Cytokinesis, the division of a cell into two daughter cells, serves as an elegant cell behavior that highlights the biomechanical systems required for many cell shape change processes. Over the life of this grant, we have demonstrated how an interplay of active force production, cortical tension, surface curvature, and viscoelasticity drive cytokinesis furrow ingression. We identified key molecular pathways that control these properties and found that the circuitry is wired like a control system complete with feedback loops that allows mechanical and chemical signals to tune the accumulation of the contractile machinery. Finally, we have applied these concepts to other systems, such as myoblast fusion, entosis, hepatocyte mechanics, pancreatic cancer, and lung biology, demonstrating the power of using a model organism (Dictyostelium) and a model process (cytokinesis) as a concept generator for more complex systems. In this proposal, we continue to build upon our understanding of cytokinesis and the mechanosensitive contractile network by developing a biochemically grounded map of the protein interactions that constitute this network (Aim 1) and discern the functional roles of five unusual suspects in cell shape control (Aim 2).
In Aim 1, we will map the biochemical interactions revealed through a combination of proteomics and genetic analyses using several state-of-the-art methodologies. We will use fluorescence cross-correlation spectroscopy to measure cellular concentrations, complex sizes (reflected in diffusion coefficients), and the strengths of the biochemical interactions (`in vivo Kd'). Using Single Molecule Pulldown, we will measure the stoichiometry of each component. Finally, using a combination of Structured Illumination Microscopy, Lattice Light Sheet Microscopy, and confocal imaging, we will develop a more complete picture of the sub-cellular distribution and architecture of the system. These studies will then give us a physical biochemical interaction map of the contractile network.
In Aim 2, we will pursue functional studies of five unexpected proteins implicated in the mechanosensory contractile system that have been revealed through two or more proteomics and/or genetics strategies. These proteins include adenine nucleotide translocase (AncA), methylmalonate semialdehyde dehydrogenase (Mmsdh), two ribonucleotide proteins (RNP1A and RNP1B), and discoidin complex. Each protein offers a unique entry point into deciphering new mechanisms of cell shape control. AncA provides an in-road into the interface between cell mechanics and metabolism. Mmsdh suggests a possible mode of regulation of contractility through propionylation. The RNP1s are predicted to be intrinsically disordered, but interact genetically and biochemically with one of the main nodes of the contractility network. Finally, the discoidin complex is a lectin, which may provide part of the anchoring complex that links the contractile network to the plasma membrane. Overall, the studies will yield a quantitative wiring diagram and provide novel insights into the mechanisms of cytokinesis and the mechanosensory contractile system that governs cell shape change more generally.
Proper cell shape control is essential for the proliferation of cells, normal development and maintenance of a healthy organism. We study how cytokinesis, the physical process in which one cell splits into two, works at a fundamental level in order to understand how cells control cell shape more generally. Ultimately, with a rigorous understanding and a complete molecular handle on these processes, it should be possible to develop better cancer therapies that are tailored to the properties of specific types of cancer cells.
| Evans, Janice P; Robinson, Douglas N (2018) Micropipette Aspiration of Oocytes to Assess Cortical Tension. Methods Mol Biol 1818:163-171 |
| Kothari, Priyanka; Srivastava, Vasudha; Aggarwal, Vasudha et al. (2018) Contractility kits promote assembly of the mechanoresponsive cytoskeletal network. J Cell Sci : |
| Liu, Yinan; Robinson, Douglas (2018) Recent advances in cytokinesis: understanding the molecular underpinnings. F1000Res 7: |
| Duan, Rui; Kim, Ji Hoon; Shilagardi, Khurts et al. (2018) Spectrin is a mechanoresponsive protein shaping fusogenic synapse architecture during myoblast fusion. Nat Cell Biol 20:688-698 |
| West-Foyle, Hoku; Kothari, Priyanka; Osborne, Jonathan et al. (2018) 14-3-3 proteins tune non-muscle myosin II assembly. J Biol Chem 293:6751-6761 |
| Kothari, P; Schiffhauer, E S; Robinson, D N (2017) Cytokinesis from nanometers to micrometers and microseconds to minutes. Methods Cell Biol 137:307-322 |
| Schiffhauer, Eric S; Robinson, Douglas N (2017) Mechanochemical Signaling Directs Cell-Shape Change. Biophys J 112:207-214 |
| Hamann, Jens C; Surcel, Alexandra; Chen, Ruoyao et al. (2017) Entosis Is Induced by Glucose Starvation. Cell Rep 20:201-210 |
| Nishida, Kristine; Brune, Kieran A; Putcha, Nirupama et al. (2017) Cigarette smoke disrupts monolayer integrity by altering epithelial cell-cell adhesion and cortical tension. Am J Physiol Lung Cell Mol Physiol 313:L581-L591 |
| Thomas, Dustin G; Robinson, Douglas N (2017) The fifth sense: Mechanosensory regulation of alpha-actinin-4 and its relevance for cancer metastasis. Semin Cell Dev Biol 71:68-74 |
Showing the most recent 10 out of 56 publications
