Filopodia are actin-based finger-like protrusions from the plasma membrane, and are used for multiple functions in cells including motility, cell-cell adhesion, cell-substratum adhesion, and viral infection. For many of these processes, the filopodia involved are highly dynamic, assembling and disassembling on a time scale of minutes. At least three actin-based activities are required for filopodial assembly: filament nucleation activity, filament elongation activity, and filament bundling activity. Two competing models for filopodial assembly differ in the nature of the nucleation and elongation activities. In the tip nucleation model, proteins such as formins act as both nucleation and elongation factors. In the convergent elongation model, Arp2/3 complex is the nucleation factor, with formins subsequently acting as elongation factors that also help re-model the branched Arp2/3-generated actin network. Our results show that the formin FMNL3 is a potent filopodial assembly factor, and we propose a novel extension to the convergent elongation model - that FMNL3 can remodel any existing filaments (Arp2/3-dependent or Arp2/3-independent) to filopodia, provided they abut the plasma membrane. In addition, we show that FMNL3 acts in cell-cell adhesion. In this proposal, we study FMNL3 in mammalian cells, focusing on the following aims.
Aim 1 uses cell-based assays to define the mechanism of FMNL3-mediated filopodial assembly. We use live-cell microscopy, inhibitor treatments and siRNA to test the ability of FMNL3 to re-model stress fiber/focal adhesion-associated actin filaments into filopodia.
Aim 2 uses a cell-free system to reconstitute filopodial assembly on supported lipid bilayers using purified proteins (FMNL3, Arp2/3 complex, capping protein, profilin, fascin). With this system, we will test assembly principles in a controlled manner and investigate the contributions of other molecules (VASP, cofilin).
Aim 3 investigates FMNL3 function in cell-cell adhesion, focusing on FMNL3's transit from intracellular storage sites to the plasma membrane, and activation at the plasma membrane during this process. Overall, this project will provide fundamentally novel mechanistic information on filopodial assembly, as well as providing novel molecular connections between actin dynamics and early events in cell-cell adhesion.

Public Health Relevance

This application investigates the basic cell biology of filopodia, which are finger-like protrusions from the plasma membrane found on a wide variety of mammalian cells. Filopodia are used in many ways in normal cellular physiology (adhesion, cell migration, nutrient up-take), and are also hijacked by many viruses for entry into cells and for cell-to-cell transmission. We will elucidate how filopodia are made through a specific protein, the formin FMNL3. In addition, we will investigate how FMNL3-mediated filopodia contribute to cell-to-cell adhesion. Our work has broad-ranging implications, with two clear applications being cancer (metastasis) and viral infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM109965-03
Application #
9181427
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Nie, Zhongzhen
Project Start
2015-01-01
Project End
2018-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Gauvin, Timothy J; Young, Lorna E; Higgs, Henry N (2015) The formin FMNL3 assembles plasma membrane protrusions that participate in cell-cell adhesion. Mol Biol Cell 26:467-77
Young, Lorna E; Heimsath, Ernest G; Higgs, Henry N (2015) Cell type-dependent mechanisms for formin-mediated assembly of filopodia. Mol Biol Cell 26:4646-59
Guo, Bingqian; Gurel, Pinar S; Shu, Rui et al. (2014) Monitoring ATP hydrolysis and ATPase inhibitor screening using (1)H NMR. Chem Commun (Camb) 50:12037-9