The goal of this research is to determine how both the dynamics and spatial organization of the actin and microtubule cytoskeletons are coordinated by a group of five interacting mammalian proteins: APC, Dia1, EB1, CLIP-170, and capping protein. This work will define the functions and mechanisms of these proteins in microtubule-actin cross-regulation, and will thereby provide a deeper understanding of the molecular activities and interactions that underlie such processes as cell migration, cell adhesion, and cell and tissue morphogenesis. This project uses bulk biochemical experiments combined with novel multi-wavelength single molecule fluorescence methods that we have tailored to directly observe the mechanisms of complex multi- component regulatory systems in vitro. Further, the mechanisms deduced from the experiments in vitro will be tested in vivo to confirm that they are important for specific cellular functions of these proteins.
The Specific Aims are: (1) Test the hypothesis that the microtubule plus end-binding protein EB1 directly regulates nucleation of actin filaments by APC-Dia1; (2) Test the hypothesis that the rate and duration of actin filament elongation is controlled by dynamic binding interactions of Dia1, capping protein, microtubules, and/or EB1 at barbed ends; and (3) Define the roles of APC, Dia1, EB1, and CLIP-170 in controlling microtubule plus end dynamics and in triggering ultrafast actin polymerization from microtubule plus ends.

Public Health Relevance

This grant is an investigation of the organization and regulation of actin and microtubule cytoskeletons by the combined effects of five proteins: APC, Capping protein, Dia1, EB1, and CLIP-170. APC (Adenomatous polyposis coli) is a tumor suppressor. Mutation of the human Apc gene is an early step in the progression of over 80% of colorectal cancers, inherited and sporadic. Interactions with disease-associated proteins have implicated Kinesin-1 in Alzheimer's, Huntington's, and Parkinson's diseases. Further, fusion of the EB1 and MLL genes can lead to acute lymphoblastic leukemia. For these reasons, this research may provide new insights into the underlying mechanisms of tumorigenesis. In addition, the work is designed to uncover basic mechanisms of cellular biochemistry that will contribute to understanding disease states in as-yet-unanticipated ways.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098143-07
Application #
9432512
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Gindhart, Joseph G
Project Start
2012-05-01
Project End
2020-02-29
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
7
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Brandeis University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
Guo, Siyang; Sokolova, Olga S; Chung, Johnson et al. (2018) Abp1 promotes Arp2/3 complex-dependent actin nucleation and stabilizes branch junctions by antagonizing GMF. Nat Commun 9:2895
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Shekhar, Shashank (2017) Microfluidics-Assisted TIRF Imaging to Study Single Actin Filament Dynamics. Curr Protoc Cell Biol 77:12.13.1-12.13.24
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Daou, Pascale; Hasan, Salma; Breitsprecher, Dennis et al. (2014) Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration. Mol Biol Cell 25:658-68
Jaiswal, Richa; Breitsprecher, Dennis; Collins, Agnieszka et al. (2013) The formin Daam1 and fascin directly collaborate to promote filopodia formation. Curr Biol 23:1373-9

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