Profilin Promotes Barbed-end Actin Filament Assembly without Lowering the Critical Concentration

Abstract

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin⅐4. Determining how cells regulate actin assembly is vital for understanding motility. Resting cells contain high concentrations of unpolymerized actin, typically 200 -400 M, or about 600 -1200 times greater than the critical concentration for assembly of pure actin (1, 2). Actin-sequestering proteins prevent spontaneous assembly of monomeric actin, and nonmuscle cells contain two such sequestering proteins: the 15-kDa protein profilin and the 5-kDa thymosin-␤4 (3, 4). These proteins were mainly thought to bind actin monomers with a one-to-one stoichiometry and to sequester actin monomers, and both have a higher affinity toward Actin⅐ATP as compared with Actin⅐ADP. The current view is that profilin and thymosin-␤4 are likely to play complementary roles in the cell. Thymosin-␤4 has a single mode of action (5-8), namely binding actin monomers to create a sequestered pool of monomers to supply Actin⅐ATP during periods of active filament growth. The mode of action of profilin, however, is more complex and in many respects has remained controversial and elusive. Although the Profilin⅐Actin complex was initially also thought to be strictly a monomersequestering protein (9 -11), Pollard and Cooper (12) later confirmed the inference that it adds to the barbed ends of actin filaments (13). Initial rate and steady-state assembly measurements showed that profilin can efficiently bind actin monomers (K d Ͻ 5 M) and can weakly interact with filament barbed ends (K capping ϳ 100 M). Profilin catalyzes exchange of free ATP with actin-bound ADP to form actin-bound ATP and free ADP (14 -16). Profilin also exhibits affinity toward oligoproline modules (17), another unique property that facilitates transfer of actin monomers to the polymerization zone (16) lying immediately behind motile Listeria (18), Shigella (19), and vaccinia (20). By concentrating Profilin⅐Actin complex in regions of active filament assembly, explosive rates of filament growth (Ն500 monomers s Ϫ1 ) are readily catalyzed. By contrast, thymosin-␤4 does not bind to oligoproline and fails to concentrate in the polymerization zone.Highly motile cells contain high concentrations of both profilin and thymosin-␤4, and these proteins probably act in concert to promote rapid filament assembly. Pantaloni and Carlier (21) investigated the role of profilin in the absence and presence of thymosin-␤4. They suggested that profilin reduces the critical monomer concentration needed for filament assembly, and they suggested that this is accomplished by accelerating the irreversible hydrolysis of polymer-bound Profilin⅐Actin⅐ATP complex. However, their experimental design was compromised in several significant ways: 1) they used pyrenyl-actin polymerization and had to correct the observed data for the low affinity of profilin for pyrenyl-actin (9,22,23); 2) all of their determinations of the steady-state extent of polymerization rested on th...

TetraThymosinβ Is Required for Actin Dynamics inCaenorhabditis elegansand Acts via Functionally Different Actin-binding Repeats

“…In contrast, profilin only effectively sequesters actin monomers in the presence of barbed end capped filaments (Pantaloni and Carlier, 1993;Kang et al, 1999), a property used to demonstrate its ability to deliver actin monomers to free barbed ends and hence promote barbed end elongation. A same effect has been described for tetraThymosin␤ homologues ciboulot and actobindin (Boquet et al, 2000;Herzog et al, 2002).…”

“…E04 -03-0225. for profilin (Boquet et al, 2000;Hertzog et al, 2002). Indeed, profilin serves as a polymerization catalyst by capturing an actin monomer and ushering the actin onto the growing filament barbed end as a 1:1 profilin:actin complex, whereupon profilin itself is released (Pantaloni and Carlier, 1993;Kang et al, 1999). Ciboulot is suggested to act on elongation via the same mechanism as profilin by forming a 1:1 complex with an actin monomer (Boquet et al, 2000).…”

Relationships between Actin Regulatory Mechanisms and Measurable State Variables

“…Thymosin b4 inhibits nucleotide exchange on bound monomers, but also has a 100-fold higher affinity for ATP-bound monomers than ADP-bound monomers, and therefore generally binds monomers after they have exchanged ADP for ATP. 10,25 The differences between Fig. 2 (with 100 lM thymosin b4 ) and Fig.…”

“…Equilibrium disassociation constant for thymosin B4 and ATP-G-actin 0.9 lM k + BT = 1 /(lM s) and k)BT = /0.9 s assigned; K BT from Kang et al 25 …”

Neuronal Differentiation

“…Profilin was originally described as an actinsequestering molecule (Carlsson et al, 1977), but was more recently found to enhance actin polymerization by adding monomers to the fast-growing ends of actin filaments (Pantaloni and Carlier, 1993;Kang et al, 1999) and catalyzing nucleotide exchange (Goldschmidt-Clermont et al, 1992;Selden et al, 1999). Thereby, profilins contribute to actin polymerization at the leading edge of lamellipodia and in the tips of filopodia.…”