Cytokinesis, the separation of a mother cell into two daughters, is an essential life process. Cytokinesis failure leads to tetraploidy then aneuploidy, an early event in tumor formation. We have been striving to understand how cells use proteins to generate the relevant cellular physical properties that drive cytokinesis contractility. We are also interested in developing small molecule inhibitors to aid in gene function identification and pathway dissection, but with the ultimate goal that some of these small molecule inhibitors will have clinical applications. In this proposal, we will build on the analytical framework that we initiated in the first cycle of the grant, but we will also expand our effort in several ways.
In Aim 1, we will measure the lifetimes of myosin-II and various actin crosslinkers in different mutant backgrounds where the mechanics are known in order to assess the consequences of mechanical strain on crosslinker lifetimes. We will test our understanding of the molecular control of cytokinesis mechanics and dynamics by measuring cortical mechanics of dividing mutant strains where we have specific predictions of the mechanics. We will also begin studying lower hierarchical levels of cytoskeletal function by reconstituting crosslinked actin networks, applying mechanical strain to them, and studying the behavior of the crosslinkers and the network. We will use either FRAP or single particle analysis to assess the strain dependency of crosslinker lifetimes;we have several predictions based on our in vivo studies.
In Aim 2, we will draw upon our observations that RacE is responsible for generating resistive stresses during cytokinesis and for restricting the mechanosensory system that we discovered to cytokinesis. In this Aim, we will identify RacE effectors to flesh out this pathway. We will study 14-3-3, which was identified as a suppressor of nocodazole. Genetic interactions between RacE and 14-3-3 further point towards a pathway in which microtubules regulate RacE and/or 14-3-3, which in turn regulate global actin crosslinkers to control the dynamics and mechanics of cytokinesis contractility.
In Aim 3, we will expand our molecular inquiry by identifying the affected genes in the REMI mutants we have already recovered.

Public Health Relevance

Of great importance for normal cell growth and disease processes such as cancer, cytokinesis has the promise of providing a rich source of new anti-cancer drug targets. We are striving to understand how cytokinesis works at a fundamental level and how the cell uses proteins to generate the physical features of contractility. 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 #
5R01GM066817-09
Application #
8269834
Study Section
Cell Structure and Function (CSF)
Program Officer
Gindhart, Joseph G
Project Start
2003-08-01
Project End
2013-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
9
Fiscal Year
2012
Total Cost
$329,510
Indirect Cost
$128,589
Name
Johns Hopkins University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Schiffhauer, Eric S; Luo, Tianzhi; Mohan, Krithika et al. (2016) Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton. Curr Biol 26:1473-9
Srivastava, Vasudha; Iglesias, Pablo A; Robinson, Douglas N (2016) Cytokinesis: Robust cell shape regulation. Semin Cell Dev Biol 53:39-44
Ngo, Thu; Miao, Xin; Robinson, Douglas N et al. (2016) An RNA-binding protein, RNP-1, protects microtubules from nocodazole and localizes to the leading edge during cytokinesis and cell migration in Dictyostelium cells. Acta Pharmacol Sin 37:1449-1457
Mackenzie, Amelia C L; Kyle, Diane D; McGinnis, Lauren A et al. (2016) Cortical mechanics and myosin-II abnormalities associated with post-ovulatory aging: implications for functional defects in aged eggs. Mol Hum Reprod 22:397-409
Surcel, Alexandra; Ng, Win Pin; West-Foyle, Hoku et al. (2015) Pharmacological activation of myosin II paralogs to correct cell mechanics defects. Proc Natl Acad Sci U S A 112:1428-33
Luo, Tianzhi; Robinson, Douglas N (2015) Kinetic Monte Carlo simulations of the assembly of filamentous biomacromolecules by dimer addition mechanism. RSC Adv 5:3922-3929
Kim, Ji Hoon; Ren, Yixin; Ng, Win Pin et al. (2015) Mechanical tension drives cell membrane fusion. Dev Cell 32:561-73
Mohan, Krithika; Luo, Tianzhi; Robinson, Douglas N et al. (2015) Cell shape regulation through mechanosensory feedback control. J R Soc Interface 12:20150512

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