Microtubules are dynamic polymers central to many processes in eukaryotic cells, including chromosome segregation, intracellular transport, and cell shape remodeling. Many of these microtubule functions are mediated by protein interactions at growing microtubule plus ends. End-binding proteins (EBs) directly recognize a structural feature of growing microtubule ends. A diverse group of proteins, called +TIPs, bind to EBs through short, conserved SxIP peptide motifs in intrinsically disordered, positively charged protein regions. These characteristics led to the identification of over thirty structurally highly heterogeneous +TIPs, but intracellular +TIP functions remain incompletely understood. A long-term goal of this project is to answer fundamental, unresolved questions of +TIP cell biology: What are molecular functions of specific +TIPs? How is microtubule plus-end-association important for these functions? How are +TIPs spatially and temporally controlled such that different +TIP complexes mediate specific microtubule activities? Based on the unexpected diversity of SxIP-motif-containing +TIPs and findings in the previous funding period, a central hypothesis of this project is that many +TIPs act as adaptors that promote spatially and temporally controlled capture of EB-positive growing microtubule ends to polarize microtubule-dependent activities, which extends the classic search-and-capture theory by providing intracellular receptors to facilitate specific interactions with EB-covered growing microtubule ends. The current application focuses on the function of CLASPs and other +TIPs that are associated with focal adhesions (FAs), multi-layered macromolecular assemblies that mediate dynamic cell interactions with the extracellular matrix (ECM). Based on preliminary data that CLASPs cluster around FAs, tether microtubules to FAs, and facilitate FA turnover, a novel mechanism is proposed in which FA-associated +TIPs establish vesicle transport tracks toward FAs to promote localized ECM remodeling and facilitate outside-in FA disassembly. Experiments in this application will define how +TIPs and microtubules control cell-matrix adhesion remodeling by employing biochemical, cell biological and advanced live cell imaging approaches:
Aim 1 asks how growing and mature FAs generate a signal that recruits specific +TIPs to a zone adjacent to FAs.
Aim 2 defines molecular mechanisms by which CLASPs capture and link MTs to FAs, and investigates a novel mechanism of EB regulation.
Aim 3 asks how localized exocytosis promotes FA turnover, tests whether cell-matrix release is sufficient to trigger FA disassembly by developing a highly innovative light-controlled cell adhesion substrate, and analyzes the consequences of +TIP-mediated local ECM remodeling during epithelial remodeling in a physiological 3D environment. Because abnormal cell-matrix interactions contribute to cancer metastasis, and EBs are overexpressed in cancer cells indicating increased +TIP activity, in addition to establishing new paradigms relating to +TIP function, our studies are highly relevant to understanding pathological cell behavior.

Public Health Relevance

Microtubules are components of the cytoskeleton that form a dynamic filament system in cells, required for accurate chromosome segregation, intracellular transport, and cell shape changes. The long-term goal of this project is to understand the molecular functions and regulation of a class of diverse proteins that specifically bind to growing microtubule ends (+TIPs). Alterations in expression levels and/or activities of +TIPs are associated with a variety of human diseases and unraveling +TIP cell biology is essential to expose underlying roles in pathological conditions. In the current project, we aim to understand how +TIP-mediated local secretion of enzymes that remodel the extracellular matrix contribute to physiological cell migration and tissue remodeling, processes that are critically involved in cancer development, metastasis and malignancy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079139-09
Application #
9194410
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Nie, Zhongzhen
Project Start
2008-05-01
Project End
2017-12-31
Budget Start
2017-01-01
Budget End
2017-12-31
Support Year
9
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Anatomy/Cell Biology
Type
Schools of Dentistry/Oral Hygn
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94118
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Webb, Bradley A; Dosey, Anne M; Wittmann, Torsten et al. (2017) The glycolytic enzyme phosphofructokinase-1 assembles into filaments. J Cell Biol 216:2305-2313
Ettinger, Andreas; van Haren, Jeffrey; Ribeiro, Susana A et al. (2016) Doublecortin Is Excluded from Growing Microtubule Ends and Recognizes the GDP-Microtubule Lattice. Curr Biol 26:1549-1555
Kenific, Candia M; Stehbens, Samantha J; Goldsmith, Juliet et al. (2016) NBR1 enables autophagy-dependent focal adhesion turnover. J Cell Biol 212:577-90
Kenific, Candia M; Wittmann, Torsten; Debnath, Jayanta (2016) Autophagy in adhesion and migration. J Cell Sci 129:3685-3693
Stehbens, Samantha J; Paszek, Matthew; Pemble, Hayley et al. (2014) CLASPs link focal-adhesion-associated microtubule capture to localized exocytosis and adhesion site turnover. Nat Cell Biol 16:561-73
Ettinger, Andreas; Wittmann, Torsten (2014) Fluorescence live cell imaging. Methods Cell Biol 123:77-94
Stehbens, Samantha J; Wittmann, Torsten (2014) Analysis of focal adhesion turnover: a quantitative live-cell imaging example. Methods Cell Biol 123:335-46
Basu, Sreya; Sladecek, Stefan; Pemble, Hayley et al. (2014) Acetylcholine receptor (AChR) clustering is regulated both by glycogen synthase kinase 3? (GSK3?)-dependent phosphorylation and the level of CLIP-associated protein 2 (CLASP2) mediating the capture of microtubule plus-ends. J Biol Chem 289:30857-67
Zhao, Xiaoyan; Kumar, Praveen; Shah-Simpson, Sheena et al. (2013) Host microtubule plus-end binding protein CLASP1 influences sequential steps in the Trypanosoma cruzi infection process. Cell Microbiol 15:571-84

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