Kinesin-binding protein remodels the kinesin motor to prevent microtubule binding.
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ABSTRACT:
SUBMITTER: Solon AL
PROVIDER: S-EPMC8604404 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Neuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1-4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites.
| S-EPMC7063570 | biostudies-literature
Project description: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.
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Project description:Kinesin-12 (also called Kif15) is a mitotic motor protein that continues to be expressed in developing neurons. Depletion of kinesin-12 causes axons to grow faster, more than doubles the frequency of microtubule transport in both directions in the axon, prevents growth cones from turning properly, and enhances the invasion of microtubules into filopodia. These results are remarkably similar to those obtained in previous studies in which neurons were depleted of kinesin-5 (also called Eg5 or Kif11), another mitotic motor protein that continues to be expressed in developing neurons. However, there are also notable differences in the phenotypes obtained with depleting each of these motors. Depleting kinesin-12 decreases axonal branching and growth cone size, whereas inhibiting kinesin-5 increases these parameters. In addition, depleting kinesin-12 diminishes the appearance of growth-cone-like waves along the length of the axon, an effect not observed with depletion of kinesin-5. Finally, depletion of kinesin-12 abolishes the "waggling" behavior of microtubules that occurs as they assemble along actin bundles within filopodia, whereas inhibition of kinesin-5 does not. Interestingly, and perhaps relevant to these differences in phenotype, in biochemical studies, kinesin-12 coimmunoprecipitates with actin but kinesin-5 does not. Collectively, these findings support a scenario whereby kinesin-12 shares functions with kinesin-5 related to microtubule-microtubule interactions, but kinesin-12 has other functions not shared by kinesin-5 that are related to the ability of kinesin-12 to interact with actin.
| S-EPMC3064264 | biostudies-literature
Project description:Kinesin motors and their associated filaments, microtubules, are essential to many biological processes. The motor and filament system can be reconstituted in vitro with the surface-adhered motors transporting the filaments along the surface. In this format, the system has been used to study active self-assembly and to power microdevices or perform analyte detection. However, fundamental properties of the system, such as the spacing of the kinesin motors bound to the microtubule and the dynamics of binding, remain poorly understood. We show that Fluorescence Interference Contrast (FLIC) microscopy can illuminate the exact height of the microtubule, which for a sufficiently low surface density of kinesin, reveals the locations of the bound motors. We examine the spacing of the kinesin motors on the microtubules at various kinesin surface densities and compare the results with theory. FLIC reveals that the system is highly dynamic, with kinesin binding and unbinding along the length of the microtubule as it is transported along the surface.
| S-EPMC6510761 | biostudies-literature
Project description:Kinesin-1 is a major microtubule motor that drives transport of numerous cellular cargoes toward the plus ends of microtubules. In the cell, kinesin-1 exists primarily in an inactive, autoinhibited state, and motor activation is thought to occur upon binding to cargo through the C terminus. Using RNAi-mediated depletion in Drosophila S2 cells, we demonstrate that kinesin-1 requires ensconsin (MAP7, E-MAP-115), a ubiquitous microtubule-associated protein, for its primary function of organelle transport. We show that ensconsin is required for organelle transport in Drosophila neurons and that Drosophila homozygous for ensconsin gene deletion are unable to survive to adulthood. An ensconsin N-terminal truncation that cannot bind microtubules is sufficient to activate organelle transport by kinesin-1, indicating that this activating domain functions independently of microtubule binding. Interestingly, ens mutant flies retaining expression of this truncation show normal viability. A "hingeless" mutant of kinesin-1, which mimics the active conformation of the motor, does not require ensconsin for transport in S2 cells, suggesting that ensconsin plays a role in relieving autoinhibition of kinesin-1. Together with other recent work, our study suggests that ensconsin is an essential cofactor for all known functions of kinesin-1.
| S-EPMC3580027 | biostudies-literature
Project description:The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail.
