TY - JOUR
T1 - Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling
AU - Arora, Pamma D.
AU - Wang, Yongqiang
AU - Bresnick, Anne
AU - Janmey, Paul A.
AU - McCulloch, Christopher A.
N1 - Publisher Copyright:
© 2015 Arora et al.
PY - 2015/6/15
Y1 - 2015/6/15
N2 - We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1-6 capped actin filaments ejficiently, whereas FliI GLD 2-6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1-6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2-6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2-6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1-6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts.
AB - We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1-6 capped actin filaments ejficiently, whereas FliI GLD 2-6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1-6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2-6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2-6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1-6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts.
UR - http://www.scopus.com/inward/record.url?scp=84930884736&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84930884736&partnerID=8YFLogxK
U2 - 10.1091/mbc.E14-11-1536
DO - 10.1091/mbc.E14-11-1536
M3 - Article
C2 - 25877872
AN - SCOPUS:84930884736
SN - 1059-1524
VL - 26
SP - 2279
EP - 2297
JO - Molecular biology of the cell
JF - Molecular biology of the cell
IS - 12
ER -