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Larisa Gheber - One of the best experts on this subject based on the ideXlab platform.
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drag induced directionality switching of Kinesin 5 cin8 revealed by cluster motility analysis
Science Advances, 2021Co-Authors: Himanshu Pandey, Jawdat Albassam, Emanuel Reithmann, Alina Goldsteinlevitin, Erwin Frey, Larisa GheberAbstract:Directed active motion of motor proteins is a vital process in virtually all eukaryotic cells. Nearly a decade ago, the discovery of directionality switching of mitotic Kinesin-5 motors challenged the long-standing paradigm that individual Kinesin motors are characterized by an intrinsic directionality. The underlying mechanism, however, remains unexplained. Here, we studied clustering-induced directionality switching of the bidirectional Kinesin-5 Cin8. Based on the characterization of single-molecule and cluster motility, we developed a model that predicts that directionality switching of Cin8 is caused by an asymmetric response of its active motion to opposing forces, referred to as drag. The model shows excellent quantitative agreement with experimental data obtained under high and low ionic strength conditions. Our analysis identifies a robust and general mechanism that explains why bidirectional motor proteins reverse direction in response to seemingly unrelated experimental factors including changes in motor density and molecular crowding, and in multimotor motility assays.
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drag induced directionality switching of Kinesin 5 cin8 revealed by cluster motility analysis
bioRxiv, 2020Co-Authors: Himanshu Handey, Jawdat Albassam, Emanuel Reithmann, Alina Goldsteinlevitin, Erwin Frey, Larisa GheberAbstract:Directed active motion of motor proteins is a vital process in virtually all eukaryotic cells. Nearly a decade ago, the discovery of directionality switching of mitotic Kinesin-5 motors challenged the long-standing paradigm that individual Kinesin motors are characterized by an intrinsic directionality. While several Kinesin motors have now been shown to exhibit context-dependent directionality that can be altered under diverse experimental conditions, the underlying mechanism remains unknown. Here, we studied clustering-induced directionality switching of the mitotic Kinesin-5 Cin8, using a fluorescence-based single-molecule motility assay combined with biophysical theory. Based on the detailed characterization of the motility of single motors and clusters of Cin8, we developed a predictive molecular model, that quantitatively agrees with experimental data. This combined approach allowed us to quantify the response of Cin8 motors to external forces as well as the interactions between Cin8 motors, and thereby develop a detailed understanding of the molecular mechanism underlying directionality switching. The main insight is that directionality switching is caused by a single feature of Cin8: an asymmetric response of active motion to forces that oppose motion, here referred to as drag. This general mechanism explains why bidirectional motor proteins are capable of reversing direction in response to seemingly unrelated experimental factors including clustering, changes in the ionic strength of the buffer, increased motor density and molecular crowding, and in motility assays.
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Synthetic-evolution reveals that phosphoregulation of the mitotic Kinesin-5 Cin8 is constrained
2018Co-Authors: Alina Goldstein, Mart Loog, Darya Goldman, Ervin Valk, Liam J. Holt, Larisa GheberAbstract:Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions. These phosphorylation sites do not appear to require precise positioning for their function. The mitotic Kinesin-5 Cin8 was shown to be phosphoregulated at three Cdk1 sites in disordered loops within its catalytic motor domain. Here, we examined the flexibility of Cin8 phosphoregulation by analyzing the phenotypes of synthetic Cdk1-sites that were systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites that we created, eight were non-functional; 19 were neutral, similar to the phosphodeficient variant; and two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one site resulted in novel phosphoregulation, and only one site, immediately adjacent to a native Cdk1 site, produced phosphoregulation similar to wild-type. This study shows that, while the gain of a single phosphorylation site can confer regulation and modulate the dynamics of the spindle, to achieve optimal regulation of a mitotic Kinesin-5 motor protein, phosphoregulation has to be site-specific and precise.
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bidirectional motility of Kinesin 5 motor proteins structural determinants cumulative functions and physiological roles
Cellular and Molecular Life Sciences, 2018Co-Authors: Sudhir Kumar Singh, Himanshu Pandey, Jawdat Albassam, Larisa GheberAbstract: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.
