This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The objective of this project is to acquire 3D structural information about the organization of the actin-myosin-II network in the cleavage furrow cortex of Dictyostelium cells at different stages f furrow ingression. In our work, we have been developing a quantitative framework for cytokinesis in order to understand the molecular mechanisms that govern the dynamics of cell shape change. Myosin-II and actin are central to this process. Many textbook models of cytokinesis invoke a circumferential belt of actin filaments with the myosin-II thick filaments arranged so that the cleavage furrow is constricted like a purse string. While this organization is likely to be correct for some organisms, several lines of evidence challenge this as a general framework. First, myosin-II mutant Dictyostelium cells undergo mitotic cell division nearly as fast as wild-type cells. Second, Dictyostelium myosin-II is kinetically tuned and assembled into thick filaments in such a way as to make it difficult to conceptualize a simple sarcomeric-like contraction mechanism; some mammalian nonmuscle myosin-IIs are similarly tuned. Third, in mammalian and Dictyostelium, cells, a clear ring of actin filaments is not clearly detectable. High resolution structural information would allow us to considerably refine our current models for cytokinesis in Dictyostelium. The initial goal of this project is to determine the orientation of actin filaments and myosin-II thick filaments at various stages of furrow ingression. We have prepared cells by rapidly plunge freezing formvar-coated gold EM grids with attached cells and are imaging these for tomographic reconstruction both as whole mounts in the frozen-hydrated state and as plastic sections after freeze-substitution.
| Giddings Jr, Thomas H; Morphew, Mary K; McIntosh, J Richard (2017) Preparing Fission Yeast for Electron Microscopy. Cold Spring Harb Protoc 2017: |
| Zhao, Xiaowei; Schwartz, Cindi L; Pierson, Jason et al. (2017) Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium ""Candidatus Pelagibacter ubique"". Appl Environ Microbiol 83: |
| Brown, Joanna R; Schwartz, Cindi L; Heumann, John M et al. (2016) A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc. J Struct Biol 194:38-48 |
| Saheki, Yasunori; Bian, Xin; Schauder, Curtis M et al. (2016) Control of plasma membrane lipid homeostasis by the extended synaptotagmins. Nat Cell Biol 18:504-15 |
| Höög, Johanna L; Lacomble, Sylvain; Bouchet-Marquis, Cedric et al. (2016) 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction. PLoS Negl Trop Dis 10:e0004312 |
| Park, J Genevieve; Palmer, Amy E (2015) Properties and use of genetically encoded FRET sensors for cytosolic and organellar Ca2+ measurements. Cold Spring Harb Protoc 2015:pdb.top066043 |
| McCoy, Kelsey M; Tubman, Emily S; Claas, Allison et al. (2015) Physical limits on kinesin-5-mediated chromosome congression in the smallest mitotic spindles. Mol Biol Cell 26:3999-4014 |
| Höög, Johanna L; Lötvall, Jan (2015) Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy. J Extracell Vesicles 4:28680 |
| Marc, Robert E; Anderson, James R; Jones, Bryan W et al. (2014) The AII amacrine cell connectome: a dense network hub. Front Neural Circuits 8:104 |
| Weber, Britta; Tranfield, Erin M; Höög, Johanna L et al. (2014) Automated stitching of microtubule centerlines across serial electron tomograms. PLoS One 9:e113222 |
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