Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling

Pamma D. Arora, Yongqiang Wang, Anne R. Bresnick, Paul A. Janmey, Christopher A. McCulloch

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Abstract

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 (k<inf>D</inf> = 0.146 μM), whereas FliI GLD 2-6 showed lower binding affinity (k<inf>D</inf> = 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.

Original languageEnglish (US)
Pages (from-to)2279-2297
Number of pages19
JournalMolecular Biology of the Cell
Volume26
Issue number12
DOIs
StatePublished - Jun 15 2015

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Nonmuscle Myosin Type IIA
Gelsolin
Collagen
Actin Capping Proteins
Fibroblasts
Actin Cytoskeleton
Immunoprecipitation
Transfection
Mass Spectrometry

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling. / Arora, Pamma D.; Wang, Yongqiang; Bresnick, Anne R.; Janmey, Paul A.; McCulloch, Christopher A.

In: Molecular Biology of the Cell, Vol. 26, No. 12, 15.06.2015, p. 2279-2297.

Research output: Contribution to journalArticle

Arora, Pamma D. ; Wang, Yongqiang ; Bresnick, Anne R. ; Janmey, Paul A. ; McCulloch, Christopher A. / Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling. In: Molecular Biology of the Cell. 2015 ; Vol. 26, No. 12. pp. 2279-2297.
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