| S-EPMC2832967 | biostudies-literature
Project description:Bidirectional vesicle transport along microtubules is necessary for cell viability and function, particularly in neurons. When multiple motors are attached to a vesicle, the distance a vesicle travels before dissociating is determined by the race between detachment of the bound motors and attachment of the unbound motors. Motor detachment rate constants (k off) can be measured via single-molecule experiments, but motor reattachment rate constants (k on) are generally unknown, as they involve diffusion through the bilayer, geometrical considerations of the motor tether length, and the intrinsic microtubule binding rate of the motor. To understand the attachment dynamics of motors bound to fluid lipid bilayers, we quantified the microtubule accumulation rate of fluorescently labeled kinesin-1 motors in a 2-dimensional (2D) system where motors were linked to a supported lipid bilayer. From the first-order accumulation rate at varying motor densities, we extrapolated a k off that matched single-molecule measurements and measured a 2D k on for membrane-bound kinesin-1 motors binding to the microtubule. This k on is consistent with kinesin-1 being able to reach roughly 20 tubulin subunits when attaching to a microtubule. By incorporating cholesterol to reduce membrane diffusivity, we demonstrate that this k on is not limited by the motor diffusion rate, but instead is determined by the intrinsic motor binding rate. For intracellular vesicle trafficking, this 2D k on predicts that long-range transport of 100-nm-diameter vesicles requires 35 kinesin-1 motors, suggesting that teamwork between different motor classes and motor clustering may play significant roles in long-range vesicle transport.
| S-EPMC6936695 | biostudies-literature
Project description: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.
| S-EPMC4198420 | biostudies-literature
Project description:Kinesin is a motor protein essential in cellular functions, such as intracellular transport and cell-division, as well as for enabling nanoscopic transport in bio-nanotechnology. Therefore, for effective control of function for nanotechnological applications, it is important to be able to modify the function of kinesin. To circumvent the limitations of chemical modifications, here we identify another potential approach for kinesin control: the use of electric forces. Using full-atom molecular dynamics simulations (247,358 atoms, total time ∼ 4.4 μs), we demonstrate, for the first time, that the kinesin-1 motor domain can be detached from a microtubule by an intense electric field within the nanosecond timescale. We show that this effect is field-direction dependent and field-strength dependent. A detailed analysis of the electric forces and the work carried out by electric field acting on the microtubule-kinesin system shows that it is the combined action of the electric field pulling on the β-tubulin C-terminus and the electric-field-induced torque on the kinesin dipole moment that causes kinesin detachment from the microtubule. It is shown, for the first time in a mechanistic manner, that an electric field can dramatically affect molecular interactions in a heterologous functional protein assembly. Our results contribute to understanding of electromagnetic field-biomatter interactions on a molecular level, with potential biomedical and bio-nanotechnological applications for harnessing control of protein nanomotors.
| S-EPMC9939557 | biostudies-literature
Project description:An expanding collection of proteins localises to microtubule ends to regulate cytoskeletal dynamics and architecture by unknown molecular mechanisms. Electron microscopy is invaluable for studying microtubule structure, but because microtubule ends are heterogeneous, their structures are difficult to determine. We therefore investigated whether tubulin oligomers induced by the drug dolastatin could mimic microtubule ends. The microtubule end-dependent ATPase of kinesin-13 motors is coupled to microtubule depolymerisation. Significantly, kinesin-13 motor ATPase activity is stimulated by dolastatin-tubulin oligomers, suggesting, first, that these oligomers share properties with microtubule ends and, second, that the physical presence of an end is less important than terminal tubulin flexibility for microtubule end recognition by the kinesin-13 motor. Using electron microscopy, we visualised the kinesin-13 motor-dolastatin-tubulin oligomer interaction in nucleotide states mimicking steps in the ATPase cycle. This enabled us to detect conformational changes that the motor undergoes during depolymerisation. Our data suggest that such tubulin oligomers can be used to examine other microtubule end-binding proteins.
| S-EPMC2396873 | biostudies-literature