Profilin induces lamellipodia by growth factor independent mechanism

Abstract

Profilin has been implicated in cell motility and in a variety of cellular processes, such as membrane extension, endocytosis, and formation of focal complexes. In vivo, profilin replenish the pool of ATP-actin monomers by increasing the rate of nucleotide exchange of ADP-actin for ATP-actin, promoting the incorporation of new actin monomers at the barbed end of actin filaments. For this report, we generated a membrane-permeable version of profilin I (PTD4-PfnI) for the alteration of intracellular profilin levels taking advantage of the protein transduction technique. We show that profilin I induces lamellipodia formation independently of growth factor presence in primary bovine trabecular meshwork (BTM) cells. The effects are time- and concentration-dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. H133S, a mutation in the polyproline binding domain, showed a reduced ability to induce lamellipodia. H199E, mutation in the actin binding domain failed to induce membrane spreading and inhibit fetal bovine serum (FBS) -induced lamellipodia extension. Incubation with a synthetic polyproline domain peptide (GP5)3, fused to a transduction domain, abolished lamellipodia induction by profilin or FBS. Time-lapse microscopy confirmed the effects of profilin on lamellipodia extension with a higher spreading velocity than FBS. PTD4-Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS-induced lamellipodia formation activates Rac1, PTD4-Pfn I stimulation did not induce Rac1 activation. We propose a role of profilin I favoring lamellipodia formation by a mechanism downstream of growth factor.

Structure and functions of profilins

“…The results from experiments using membranepermeable profilin-I (PTD4-PfnI) suggest that profilin-I induces lamellipodia formation independently of the presence of EGF in primary bovine trabecular meshwork cells (Syriani et al 2008). The effects are time-and dosagedependent and specific to the profilin-I isoform: the H133S mutation in the poly-L-proline binding domain showed a reduced ability to induce lamellipodia, and the H199E mutation in the actin binding domain failed to induce membrane spreading (Syriani et al 2008).…”

“…The effects are time-and dosagedependent and specific to the profilin-I isoform: the H133S mutation in the poly-L-proline binding domain showed a reduced ability to induce lamellipodia, and the H199E mutation in the actin binding domain failed to induce membrane spreading (Syriani et al 2008). Profilin-I levels were found to be lower in hepatocarcinoma (SMMC-7721) cells and the all-trans retinoic acid-induced inhibition of proliferation and migration was found to be mediated through profilin-I expression.…”

Profilin 1 is required for peripheral nervous system myelination

“…Pfn1 is a ROCK substrate (Shao et al, 2008) known to promote lamellipodia formation in different cell types (Cao et al, 1992;Syriani et al, 2008). Accordingly, we show that in SCs exposed to LPA or in Ilk mutant sciatic nerves in which ROCK activation is increased, Pfn1 is phosphorylated on Ser-137 by ROCK.…”

“…Pfn1 is an actin remodeling protein that is capable of regulating lamellipodia formation in different cell types (Cao et al, 1992;Syriani et al, 2008), and is a ROCK substrate (Shao et al, 2008). Therefore, it is a strong candidate in mediating lamellipodia formation downstream of Rho/ROCK in SCs.…”

“…ROCK-mediated Pfn1 phosphorylation on Ser137 leads to its inactivation in both HEK293 cells and primary neurons (Shao et al, 2008). In light of the central role of Pfn in the regulation of actin dynamics and lamellipodia formation (Cao et al, 1992;Syriani et al, 2008), Pfn1 is poised as an excellent candidate to regulate myelination downstream of Rho/ROCK by potentially modulating SC radial lamellipodia formation. Using mouse genetics and in vitro experiments, we demonstrate a novel crucial function for Pfn1 in radial sorting and ensheathment during PNS development.…”