically disordered proteins or such domains in globular proteins are believed to be playing important roles in protein functions by virtue of their ability to adapt themselves to requirements of different binding partners and thereby accord high specificity to the interaction. Eukaryotic ribosomal stalk is made up of a supramolecular assembly of P0, P1 and P2 proteins. In Plasmodium falciparum, homo-oligomers of P2 are also seen which seem to be involved in many non-ribosomal functions of the protein in the parasite, and in all of these the protein interacts with different interactors. Here we show by extensive (15)N NMR relaxation studies that the C-terminal stretch of about 45 residues of the protein always remains as a flexible disordered domain, regardless of the state of association of the protein. The relaxation behaviors and the derived rotational correlation times for this portion of the protein are essentially the same in the presence of different concentrations of urea which produce different mixtures of PfP2 oligomers in rapid exchange, whereas the rest of the protein shows substantial variations with urea concentration in the relaxation behaviors. In other words, the C-terminal domain behaves as if it were an independent intrinsically disordered peptide. This would augment the notion that the C-terminal domain of PfP2 would be acting as a scavenger for different interactors depending upon the different functions of the protein inside the parasite.

Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.

PubMed

Bermudez, Jessica G; Chen, Hui; Einstein, Lily C; Good, Matthew C

2017-01-01

Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery. © 2017 Wiley Periodicals, Inc.

GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

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

Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of thismore » domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation*

PubMed Central

Chen, Zan; Dempsey, Daniel R.; Thomas, Stefani N.; Hayward, Dawn; Bolduc, David M.; Cole, Philip A.

2016-01-01

PTEN is a tumor suppressor that functions to negatively regulate the PI3K/AKT pathway as the lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate. Phosphorylation of a cluster of Ser/Thr residues (amino acids 380–385) on the C-terminal tail serves to alter the conformational state of PTEN from an open active state to a closed inhibited state, resulting in a reduction of plasma membrane localization and inhibition of enzyme activity. The relative contribution of each phosphorylation site to PTEN autoinhibition and the structural basis for the conformational closure is still unclear. To further the structural understanding of PTEN regulation by C-terminal tail phosphorylation, we used protein semisynthesis to insert stoichiometric and site-specific phospho-Ser/Thr(s) in the C-terminal tail of PTEN. Additionally, we employed photo-cross-linking to map the intramolecular PTEN interactions of the phospho-tail. Systematic evaluation of the PTEN C-tail phospho-cluster showed autoinhibition, and conformational closure was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. Moreover, photo-cross-linking suggested a direct interaction between the PTEN C-tail and a segment in the N-terminal region of the catalytic domain. Mutagenesis experiments provided additional insights into how the PTEN phospho-tail interacts with both the C2 and catalytic domains. PMID:27226612

Parachute-Deployment Flight Termination System on X-48C

NASA Image and Video Library

2013-02-28

The X-48C Hybrid Wing Body aircraft flew over Rogers Dry Lake on Feb. 28, 2013, from NASA's Dryden Flight Research Center, Edwards, CA. The long boom protruding from between the tails was part of the aircraft's parachute-deployment flight termination system.

The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

NASA Astrophysics Data System (ADS)

Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa

2015-10-01

Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.

7 CFR 1c.123 - Early termination of research support: Evaluation of applications and proposals.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 1 2011-01-01 2011-01-01 false Early termination of research support: Evaluation of applications and proposals. 1c.123 Section 1c.123 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.123 Early termination of research support: Evaluation of applications and...

7 CFR 1c.123 - Early termination of research support: Evaluation of applications and proposals.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 1 2010-01-01 2010-01-01 false Early termination of research support: Evaluation of applications and proposals. 1c.123 Section 1c.123 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.123 Early termination of research support: Evaluation of applications and...

The C- and N-Terminal Residues of Synthetic Heptapeptide Ion Channels Influence Transport Efficacy Through Phospholipid Bilayers

PubMed Central

Djedovič, Natasha; Ferdani, Riccardo; Harder, Egan; Pajewska, Jolanta; Pajewski, Robert; Weber, Michelle E.; Schlesinger, Paul H.; Gokel, George W.

2008-01-01

The synthetic peptide, R2N-COCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR’, was shown to be selective for Cl- over K+ when R is n-octadecyl and R’ is benzyl. Nineteen heptapeptides have now been prepared in which the N-terminal and C-terminal residues have been varied. All of the N-terminal residues are dialkyl but the C-terminal chains are esters, 2° amides, or 3° amides. The compounds having varied N-terminal anchors and C-terminal benzyl groups are as follows: 1, R = n-propyl; 2, R = n-hexyl; 3, R = n-octyl; 4, R = n-decyl; 5, R = n-dodecyl; 6, R = n-tetradecyl; 7, R = n-hexadecyl; 8, R = n-octadecyl. Compounds 9-19 have R = n-octadecyl and C-terminal residues as follows: 9, OR’ = OCH2CH3; 10, OR’ = OCH(CH3)2; 11, OR’ = O(CH2)6CH3; 12, OR’ = OCH2-c-C6H11; 13, OR’ = O(CH2)9CH3; 14, OR’ = O (CH2)17CH3; 15, NR’2 = N[(CH2)6CH3]2; 16, NHR’ = NH(CH2)9CH3; 17, NR’2 = N[(CH2)9CH3]2; 18, NHR’ = NH(CH2)17CH3; 19, NR’2 = N[(CH2)17CH3]2. The highest anion transport activities were observed as follows. For the benzyl esters whose N-terminal residues were varied, i.e. 1-8, compound 3 was most active. For the C18 anchored esters 10-14, n-heptyl ester 11 was most active. For the C18 anchored, C-terminal amides 15-19, di-n-decylamide 17 was most active. It was concluded that both the C- and N-terminal anchors were important for channel function in the bilayer but that activity was lost unless only one of the two anchoring groups was dominant. PMID:19633728

C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus.

