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David Pellman - One of the best experts on this subject based on the ideXlab platform.
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multimodal tubulin binding by the yeast Kinesin 8 kip3 underlies its motility and depolymerization
bioRxiv, 2021Co-Authors: Hugo Arellanosantoyo, Ema Stokasimov, David Pellman, Rogelio A Hernandezlopez, Ray Yr Wang, Andres E LeschzinerAbstract:The microtubule (MT) cytoskeleton is central to cellular processes including axonal growth, intracellular transport, and cell division, all of which rely on precise spatiotemporal control of MT organization. Kinesin-8s play a key role in regulating MT length by combining highly processive directional motility with MT-end disassembly. However, how Kinesin-8 switches between these two apparently opposing activities remains unclear. Here, we define the structural features underlying this molecular switch through cryo-EM analysis of the yeast Kinesin-8, Kip3 bound to MTs, and molecular dynamics simulations to approximate the complex of Kip3 with the curved tubulin state found at the MT plus-end. By integrating biochemical and single-molecule biophysical assays, we identified specific intra- and intermolecular interactions that modulate processive motility and MT disassembly. Our findings suggest that Kip3 undergoes conformational changes in response to tubulin curvature that underlie its unique ability to interact differently with the MT lattice than with the MT-end.
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a tubulin binding switch underlies kip3 Kinesin 8 depolymerase activity
Developmental Cell, 2017Co-Authors: Hugo Arellanosantoyo, Elisabeth A Geyer, Ema Stokasimov, Gengyuan Chen, William O Hancock, Luke M Rice, David PellmanAbstract:Summary Kinesin-8 motors regulate the size of microtubule structures, using length-dependent accumulation at the plus end to preferentially disassemble long microtubules. Despite extensive study, the Kinesin-8 depolymerase mechanism remains under debate. Here, we provide evidence for an alternative, tubulin curvature-sensing model of microtubule depolymerization by the budding yeast Kinesin-8, Kip3. Kinesin-8/Kip3 uses ATP hydrolysis, like other Kinesins, for stepping on the microtubule lattice, but at the plus end Kip3 undergoes a switch: its ATPase activity is suppressed when it binds tightly to the curved conformation of tubulin. This prolongs plus-end binding, stabilizes protofilament curvature, and ultimately promotes microtubule disassembly. The tubulin curvature-sensing model is supported by our identification of Kip3 structural elements necessary and sufficient for plus-end binding and depolymerase activity, as well as by the identification of an α-tubulin residue specifically required for the Kip3-curved tubulin interaction. Together, these findings elucidate a major regulatory mechanism controlling the size of cellular microtubule structures.
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microtubule sliding activity of a Kinesin 8 promotes spindle assembly and spindle length control
Nature Cell Biology, 2013Co-Authors: Hugo Arellanosantoyo, David Pellman, Xiaolei Su, Didier Portran, Jeremie Gaillard, Marylin Vantard, Manuel TheryAbstract:Pellman and colleagues report that the Kip3 Kinesin-8 has antiparallel microtubule-sliding activity. The ability of Kip3 to slide antiparallel microtubules, along with its known role in destabilizing microtubules, are important for regulating spindle length.
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Microtubule-sliding activity of a Kinesin-8 promotes spindle assembly and spindle-length control.
Nature Cell Biology, 2013Co-Authors: Hugo Arellano-santoyo, Didier Portran, Jeremie Gaillard, Marylin Vantard, Manuel Thery, David PellmanAbstract:Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by crosslinking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report an antiparallel microtubule-sliding activity of the budding yeast Kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule-destabilizing activity. In conjunction with Cin8, a Kinesin-5 family member, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a slide-disassemble model where the sliding and destabilizing activity of Kip3 balance during pre-anaphase. This facilitates normal spindle assembly. However, the destabilizing activity of Kip3 dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.
