The broad objective of this proposal is to understand how cells explore the environment and respond to it in a meaningful way. The specific focus of the proposal is on investigating the molecular design of the cell's sensory and guiding organelles, filopodia. Filopodia play critical roles in a variety of cellular processes including the navigation of neurons and metastasis of cancer cells. The conceptual framework of the proposal is based on the recently formulated convergent elongation hypothesis for filopodia initiation. This hypothesis states that the actin filament bundle comprising the structural and functional core of a filopodium is formed by reorganization of the dendritic actin network. Reorganization is driven by the filopodial tip complex, which controls elongation and physical interaction of filament growing ends and initiates filament bundling. We propose to investigate specific elements of this model including: (1) whether Arp2/3- dependent nucleation supplies filaments for filopodial initiation; (2) the molecular mechanism of persistent filament elongation in filopodia; (3) the mechanism of barbed end interaction; (4) the mechanism of filament bundling and its role in filopodial protrusion. The proposed research strategy employs a combination of structural, dynamic, and functional approaches applied at the subcellular and supramolecular levels. The general procedure will be to identify essential molecular players, to determine their place in the supramolecular organization of filopodia, to analyze their dynamics, and to establish their functions in filopodia formation and cell behavior. This strategy uniquely bridges the gap between properties of individual molecules and the behavior of a whole cell. Correlative light and platinum replica electron microscopy will be used to relate structure to cell behavior. Kinetic studies will use fluorescence microscopy, fluorescence recovery after photobleaching and fluorescence resonance energy transfer techniques. Functional assays will be largely based on the novel RNA interference approach. Experiments will be conducted using particularly favorable cell model systems: a highly metastatic mouse melanoma cell line and cultured neuronal cells. The results will contribute to an understanding of the mechanism of filopodia formation and the roles of filopodia in such fundamental processes in development and disease as cell migration, cell-cell communication, and tissue morphogenesis. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM070898-01
Application #
6762721
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rodewald, Richard D
Project Start
2004-05-01
Project End
2009-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
1
Fiscal Year
2004
Total Cost
$295,035
Indirect Cost
Name
University of Pennsylvania
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Svitkina, Tatyana (2016) Imaging Cytoskeleton Components by Electron Microscopy. Methods Mol Biol 1365:99-118
Jones, Steven L; Korobova, Farida; Svitkina, Tatyana (2014) Axon initial segment cytoskeleton comprises a multiprotein submembranous coat containing sparse actin filaments. J Cell Biol 205:67-81
Zwolak, Adam; Yang, Changsong; Feeser, Elizabeth A et al. (2013) CARMIL leading edge localization depends on a non-canonical PH domain and dimerization. Nat Commun 4:2523
Svitkina, Tatyana M (2013) Ultrastructure of protrusive actin filament arrays. Curr Opin Cell Biol 25:574-81
Hu, Jianli; Bai, Xiaobo; Bowen, Jonathan R et al. (2012) Septin-driven coordination of actin and microtubule remodeling regulates the collateral branching of axons. Curr Biol 22:1109-15
Svitkina, Tatyana M (2012) Actin bends over backward for directional branching. Proc Natl Acad Sci U S A 109:2693-4
Wang, Yu-Hsiu; Collins, Agnieszka; Guo, Lin et al. (2012) Divalent cation-induced cluster formation by polyphosphoinositides in model membranes. J Am Chem Soc 134:3387-95
Shutova, Maria; Yang, Changsong; Vasiliev, Jury M et al. (2012) Functions of nonmuscle myosin II in assembly of the cellular contractile system. PLoS One 7:e40814
Hoelzle, Matthew K; Svitkina, Tatyana (2012) The cytoskeletal mechanisms of cell-cell junction formation in endothelial cells. Mol Biol Cell 23:310-23
Liu, Jianglan; Zhao, Yuting; Sun, Yujie et al. (2012) Exo70 stimulates the Arp2/3 complex for lamellipodia formation and directional cell migration. Curr Biol 22:1510-5

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