PubMed

Li, Zhong; Yalcin, Talat; Cassady, Carolyn J

2006-07-01

Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode.

Lack of association between the Trp719Arg polymorphism in kinesin-like protein 6 and coronary artery disease in 19 case-control studies

PubMed Central

Assimes, Themistocles L; Hólm, Hilma; Kathiresan, Sekar; Reilly, Muredach P; Thorleifsson, Gudmar; Voight, Benjamin F; Erdmann, Jeanette; Willenborg, Christina; Vaidya, Dhananjay; Xie, Changchun; Patterson, Chris C; Morgan, Thomas M; Burnett, Mary Susan; Li, Mingyao; Hlatky, Mark A; Knowles, Joshua W; Thompson, John R; Absher, Devin; Iribarren, Carlos; Go, Alan; Fortmann, Stephen P; Sidney, Stephen; Risch, Neil; Tang, Hua; Myers, Richard M; Berger, Klaus; Stoll, Monika; Shah, Svati H.; Thorgeirsson, Gudmundur; Andersen, Karl; Havulinna, Aki S; Herrera, J. Enrique; Faraday, Nauder; Kim, Yoonhee; Kral, Brian G.; Mathias, Rasika; Ruczinski, Ingo; Suktitipat, Bhoom; Wilson, Alexander F; Yanek, Lisa R.; Becker, Lewis C; Linsel-Nitschke, Patrick; Lieb, Wolfgang; König, Inke R; Hengstenberg, Christian; Fischer, Marcus; Stark, Klaus; Reinhard, Wibke; Winogradow, Janina; Grassl, Martina; Grosshennig, Anika; Preuss, Michael; Eifert, Sandra; Schreiber, Stefan; Wichmann, H-Erich; Meisinger, Christa; Yee, Jean; Friedlander, Yechiel; Do, Ron; Meigs, James B; Williams, Gordon; Nathan, David M; MacRae, Calum A; Qu, Liming; Wilensky, Robert L; Matthai, William H.; Qasim, Atif N; Hakonarson, Hakon; Pichard, Augusto D; Kent, Kenneth M; Satler, Lowell; Lindsay, Joseph M; Waksman, Ron; Knouff, Christopher W; Waterworth, Dawn M; Walker, Max C; Mooser, Vincent; Marrugat, Jaume; Lucas, Gavin; Subirana, Isaac; Sala, Joan; Ramos, Rafael; Martinelli, Nicola; Olivieri, Oliviero; Trabetti, Elisabetta; Malerba, Giovanni; Pignatti, Pier Franco; Guiducci, Candace; Mirel, Daniel; Parkin, Melissa; Hirschhorn, Joel N; Asselta, Rosanna; Duga, Stefano; Musunuru, Kiran; Daly, Mark J; Purcell, Shaun; Braund, Peter S; Wright, Benjamin J; Balmforth, Anthony J; Ball, Stephen G; Ouwehand, Willem H; Deloukas, Panos; Scholz, Michael; Cambien, Francois; Huge, Andreas; Scheffold, Thomas; Salomaa, Veikko; Girelli, Domenico; Granger, Christopher B.; Peltonen, Leena; McKeown, Pascal P; Altshuler, David; Melander, Olle; Devaney, Joseph M; Epstein, Stephen E; Rader, Daniel J; Elosua, Roberto; Engert, James C; Anand, Sonia S; Hall, Alistair S; Ziegler, Andreas; O’Donnell, Christopher J; Spertus, John A; Siscovick, David; Schwartz, Stephen M; Becker, Diane; Thorsteinsdottir, Unnur; Stefansson, Kari; Schunkert, Heribert; Samani, Nilesh J; Quertermous, Thomas

2011-01-01

Objectives We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455) and clinical coronary artery disease (CAD). Background Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with non-carriers. Methods The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in nineteen case-control studies of non-fatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports. Results Over 17 000 cases and 39 000 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the nineteen studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with non-carriers. Regression analyses and fixed effect meta-analyses ruled out with high degree of confidence an increase of ≥2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early onset disease (<50 years of age for males and <60 years for females) compared with similarly aged controls as well as all non-European subgroups. Conclusions The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study. PMID:20933357

A novel Zea mays ssp. mexicana L. MYC-type ICE-like transcription factor gene ZmmICE1, enhances freezing tolerance in transgenic Arabidopsis thaliana.

PubMed

Lu, Xiang; Yang, Lei; Yu, Mengyuan; Lai, Jianbin; Wang, Chao; McNeil, David; Zhou, Meixue; Yang, Chengwei

2017-04-01

The annual Zea mays ssp. mexicana L., a member of the teosinte group, is a close wild relative of maize and thus can be effectively used in maize improvement. In this study, an ICE-like gene, ZmmICE1, was isolated from a cDNA library of RNA-Seq from cold-treated seedling tissues of Zea mays ssp. mexicana L. The deduced protein of ZmmICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE-like proteins. The ZmmICE1 protein localizes to the nucleus and shows sumoylation when expressed in an Escherichia coli reconstitution system. In addition, yeast one hybrid assays indicated that ZmmICE1 has transactivation activities. Moreover, ectopic expression of ZmmICE1 in the Arabidopsis ice1-2 mutant increased freezing tolerance. The ZmmICE1 overexpressed plants showed lower electrolyte leakage (EL), reduced contents of malondialdehyde (MDA). The expression of downstream cold related genes of Arabidopsis C-repeat-binding factors (AtCBF1, AtCBF2 and AtCBF3), cold-responsive genes (AtCOR15A and AtCOR47), kinesin-1 member gene (AtKIN1) and responsive to desiccation gene (AtRD29A) was significantly induced when compared with wild type under low temperature treatment. Taken together, these results indicated that ZmmICE1 is the homolog of Arabidopsis inducer of CBF expression genes (AtICE1/2) and plays an important role in the regulation of freezing stress response. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea).

