Anastral spindle assembly and γ-tubulin in Drosophila oocytes

<p>Abstract</p> <p>Background</p> <p>Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether γ-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for γ-tubulin to form anastral <it>Drosophila </it>oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain.</p> <p>Results</p> <p>We show, for the first time, using a bright GFP fusion protein and live imaging, that the <it>Drosophila </it>maternally-expressed γTub37C is present at low levels in oocyte meiosis I spindles. Despite this, we find that formation of bipolar meiosis I spindles does not require functional γTub37C, extending previous findings by others. Fluorescence photobleaching assays show rapid recovery of γTub37C in the meiosis I spindle, similar to the cytoplasm, indicating weak binding by γTub37C to spindles, and fits of a new, potentially more accurate model for fluorescence recovery yield kinetic parameters consistent with transient, diffusional binding.</p> <p>Conclusions</p> <p>The FRAP results, together with its mutant effects late in meiosis I, indicate that γTub37C may perform a role subsequent to metaphase I, rather than nucleating microtubules for meiosis I spindle formation. Weak binding to the meiosis I spindle could stabilize pre-existing microtubules or position γ-tubulin for function during meiosis II spindle assembly, which follows rapidly upon oocyte activation and completion of the meiosis I division.</p

Chromosome Congression by Kinesin-5 Motor-Mediated Disassembly of Longer Kinetochore Microtubules

During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus-ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely-conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus-end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression

Self-organization of Microtubule and Associated Protein Map65

One of the most fascinating micron-scale structures in the biological world is the cell cytoskele- ton, with numerous components of different sizes, shapes, and geometries working together. How the local interactions of these macromolecules and polymers in nano-scale help self- organize into this higher-order micron-scale structure remains an open question in the field of biophysics. In this thesis, via a minimal two-protein in vitro system containing cytoskeletal fiber microtubule and associated protein MAP65, I try to address self-organization through passive entropic force mechanisms. I use techniques like protein purification, light microscopy, and image analysis to quantify my results. First, I show how crowded environments contribute to microtubule nucleation, polymerization, and bundling via experiments and simulations. Next, I discuss a self-assembled limited-size microtubule bundle that is reminiscent of the mitotic spindle. I characterize these \u27tactoids\u27 and show that these are homogeneous and jammed inside. Lastly, I present results of the MAP65 condensate formation via liquid-liquid phase separation and quantification of their properties. Additionally, I elucidate a mechanism of non-centrosomal microtubule nucleation that may play an essential role in the mitotic spindle