Mechanics of Mitotic Spindle Poles and Polymerization Dynamics of Microtubules.

Optical Tweezers;Mitosis;Mitotic Spindle;Cytoskeleton;Microtubule Polymerization Dynamics;Kinesin-5;Biomedical Engineering;Engineering;Biomedical Engineering

Charlebois, Blake D.Verhey, Kristen J. ;

University of Michigan

关键词: Optical Tweezers;    Mitosis;    Mitotic Spindle;    Cytoskeleton;    Microtubule Polymerization Dynamics;    Kinesin-5;    Biomedical Engineering;    Engineering;    Biomedical Engineering;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/89627/charleb_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

During cell division, chromosomes must faithfully segregate to maintain genome integrity,and this dynamic mechanical process is driven by the macromolecular machineryof the mitotic spindle. However, little is known about spindle mechanics.For example, spindle microtubules are organized by numerous cross-linking proteinsyet the mechanical properties of those cross-links remain unexplored. To examinethe mechanical properties of microtubule cross-links we applied optical trapping tomitotic asters that form in mammalian mitotic extracts. These asters are foci ofmicrotubules, motors, and microtubule-associated proteins that reflect many of thefunctional properties of spindle poles and represent centrosome-independent spindlepoleanalogs. We observed bidirectional motor-driven microtubule movements, showingthat microtubule linkages within asters are remarkably compliant (mean stiffness0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motorEg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical propertiesof microtubule asters in a manner consistent with its localization to spindle polesin cells. We propose that compliant linkages among microtubules provide a mechanicalarchitecture capable of accommodating microtubule movements and distributingforce among microtubules without loss of pole integrity—a mechanical paradigm thatmay be important throughout the spindle.Microtubule assembly and disassembly are vital for many fundamental cellularprocesses. Our current understanding of microtubule assembly kinetics is based ona one-dimensional assembly model, which assumes identical energetics for subunitsexchanging at the tip. In this model, the subunit disassociation rate from a microtubuletip is independent of free subunit concentration. Using total-internal-reflectionfluorescence (TIRF) microscopy and an optical tweezers assay to measure in vitro microtubuleassembly with nanometer resolution, we find that the subunit dissociationrate from a microtubule tip increases at higher free subunit concentrations. Thisis because, as predicted by Hill, there is a shift in microtubule tip structure fromrelatively blunt at low free subunit concentrations to relatively tapered at high concentrations,which we confirmed experimentally by TIRF microscopy. Because boththe association and the dissociation rates increase with free subunit concentrations,we find that the kinetics of microtubule assembly are an order of magnitude fasterthan currently estimated in the literature.