PubMed

Ao, Jingqun; Ding, Yang; Chen, Yuanyuan; Mu, Yinnan; Chen, Xinhua

2015-12-10

The C-type lectin-like receptors (CTLRs) play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR) from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD), an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1-C6), a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp) and WYD (Trp-Tyr-Asp). Ca(2+) binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus) and guppy (Poecilia retitculata) CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR) protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca(2+)-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish.

Discovery of new molecular entities able to strongly interfere with Hsp90 C-terminal domain.

PubMed

Terracciano, Stefania; Russo, Alessandra; Chini, Maria G; Vaccaro, Maria C; Potenza, Marianna; Vassallo, Antonio; Riccio, Raffaele; Bifulco, Giuseppe; Bruno, Ines

2018-01-26

Heat shock protein 90 (Hsp90) is an ATP dependent molecular chaperone deeply involved in the complex network of cellular signaling governing some key functions, such as cell proliferation and survival, invasion and angiogenesis. Over the past years the N-terminal protein domain has been fully investigated as attractive strategy against cancer, but despite the many efforts lavished in the field, none of the N-terminal binders (termed "classical inhibitors"), currently in clinical trials, have yet successfully reached the market, because of the detrimental heat shock response (HSR) that showed to induce; thus, recently, the selective inhibition of Hsp90 C-terminal domain has powerfully emerged as a more promising alternative strategy for anti-cancer therapy, not eliciting this cell rescue cascade. However, the structural complexity of the target protein and, mostly, the lack of a co-crystal structure of C-terminal domain-ligand, essential to drive the identification of new hits, represent the largest hurdles in the development of new selective C-terminal inhibitors. Continuing our investigations on the identification of new anticancer drug candidates, by using an orthogonal screening approach, here we describe two new potent C-terminal inhibitors able to induce cancer cell death and a considerable down-regulation of Hsp90 client oncoproteins, without triggering the undesired heat shock response.

Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein.

PubMed

Duncan, R; Horne, D; Strong, J E; Leone, G; Pon, R T; Yeung, M C; Lee, P W

1991-06-01

We have been investigating structure-function relationships in the reovirus cell attachment protein sigma 1 using various deletion mutants and protease analysis. In the present study, a series of deletion mutants were constructed which lacked 90, 44, 30, 12, or 4 amino acids from the C-terminus of the 455-amino acid-long reovirus type 3 (T3) sigma 1 protein. The full-length and truncated sigma 1 proteins were expressed in an in vitro transcription/translation system and assayed for L cell binding activity. It was found that the removal of as few as four amino acids from the C-terminus drastically affected the cell binding function of the sigma 1 protein. The C-terminal-truncated proteins were further characterized using trypsin, chymotrypsin, and monoclonal and polyclonal antibodies. Our results indicated that the C-terminal portions of the mutant proteins were misfolded, leading to a loss in cell binding function. The N-terminal fibrous tail of the proteins was unaffected by the deletions as was sigma 1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of sigma 1. In an attempt to identify smaller, functional peptides, full-length sigma 1 expressed in vitro was digested with trypsin and subsequently with chymotrypsin under various conditions. The results clearly demonstrated the highly stable nature of the C-terminal globular head of sigma 1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of sigma 1 exists as a compact, protease-resistant oligomeric structure; (2) an intact C-terminus is required for proper head folding and generation of the conformationally dependent cell binding domain.

Biochemical study of prolactin binding sites in Xenopus laevis brain and choroid plexus

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

Muccioli, G.; Guardabassi, A.; Pattono, P.

1990-03-01

The occurrence of prolactin binding sites in some brain structures (telencephalon, ventral hypothalamus, myelencephalon, hypophysis, and choroid plexus) from Xenopus laevis (anuran amphibian) was studied by the in vitro biochemical technique. The higher binding values were obtained at the level of the choroid plexus and above all of the hypothalamus. On the bases of hormonal specificity and high affinity, these binding sites are very similar to those of prolactin receptors of classical target tissues as well as of those described by us in other structures from Xenopus. To our knowledge, the present results provide the first demonstration of the occurrencemore » of prolactin specific binding sites in Xenopus laevis choroid plexus cells.« less

4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

NASA Astrophysics Data System (ADS)

Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

2017-07-01

γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

75 FR 1799 - Terminate Long Range Aids to Navigation (Loran-C) Signal

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-13

... to Navigation (Loran-C) Signal AGENCY: U.S. Coast Guard, DHS. ACTION: Notice; correction. SUMMARY... 998). The document announced termination of the Long Range Aids to Navigation (Loran-C) Signal...

Role of C-terminal heptapeptide in pore-forming activity of antimicrobial agent, gaegurin 4.