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Deletion of the tail domain of the Kinesin-5 Cin8 affects its directionality
The Journal of biological chemistry, 2015Co-Authors: André Düselder, Vladimir Fridman, Larisa Gheber, Christina Thiede, Dieter R. Klopfenstein, Alice Wiesbaum, Alina Goldstein, Olga Zaitseva, Marcel E. Janson, Christoph F. SchmidtAbstract:Abstract 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.
Christoph F. Schmidt - One of the best experts on this subject based on the ideXlab platform.
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The natural diterpene tonantzitlolone A and its synthetic enantiomer inhibit cell proliferation and Kinesin-5 function.
European journal of medicinal chemistry, 2016Co-Authors: Tobias J. Pfeffer, Christoph F. Schmidt, Stefan Lakämper, Florenz Sasse, Andreas Kirschning, Tim ScholzAbstract:Tonantzitlolone A, a diterpene isolated from the Mexican plant Stillingia sanguinolenta, shows cytostatic activity. Both the natural product tonantzitlolone A and its synthetic enantiomer induce monoastral spindle formation in cell experiments which indicates inhibitory activity on Kinesin-5 mitotic motor molecules. These inhibitory effects on Kinesin-5 could be verified in in vitro single-molecule motility assays, where both tonantzitlolones interfered with Kinesin-5 binding to its cellular interaction partner microtubules in a concentration-dependent manner, yet with a larger effect of the synthetic enantiomer. In contrast to Kinesin-5 inhibition, both tonantzitlolone A enantiomers did not affect conventional Kinesin-1 function; hence tonantzitlolones are not unspecific Kinesin inhibitors. The observed stronger inhibitory effect of the synthetic enantiomer demonstrates the possibility to enhance the overall moderate anti-proliferative effect of the lead compound tonantzitlolon A by chemical modification.
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Deletion of the tail domain of the Kinesin-5 Cin8 affects its directionality
The Journal of biological chemistry, 2015Co-Authors: André Düselder, Vladimir Fridman, Larisa Gheber, Christina Thiede, Dieter R. Klopfenstein, Alice Wiesbaum, Alina Goldstein, Olga Zaitseva, Marcel E. Janson, Christoph F. SchmidtAbstract:Abstract 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.
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A chimeric Kinesin-1 head/Kinesin-5 tail motor switches between diffusive and processive motility.
Biophysical journal, 2013Co-Authors: Christina Thiede, Stefanie Kramer, Stefan Lakämper, Alok D. Wessel, Christoph F. SchmidtAbstract: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.
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a chimeric Kinesin 1 head Kinesin 5 tail motor switches between diffusive and processive motility
Biophysical Journal, 2013Co-Authors: Christina Thiede, Stefanie Kramer, Stefan Lakämper, Alok D. Wessel, Christoph F. SchmidtAbstract: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.
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Neck-linker length dependence of processive Kinesin-5 motility.
Journal of molecular biology, 2012Co-Authors: André Düselder, Christina Thiede, Stefanie Kramer, Christoph F. Schmidt, Stefan LakämperAbstract:Abstract Processive motility of individual molecules is essential for the function of many Kinesin motors. Processivity for Kinesins relies on communication between the two heads of a dimeric molecule, such that binding strictly alternates. The main communicating elements are believed to be the two neck linkers connecting the motors' stalks and heads. A proposed mechanism for coordination is the transmission of stress through the neck linkers. It is believed that the efficiency of gating depends on the length of the neck linker. Recent studies have presented support for a simple model in which the length of the neck linker directly controls the degree of processivity. Based on a previously published Kinesin-1/Kinesin-5 chimera, Eg5Kin, we have analyzed the motility of 12 motor constructs: we have varied the length of the neck linker in the range between 9 and 21 amino acids using the corresponding native Kinesin-5 sequence ( Xenopus laevis Eg5). We found, surprisingly, that neither velocity nor force generation depended on neck-linker length. We also found that constructs with short neck linkers, down to 12 amino acids, were still highly processive, while processivity was lost at a length of 9 amino acids. Run lengths were maximal with neck linkers close to the native Kinesin-5 length and decreased beyond that length. This finding generally confirms the coordinating role of the neck linker for Kinesin motility but challenges the simplest model postulating a motor-type‐independent optimal length. Instead, our results suggest that different Kinesins might be optimized for different neck-linker lengths.
Vladimir Fridman - One of the best experts on this subject based on the ideXlab platform.
