Dynamics of myosin, microtubules, and Kinesin-6 at the cortex during cytokinesis in Drosophila S2 cells

© The Authors, 2009 . This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of Cell Biology 186 (2009): 727-738, doi:10.1083/jcb.200902083.Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein–tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.This work was supported by a National Institutes of Health grant NIH 38499 to R.D. Vale

Saving Face: Inclusive Communication With College Students With Disabilities Using Politeness And Face Negotiation

Have I offended anyone today? Have I been insensitive? Creating welcoming inclusive environments for students with and without disabilities is a higher education imperative. The academy strives to create diverse and welcoming atmospheres for students and educators and employ social justice and face saving measures to encourage respectful communication and discourage discriminatory behaviors. With the increase of college students with disabilities, professionals need to be comfortable and confident in their communication tactics. Applying politeness and face-negotiation theories to the communication preferences and behaviors of college students with disabilities, this article offers practice-oriented applications for respectful inclusive communication

Effects of super−shear rupture speed on the high frequency content of S−waves investigated using spontaneous dynamic rupture models and isochrone theory

This paper achieves three goals: 1) It demonstrates that crack tips governed by friction laws including slip–weakening, rate–and state–dependent laws, and thermal pressurization of pore fluids, propagating at super–shear speed have slip velocity functions with reduced high frequency content compared to crack tips traveling at sub–shear speeds. This is demonstrated using a fully dynamic, spontaneous, 3–D earthquake model, in which we calculate fault slip velocity at nine points (locations) distributed along a quarter–circle on the fault where the rupture is traveling at super–shear speed in the in–plane direction and sub–shear speed in the anti–plane direction. This holds for a fault governed by the linear slip–weakening constitutive equation, by slip–weakening with thermal pressurization of pore fluid and by rate– and state–dependent laws with thermal pressurization. The same is also true even assuming a highly heterogeneous initial shear stress field on the fault. 2) Using isochrone theory we derive a general expressions for the spectral characteristics and geometric spreading of two pulses arising from super–shear rupture, the well–known Mach wave, and a second lesser known pulse caused by rupture acceleration. 3) The paper demonstrates that the Mach cone amplification of high frequencies overwhelms the deamplification of high frequency content in the slip velocity functions in super–shear ruptures. Consequently, when earthquake ruptures travel at super–shear speed, a net enhancement of high frequency radiation is expected, and the alleged “low” peak accelerations observed for the 2002 Denali and other large earthquakes are probably not caused by diminished high frequency content in the slip velocity function, as has been speculated

A global search inversion for earthquake kinematic rupture history: Application to the 2000 western Tottori, Japan earthquake

We present a two-stage nonlinear technique to invert strong motions records and geodetic data to retrieve the rupture history of an earthquake on a finite fault. To account for the actual rupture complexity, the fault parameters are spatially variable peak slip velocity, slip direction, rupture time and risetime. The unknown parameters are given at the nodes of the subfaults, whereas the parameters within a subfault are allowed to vary through a bilinear interpolation of the nodal values. The forward modeling is performed with a discrete wave number technique, whose Green’s functions include the complete response of the vertically varying Earth structure. During the first stage, an algorithm based on the heat-bath simulated annealing generates an ensemble of models that efficiently sample the good data-fitting regions of parameter space. In the second stage (appraisal), the algorithm performs a statistical analysis of the model ensemble and computes a weighted mean model and its standard deviation. This technique, rather than simply looking at the best model, extracts the most stable features of the earthquake rupture that are consistent with the data and gives an estimate of the variability of each model parameter. We present some synthetic tests to show the effectiveness of the method and its robustness to uncertainty of the adopted crustal model. Finally, we apply this inverse technique to the well recorded 2000 western Tottori, Japan, earthquake (Mw 6.6); we confirm that the rupture process is characterized by large slip (3-4 m) at very shallow depths but, differently from previous studies, we imaged a new slip patch (2-2.5 m) located deeper, between 14 and 18 km depth

Crystal structure of the Anabaena sensory rhodopsin transducer.

We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured beta-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal alpha-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the alpha-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K(d) of 8 microM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible alpha-face to mediate transduction of the light signal are discussed

Single molecule biochemistry using optical tweezers

AbstractThe use of optical trapping to create extremely compliant mechanical probes has ushered in a new field of biological inquiry, the mechanical and kinetic study of proteins at the single molecule level. This review focuses on three examples of such study and includes methods of extracting parameters of interest from the raw data such experiments generate

One must reconstitute the functions of interest from purified proteins

I am often asked by students and younger colleagues and now by the editors of this issue to tell the history of the development of the in vitro motility assay and the dual-beam single-molecule laser trap assay for myosin-driven actin filament movement, used widely as key assays for understanding how both muscle and nonmuscle myosin molecular motors work. As for all discoveries, the history of the development of the myosin assays involves many people who are not authors of the final publications, but without whom the assays would not have been developed as they are. Also, early experiences shape how one develops ideas and experiments, and influence future discoveries in major ways. I am pleased here to trace my own path and acknowledge the many individuals involved and my early science experiences that led to the work I and my students, postdoctoral fellows, and sabbatical visitors did to develop these assays. Mentors are too often overlooked in historical descriptions of discoveries, and my story starts with those who mentored me

Nonlinear Relaxation Dynamics in Elastic Networks and Design Principles of Molecular Machines

Analyzing nonlinear conformational relaxation dynamics in elastic networks corresponding to two classical motor proteins, we find that they respond by well-defined internal mechanical motions to various initial deformations and that these motions are robust against external perturbations. We show that this behavior is not characteristic for random elastic networks. However, special network architectures with such properties can be designed by evolutionary optimization methods. Using them, an example of an artificial elastic network, operating as a cyclic machine powered by ligand binding, is constructed.Comment: 12 pages, 9 figure

Internal Motility in Stiffening Actin-Myosin Networks

We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and suggest a "zipping" mechanism to explain the filament motion in the vicinity of the sol-gel transition.Comment: 4 pages, 3 figure

Quantitation of the distribution and flux of myosin-II during cytokinesis

BACKGROUND: During cytokinesis, the cell's equator contracts against the cell's global stiffness. Identifying the biochemical basis for these mechanical parameters is essential for understanding how cells divide. To achieve this goal, the distribution and flux of the cell division machinery must be quantified. Here we report the first quantitative analysis of the distribution and flux of myosin-II, an essential element of the contractile ring. RESULTS: The fluxes of myosin-II in the furrow cortex, the polar cortex, and the cytoplasm were examined using ratio imaging of GFP fusion proteins expressed in Dictyostelium. The peak concentration of GFP-myosin-II in the furrow cortex is 1.8-fold higher than in the polar cortex and 2.0-fold higher than in the cytoplasm. The myosin-II in the furrow cortex, however, represents only 10% of the total cellular myosin-II. An estimate of the minimal amount of this motor needed to produce the required force for cell cleavage fits well with this 10% value. The cell may, therefore, regulate the amount of myosin-II sent to the furrow cortex in accordance with the amount needed there. Quantitation of the distribution and flux of a mutant myosin-II that is defective in phosphorylation-dependent thick filament disassembly confirms that heavy chain phosphorylation regulates normal recruitment to the furrow cortex. CONCLUSION: The analysis indicates that myosin-II flux through the cleavage furrow cortex is regulated by thick filament phosphorylation. Further, the amount of myosin-II observed in the furrow cortex is in close agreement with the amount predicted to be required from a simple theoretical analysis