PubMed

Kim, H J; Kim, S S; Lee, M H; Lee, B J; Ryu, P D

2004-10-01

Gaegurin 4 (GGN4) is an antimicrobial peptide of 37 amino acids isolated from the skin of a frog, Rana rugosa. GGN4 has a disulfide bond between the residues 31 and 37, which is highly conserved among the antimicrobial peptides isolated from skin of the genus, Rana. However, the role of this C-terminal heptapeptide motif is not well understood. In this work, we compared the membrane effects of the full-length GGN4 (C37) and GGN4 1-30 (C30), which is devoid of the C-terminal seven amino acids to elucidate the function of the C-terminal motif. C37 induced significantly larger membrane conductance (>10x) in the model lipid bilayers formed with acidic and neutral phospholipids and larger K+ efflux from gram-positive (>30x) and gram-negative bacteria. However, the pores induced by C37 and C30 were not different in their permeability to K+ over Cl- (permeability ratio of K+ to Cl- = 4.8-7.1). In addition, the pore-forming effect of C37 or C30 in acidic membranes was not different from that in neutral membranes. Furthermore, C37-induced K+ efflux was not significantly decreased by the reducing agent, dithiothreitol. The results indicate that C-terminal heptapeptide sequence plays an important role in maintaining the high pore-forming activity of GGN4, but does not participate in forming GGN4-induced pore structure. The disulfide bond in this region does not appear critical for such high ionophoric activity of GGN4.

Diverse C-Terminal Sequences Involved in Flavobacterium johnsoniae Protein Secretion

PubMed Central

Kulkarni, Surashree S.; Zhu, Yongtao; Brendel, Colton J.

2017-01-01

ABSTRACT Flavobacterium johnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to the family TIGR04183 (type A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign superfolder green fluorescent protein (sfGFP) that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case, approximately 80 to 100 amino acids from the extreme carboxy termini were needed for efficient secretion. Several type A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting the secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type A CTD. It has a conserved C-terminal domain belonging to the family TIGR04131, which we refer to as a type B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1,182 amino acids to sfGFP failed to result in secretion. Additional features outside the C-terminal region of SprB may be required for its secretion. IMPORTANCE Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes. Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to the protein domain family TIGR04183 (type A CTDs) or TIGR04131 (type B CTDs). Here, we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the

XENOPUS LAEVIS: A CULTURING AND REARING PROTOCOL

EPA Science Inventory

Xenopus laevis are used extensively here at MED-Duluth as a model for assessing development toxicity to xenobiotics. As a result, a culturing system has been developed that provides eggs and tadpoles of consistent high quality for use by researchers at the facility. The methods ...

Ketamine inhibits tumor necrosis factor-{alpha} and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

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

Wu, G.-J.; Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 {mu}M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 {mu}M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-{alpha}more » and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-{alpha} and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 {mu}M) significantly inhibited LPS-induced TNF-{alpha} and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-{alpha} and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-{alpha} and IL-6 gene expressions in LPS-activated macrophages. The suppressive

Ketamine inhibits tumor necrosis factor-alpha and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation.

PubMed

Wu, Gone-Jhe; Chen, Ta-Liang; Ueng, Yune-Fang; Chen, Ruei-Ming

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 microM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 microM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-alpha and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-alpha and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 microM) significantly inhibited LPS-induced TNF-alpha and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-alpha and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-alpha and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through

Adenosine A1 receptors modulate high voltage-activated Ca2+ currents and motor pattern generation in the Xenopus embryo

PubMed Central

Brown, Paul; Dale, Nicholas

2000-01-01

Adenosine causes voltage- and non-voltage-dependent inhibition of high voltage-activated (HVA) Ca2+ currents in Xenopus laevis embryo spinal neurons. As this inhibition can be blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and mimicked by N6-cyclopentyladenosine (CPA) it appears to be mediated by A1 receptors. Agents active at A2 receptors either were without effect or could be blocked by DPCPX. AMP had no agonist action on these receptors. By using ω-conotoxin GVIA we found that adenosine inhibited an N-type Ca2+ current as well as a further unidentified HVA current that was insensitive to dihydropyridines, ω-agatoxin TK and ω-conotoxin MVIIC. Both types of current were subject to voltage- and non-voltage-dependent inhibition. We used CPA and DPCPX to test whether A1 receptors regulated spinal motor pattern generation in spinalized Xenopus embryos. DPCPX caused a near doubling of, while CPA greatly shortened, the length of swimming episodes. In addition, DPCPX slowed, while CPA greatly speeded up, the rate of run-down of motor activity. Our results demonstrate a novel action of A1 receptors in modulating spinal motor activity. Furthermore they confirm that adenosine is produced continually throughout swimming episodes and acts to cause the eventual termination of activity. PMID:10856119

Xenopus laevis in Developmental and Molecular Biology.

ERIC Educational Resources Information Center

Dawid, Igor B.; Sargent, Thomas D.

1988-01-01

Discusses the advantages of Xenopus laevis as an experimental animal in the study of embryogenesis in vertebrates. Summarizes the contributions of this system to the analysis of ribosomal and 5S RNA genes, and the diverse and highly productive applications of the oocyte injection technology. (RT)

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells.

PubMed

Ding, J; Takano, T; Hermann, P; Gao, S; Han, W; Noda, C; Yanagi, S; Yamamura, H

2000-05-01

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.

Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein.