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Deletion of the tail domain of the Kinesin-5 Cin8 affects its directionality
The Journal of biological chemistry, 2015Co-Authors: André Düselder, Vladimir Fridman, Larisa Gheber, Christina Thiede, Dieter R. Klopfenstein, Alice Wiesbaum, Alina Goldstein, Olga Zaitseva, Marcel E. Janson, Christoph F. SchmidtAbstract:Abstract 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.
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Regulation of bi-directional movement of single Kinesin-5 Cin8 molecules.
Bioarchitecture, 2012Co-Authors: Christina Thiede, Vladimir Fridman, Adina Gerson-gurwitz, Larisa Gheber, Christoph F. SchmidtAbstract:Kinesin-5 mechanoenzymes drive mitotic spindle dynamics as slow, processive microtubule (MT)-plus-end directed motors. Surprisingly, the Saccharomyces cerevisiae Kinesin-5 Cin8 was recently found to be bi-directional: it can move processively in both directions on MTs. Two hypotheses have been suggested for the mechanism of the directionality switch: (1) single molecules of Cin8 are intrinsically minus-end directed, but mechanical coupling between two or more motors triggers the switch; (2) a single motor can switch direction, and “cargo binding” i.e., binding between two MTs triggers the switch to plus-end motility. Single-molecule fluorescence data we published recently, and augment here, favor hypothesis (2). In low-ionic-strength conditions, single molecules of Cin8 move in both minus- and plus-end directions. Fluorescence photo bleaching data rule out aggregation of Cin8 while they move in the plus and in the minus direction. The evidence thus points toward cargo regulation of directionality, which i...
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Control of Directionality of Individual Kinesin-5 Motors
Biophysical Journal, 2012Co-Authors: Adina Gerson-gurwitz, Natalia Movshovich, Vladimir Fridman, Christina Thiede, Christoph F. Schmidt, Stefan Lakämper, Maria Podolskaya, Dieter R. Klopfenstein, Larisa GheberAbstract:Kinesin-5 motors fulfill essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT)-plus-end directed motors. The Saccharomyces cerevisiae Kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here we have examined Cin8 directionality control using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly towards the midzone, in the plus-end direction of the interpolar MTs. Occasionally, Cin8 also moved faster towards the spindle poles, in the minus-end direction of the MTs. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel microtubules, while driving steady plus-end relative sliding. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic-strength dependent directional switching of Cin8 in vitro. In vivo, the deletion mutant exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.
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Loop 8 Plays a Role in Controlling S. Cerevisiae Kinesin-5 Cin8 Motility and Function
Biophysical Journal, 2012Co-Authors: Adina Gerson Gurwitz, Natalia Movshovich, Vladimir Fridman, Christina Thiede, Christoph F. Schmidt, Stefan Lakämper, Maria Podolskaya, Dieter R. Klopfenstein, Larisa GheberAbstract:Kinesin-5 proteins are microtubule associated motors, which are highly conserved from yeast to human cells. They share high homology in their catalytic motor domain sequence, fulfill similar essential mitotic roles in spindle assembly and anaphase B spindle elongation and, until recently (Roostalu et al., Science, 2011), were all thought to move towards plus ends of microtubules. Mechanisms that regulate Kinesin-5 function, specifically during anaphase B, are not well understood.S. cerevisiae cells express two Kinesin-5 homologues, Cin8 and Kip1, which overlap in function. Here we have examined in vitro and in vivo functions and regulation of Cin8 during anaphase B. We followed Cin8 localization and carried out single molecule fluorescence motility assays to study Cin8 motile properties. We found that in vitro, Cin8 molecules are able to switch directionality along a single microtubule as a function of ionic strength conditions and that during anaphase B, Cin8 moves not only towards the plus, but also towards the minus ends of spindle microtubules.Compared to Kinesin-5 homologues of higher eukaryotes, S. cerevisiae Cin8 carries a uniquely large insert in loop 8 in its motor domain. To probe the role of the large loop 8 in the directionality switch of Cin8, we studied a construct in which this segment was replaced with the seven amino acids of loop 8 in the related S. cerevisiae Kinesin-5 Kip1 (Cin8Δ99) (Hoyt et al.,J Cell Biol, 1992). We examined the anaphase B localization and in vitro motile properties of the Cin8Δ99 variant. Using combined in vitro and in vivo approaches, we were able to characterize the role of loop 8 in controlling Cin8 motility and function during S. cerevisiae anaphase.
