Self-repair protects microtubules from destruction by molecular motors.
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ABSTRACT:
SUBMITTER: Triclin S
PROVIDER: S-EPMC7611741 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Despite the crowdedness of the interior of cells, microtubule-based motor proteins are able to deliver cargoes rapidly and reliably throughout the cytoplasm. We hypothesize that motor proteins may be adapted to operate in crowded environments by having molecular properties that prevent them from forming traffic jams. To test this hypothesis, we reconstituted high-density traffic of purified kinesin-8 motor protein, a highly processive motor with long end-residency time, along microtubules in a total internal-reflection fluorescence microscopy assay. We found that traffic jams, characterized by an abrupt increase in the density of motors with an associated abrupt decrease in motor speed, form even in the absence of other obstructing proteins. To determine the molecular properties that lead to jamming, we altered the concentration of motors, their processivity, and their rate of dissociation from microtubule ends. Traffic jams occurred when the motor density exceeded a critical value (density-induced jams) or when motor dissociation from the microtubule ends was so slow that it resulted in a pileup (bottleneck-induced jams). Through comparison of our experimental results with theoretical models and stochastic simulations, we characterized in detail under which conditions density- and bottleneck-induced traffic jams form or do not form. Our results indicate that transport kinesins, such as kinesin-1, may be evolutionarily adapted to avoid the formation of traffic jams by moving only with moderate processivity and dissociating rapidly from microtubule ends.
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Project description:Mixtures of filaments and molecular motors form active materials with diverse dynamical behaviors that vary based on their constituents' molecular properties. To develop a multiscale of these materials, we map the nonequilibrium phase diagram of microtubules and tip-accumulating kinesin-4 molecular motors. We find that kinesin-4 can drive either global contractions or turbulentlike extensile dynamics, depending on the concentrations of both microtubules and a bundling agent. We also observe a range of spatially heterogeneous nonequilibrium phases, including finite-sized radial asters, 1D wormlike chains, extended 2D bilayers, and system-spanning 3D active foams. Finally, we describe intricate kinetic pathways that yield microphase separated structures and arise from the inherent frustration between the orientational order of filamentous microtubules and the positional order of tip-accumulating molecular motors. Our work reveals a range of novel active states. It also shows that the form of active stresses is not solely dictated by the properties of individual motors and filaments, but is also contingent on the constituent concentrations and spatial arrangement of motors on the filaments.
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Project description:Microtubules--which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport--can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses.
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Project description:Mammalian centrosomes and cilia play key roles in many cellular processes and their deregulation is linked to cancer and ciliopathies. Spatiotemporal regulation of their biogenesis and function in response to physiological stimuli requires timely protein targeting. This can occur by different pathways, including microtubule-dependent active transport and via centriolar satellites, which are key regulators of cilia assembly and signaling. How satellites mediate their functions and their relationship with other targeting pathways is currently unclear. To address this, we studied retinal degeneration gene product CCDC66, which localizes to centrosomes, cilia, satellites and microtubules and functions in ciliogenesis. FRAP experiments showed that its centrosomal pool was dynamic and the ciliary pool associated with the ciliary axoneme and was stable. Centrosomal CCDC66 abundance and dynamics required microtubule-dependent active transport and tethering, and was inhibited by sequestration at satellites. Systematic quantitation of satellite dynamics identified only a small fraction to display microtubule-based bimodal motility, consistent with trafficking function. Majority displayed diffusive motility with unimodal persistence, supporting sequestration function. Together, our findings reveal new mechanisms of communication between membrane-less compartments.
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Project description:During metaphase, sister chromatids are positioned at the midpoint of the microtubule-based mitotic spindle in preparation for their segregation. The onset of anaphase triggers inactivation of the key mitotic kinase cyclin-dependent kinase 1 (CDK1) and the polewards movement of sister chromatids. During anaphase, the mitotic spindle reorganizes in preparation for cytokinesis. Kinesin motor proteins and microtubule-associated proteins bundle the plus ends of interpolar microtubules and generate the central spindle, which regulates cleavage furrow initiation and the completion of cytokinesis. Complementary approaches, including cell biology, genetics and computational modelling, have provided new insights into the mechanism and regulation of central spindle assembly.