PubMed

Monteiro, Rose A; Souza, Emanuel M; Geoffrey Yates, M; Steffens, M Berenice R; Pedrosa, Fábio O; Chubatsu, Leda S

2003-02-01

The Herbaspirillum seropedicae NifA protein is responsible for nif gene expression. The C-terminal domain of the H. seropedicae NifA protein, fused to a His-Tag sequence (His-Tag-C-terminal), was over-expressed and purified by metal-affinity chromatography to yield a highly purified and active protein. Band-shift assays showed that the NifA His-Tag-C-terminal bound specifically to the H. seropedicae nifB promoter region in vitro. In vivo analysis showed that this protein inhibited the Central + C-terminal domains of NifA protein from activating the nifH promoter of K. pneumoniae in Escherichia coli, indicating that the protein must be bound to the NifA-binding site (UAS site) at the nifH promoter region to activate transcription. Copyright 2002 Elsevier Science (USA)

Accelerated Gene Evolution and Subfunctionalization in thePseudotetraploid Frog Xenopus Laevis

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

Hellsten, Uffe; Khokha, Mustafa K.; Grammar, Timothy C.

2007-03-01

Ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. The African clawed frog Xenopus laevis experienced a relatively recent tetraploidization {approx} 40 million years ago. Analysis of the considerable amount of EST sequence available for this species together with the genome sequence of the related diploid Xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication.

Activin-like signal activates dorsal-specific maternal RNA between 8- and 16-cell stages of Xenopus.

PubMed

Hainski, A M; Moody, S A

1996-01-01

In many animals the dorsal-ventral axis forms by an initial localization of maternal molecules, which then regulate the spatial location of signals that directly influence the expression of axis-specific fates. Several recent studies have demonstrated that dorsal-animal blastomeres of the Xenopus morula (8-32 cells) are biased toward dorsal fates prior to mesoderm inductive signaling. In this study we ask whether the dorsal bias is the result of autonomous expression of maternal molecules specifically localized within dorsal cells or of early activating signals. It was found that although 16-cell dorsal-animal blastomeres (D1.1) can differentiate into dorsal tissues when cultured alone, the 8-cell mothers (D1) can not. Likewise, although RNA extracted from D1.1 can induce an extra dorsal axis when injected into vegetal blastomeres, RNA extracted from D1 can not. However, D1 does express dorsal tissues if co-cultured with dorsal-vegetal cells or with culture medium containing a mixture of activins (PIF-medium). Furthermore, short-term culture of D1 in PIF-medium enables the D1 RNA to induce an ectopic dorsal axis. Ventral-animal blastomeres also can express dorsal axial tissues when co-cultured with dorsal-vegetal blastomeres or in PIF-medium, but the RNA from the activin-treated ventral cells cannot induce ectopic dorsal axes. These studies demonstrate that there are maternal RNAs that, shortly after fertilization, are present only in the dorsal-animal region. They do not act cell autonomously, but require an activin-like signal. These RNAs may function by increasing the responsiveness of dorsal-animal blastomeres to the mesoderm inductive signals present in both the morula and the blastula.

Expression of LRRC8/VRAC Currents in Xenopus Oocytes: Advantages and Caveats.

PubMed

Gaitán-Peñas, Héctor; Pusch, Michael; Estévez, Raúl

2018-03-02

Volume-regulated anion channels (VRACs) play a role in controlling cell volume by opening upon cell swelling. Apart from controlling cell volume, their function is important in many other physiological processes, such as transport of metabolites or drugs, and extracellular signal transduction. VRACs are formed by heteromers of the pannexin homologous protein LRRC8A (also named Swell1) with other LRRC8 members (B, C, D, and E). LRRC8 proteins are difficult to study, since they are expressed in all cells of our body, and the channel stoichiometry can be changed by overexpression, resulting in non-functional heteromers. Two different strategies have been developed to overcome this issue: complementation by transient transfection of LRRC8 genome-edited cell lines, and reconstitution in lipid bilayers. Alternatively, we have used Xenopus oocytes as a simple system to study LRRC8 proteins. Here, we have reviewed all previous experiments that have been performed with VRAC and LRRC8 proteins in Xenopus oocytes. We also discuss future strategies that may be used to perform structure-function analysis of the VRAC in oocytes and other systems, in order to understand its role in controlling multiple physiological functions.

A Convenient Approach to Synthesizing Peptide C-Terminal N-Alkyl Amides

PubMed Central

Fang, Wei-Jie; Yakovleva, Tatyana; Aldrich, Jane V.

2014-01-01

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the PAL-PEG-PS (Peptide Amide Linker-polyethylene glycol-polystyrene) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides. PMID:22252422

Uncovering the role of the flexible C-terminal tail: A model study with Strep-tagged GFP.

PubMed

Lassalle, Michael W; Kondou, Shinobu

2016-06-01

Recently, it has been recognized that, much like an electric current in an electric circuit, dynamic disruptions from flexible, unstructured regions distal to the active region are transferred through the contact network to the active site and influence protein stability and/or function. As transmembrane proteins frequently possess the β-barrel structure, studies of proteins with this topology are required. The unstructured lid segments of the β-barrel GFP protein are conserved and could play a role in the backbone stabilization required for chromophore function. A study of the disordered C-terminus and the function within the lid is necessary. In this study, we entirely truncated the flexible C-terminal tail and investigated the N-terminal Strep-tagged GFP by fluorescence spectroscopy, and the temperature- and GdnHCl-induced unfolding by circular dichroism. The introduction of the unstructured Strep-tag itself changed the unfolding pathway. Truncating the entire flexible tail did not decrease the fluorescence intensity to a large extent; however, the protein stability changed dramatically. The temperature for half-denaturation T 1/2 changed significantly from 79 °C for the wild-type to 72.8 °C for the mutant. Unfolding kinetics at different temperatures have been induced by 4 M GdnHCl, and the apparent Arrhenius activation energy decreased by 40% as compared to the wild-type.

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Dupré, Mathieu; Cantel, Sonia; Martinez, Jean; Enjalbal, Christine

2012-02-01

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1 + H2O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2O and NH3) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in

C-terminal Lysine-Linked Magainin 2 with Increased Activity Against Multidrug-Resistant Bacteria.