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novel roles of Kinesin 8 in organizing mitotic spindles
Biophysical Journal, 2012Co-Authors: David PellmanAbstract:The Kinesin-8 family of microtubule motors plays a critical role in microtubule length control in cells. The budding yeast Kinesin-8 Kip3 is a microtubule plus end-specific depolymerase, which apparently destabilizes microtubules and mitotic spindles. We identified a secondary tubulin-binding domain on the C-terminal tail of Kip3. With the tail-binding to tubulin, Kip3 can transport tubulin dimers along microtubules. Kip3 can also slide apart anti-parallel microtubules whereas parallel microtubules display a tug-of-war behavior in the presence of Kip3. To investigate the physiological role of this newly found microtubule-crosslinking activity of Kip3, we made a tail-less mutant form of Kip3, which does not contain the crosslinking activity. We expressed this mutant at the depolymerase activity similar to the wild-type Kip3. We found spindles are fragile and prematurely broke during anaphase. On the other hand, cells expressing a Kip3 mutant that loses the depolymerase activity but maintains motility and the crosslinking-activity, have more stabilized spindles, compared to kip3-null cells. These data suggest that Kip3 has a tail-mediated stabilizing effect on mitotic spindles. Combining the previously found depolymerase activity, we propose a “slide, crosslink and chew” model to describe the roles of Kip3/Kinesin-8 in organizing mitotic spindles.
Erik Schaffer - One of the best experts on this subject based on the ideXlab platform.
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the Kinesin 8 kip3 depolymerizes microtubules with a collective force dependent mechanism
Biophysical Journal, 2020Co-Authors: Michael Bugiel, Erik Schaffer, Mayank Chugh, Tobias Jorg Jachowski, Anita JannaschAbstract:Abstract Microtubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes, in particular during cell division. Motor proteins from the Kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how Kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast Kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors’ residence time at the microtubule end. On the microtubule lattice, loads also exponentially decreased the run length and time. However, for the same load, lattice run times were significantly longer compared to end residence times, suggesting the presence of a distinct force-dependent detachment mechanism at the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism that unifies the so-called “bump-off” and “switching” models. Understanding the mechanics of Kinesin-8’s microtubule end activity will provide important insights into cell division with implications for cancer research.
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the Kinesin 8 kip3 depolymerizes microtubules with a collective force dependent mechanism
bioRxiv, 2019Co-Authors: Michael Bugiel, Erik Schaffer, Mayank Chugh, Tobias Jorg Jachowski, Anita JannaschAbstract:Microtubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes in particular, during cell division. Motor proteins from the Kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how Kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast Kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors9 residence time at the microtubule end. On the microtubule lattice, Kip3 had a different binding behavior suggesting that the observations are distinct for the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism. Understanding the mechanics of Kinesin-89s microtubule end activity will provide important insights into cell division with implications for cancer research.
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three dimensional optical tweezers tracking resolves random sideward steps of the Kinesin 8 kip3
Biophysical Journal, 2018Co-Authors: Michael Bugiel, Erik SchafferAbstract:The budding yeast Kinesin-8 Kip3 is a highly processive motor protein that walks to the ends of cytoskeletal microtubules and shortens them in a collective manner. However, how exactly Kip3 reaches the microtubule end is unclear. Although rotations of microtubules in multimotored Kip3 gliding assays implied directed sideward switching between microtubule protofilaments, two-dimensional, single-molecule, optical-tweezers assays indicated that Kip3 randomly switched protofilaments. Here, we topographically suspended microtubules such that Kip3 motors could freely access the microtubules in three dimensions. Tracking single-motor-driven microspheres with a three-dimensional, zero-load, optical-tweezers-based force clamp showed that Kip3 switched protofilaments in discrete steps equally frequent in both directions. A statistical analysis confirmed the diffusive sideward motion of Kip3, consistent with the two-dimensional single-molecule results. Furthermore, we found that motors were in one of three states: either not switching protofilaments or switching between them with a slow or fast sideward-stepping rate. Interestingly, this sideward diffusion was limited to one turn, suggesting that motors could not cross the microtubule seam. The diffusive protofilament switching may enable Kip3 to efficiently bypass obstacles and reach the microtubule end for length regulation.
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the Kinesin 8 kip3 switches protofilaments in a sideward random walk asymmetrically biased by force
Biophysical Journal, 2015Co-Authors: Michael Bugiel, Elisa Bohl, Erik SchafferAbstract:Molecular motors translocate along cytoskeletal filaments, as in the case of Kinesin motors on microtubules. Although conventional Kinesin-1 tracks a single microtubule protofilament, other Kinesins, akin to dyneins, switch protofilaments. However, the molecular trajectory—whether protofilament switching occurs in a directed or stochastic manner—is unclear. Here, we used high-resolution optical tweezers to track the path of single budding yeast Kinesin-8, Kip3, motor proteins. Under applied sideward loads, we found that individual motors stepped sideward in both directions, with and against loads, with a broad distribution in measured step sizes. Interestingly, the force response depended on the direction. Based on a statistical analysis and simulations accounting for the geometry, we propose a diffusive sideward stepping motion of Kip3 on the microtubule lattice, asymmetrically biased by force. This finding is consistent with previous multimotor gliding assays and sheds light on the molecular switching mechanism. For Kinesin-8, the diffusive switching mechanism may enable the motor to bypass obstacles and reach the microtubule end for length regulation. For other motors, such a mechanism may have implications for torque generation around the filament axis.
