Cell migration plays a central role in embryonic development, wound healing, inflammation and tumor metastasis. Our long-term goal is to understand how cell-extracellular matrix interactions and integrinmediated signaling regulates these events. In this proposal, we will elucidate the mechanisms by which α4β1 integrin regulates directionally persistent cell migration. We discovered that non-muscle myosin II heavy chain (referred to as MIIA) co-immunoprecipitates with α4 integrin and can be pulled down from cell lysates by the cytoplasmic tail of α4 integrin, indicating that these two proteins are physically associated. This proposal will focus on this association (referred to as α4/MIIA association). We propose to accomplish three specific aims.
Aim 1. Characterize α4/MIIA association. We will (A) determine if MIIA binds directly to the α4 tail, using GST-α4 tail or an α4 tail peptide and recombinant MIIA expressed in and purified from baculovirus;(B) use mass spectrometry analysis to identify additional proteins that are associated with the α4/MIIA complex;(C) characterize mutations in the α4 tail that disrupt α4/MIIA association.
Aim 2. . Determine how α4/MIIA association is regulated during cell migration. Two sub-aims are proposed.
In Aim 2 A, we will examine the temperospatial dynamics of α4 integrin and MIIA in migrating cells. We will also use a bead assay to test asymmetrical forces imposed onto the front and back of the cell by shear flow have differential effects on α4/MIIA association.
In Aim 2 B, we will determine how α4/MIIA association is regulated, if α4/MIIA association requires (a) α4 phosphorylation, (b) RhoA, (c) the ATPase activity, (d) MIIA filament assembly.
Aim 3. Identify pathways/events that are downstream of α4/MIIA association. Two sub-aims are proposed.
In Aim 3 A, we will test the role of α4/MIIA association in regulating the functions of MIIA. We will test the role of α4/MIIA association in (a) localization and organization of MIIA;(b) focal adhesion turnover;(c) regulating MLC phosphorylation and filament assembly;and (d) regulating RhoAand FAK-mediated pathways.
In Aim 3 B, we will test the role of α4/MIIA association in the ligandbinding and adhesive activities of α4β1 integrin. These studies will provide a new mechanism by which α4β1 transduce signals into cells to regulate cell migration. An understanding of this signaling pathway will eventually allow us to delineate the functions of α4β1 integrin in vivo and in pathological processes such as inflammation and tumor metastasis..
The proposed studies will facilitate our understanding on the cellular and molecular mechanisms of cell migration, which is critical to the discovery of novel therapeutics for treating cancer. The proposed studies will also provide new knowledge on an ?4?1 integrin-mediated signaling pathway. This integrin is an excellent drug target for multiple sclerosis, inflammatory bowel disease and other inflammatory disorders. Our studies may reveal new drug targets that block the ?4?1-mediated signaling pathway.
| Hung, Wei-Chien; Chen, Shih-Hsun; Paul, Colin D et al. (2013) Distinct signaling mechanisms regulate migration in unconfined versus confined spaces. J Cell Biol 202:807-24 |
| Rivera Rosado, Leslie A; Horn, Troy A; McGrath, Sara C et al. (2011) Association between ýý4 integrin cytoplasmic tail and non-muscle myosin IIA regulates cell migration. J Cell Sci 124:483-92 |
| Dikeman, Dustin A; Rivera Rosado, Leslie A; Horn, Troy A et al. (2008) alpha4 beta1-Integrin regulates directionally persistent cell migration in response to shear flow stimulation. Am J Physiol Cell Physiol 295:C151-9 |
| Grazioli, Alison; Alves, Christina S; Konstantopoulos, Konstantinos et al. (2006) Defective blood vessel development and pericyte/pvSMC distribution in alpha 4 integrin-deficient mouse embryos. Dev Biol 293:165-77 |
