Effect of Kinesin-5 Tail Domain on Motor Dynamics for Antiparallel Microtubule Sliding.
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ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC8345995 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.
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Project description:Biopolymers serve as one-dimensional tracks on which motor proteins move to perform their biological roles. Motor protein phenomena have inspired theoretical models of one-dimensional transport, crowding, and jamming. Experiments studying the motion of Xklp1 motors on reconstituted antiparallel microtubule overlaps demonstrated that motors recruited to the overlap walk toward the plus end of individual microtubules and frequently switch between filaments. We study a model of this system that couples the totally asymmetric simple exclusion process for motor motion with switches between antiparallel filaments and binding kinetics. We determine steady-state motor density profiles for fixed-length overlaps using exact and approximate solutions of the continuum differential equations and compare to kinetic Monte Carlo simulations. Overlap motor density profiles and motor trajectories resemble experimental measurements. The phase diagram of the model is similar to the single-filament case for low switching rate, while for high switching rate we find a new (to our knowledge) low density-high density-low density-high density phase. The overlap center region, far from the overlap ends, has a constant motor density as one would naïvely expect. However, rather than following a simple binding equilibrium, the center motor density depends on total overlap length, motor speed, and motor switching rate. The size of the crowded boundary layer near the overlap ends is also dependent on the overlap length and switching rate in addition to the motor speed and bulk concentration. The antiparallel microtubule overlap geometry may offer a previously unrecognized mechanism for biological regulation of protein concentration and consequent activity.
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Project description:Microtubule-crosslinking motor proteins, which slide antiparallel microtubules, are required for the remodeling of microtubule networks. Hitherto, all microtubule-crosslinking motors have been shown to slide microtubules at a constant velocity until no overlap remains between them, leading to the breakdown of the initial microtubule geometry. Here, we show in vitro that the sliding velocity of microtubules, driven by human kinesin-14 HSET, decreases when microtubules start to slide apart, resulting in the maintenance of finite-length microtubule overlaps. We quantitatively explain this feedback using the local interaction kinetics of HSET with overlapping microtubules that cause retention of HSET in shortening overlaps. Consequently, the increased HSET density in the overlaps leads to a density-dependent decrease in sliding velocity and the generation of an entropic force that antagonizes the force exerted by the motors. Our results demonstrate that a spatial arrangement of microtubules can regulate the collective action of molecular motors through the local alteration of their individual interaction kinetics.
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Project description:Recent experimental studies of the role of microtubule sliding in neurite outgrowth suggested a qualitative model, according to which kinesin-1 motors push the minus-end-out microtubules against the cell membrane and generate the early cell processes. At the later stage, dynein takes over the sliding, expels the minus-end-out microtubules from the neurites, and pulls in the plus-end-out microtubules that continue to elongate the nascent axon. This model leaves unanswered a number of questions: why is dynein unable to generate the processes alone, whereas kinesin-1 can? What is the role of microtubule dynamics in process initiation and growth? Can the model correctly predict the rates of process growth in control and dynein-inhibited cases? What triggers the transition from kinesin-driven to dynein-driven sliding? To answer these questions, we combine computational modeling of a network of elastic dynamic microtubules and kinesin-1 and dynein motors with measurements of the process growth kinetics and pharmacological perturbations in Drosophila S2 cells. The results verify quantitatively the qualitative model of the microtubule polarity sorting and suggest that dynein-powered elongation is effective only when the processes are longer than a threshold length, which explains why kinesin-1 alone, but not dynein, is sufficient for the process growth. Furthermore, we show that the mechanism of process elongation depends critically on microtubule dynamic instability. Both modeling and experimental measurements show, surprisingly, that dynein inhibition accelerates the process extension. We discuss implications of the model for the general problems of cell polarization, cytoskeletal polarity emergence, and cell process protrusion.
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Project description:Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, we constructed a minimal kinesin-5 tetramer (mini-tetramer). We determined the x-ray structure of the extended, 34-nm BASS domain. Guided by these structural studies, we generated active bipolar kinesin-5 mini-tetramer motors from Drosophila melanogastor and human orthologues which are half the length of native kinesin-5. We then used these kinesin-5 mini-tetramers to examine the role of two unique structural adaptations of kinesin-5: 1) the length and flexibility of the tetramer, and 2) the C-terminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, we find that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.
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Project description:Kinesins-13s are members of the kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other kinesins, kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct kinesin-13 functionality, we used ensemble and single-molecule fluorescence polarization microscopy to analyze the behavior and conformation of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to the MT lattice. We found that KLP10A interacts with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs we conclude that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile kinesins.
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Project description:The formation and maintenance of the mitotic spindle during cell division requires several microtubule-interacting motor proteins. Members of the kinesin-5 family play an essential role in the bipolar organization of the spindle. These highly conserved, homotetrameric proteins cross-link anti-parallel microtubules and slide them apart to elongate the spindle during the equal separation of chromosomes. Whereas vertebrate kinesin-5 proteins are well studied, knowledge about the biochemical properties and the function of plant kinesin-5 proteins is still limited. Here, we characterized the properties of AtKRP125b, one of four kinesin-5 proteins in Arabidopsis thaliana. In in vitro motility assays, AtKRP125b displayed the archetypal characteristics of a kinesin-5 protein, a low velocity of about 20 nm·s-1, and a plus end-directed, processive movement. Moreover, AtKRP125b was able to cross-link microtubules and to slide them apart, as required for developing and maintaining the mitotic spindle. In line with such a function, GFP-AtKRP125b fusion proteins were predominantly detected in the nucleus when expressed in Arabidopsis thaliana leaf protoplasts or Nicotiana benthamiana epidermis cells and analyzed by confocal microscopy. However, we also detected GFP signals in the cytoplasm, suggesting additional functions. By generating and analyzing AtKRP125b promoter-reporter lines, we showed that the AtKRP125b promoter was active in the vascular tissue of roots, lateral roots, cotyledons, and true leaves. Remarkably, we could not detect promoter activity in meristematic tissues. Taken together, our biochemical data support a role of AtKRP125b in mitosis, but it may also have additional functions outside the nucleus and during interphase.
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Project description:Motor and non-motor crosslinking proteins play critical roles in determining the size and stability of microtubule-based architectures. Currently, we have a limited understanding of how geometrical properties of microtubule arrays, in turn, regulate the output of crosslinking proteins. Here we investigate this problem in the context of microtubule sliding by two interacting proteins: the non-motor crosslinker PRC1 and the kinesin Kif4A. The collective activity of PRC1 and Kif4A also results in their accumulation at microtubule plus-ends ('end-tag'). Sliding stalls when the end-tags on antiparallel microtubules collide, forming a stable overlap. Interestingly, we find that structural properties of the initial array regulate microtubule organization by PRC1-Kif4A. First, sliding velocity scales with initial microtubule-overlap length. Second, the width of the final overlap scales with microtubule lengths. Our analyses reveal how micron-scale geometrical features of antiparallel microtubules can regulate the activity of nanometer-sized proteins to define the structure and mechanics of microtubule-based architectures.
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