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Kinesin 8 is a low force motor protein with a weakly bound slip state
Biophysical Journal, 2013Co-Authors: Anita Jannasch, Volker Bormuth, Jonathon Howard, Marko Storch, Erik SchafferAbstract:During the cell cycle, Kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) Kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both Kinesin-8 motors were much weaker compared with other Kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with Kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity.
Jonathon Howard - One of the best experts on this subject based on the ideXlab platform.
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Kinesin 8 is a low force motor protein with a weakly bound slip state
Biophysical Journal, 2013Co-Authors: Anita Jannasch, Volker Bormuth, Jonathon Howard, Marko Storch, Erik SchafferAbstract:During the cell cycle, Kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) Kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both Kinesin-8 motors were much weaker compared with other Kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with Kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity.
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the highly processive Kinesin 8 kip3 switches microtubule protofilaments with a bias toward the left
Biophysical Journal, 2012Co-Authors: Volker Bormuth, Aniruddha Mitra, Felix Ruhnow, Bert Nitzsche, Marko Storch, Burkhard Rammner, Jonathon HowardAbstract:Kinesin-1 motor proteins walk parallel to the protofilament axes of microtubules as they step from one tubulin dimer to the next. Is protofilament tracking an inherent property of processive Kinesin motors, like Kinesin-1, and what are the structural determinants underlying protofilament tracking? To address these questions, we investigated the tracking properties of the processive Kinesin-8, Kip3. Using in vitro gliding motility assays, we found that Kip3 rotates microtubules counterclockwise around their longitudinal axes with periodicities of ∼1 μm. These rotations indicate that the motors switch protofilaments with a bias toward the left. Molecular modeling suggests 1), that the protofilament switching may be due to Kinesin-8 having a longer neck linker than Kinesin-1, and 2), that the leftward bias is due the asymmetric geometry of the motor neck linker complex.
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Kinesin 8 is a weak motor protein with a weakly bound slip state
Biophysical Journal, 2012Co-Authors: Anita Jannasch, Jonathon Howard, Marko Storch, Erik SchafferAbstract:Kinesin-8 is a highly processive plus-end directed motor protein that is conserved in eukaryotes from yeast to human. Different members of the Kinesin-8 family have been shown in vivo to control the length of microtubules by interacting with their plus ends. High processivity is crucial for these motors to reach the microtubule plus ends, where they induce catastrophes or otherwise interfere with microtubule dynamics. In this study, we characterized yeast (Kip3) and human (Kif18A) Kinesin-8s using optical tweezers. Both Kinesin-8 motors stalled under load forces of only ∼1 pN, much less than the stall forces measured for other Kinesins. Furthermore, we found that higher load forces caused the motor to slip backwards on the microtubule towards the minus end; the motor could recover from this state and resume plus-end motility. The low forces are consistent with Kinesin-8s being regulators of microtubule dynamics rather than cargo transporters; the slip state, in which the motor remains in a weakly bound state, may be an adaptation for high processivity.
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depolymerizing Kinesins kip3 and mcak shape cellular microtubule architecture by differential control of catastrophe
Cell, 2011Co-Authors: Melissa K Gardner, Volker Bormuth, Christopher Gell, Marija Zanic, Jonathon HowardAbstract:Microtubules are dynamic filaments whose ends alternate between periods of slow growth and rapid shortening as they explore intracellular space and move organelles. A key question is how regulatory proteins modulate catastrophe, the conversion from growth to shortening. To study this process, we reconstituted microtubule dynamics in the absence and presence of the Kinesin-8 Kip3 and the Kinesin-13 MCAK. Surprisingly, we found that, even in the absence of the Kinesins, the microtubule catastrophe frequency depends on the age of the microtubule, indicating that catastrophe is a multistep process. Kip3 slowed microtubule growth in a length-dependent manner and increased the rate of aging. In contrast, MCAK eliminated the aging process. Thus, both Kinesins are catastrophe factors; Kip3 mediates fine control of microtubule length by narrowing the distribution of maximum lengths prior to catastrophe, whereas MCAK promotes rapid restructuring of the microtubule cytoskeleton by making catastrophe a first-order random process.