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Phospho-regulation of Kinesin-5 during anaphase spindle elongation.
Journal of Cell Science, 2011Co-Authors: Rachel Avunie-masala, Natalia Movshovich, Vladimir Fridman, Adina Gerson-gurwitz, M. Andrew Hoyt, Yael Nissenkorn, Mardo Kõivomägi, Mart Loog, Arieh Zaritsky, Larisa GheberAbstract:The Kinesin-5 Saccharomyces cerevisiae homologue Cin8 is shown here to be differentially phosphorylated during late anaphase at Cdk1-specific sites located in its motor domain. Wild-type Cin8 binds to the early-anaphase spindles and detaches from the spindles at late anaphase, whereas the phosphorylation-deficient Cin8-3A mutant protein remains attached to a larger region of the spindle and spindle poles for prolonged periods. This localization of Cin8-3A causes faster spindle elongation and longer anaphase spindles, which have aberrant morphology. By contrast, the phospho-mimic Cin8-3D mutant exhibits reduced binding to the spindles. In the absence of the Kinesin-5 homologue Kip1, cells expressing Cin8-3D exhibit spindle assembly defects and are not viable at 37°C as a result of spindle collapse. We propose that dephosphorylation of Cin8 promotes its binding to the spindle microtubules before the onset of anaphase. In mid to late anaphase, phosphorylation of Cin8 causes its detachment from the spindles, which reduces the spindle elongation rate and aids in maintaining spindle morphology.
Christina Thiede - One of the best experts on this subject based on the ideXlab platform.
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Deletion of the tail domain of the Kinesin-5 Cin8 affects its directionality
The Journal of biological chemistry, 2015Co-Authors: André Düselder, Vladimir Fridman, Larisa Gheber, Christina Thiede, Dieter R. Klopfenstein, Alice Wiesbaum, Alina Goldstein, Olga Zaitseva, Marcel E. Janson, Christoph F. SchmidtAbstract:Abstract 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.
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A chimeric Kinesin-1 head/Kinesin-5 tail motor switches between diffusive and processive motility.
Biophysical journal, 2013Co-Authors: Christina Thiede, Stefanie Kramer, Stefan Lakämper, Alok D. Wessel, Christoph F. SchmidtAbstract: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.
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a chimeric Kinesin 1 head Kinesin 5 tail motor switches between diffusive and processive motility
Biophysical Journal, 2013Co-Authors: Christina Thiede, Stefanie Kramer, Stefan Lakämper, Alok D. Wessel, Christoph F. SchmidtAbstract: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.
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Neck-linker length dependence of processive Kinesin-5 motility.
Journal of molecular biology, 2012Co-Authors: André Düselder, Christina Thiede, Stefanie Kramer, Christoph F. Schmidt, Stefan LakämperAbstract:Abstract Processive motility of individual molecules is essential for the function of many Kinesin motors. Processivity for Kinesins relies on communication between the two heads of a dimeric molecule, such that binding strictly alternates. The main communicating elements are believed to be the two neck linkers connecting the motors' stalks and heads. A proposed mechanism for coordination is the transmission of stress through the neck linkers. It is believed that the efficiency of gating depends on the length of the neck linker. Recent studies have presented support for a simple model in which the length of the neck linker directly controls the degree of processivity. Based on a previously published Kinesin-1/Kinesin-5 chimera, Eg5Kin, we have analyzed the motility of 12 motor constructs: we have varied the length of the neck linker in the range between 9 and 21 amino acids using the corresponding native Kinesin-5 sequence ( Xenopus laevis Eg5). We found, surprisingly, that neither velocity nor force generation depended on neck-linker length. We also found that constructs with short neck linkers, down to 12 amino acids, were still highly processive, while processivity was lost at a length of 9 amino acids. Run lengths were maximal with neck linkers close to the native Kinesin-5 length and decreased beyond that length. This finding generally confirms the coordinating role of the neck linker for Kinesin motility but challenges the simplest model postulating a motor-type‐independent optimal length. Instead, our results suggest that different Kinesins might be optimized for different neck-linker lengths.
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Regulation of bi-directional movement of single Kinesin-5 Cin8 molecules.
