Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Dynamic cell-extracellular matrix (ECM) interactions, including cell adhesion and migration, require binding of ECM proteins by integrins and subsequent intracellular biochemical signalling. Integrin function is dependent on the intracellular trafficking of integrins and associated proteins, and this traffic is mediated by membrane-anchored proteins called SNAREs ( S oluble N SF- A ttachment protein Re ceptors). We recently discovered that the function of a SNARE called Syntaxin4 (Stx4) is required for normal integrin-mediated cell-ECM interactions, and that an associated protein, Munc18c, regulates Stx4 in this context. The interaction of Stx4 and Munc18c represents a novel regulatory point in cell-ECM interactions, and analysis of this regulation will reveal molecular mechanisms that underlie the development and maintenance of tissue architecture and function.

In the current proposal, we aim to define the mechanisms by which Stx4 and Munc18c control cell adhesion and migration, and to determine how the functions of Stx4 and Munc18c are regulated in this context. The proposal contains three major aims.

(1) To elucidate the functions of Syntaxin4 and Munc18c in cell adhesion and migration.
Trafficking and recycling of B1 integrins, and the targeting of FA components (e.g. Src, FAK, paxillin) to sites of ECM contact, will be analyzed. We have developed stable cell lines, which express constructs to inhibit Stx4 or Munc18c, to facilitate both biochemical and microscopic analyses, as well as cell-based assays to examine cell-ECM interactions.

(2) To examine the regulation of Syntaxin4 and Munc18c by phosphorylation.
The relationship between phosphorylation of Stx4 and/or Munc18c and cell-ECM interactions will be defined. Stx4 or Munc18c will be immunoprecipitated from cells in a ‘resting’ state (plated on poly-L-lysine) or an ‘adhesive’ state (plated on ECM substrate), and the phosphorylated site(s) in Syntaxin4 and Munc18c will be determined through phosphoproteomic analysis. Phosphorylation at these sites during cell-ECM interactions will then be characterized using biochemical and molecular approaches.

(3) To identify novel regulators of SNARE function that control cell-ECM interactions.
Cells will be plated on ECM substrates, and SNARE complexes will then be immunoprecipitated. Proteins, captured along with SNARE complexes, that are differentially represented in cells plated on ECM vs. control cells will be identified by proteomic analyses and subsequently characterized.

The above aims include several projects that offer collaborative training opportunities for graduate and undergraduate students. The research will benefit cell biologists, and other researchers across Canada and abroad, both through the training of highly qualified personnel and by advancing our understanding of the molecular underpinnings of cellular function within tissues and organs.