PubMed

Lorenzón, Esteban N; Santos-Filho, Norival A; Ramos, Matheus A S; Bauab, Tais M; Camargo, Ilana L B C; Cilli, Eduardo M

2016-01-01

Due to the growing problem of antibiotic-resistant microorganisms, the development of novel antimicrobial agents is a very important challenge. Dimerization of cationic antimicrobial peptides (cAMPs) is a potential strategy for enhancing antimicrobial activity. Here, we studied the effects of magainin 2 (MG2) dimerization on its structure and biological activity. Lysine and glutamic acid were used to synthesize the C- and N-terminal dimers of MG2, respectively, in order to evaluate the impact of linker position used to obtain the dimers. Both MG2 and its dimeric versions showed a random coil structure in aqueous solution. However, in the presence of a structure-inducing solvent or a membrane mimetic, all peptides acquired helical structure. N-terminal dimerization did not affect the biological activity of the peptide. On the other hand, the C-terminal dimer, (MG2)2K, showed antimicrobial activity 8-16 times higher than that of MG2, and the time required to kill Escherichia coli was lower. The enhanced antimicrobial activity was related to membrane permeabilization. (MG2)2K was also more active against multidrug-resistant bacteria of clinical origin. Overall, the results presented here demonstrate that C-terminal lysine-linked dimerization improve the activity of MG2, and (MG2)2K can be considered as a potential antimicrobial agent.

KIFC1-Like Motor Protein Associates with the Cephalopod Manchette and Participates in Sperm Nuclear Morphogenesis in Octopus tankahkeei

PubMed Central

Tan, Fu-Qing; Yang, Wan-Xi

2010-01-01

Background Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. Methodology/Principal Findings We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level. Conclusions/Significance The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod. PMID:21187923

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2015-03-01

1 AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER... Lateral   Sclerosis ”   Final  Report:  Project  Period  Sept  2012-­‐Dec  2014     Personnel  List:     Feng,  Yangbo

The C-terminal region of Ge-1 presents conserved structural features required for P-body localization.

PubMed

Jinek, Martin; Eulalio, Ana; Lingel, Andreas; Helms, Sigrun; Conti, Elena; Izaurralde, Elisa

2008-10-01

The removal of the 5' cap structure by the DCP1-DCP2 decapping complex irreversibly commits eukaryotic mRNAs to degradation. In human cells, the interaction between DCP1 and DCP2 is bridged by the Ge-1 protein. Ge-1 contains an N-terminal WD40-repeat domain connected by a low-complexity region to a conserved C-terminal domain. It was reported that the C-terminal domain interacts with DCP2 and mediates Ge-1 oligomerization and P-body localization. To understand the molecular basis for these functions, we determined the three-dimensional crystal structure of the most conserved region of the Drosophila melanogaster Ge-1 C-terminal domain. The region adopts an all alpha-helical fold related to ARM- and HEAT-repeat proteins. Using structure-based mutants we identified an invariant surface residue affecting P-body localization. The conservation of critical surface and structural residues suggests that the C-terminal region adopts a similar fold with conserved functions in all members of the Ge-1 protein family.

C-Terminal End-Directed Protein Elimination by CRL2 Ubiquitin Ligases.

PubMed

Lin, Hsiu-Chuan; Yeh, Chi-Wei; Chen, Yen-Fu; Lee, Ting-Ting; Hsieh, Pei-Yun; Rusnac, Domnita V; Lin, Sung-Ya; Elledge, Stephen J; Zheng, Ning; Yen, Hsueh-Chi S

2018-05-17

The proteolysis-assisted protein quality control system guards the proteome from potentially detrimental aberrant proteins. How miscellaneous defective proteins are specifically eliminated and which molecular characteristics direct them for removal are fundamental questions. We reveal a mechanism, DesCEND (destruction via C-end degrons), by which CRL2 ubiquitin ligase uses interchangeable substrate receptors to recognize the unusual C termini of abnormal proteins (i.e., C-end degrons). C-end degrons are mostly less than ten residues in length and comprise a few indispensable residues along with some rather degenerate ones. The C-terminal end position is essential for C-end degron function. Truncated selenoproteins generated by translation errors and the USP1 N-terminal fragment from post-translational cleavage are eliminated by DesCEND. DesCEND also targets full-length proteins with naturally occurring C-end degrons. The C-end degron in DesCEND echoes the N-end degron in the N-end rule pathway, highlighting the dominance of protein "ends" as indicators for protein elimination. Copyright © 2018 Elsevier Inc. All rights reserved.

Dynamic expression of the LAP family of genes during early development of Xenopus tropicalis.

PubMed

Yang, Qiutan; Lv, Xiaoyan; Kong, Qinghua; Li, Chaocui; Zhou, Qin; Mao, Bingyu

2011-10-01

The leucine-rich repeats and PDZ (LAP) family of genes are crucial for the maintenance of cell polarity as well as for epithelial homeostasis and tumor suppression in both vertebrates and invertebrates. Four members of this gene family are known: densin, erbin, scribble and lano. Here, we identified the four members of the LAP gene family in Xenopus tropicalis and studied their expression patterns during embryonic development. The Xenopus LAP proteins show a conserved domain structure that is similar to their homologs in other vertebrates. In Xenopus embryos, these genes were detected in animal cap cells at the early gastrula stage. At later stages of development, they were widely expressed in epithelial tissues that are highly polar in nature, including the neural epithelia, optic and otic vesicles, and in the pronephros. These data suggest that the roles of the Xenopus LAP genes in the control of cell polarity and morphogenesis are conserved during early development. Erbin and lano show similar expression patterns in the developing head, suggesting potential functional interactions between the two molecules in vivo.