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a non motor microtubule binding site is essential for the high processivity and mitotic function of Kinesin 8 kif18a
PLOS ONE, 2011Co-Authors: Monika I Mayr, Jonathon Howard, Marko Storch, Thomas U MayerAbstract:Background Members of the Kinesin-8 subfamily are plus end-directed molecular motors that accumulate at the plus-ends of kinetochore-microtubules (kt-MTs) where they regulate MT dynamics. Loss of vertebrate Kinesin-8 function induces hyperstable MTs and elongated mitotic spindles accompanied by severe chromosome congression defects. It has been reported that the motility of human Kinesin-8, Kif18A, is required for its accumulation at the plus tips of kt-MTs.
Stefan Diez - One of the best experts on this subject based on the ideXlab platform.
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kinetically distinct phases of tau on microtubules regulate Kinesin motors and severing enzymes
Nature Cell Biology, 2019Co-Authors: Valerie Siahaan, Amayra Hernandezvega, Stefan Diez, Jochen Krattenmacher, Zdenek Lansky, Anthony A Hyman, Marcus BraunAbstract:Tau is an intrinsically disordered protein, which diffuses on microtubules1. In neurodegenerative diseases, collectively termed tauopathies, malfunction of tau and its detachment from axonal microtubules are correlated with axonal degeneration2. Tau can protect microtubules from microtubule-degrading enzymes such as katanin3. However, how tau carries out this regulatory function is still unclear. Here, using in vitro reconstitution, we show that tau molecules on microtubules cooperatively form cohesive islands that are kinetically distinct from tau molecules that individually diffuse on microtubules. Dependent on the tau concentration in solution, the islands reversibly grow or shrink by addition or release of tau molecules at their boundaries. Shielding microtubules from Kinesin-1 motors and katanin, the islands exhibit regulatory qualities distinct from a comparably dense layer of diffusible tau. Superprocessive Kinesin-8 motors penetrate the islands and cause their disassembly. Our results reveal a microtubule-dependent phase of tau that constitutes an adaptable protective layer on the microtubule surface. We anticipate that other intrinsically disordered axonal proteins display a similar cooperative behaviour and potentially compete with tau in regulating access to the microtubule surface.
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kinetically distinct phases of tau on microtubules regulate Kinesin motors and severing enzymes
bioRxiv, 2018Co-Authors: Valerie Siahaan, Amayra Hernandezvega, Stefan Diez, Jochen Krattenmacher, Zdenek Lansky, Anthony A Hyman, Marcus BraunAbstract:Tau is an intrinsically disordered protein, which diffuses on microtubules. In neurodegenerative diseases collectively termed tauopathies, tau malfunction and its detachment from axonal microtubules is correlated with microtubule degradation. It is known that tau can protect microtubules from microtubule-degrading enzymes, such as katanin. However, how tau can fulfill such regulative function is still unclear. Using in vitro reconstitution, we here show that tau molecules on microtubules cooperatively form islands of an ordered layer with regulatory qualities distinct from a comparably dense layer of diffusible tau. These islands shield the microtubules from katanin and Kinesin-1 but are penetrable by Kinesin-8 which causes the islands to disassemble. Our results indicate a new phase of tau, constituting an adjustable protective sheath around microtubules.
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directionally biased sidestepping of kip3 Kinesin 8 is regulated by atp waiting time and motor microtubule interaction strength
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Aniruddha Mitra, Stefan Diez, Felix Ruhnow, Salvatore GirardoAbstract:Kinesin-8 motors, which move in a highly processive manner toward microtubule plus ends where they act as depolymerases, are essential regulators of microtubule dynamics in cells. To understand their navigation strategy on the microtubule lattice, we studied the 3D motion of single yeast Kinesin-8 motors, Kip3, on freely suspended microtubules in vitro. We observed short-pitch, left-handed helical trajectories indicating that Kinesin-8 motors frequently switch protofilaments in a directionally biased manner. Intriguingly, sidestepping was not directly coupled to forward stepping but rather depended on the average dwell time per forward step under limiting ATP concentrations. Based on our experimental findings and numerical simulations we propose that effective sidestepping toward the left is regulated by a bifurcation in the Kip3 step cycle, involving a transition from a two-head–bound to a one-head–bound conformation in the ATP-waiting state. Results from a Kinesin-1 mutant with extended neck linker hint toward a generic sidestepping mechanism for processive Kinesins, facilitating the circumvention of intracellular obstacles on the microtubule surface.
