LIN7 regulates the filopodia and neurite promoting activity of IRSp53

Abstract

SummaryThe insulin receptor substrate protein of 53 kDa (IRSp53) is crucially involved in the formation of filopodia and neurites through mechanisms that have only partially been clarified. We have investigated the role of the small scaffold protein LIN7, which interacts with IRSp53. We found that formation of actin-filled protrusions in neuronal NSC34 cells and neurites in neuroblastoma N2A cells depends on motifs mediating the LIN7:IRSp53 association, as both the coexpression of LIN7 with IRSp53 or the expression of the L27-IRSp53 chimera (a fusion protein between IRSp53 and the LIN7L27 domain for plasma membrane protein complexes association) prevented actin-deficient protrusions induced by overexpressed IRSp53, and enhanced the formation of actin-filled protrusions. The regulatory role of LIN7 in IRSp53-mediated extension of filopodia in neuronal N2A cells was demonstrated by live-cell imaging experiments. Moreover, LIN7 silencing prevented the extension of filopodia and neurites, induced by ectopic expression of IRSp53 or serum starvation, respectively, in undifferentiated and differentiated N2A cells. The expression of full-length IRSp53 or the LIN7DPDZ mutant lacking the domain for association with IRSp53 was unable to restore neuritogenesis in LIN7-silenced cells. Conversely, defective neuritogenesis could be rescued by the expression of RNAi-resistant full-length LIN7 or chimeric L27-IRSp53. Finally, LIN7 silencing prevented the recruitment of IRSp53 in Triton X-100-insoluble complexes, otherwise occurring in differentiated cells. Collectively these data indicate that LIN7 is a novel regulator of IRSp53, and that the association of these proteins is required to promote the formation of actin-dependent filopodia and neurites.

SOD1 stimulates lamellipodial protrusions in Neuro 2A cell lines

“…SOD1-mediated increase in lamellipodia was greatly prevented in N2A cells downregulated for IRSp53 or LIN7C, the LIN7 form mainly expressed by these cells [16]. In line with a role for the IRSp53-LIN7 complex in lamellipodia formation, the percentage of SOD1-expressing cells with lamellipodia (~45%) dropped to ~21% and ~17% in cells downregulated for IRSp53 or LIN7, respectively.…”

“…The constructs GFP-IRSp53, GFP-LIN7A and shRNA LIN7C have been previously described [15,16]; shRNA IRSp53 was purchased from Sigma Aldrich. A 50% reduction of LIN7 was achieved by shRNA LIN7C [16], while a slightly lower downregulation of IRSp53 was detected by Western blot in cells transfected with shRNA IRSp53 (Figure S2).…”

“…A 50% reduction of LIN7 was achieved by shRNA LIN7C [16], while a slightly lower downregulation of IRSp53 was detected by Western blot in cells transfected with shRNA IRSp53 (Figure S2). cDNAs for human SOD1 and SOD1G93A were a kind gift of Dr. C. Bendotti [21].…”

POF1B Localizes to Desmosomes and Regulates Cell Adhesion in Human Intestinal and Keratinocyte Cell Lines

“…Immunoprecipitation in Caco-2 cells using POF1B antibody was performed as previously described (Crespi et al, 2012). Cell extracts and immunoprecipitates were solubilized in SDS denaturation buffer.…”

Novel localisation and possible function of LIN7 and IRSp53 in mitochondria of HeLa cells

“…Partners of the L27 domain of LIN7 such as the calcium/calmodulin-dependent serine protein kinase CASK may recruit IRSp53 to molecular complexes known to traffic proteins to specific subcellular locations or to anchor and stabilise them on the PM (Borg et al, 1999;Butz et al, 1998;Crespi et al, 2012;Massari et al, 2009;Perego et al, 1999;Setou et al, 2000).…”