Modulation of K+ currents in Xenopus spinal neurons by p2y receptors: a role for ATP and ADP in motor pattern generation

PubMed Central

Brown, Paul; Dale, Nicholas

2002-01-01

We have investigated the pharmacological properties and targets of p2y purinoceptors in Xenopus embryo spinal neurons. ATP reversibly inhibited the voltage-gated K+ currents by 10 ± 3 %. UTP and the analogues α,β-methylene-ATP and 2-methylthio-ATP also inhibited K+ currents. This agonist profile is similar to that reported for a p2y receptor cloned from Xenopus embryos. Voltage-gated K+ currents could be inhibited by ADP (9 ± 0.8 %) suggesting that a further p2y1-like receptor is also present in the embryo spinal cord. Unexpectedly we found that α,β-methylene-ADP, often used to block the ecto-5′-nucleotidase, also inhibited voltage-gated K+ currents (7 ± 2.3 %). This inhibition was occluded by ADP, suggesting that α,β-methylene-ADP is an agonist at p2y1 receptors. We have directly studied the properties of the ecto-5′-nucleotidase in Xenopus embryo spinal cord. Although ADP inhibited this enzyme, α,β-methylene-ADP had no action. Caution therefore needs to be used when interpreting the actions of α,β-methylene-ADP as it has previously unreported agonist activity at P2 receptors. Xenopus spinal neurons possess fast and slow voltage-gated K+ currents. By using catechol to selectively block the fast current, we completely occluded the actions of ATP and ADP. Furthermore, the purines appeared to block only the fast relaxation component of the tail currents. We therefore conclude that the p2y receptors target only the fast component of the delayed rectifier. As ATP breakdown to ADP is rapid and ADP may accumulate at higher levels than ATP, the contribution of ADP acting through p2y1-like receptors may be an important additional mechanism for the control of spinal motor pattern generation. PMID:11986373

Characterization and identification of a C-terminal amidated mechano growth factor (MGF) analogue in black market products.

PubMed

Esposito, Simone; Deventer, Koen; Van Eenoo, Peter

2012-03-30

Mechano growth factor (MGF) is a splice variant of insulin-like growth factor that possesses anabolic properties and has not yet been approved for therapeutic use. Nevertheless, it is readily available on the black market. Although the World Anti-Doping Agency (WADA) has banned the use of MGF in sports, no routinely performed methods have been reported for its detection. In this work, two preparations from the black market containing an unknown MGF analogue were characterized. Mass spectrometry characterizations of unknown preparations and a reference human MGF were performed on an Orbitrap and a triple quadrupole mass spectrometers after separation by liquid chromatography. High accuracy measurements allowed protein identification from full scan MS data, and low-resolution full scan MS/MS provided further information on fragmentation. HCD scans of the analytes showed the presence of common b series product ions in the black market preparations and the human MGF reference standard, but all the y series ions starting from (y(1))(+) exhibited a difference of 1 m/z unit in nominal mass. This difference was demonstrated to be due to a C-terminal amidation of MGF. High-resolution data demonstrated that the black market products were both C-terminal amidated analogues of human MGF. In addition, low-resolution MS/MS characterization revealed a potentially diagnostic transition (m/z 717.8 → 431.1) for the discrimination of C-amidated MGF from the endogenous form. Qualitative identification of a MGF C-terminal amidated analogue in two black market products was successfully achieved. This report demonstrates that illegal MGF preparations are commercially available for use as doping agent in sports. Copyright © 2012 John Wiley & Sons, Ltd.

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica

PubMed Central

Wong, Ka H.; Tan, Wei Liang; Kini, Shruthi G.; Xiao, Tianshu; Serra, Aida; Sze, Sui Kwan; Tam, James P.

2017-01-01

Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40–41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics. PMID:28680440

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica.

PubMed

Wong, Ka H; Tan, Wei Liang; Kini, Shruthi G; Xiao, Tianshu; Serra, Aida; Sze, Sui Kwan; Tam, James P

2017-01-01

Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40-41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC 50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics.

A domain-based approach for analyzing the function of aluminum-activated malate transporters from wheat (Triticum aestivum) and Arabidopsis thaliana in Xenopus oocytes.

PubMed

Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Furuichi, Takuya; Yamamoto, Yoko

2014-12-01

Wheat and Arabidopsis plants respond to aluminum (Al) ions by releasing malate from their root apices via Al-activated malate transporter. Malate anions bind with the toxic Al ions and contribute to the Al tolerance of these species. The genes encoding the transporters in wheat and Arabidopsis, TaALMT1 and AtALMT1, respectively, were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the two-electrode voltage clamp system. The Al-activated currents generated by malate efflux were detected for TaALMT1 but not for AtALMT1. Chimeric proteins were generated by swapping the N- and C-terminal halves of TaALMT1 and AtALMT1 (Ta::At and At::Ta). When these chimeras were characterized in oocytes, Al-activated malate efflux was detected for the Ta::At chimera but not for At::Ta, suggesting that the N-terminal half of TaALMT1 is necessary for function in oocytes. An additional chimera, Ta(48)::At, generated by swapping 17 residues from the N-terminus of AtALMT1 with the equivalent 48 residues from TaALMT1, was sufficient to support transport activity. This 48 residue region includes a helical region with a putative transmembrane domain which is absent in AtALMT1. The deletion of this domain from Ta(48)::At led to the complete loss of transport activity. Furthermore, truncations and a deletion at the C-terminal end of TaALMT1 indicated that a putative helical structure in this region was also required for transport function. This study provides insights into the structure-function relationships of Al-activated ALMT proteins by identifying specific domains on the N- and C-termini of TaALMT1 that are critical for basal transport function and Al responsiveness in oocytes. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Multiple-interactions among EMILIN1 and EMILIN2 N- and C-terminal domains.