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impact free measurement of microtubule rotations on Kinesin and cytoplasmic dynein coated surfaces
PLOS ONE, 2015Co-Authors: Aniruddha Mitra, Felix Ruhnow, Bert Nitzsche, Stefan DiezAbstract:Knowledge about the three-dimensional stepping of motor proteins on the surface of microtubules (MTs) as well as the torsional components in their power strokes can be inferred from longitudinal MT rotations in gliding motility assays. In previous studies, optical detection of these rotations relied on the tracking of rather large optical probes present on the outer MT surface. However, these probes may act as obstacles for motor stepping and may prevent the unhindered rotation of the gliding MTs. To overcome these limitations, we devised a novel, impact-free method to detect MT rotations based on fluorescent speckles within the MT structure in combination with fluorescence-interference contrast microscopy. We (i) confirmed the rotational pitches of MTs gliding on surfaces coated by Kinesin-1 and Kinesin-8 motors, (ii) demonstrated the superiority of our method over previous approaches on Kinesin-8 coated surfaces at low ATP concentration, and (iii) identified MT rotations driven by mammalian cytoplasmic dynein, indicating that during collective motion cytoplasmic dynein side-steps with a bias in one direction. Our novel method is easy to implement on any state-of-the-art fluorescence microscope and allows for high-throughput experiments.
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Kinesin-8 Motors Act Cooperatively to Mediate Length-Dependent Microtubule Depolymerization
Cell, 2009Co-Authors: Vladimir Varga, Stefan Diez, Volker Bormuth, Cécile Leduc, Jonathon HowardAbstract:Summary Motor proteins in the Kinesin-8 family depolymerize microtubules in a length-dependent manner that may be crucial for controlling the length of organelles such as the mitotic spindle. We used single-molecule microscopy to understand the mechanism of length-dependent depolymerization by the budding yeast Kinesin-8, Kip3p. We found that after binding at a random position on a microtubule and walking to the plus end, an individual Kip3p molecule pauses there until an incoming Kip3p molecule bumps it off. Kip3p dissociation is accompanied by removal of just one or two tubulin dimers (on average). Such a cooperative mechanism leads to a depolymerization rate that is proportional to the flux of motors to the microtubule end and accounts for the length dependence of depolymerization. This type of feedback between length and disassembly may serve as a model for understanding how an ensemble of molecules can measure and control polymer length.
Gohta Goshima - One of the best experts on this subject based on the ideXlab platform.
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Kinesin-13 and Kinesin-8 Function during Cell Growth and Division in the Moss Physcomitrella patens.
The Plant Cell, 2020Co-Authors: Shu Yao Leong, Tomoya Edzuka, Gohta Goshima, Moé YamadaAbstract:Kinesin-13 and Kinesin-8 are well-known microtubule (MT) depolymerases that regulate MT length and chromosome movement in animal mitosis. While much is unknown about plant Kinesin-8, Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) Kinesin-13 have been shown to depolymerize MTs in vitro. However, the mitotic function of both Kinesins has yet to be determined in plants. Here, we generated complete null mutants of Kinesin-13 and Kinesin-8 in moss (Physcomitrella patens). Both Kinesins were found to be nonessential for viability, but the Kinesin-13 knockout (KO) line had increased mitotic duration and reduced spindle length, whereas the Kinesin-8 KO line did not display obvious mitotic defects. Surprisingly, spindle MT poleward flux, which is mediated by Kinesin-13 in animals, was retained in the absence of Kinesin-13. MT depolymerase activity was not detectable for either Kinesin in vitro, while MT catastrophe-inducing activity (Kinesin-13) or MT gliding activity (Kinesin-8) was observed. Interestingly, both KO lines showed waviness in their protonema filaments, which correlated with positional instability of the MT foci in their tip cells. Taken together, the results suggest that plant Kinesin-13 and Kinesin-8 have diverged in both mitotic function and molecular activity, acquiring roles in regulating MT foci positioning for directed tip growth.