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Project description:Actin-based cellular protrusions are an ubiquitous feature of cells, performing a variety of critical functions ranging from cell-cell communication to cell motility. The formation and maintenance of these protrusions relies on the transport of proteins via myosin motors, to the protrusion tip. While tip-directed motion leads to accumulation of motors (and their molecular cargo) at the protrusion tip, it is observed that motors also form rearward moving, periodic and isolated aggregates. The origins and mechanisms of these aggregates, and whether they are important for the recycling of motors, remain open puzzles. Motivated by novel myosin-XV experiments, a mass conserving reaction-diffusion-advection model is proposed. The model incorporates a non-linear cooperative interaction between motors, which converts them between an active and an inactive state. Specifically, the type of aggregate formed (traveling waves or pulse-trains) is linked to the kinetics of motors at the protrusion tip which is introduced by a boundary condition. These pattern selection mechanisms are found not only to qualitatively agree with empirical observations but open new vistas to the transport phenomena by molecular motors in general.
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Project description:Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 ?m/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 ?M of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 ?M ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 ?m/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 ?M vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.
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Project description:BackgroundAt the beginning of mitosis, the cell forms a spindle made of microtubules and associated proteins to segregate chromosomes. An important part of spindle architecture is a set of antiparallel microtubule bundles connecting the spindle poles. A key question is how microtubules extending at arbitrary angles form an antiparallel interpolar bundle.ResultsHere, we show in fission yeast that microtubules meet at an oblique angle and subsequently rotate into antiparallel alignment. Our live-cell imaging approach provides a direct observation of interpolar bundle formation. By combining experiments with theory, we show that microtubules from each pole search for those from the opposite pole by performing random angular movement. Upon contact, two microtubules slide sideways along each other in a directed manner towards the antiparallel configuration. We introduce the contour length of microtubules as a measure of activity of motors that drive microtubule sliding, which we used together with observation of Cut7/kinesin-5 motors and our theory to reveal the minus-end-directed motility of this motor in vivo.ConclusionRandom rotational motion helps microtubules from the opposite poles to find each other and subsequent accumulation of motors allows them to generate forces that drive interpolar bundle formation.
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Project description:The enormous diversity of antibody specificities is generated by random rearrangement of immunoglobulin gene segments and is important for general protection against pathogens. Since random rearrangement harbors the risk of producing self-destructive antibodies, it is assumed that autoreactive antibody specificities are removed during early B-cell development leading to a peripheral compartment devoid of autoreactivity. Here, we immunized wild-type mice with insulin as a common self-antigen and monitored diabetes symptoms as a measure for autoimmune disease. Our results show that autoreactive anti-insulin IgM and IgG antibodies associated with autoimmune diabetes can readily be generated in wild-type animals. Surprisingly, recall immunizations induced increased titers of high-affinity insulin-specific IgM, which prevented autoimmune diabetes. We refer to this phenomenon as adaptive tolerance, in which high-affinity memory IgM prevents autoimmune destruction by competing with self-destructive antibodies. Together, this study suggests that B-cell tolerance is not defined by the absolute elimination of autoreactive specificities, as harmful autoantibody responses can be generated in wild-type animals. In contrast, inducible generation of autoantigen-specific affinity-matured IgM acts as a protective mechanism preventing self-destruction.
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Project description:Molecular motors are Nature's solution for (supra)molecular transport and muscle functioning and are involved in most forms of directional motion at the cellular level. Their synthetic counterparts have also found a myriad of applications, ranging from molecular machines and smart materials to catalysis and anion transport. Although light-driven rotary molecular motors are likely to be suitable for use in an artificial cell, as well as in bionanotechnology, thus far they are not readily applied under physiological conditions. This results mainly from their inherently aromatic core structure, which makes them insoluble in aqueous solution. Here, the study of the dynamic behavior of these motors in biologically relevant media is described. Two molecular motors were equipped with solubilizing substituents and studied in aqueous solutions. Additionally, the behavior of a previously reported molecular motor was studied in micelles, as a model system for the biologically relevant confined environment. Design principles were established for molecular motors in these media, and insights are given into pH-dependent behavior. The work presented herein may provide a basis for the application of the remarkable properties of molecular motors in more advanced biohybrid systems.
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