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A2934 - GMFγ Coordinates Focal Adhesion and Lamellipodial Dynamics to Regulate Human Airway Smooth Muscle Migration
Author Block: B. D. Gerlach1, G. Liao2, A. Rezey1, R. Wang3, D. D. Tang4; 1Molecular Cellular Physiology, Albany Medical College, Albany, NY, United States, 2Albany Medical College, Albany, NY, United States, 3Cardiovascular Sciences, Albany Medical College, Albany, NY, United States, 4Ctr for Cardiovascular Sciences Albany Med College, Albany, NY, United States.
Airway remodeling is a key feature in the pathogenesis of asthma. Airway smooth muscle migration plays a critical role in respiratory development; however, it’s also implicated in the pathogenesis of airway remodeling. During cell migration, a critical balance between protrusion and focal adhesion dynamics is established through applicable signaling cascades. The Arp2/3 complex and Neural Wiskott-Aldrich Syndrome protein (N-WASP) are both implicated in the formation of the lamellipodia, as well as nascent focal adhesion formation. However, the underpinning molecular mechanisms that coordinate this interdependent process have not been fully explored. Glia Maturation factor-γ (GMFγ) is a 17kD protein found in the ADF/cofilin depolymerizing factor superfamily. GMFγ is known to interact directly with Arp2 of the Arp2/3 complex to initiate actin branch disassembly. Here we demonstrate in primary human smooth muscle cells, the phosphorylation of GMFγ at tyrosine 104 by c-Abl is a critical determinant for the spatial distribution of activated N-WASP (pY256) to the leading edge and to growing focal adhesions. Using super-resolution microscopy coupled with Imaris 9.0 software analysis, we show phosphorylated GMFγ and N-WASP (pY256) were enriched at the leading edge, where it promoted extension of the lamellipodia. Conversely, non-phosphorylated GMFγ and N-WASP (pY256) localized to focal adhesions and increased growth and maturation through recruitment of vinculin and zyxin. Knockdown of GMFγ decreased the area, volume, length and recruitment of N-WASP (pY256) to growing focal adhesions. Moreover, GMFγ knockdown cells displayed less prominent leading-edge morphology and had decreased localization of N-WASP (pY256) with Arp2 along the leading edge, as compared to control shRNA expressing cells. Thus, suggesting that GMFγ is critical for both focal adhesion and leading-edge growth and N-WASP (pY256) recruitment. Time-lapse microscopy demonstrated that expression of a non-phosphorylated mutant GMFγ (Y104F) attenuated migration speed, directionality, accumulated distance, and net distance in GMFγ knockdown cells. In contrast, phosphorylated mutant GMFγ (Y104D) rescued and enhanced all migratory parameters in GMFγ shRNA expressing cells. Altogether, our findings reveal GMFγ as a key regulator of migration through balancing focal adhesion and lamellipodial dynamics.