Bioarchitecture, 2012Co-Authors: Christina Thiede, Vladimir Fridman, Adina Gerson-gurwitz, Larisa Gheber, Christoph F. SchmidtAbstract:Kinesin-5 mechanoenzymes drive mitotic spindle dynamics as slow, processive microtubule (MT)-plus-end directed motors. Surprisingly, the Saccharomyces cerevisiae Kinesin-5 Cin8 was recently found to be bi-directional: it can move processively in both directions on MTs. Two hypotheses have been suggested for the mechanism of the directionality switch: (1) single molecules of Cin8 are intrinsically minus-end directed, but mechanical coupling between two or more motors triggers the switch; (2) a single motor can switch direction, and “cargo binding” i.e., binding between two MTs triggers the switch to plus-end motility. Single-molecule fluorescence data we published recently, and augment here, favor hypothesis (2). In low-ionic-strength conditions, single molecules of Cin8 move in both minus- and plus-end directions. Fluorescence photo bleaching data rule out aggregation of Cin8 while they move in the plus and in the minus direction. The evidence thus points toward cargo regulation of directionality, which i...
Adina Gerson-gurwitz - One of the best experts on this subject based on the ideXlab platform.
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Regulation of bi-directional movement of single Kinesin-5 Cin8 molecules.
Bioarchitecture, 2012Co-Authors: Christina Thiede, Vladimir Fridman, Adina Gerson-gurwitz, Larisa Gheber, Christoph F. SchmidtAbstract:Kinesin-5 mechanoenzymes drive mitotic spindle dynamics as slow, processive microtubule (MT)-plus-end directed motors. Surprisingly, the Saccharomyces cerevisiae Kinesin-5 Cin8 was recently found to be bi-directional: it can move processively in both directions on MTs. Two hypotheses have been suggested for the mechanism of the directionality switch: (1) single molecules of Cin8 are intrinsically minus-end directed, but mechanical coupling between two or more motors triggers the switch; (2) a single motor can switch direction, and “cargo binding” i.e., binding between two MTs triggers the switch to plus-end motility. Single-molecule fluorescence data we published recently, and augment here, favor hypothesis (2). In low-ionic-strength conditions, single molecules of Cin8 move in both minus- and plus-end directions. Fluorescence photo bleaching data rule out aggregation of Cin8 while they move in the plus and in the minus direction. The evidence thus points toward cargo regulation of directionality, which i...
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Control of Directionality of Individual Kinesin-5 Motors
Biophysical Journal, 2012Co-Authors: Adina Gerson-gurwitz, Natalia Movshovich, Vladimir Fridman, Christina Thiede, Christoph F. Schmidt, Stefan Lakämper, Maria Podolskaya, Dieter R. Klopfenstein, Larisa GheberAbstract:Kinesin-5 motors fulfill essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT)-plus-end directed motors. The Saccharomyces cerevisiae Kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here we have examined Cin8 directionality control using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly towards the midzone, in the plus-end direction of the interpolar MTs. Occasionally, Cin8 also moved faster towards the spindle poles, in the minus-end direction of the MTs. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel microtubules, while driving steady plus-end relative sliding. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic-strength dependent directional switching of Cin8 in vitro. In vivo, the deletion mutant exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.
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Phospho-regulation of Kinesin-5 during anaphase spindle elongation.
Journal of Cell Science, 2011Co-Authors: Rachel Avunie-masala, Natalia Movshovich, Vladimir Fridman, Adina Gerson-gurwitz, M. Andrew Hoyt, Yael Nissenkorn, Mardo Kõivomägi, Mart Loog, Arieh Zaritsky, Larisa GheberAbstract:The Kinesin-5 Saccharomyces cerevisiae homologue Cin8 is shown here to be differentially phosphorylated during late anaphase at Cdk1-specific sites located in its motor domain. Wild-type Cin8 binds to the early-anaphase spindles and detaches from the spindles at late anaphase, whereas the phosphorylation-deficient Cin8-3A mutant protein remains attached to a larger region of the spindle and spindle poles for prolonged periods. This localization of Cin8-3A causes faster spindle elongation and longer anaphase spindles, which have aberrant morphology. By contrast, the phospho-mimic Cin8-3D mutant exhibits reduced binding to the spindles. In the absence of the Kinesin-5 homologue Kip1, cells expressing Cin8-3D exhibit spindle assembly defects and are not viable at 37°C as a result of spindle collapse. We propose that dephosphorylation of Cin8 promotes its binding to the spindle microtubules before the onset of anaphase. In mid to late anaphase, phosphorylation of Cin8 causes its detachment from the spindles, which reduces the spindle elongation rate and aids in maintaining spindle morphology.