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Novel roles of Kinesin-13 and Kinesin-8 during cell growth and division in the moss Physcomitrella patens
2019Co-Authors: Shu Yao Leong, Tomoya Edzuka, Gohta Goshima, Moé YamadaAbstract:Abstract Kinesin-13 and -8 are well-known microtubule (MT) depolymerases that regulate MT length and chromosome movement in animal mitosis. While much is unknown about plant Kinesin-8, Arabidopsis and rice Kinesin-13 have been shown to depolymerise MTs in vitro. However, mitotic function of both Kinesins has yet to be understood in plants. Here, we generated the complete null mutants in plants of Kinesin-13 and -8 in the moss Physcomitrella patens. Both Kinesins were found to be non-essential for viability, but the Kinesin-13 knockout (KO) line had increased mitotic duration and reduced spindle length, whereas the Kinesin-8 KO line did not display obvious mitotic defects. Surprisingly, spindle MT poleward flux, for which Kinesin-13 is responsible for in animals, was retained in the absence of Kinesin-13. Concurrently, MT depolymerase activity of either moss Kinesins could not be observed, with MT catastrophe inducing (Kinesin-13) or MT gliding (Kinesin-8) activity observed in vitro. Interestingly, both KO lines showed waviness in their protonema filaments, which correlated with positional instability of the MT foci in their tip cells. Taken together, the results suggest that plant Kinesin-13 and -8 have diverged in both mitotic function and molecular activity, acquiring new roles in regulating MT foci positioning for directed tip-growth. One sentence summary This study uncovered the roles of Kinesin-13 and Kinesin-8 in regulating microtubule dynamics for mitotic spindle formation and straight tip cell growth in the moss Physcomitrella patens
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drosophila Kinesin 8 stabilizes the kinetochore microtubule interaction
Journal of Cell Biology, 2019Co-Authors: Tomoya Edzuka, Gohta GoshimaAbstract:Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, it is unclear how this motor protein family controls chromosome alignment, as multiple biochemical activities, including inconsistent ones between studies, have been identified. Here, we find that Drosophila Kinesin-8 (Klp67A) possesses both microtubule (MT) plus end–stabilizing and –destabilizing activity, in addition to Kinesin-89s commonly observed MT plus end–directed motility and tubulin-binding activity in vitro. We further show that Klp67A is required for stable kinetochore–MT attachment during prometaphase in S2 cells. In the absence of Klp67A, abnormally long MTs interact in an “end-on” fashion with kinetochores at normal frequency. However, the interaction is unstable, and MTs frequently become detached. This phenotype is rescued by ectopic expression of the MT plus end–stabilizing factor CLASP, but not by artificial shortening of MTs. We show that human Kinesin-8 (KIF18A) is also important to ensure proper MT attachment. Overall, these results suggest that the MT-stabilizing activity of Kinesin-8 is critical for stable kinetochore–MT attachment.
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drosophila Kinesin 8 stabilises kinetochore microtubule interaction
bioRxiv, 2018Co-Authors: Tomoya Edzuka, Gohta GoshimaAbstract:Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, how this conserved motor protein controls chromosome alignment remains unclear, as multiple biochemical activities, including inconsistent ones between studies, have been identified for this motor family. Here, we show that Drosophila Kinesin-8 Klp67A possesses both microtubule (MT) plus-end-stabilising and -destabilising activities in addition to commonly observed MT plus-end-directed motility and tubulin-binding activity in vitro, and is required for stable kinetochore-MT attachment during prometaphase in vivo. In the absence of Kinesin-8/Klp67A, abnormally-long MTs interact in an "end-on" fashion with kinetochores at normal frequency. However, the interaction was not stable and, once-attached, MTs were frequently detached. This phenotype was rescued by ectopic expression of MT plus-end-stabilising factor CLASP, but not by artificial shortening of MTs. These results suggest that MT-stabilising activity of Kinesin-8/Klp67A is critical for stable kinetochore-MT attachment.
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length control of the metaphase spindle
Current Biology, 2005Co-Authors: Gohta Goshima, Ronald D Vale, Roy Wollman, Nico Stuurman, Jonathan M ScholeyAbstract:BACKGROUND: The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. RESULTS: Using the Drosophila S2 cell line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. CONCLUSIONS: By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.
