We have used a small angle scattering system assembled on the high flux multipole wiggler beam line at CHESS (Cornell) to make very accurate spacing measurements of certain meridional and layer-line reflections from contracting muscles. During isometric contraction, the actin 27.3 A reflection increases in spacing from its resting value by approximately 0.3%, and other actin reflections, including the 59 and 51 A off-meridional reflections, show corresponding changes in spacing. When tension is augmented or diminished by applying moderate speed length changes to a contracting muscle, changes in spacing in the range of 0.19-0.24% (when scaled to full isometric tension) can be seen. The larger difference between the resting and isometric spacings suggests either nonlinearity at low tension levels or the presence of a component related to activation itself. Myosin filaments also show similar increases in axial period during slow stretch, in addition to the well known larger change associated with activation. An actin spacing change of 0.25-0.3% can also be measured during a 2 ms time frame immediately after a quick release, showing that the elastic behavior is rapid. These observations of filament extensions totaling 2-3 nm per half-sarcomere may necessitate some significant revision of the interpretation of a number of mechanical experiments in muscle, in which it has usually been assumed that virtually all of the elasticity resides in the cross-bridges.
Kinesin motors convert chemical energy from ATP hydrolysis into unidirectional movement. To understand how kinesin motors bind to and move along microtubules, we fit the atomic structure of the motor domain of Ncd (a kinesin motor involved in meiosis and mitosis) into three-dimensional density maps of Ncd-microtubule complexes calculated by cryo-electron microscopy and image analysis. The model reveals that Ncd shares an extensive interaction surface with the microtubule, and that a portion of the binding site involves loops that contain conserved residues. In the Ncd dimer, the microtubule-bound motor domain makes intimate contact with its partner head, which is dissociated from the microtubule. This head-head interaction may be important in positioning the dissociated head to take a step to the next binding site on the microtubule protofilament.
The methods of single-molecule fluorescence spectroscopy and microscopy have been recently utilized to explore the mechanism of action of several members of the kinesin and myosin biomolecular motor protein families. Whereas ensemble averaging is removed in single-molecule studies, heterogeneity in the behavior of individual motors can be directly observed, without synchronization. Observation of translocation by individual copies of motor proteins allows analysis of step size, rate, pausing, and other statistical properties of the process. Polarization microscopy as a function of nucleotide state has been particularly useful in revealing new and highly rotationally mobile forms of particular motors. These experiments complement X-ray and biochemical studies and provide a detailed view into the local dynamical behavior of motor proteins.
Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila Katanin, Dm-Kat60, functions to generate a dynamic cortical-microtubule interface in interphase cells. In S2 cells, Dm-Kat60 concentrates at the interphase cell cortex where it suppresses the polymerization of microtubule plus-ends thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes to the leading edge migratory D17 cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes MTs from their ends. Based on these data, we propose that Dm-Kat60 removes tubulin from microtubule ends or lattice that contact specific cortical sites to preventing stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in MT behaviors involved in cell migration.
To elucidate the structural basis of the mechanism of microtubule depolymerization by kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10A by electron microscopy (EM) and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.
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