To investigate how actin and microtubules function in vivo, we are using the Natural Killer (NK) lymphoid cell as a model cell system. This system offers physiological relevance to human biology and disease, ease of use, and a multiplicity of cytoskeleton-based functions downstream of diverse signaling pathways. In previous studies, we discovered that the cortactin homologue HS1 is essential for many actin-based functions and signaling pathways. We found that Arp2/3 is necessary for assembly of the actin-based lytic synapse and that the formin Dia1 appears to capture microtubule plus ends at the lytic synapse, which is required for the polarized secretion of lytic granules onto target cells.
Aim 1. Functions of the Multi-domain Cortactin Homologue HS1. a. Distinct functions of two Src-phosphorylated tyrosines. We discovered that Tyr 378 is necessary for NK cell chemotaxis, while Tyr 397 is necessary for target cell killing. We will search for specific ligands of these individual phosphotyrosines, investigate the functional roles of the ligands and test the significance of their interactions with HS1. b. Functions of HS1 domains. HS1 has multiple domains with different interaction partners. We will study the functions of these domains and the physiological relevance of their molecular interactions.
Aim 2. Microtubule Plus-end Capture, MTOC Polarization and Lytic Granule Movement. a. Microtubule capture by the lytic synapse. The formin Dia1 appears to be necessary for the capture of dynamic microtubule plus ends at the lytic synapse, acting in concert with APC and EB1. We will investigate the molecular mechanisms that underlie microtubule capture, using a combination of molecular genetics and cell biology approaches. b. Positioning of the MTOC. We hypothesize that the MTOC moves to a forward position in association with assembly of the lytic synapse. We will determine whether microtubule capture or other mechanisms are involved in positioning the MTOC. c. Lytic granule movement. Lytic granules line up along microtubules in the NK cell, and those microtubules are directed toward the target cell. In contrast to an influential previous study, we hypothesize that plus-end directed transport of lytic granules will be necessary for their secretion onto the target cell. We will investigate the molecular mechanism of plus-end transport, testing various kinesins. d. Role of actin in granule transport and fusion. We hypothesize that actin filaments may play positive or negative roles in how lytic granules are transported to the lytic synapse and then fuse with the plasma membrane. We will investigate potential roles by observing transport and manipulating the actin cytoskeleton.

Public Health Relevance

This research will help us understand the molecular mechanisms by which certain lymphocytes, called Natural Killer cells, are able to find and kill cancer cells and virus-infected cells. This fundamental information may serve as the basis for the development of new therapies for human diseases involving the molecules and processes that are discovered and illuminated.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038542-24
Application #
8328657
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Nie, Zhongzhen
Project Start
1988-12-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
24
Fiscal Year
2012
Total Cost
$586,811
Indirect Cost
$200,751
Name
Washington University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Wang, Xinxin; Galletta, Brian J; Cooper, John A et al. (2016) Actin-Regulator Feedback Interactions during Endocytosis. Biophys J 110:1430-43
Zhou, Julie Y; Szasz, Taylor P; Stewart-Hutchinson, Phillip J et al. (2016) L-Plastin promotes podosome longevity and supports macrophage motility. Mol Immunol 78:79-88
Xu, Xiaolu; Wang, Xinxin; Todd, Elizabeth M et al. (2016) Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration. J Immunol 197:1683-91
Sherman, Marc S; Lorenz, Kim; Lanier, M Hunter et al. (2015) Cell-to-cell variability in the propensity to transcribe explains correlated fluctuations in gene expression. Cell Syst 1:315-325
Mooren, Olivia L; Kim, Joanna; Li, Jinmei et al. (2015) Role of N-WASP in Endothelial Monolayer Formation and Integrity. J Biol Chem 290:18796-805
Onken, Michael D; Winkler, Ashley E; Kanchi, Krishna-Latha et al. (2014) A surprising cross-species conservation in the genomic landscape of mouse and human oral cancer identifies a transcriptional signature predicting metastatic disease. Clin Cancer Res 20:2873-84
Aiken, Jayne; Sept, David; Costanzo, Michael et al. (2014) Genome-wide analysis reveals novel and discrete functions for tubulin carboxy-terminal tails. Curr Biol 24:1295-303
Onken, Michael D; Li, Jinmei; Cooper, John A (2014) Uveal melanoma cells utilize a novel route for transendothelial migration. PLoS One 9:e115472
Mooren, Olivia L; Li, Jinmei; Nawas, Julie et al. (2014) Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier. Mol Biol Cell 25:4115-29
Edwards, Marc; Zwolak, Adam; Schafer, Dorothy A et al. (2014) Capping protein regulators fine-tune actin assembly dynamics. Nat Rev Mol Cell Biol 15:677-89

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