Cytokinesis, the separation of a mother cell into two daughters, is an essential life process. Cytokinesis success is critical to the health and fidelity of single cells, multi-cellular development, and disease prevention. In this proposal, we build upon our framework for deciphering the molecular underpinnings of cytokinesis mechanics and mechanosensing. Using Dictyostelium, in Aim 1, we study the Myosin II-Cortexillin I-IQGAP2- Kinesin-6 pathway (the equatorial mechanosensitive pathway). This network of proteins is structured like a mechanochemical feedback system that integrates signals from the mitotic spindle and mechanical stress to tune the myosin II levels at the cleavage furrow. Using fluorescence recovery after photobleaching, we will analyze the dynamics of key proteins with and without applied mechanical stress and in wild type and selected mutants. From this, we will decipher how proteins depend on each other for mechanosensitive accumulation. We will use pull-downs followed by LC-MS to identify interacting proteins. The list of interacting proteins will then be compared to the lists of genetic interacting proteins we have already identified. Preliminary data identify important enzymes involved in post-translational modifications (PTMs), such as propionylation and acetylation. Acetylation of myosin II and other proteins has been implicated in mammalian mitosis and in cardiac contractile system function. Thus, we are interested to see if these PTMs contribute to the mechanosensory feedback system and cytokinesis cell shape change. We will also use purified proteins to determine how IQGAPs modulate cortexillin and possibly myosin II function.
In Aim 2, we will expand the Microtubule-RacE-14-3-3-Myosin II pathway, which we discovered. This pathway controls the global/polar cortex mechanics, cortical tension and cytokinesis shape change. We will draw upon genetic and biochemical methods to identify interactors of racE. We will also determine the mechanism by which 14-3-3 controls myosin II bipolar thick filament assembly. We will then determine how human 14-3-3 proteins modulate human myosin II thick filament assembly. Preliminary data points toward the conserved nature of 14-3-3-myosin II interactions and a possible similar mechanism shared between 14-3-3 and another cancer-related protein S100A4/Mts1. Overall, proposed work in this renewal application strives to develop a sophisticated understanding of the force transmission that promotes and regulates cell shape change and the pathways that control cortical tension, myosin II dynamics, and cytokinesis.

Public Health Relevance

Successful cell division is essential for the proliferation of cells, normal development and maintenance of a healthy organism. We are striving to understand how cytokinesis, the physical process in which one cell splits into two, works at a fundamental level and how chemical and mechanical signal transduction drive cell division. Ultimately, with a rigorous understanding and a complete molecular handle on the process, it should be possible to develop better cancer therapies that are tailored to the properties of specific types of cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM066817-10A1
Application #
8628296
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Gindhart, Joseph G
Project Start
2003-08-01
Project End
2017-11-30
Budget Start
2014-01-01
Budget End
2014-11-30
Support Year
10
Fiscal Year
2014
Total Cost
$327,071
Indirect Cost
$119,118
Name
Johns Hopkins University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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
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
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
Miao, Chunguang; Schiffhauer, Eric S; Okeke, Evelyn I et al. (2017) Parallel Compression Is a Fast Low-Cost Assay for the High-Throughput Screening of Mechanosensory Cytoskeletal Proteins in Cells. ACS Appl Mater Interfaces 9:28168-28179
Hamann, Jens C; Surcel, Alexandra; Chen, Ruoyao et al. (2017) Entosis Is Induced by Glucose Starvation. Cell Rep 20:201-210
Bai, Haibo; Zhu, Qingfeng; Surcel, Alexandra et al. (2016) Yes-associated protein impacts adherens junction assembly through regulating actin cytoskeleton organization. Am J Physiol Gastrointest Liver Physiol 311:G396-411
Srivastava, Vasudha; Iglesias, Pablo A; Robinson, Douglas N (2016) Cytokinesis: Robust cell shape regulation. Semin Cell Dev Biol 53:39-44
Ibo, Markela; Srivastava, Vasudha; Robinson, Douglas N et al. (2016) Cell Blebbing in Confined Microfluidic Environments. PLoS One 11:e0163866
Schiffhauer, Eric S; Luo, Tianzhi; Mohan, Krithika et al. (2016) Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton. Curr Biol 26:1473-1479

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