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Mitotic Functions and Motile Properties of the S. cerevisiae Kinesin-5 motors
Biophysical Journal, 2011Co-Authors: Vladimir Fridman, Adina Gerson-gurwitz, Christina Thiede, Christoph F. Schmidt, Stefan Lakämper, Maria Podolskaya, Movshovich Natalia, Larisa GheberAbstract:Mitotic chromosome segregation is mediated by mitotic spindle, a highly dynamic microtubule-based structure, which undergoes a distinct set of morphological changes in each mitotic cycle. Major factors that contribute to spindle morphogenesis are microtubule (MT) plus-end dynamics and function of molecular motors from the Kinesin-5 family. Kinesin-5 family members are conserved, homotetrameric motors with two catalytic domains located on opposite sides of the active complex. This special architecture enables these motors to crosslink and slide anti-parallel MTs originating from opposite spindle poles and thereby perform their essential functions in mitotic spindle morphogenesis. It was recently shown that Kinesin-5 motors affect anaphase spindle symmetry and midzone organization (1). S. cerevisiae cells express two Kinesin-5 homologues, Cin8p and Kip1p that overlap in function during spindle assembly, metaphase and anaphase B and at least one of them need to be expressed for viability. So far, the extent of redundancy between these two Kinesin-5 proteins and their motile properties in vitro have not been thoroughly investigated.In the present study, we use high temporal and spatial resolution imaging and FRAP to characterize interpolar MT (iMT) plus-end dynamics during spindle morphogenesis in S. cerevisiae cells expressing tubulin-GFP(2). This approach allowed us to study the role of the major midzone organizing protein Ase1(2) in controlling iMT plus-end dynamics and to compare between the effects of Cin8 and Kip1 on these dynamics during anaphase. In addition, in order to understand in vivo functions of the Kinesin-5 Kip1, we characterized its motile properties in single-molecule fluorescence motility assay. Results from this assay will be presented.(1) Movshovich N, et al. J Cell Sci. 2008; 121:2529.(2) Fridman V, et al. EMBO Rep. 2009; 10:387.∗ Supported by the Lower-Saxony collaboration grant between BGU and GAUG
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Slk19-dependent mid-anaphase pause in Kinesin-5-mutated cells.
Journal of Cell Science, 2008Co-Authors: Natalia Movshovich, Vladimir Fridman, Adina Gerson-gurwitz, Inbal Shumacher, Irena Gertsberg, Alexander Fich, M. Andrew Hoyt, Benjamin Katz, Larisa GheberAbstract:We examined spindle elongation in anaphase in Saccharomyces cerevisiae cells mutated for the Kinesin-5 motor proteins Cin8 and Kip1. Cells were deleted for KIP1 and/or expressed one of two motor-domain Cin8 mutants (Cin8-F467A or Cin8-R196K, which differ in their ability to bind microtubules in vitro, with Cin8-F467A having the weakest ability). We found that, in Kinesin-5-mutated cells, predominantly in kip1 Δ cin8-F467A cells, anaphase spindle elongation was frequently interrupted after the fast phase, resulting in a mid-anaphase pause. Expression of Kinesin-5 mutants also caused an asymmetric midzone location and enlarged midzone size, suggesting that proper organization of the midzone is required for continuous spindle elongation. We also examined the effects of components of the FEAR pathway, which is involved in the early-anaphase activation of Cdc14 regulatory phosphatase, on anaphase spindle elongation in kip1 Δ cin8-F467A cells. Deletion of SLK19 , but not SPO12 , eliminated the mid-anaphase pause, caused premature anaphase onset and defects in DNA division during anaphase, and reduced viability in these cells. Finally, overriding of the pre-anaphase checkpoint by overexpression of Cdc20 also eliminated the mid-anaphase pause and caused DNA deformation during anaphase in kip1 Δ cin8 - F467A cells. We propose that transient activation of the pre-anaphase checkpoint in Kinesin-5-mutated cells induces a Slk19-dependent mid-anaphase pause, which might be important for proper DNA segregation.
