Technical Reference #2009

2009.

WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells Corina Sarmiento; Weigang Wang; Athanassios Dovas; Hideki Yamaguchi; Mazen Sidani; Mirvat El-Sibai; Vera DesMarais; Holly A. Holman; Susan Kitchen; Jonathan M. Backer; Art Alberts; and John Condeelis, Albert Einstein College of Medicine, Journal of Cell Biology, 180(2009), (2008)
Link To Paper

Abstract:
We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF) a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation whereas N-WASP KD has no effect. However simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous–related formin mDia1 localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity which stimulates mDia1 nucleation was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity.

Keywords:
Arp; actin-related protein; BE; barbed end; CRIB; Cdc42/Rac interactive binding domain; DN; dominant negative; FH2; formin homology 2; KD; knockdown; N-WASP; neural WASP; RBD; Rho binding domain; WASP; Wiskott-Aldrich syndrome protein

Materials & Methods:
MTLn3 cells (rat mammary adenocarcinoma cell line) were maintained starved and stimulated as described previously ( DesMarais et al. 2004 ). For light microscopy experiments cells were plated on glass-bottom dishes (MatTek Corporation) that had been treated with 1 M HCl for 10 min followed by one wash with 75% ethanol and then one wash with PBS. Before each experiment cells were starved in L15 medium (Invitrogen) supplemented with 0.35% BSA (starvation medium) for 3 – 4 h. For stimulation MTLn3 cells were treated at 37 ° C with a bath application of 5 nM EGF (Invitrogen) for various times. Previous work has shown that serum stimulation of MTLn3 protrusion activity cell motility RhoA activity and Cdc42 activity is mediated by the EGF receptor ( Wyckoff et al. 2004 ; El-Sibai et al. 2007 2008 ). Therefore we used EGF stimulation in place of serum to examine the motility pathways in MTLn3 cells after stimulation.

Microscopic Technique
Immunofluorescence microscopy

Cell Type(s)
MTLn3 cells