PubMed

Bot, Simonetta; Andreuzzi, Eva; Capuano, Alessandra; Schiavinato, Alvise; Colombatti, Alfonso; Doliana, Roberto

2015-01-01

EMILIN1 and EMILIN2 belong to a family of extracellular matrix glycoproteins characterized by the N-terminal cysteine-rich EMI domain, a long segment with high probabilty for coiled-coil structure formation and a C-terminal gC1q domain. To study EMILIN1 and EMILIN2 interaction and assembly we have applied qualitative and quantitative two hybrid systems using constructs corresponding to the gC1q and EMI domains. The identified interactions were further confirmed in yeast extracts of co-transfected cells followed by co-immunoprecipitation. The data indicated that gC1q domains are able to self-interact as well as to interact one each other and with the EMI domains, but no self interactions were detected between the EMI domains. Furthermore EMILINs interactions were studied in 293-EBNA cells co-transfected with full lenght EMILIN1 and EMILIN2 constructs. Specific antibodies were able to co-immunoprecipitate EMILINs, indicating that also full-lenght proteins can give rise to non-covalent homo- and hetero-multimers even if reduced and alkylated before mixing. Immunofluorescence analysis on mouse cell cultures and tissues sections with specific antibodies showed co-distribution of EMILIN1 and EMILIN2. Thus, we can hypothesize that EMILINs multimers are formed by head-to-tail interaction between C-terminal and N-terminal domains of EMILIN1 and/or EMILIN2 but also by tail-to-tail interaction between gC1q domains. These multiple interactions may regulate homo-typic and/or hetero-typic linear and eventually lateral branching assemblies of EMILIN1 and EMILIN2 in tissues. Copyright © 2014. Published by Elsevier B.V.

HABP1/p32/gC1qR induces aberrant growth and morphology in Schizosaccharomyces pombe through its N-terminal {alpha} helix

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

Mallick, Jaideep; Datta, Kasturi

2005-10-01

Hyaluronan binding protein (HABP1), located on human chromosome 17p13.3, was identified and characterized as being involved in cellular signaling from our laboratory. Here, we demonstrate that HABP1 expression in Schizosaccharomyces pombe induces growth inhibition, morphological abnormalities like elongation, multinucleation and aberrant cell septum formation in several strains of S. pombe, implicating its role in cell cycle progression and cytokinesis. This argument is further strengthened by an observed delay in the maximal expression of cell cycle regulatory proteins like CDC 2 and CDC 25 coupled to the direct interaction of HABP1 with CDC 25. In order to pinpoint the interacting domainmore » of HABP1, its N- and C-terminal truncated variants ({delta}N.HABP1 and {delta}C.HABP1, respectively) were utilized which revealed that while expression of the former did not alter the phenotype, the latter generated morphological changes similar to those imparted upon HABP1 expression. It was also noted that along with HABP1, {delta}C.HABP1 too directly interacts with CDC 25 while {delta}N.HABP1 does not. Taken together, these data suggest that HABP1 induces morphological changes and modulates the cell cycle by interacting with proteins like CDC 25 through its N-terminal {alpha}-helix.« less

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones.

PubMed

Siebert, Matthias; Böhme, Mathias A; Driller, Jan H; Babikir, Husam; Mampell, Malou M; Rey, Ulises; Ramesh, Niraja; Matkovic, Tanja; Holton, Nicole; Reddy-Alla, Suneel; Göttfert, Fabian; Kamin, Dirk; Quentin, Christine; Klinedinst, Susan; Andlauer, Till Fm; Hell, Stefan W; Collins, Catherine A; Wahl, Markus C; Loll, Bernhard; Sigrist, Stephan J

2015-08-14

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

PubMed

Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

2003-08-29

One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

Recognition of the Xenopus ribosomal core promoter by the transcription factor xUBF involves multiple HMG box domains and leads to an xUBF interdomain interaction.

PubMed

Leblanc, B; Read, C; Moss, T

1993-02-01

The interaction of the ribosomal transcription factor xUBF with the RNA polymerase I core promoter of Xenopus laevis has been studied both at the DNA and protein levels. It is shown that a single xUBF-DNA complex forms over the 40S initiation site (+1) and involves at least the DNA sequences between -20 and +60 bp. DNA sequences upstream of +10 and downstream of +18 are each sufficient to direct complex formation independently. HMG box 1 of xUBF independently recognizes the sequences -20 to -1 and +1 to +22 and the addition of the N-terminal dimerization domain to HMG box 1 stabilizes its interaction with these sequences approximately 10-fold. HMG boxes 2/3 interact with the DNA downstream of +22 and can independently position xUBF across the initiation site. The C-terminal segment of xUBF, HMG boxes 4, 5 or the acidic domain, directly or indirectly interact with HMG box 1, making the core promoter sequences between -11 and -15 hypersensitive to DNase. This interaction also requires the DNA sequences between +17 and +32, i.e. the HMG box 2/3 binding site. The data suggest extensive folding of the core promoter within the xUBF complex.

A Statistical Physicist's Approach to Biological Motion: From the the Kinesin Walk to Muscle Contraction

NASA Astrophysics Data System (ADS)

Vicsek, Tamas

1997-03-01