Profilin, an essential globular-actin (as opposed to filamentous actin) binding protein, has two opposite regulatory functions in actin filament assembly. It facilitates assembly at the barbed ends by lowering the critical concentration. In contrast to this, profilin contributes to the pool of unassembled actin when barbed ends are capped. It is proposed that the first of these functions required an input of energy. How profilin uses the ATP hydrolysis that accompanies actin polymerization and whether the acceleration of nucleotide exchange on G-actin by profilin participates in its function in filament assembly are the issues addressed in this paper. It is shown that:

1. profilin increases the treadmilling rate of actin filaments in the presence of Mg2+ ions
2. When filaments are assembled from CaATP-actin (which polymerizes in a quasi-reversible fashion) profilin does not promote assembly at the barbed ends and has only a G-actin-sequestering function.
3. Plant profilins do not accelerate nucleotide exchange on G-actin, yet they promote assembly at the barbed end.

Replication-incompetent adenovirus carrying the human profilin I cDNA is a means to rapidly increase the concentration of profilin in human aortic endothelial cells 12-31-fold above baseline. The concentration of filamentous actin is not detectably affected by profilin overexpression. Actin stress fibers are, however, absent from areas of high profilin content in overexpressing cells; the bulk of filaments are located at the periphery of the cells. A gradient in the distribution of overexpressed profilin was observed in migrating endothelial cells, with most profilin molecules concentrated near the advancing edge where focal contacts are being formed and focal adhesion proteins are located. Profilin overexpression results in increased recruitment of fibronectin receptors to the plasma membrane. Adhesion of endothelial cells to fibronectin is markedly and selectively increased by profilin overexpression. It is concluded that an important role for profilin in mammalian cells may be its contribution to the formation of focal contacts, particularly those involving the fibronectin receptor (Moldovan, 1997).

The axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, was studied in the chicken sciatic nerve following injection of [35S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. The specific activity of soluble actin is two to three times that of its particulate form. Soluble actin, cofilin, actin depolymerizing factor, and profilin are transported at similar rates in slow component b of axonal flow. These data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its "inactive" phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool (Mills, 1996).

The gene encoding the actin-related protein Arp3 was first identified in the fission yeast S. pombe and is a member of an evolutionarily conserved family of actin-related proteins. Mutants in arp3 interact specifically with profilin and actin mutants. Arp3 localizes to cortical actin patches that are required for polarized cell growth. The arp3 gene is required for the reorganization of the actin cytoskeleton during the cell cycle. The Arp3 protein is present in a large protein complex. This complex may mediate the cortical functions of profilin at actin patches in S. pombe (McCollum, 1996).

Deleting proline 96 and threonine 97, which are located close to the major actin binding site on profilin, does not significantly alter the interaction between profilin and phosphatidylinositol 4,5-bisphosphate, nor does it affect the profilin:poly(L-proline) interaction. The mutant protein, however, has a lower capacity to bind to actin in vitro than wild-type profilin, though it shows a slightly increased profilin-enhanced nucleotide exchange on the actin. When microinjected into Swiss 3T3 mouse fibroblasts or porcine aortic endothelial cells, the mutant profilin does not change the organization of the microfilament system as does wild-type profilin. This provides further evidence that profilin controls microfilament organization in the cell by interacting directly with actin (Hajkova, 1997).

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP2 and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, the Saccharomyces cerevisiae profilin gene (PFY1) was extensively mutagenized and the consequences of specific point mutations on growth and actin organization were examined. The actin-binding region of profilin is critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppresses defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin is required for fast rates of actin filament turnover. However, Act1-157p circumvents the requirement for profilin. Based on the results of these studies, it is concluded that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly (Wolven, 2000).

The mechanism of control of the steady state of actin assembly by actin depolymerizing factor (ADF)/cofilin and profilin has been investigated. Using Tbeta4 as an indicator of the concentration of ATP-G-actin, it has been shown that ADF increases the concentration of ATP-G-actin at steady state. The measured higher concentration of ATP-G-actin is quantitatively consistent with the increase in treadmilling, caused by the large increase in the rate of depolymerization from the pointed ends induced by ADF. Experiments have demonstrated that profilin synergizes with ADF to further enhance the turnover of actin filaments up to a value 125-fold higher than in pure F-actin solutions. Profilin and ADF act at the two ends of filaments in a complementary fashion to increase the processivity of treadmilling. Using the capping protein CapZ, it has been shown that ADF increases the number of filaments at steady state by 1.3-fold, which cannot account for the 25-fold increase in turnover rate. Computer modeling of the combined actions of ADF and profilin on the dynamics of actin filaments using experimentally determined rate constants generates a distribution of the different actin species at steady state, which is in quantitative agreement with the data (Didry, 1998).

The experimental demonstration of the synergy between ADF and profilin in the enhancement of filament turnover is a main point of this work. Profilin alone has a weak effect on filament turnover; however, the property of profilin-actin to participate in barbed end assembly is sufficient to explain the synergy between ADF and profilin. These two proteins act each at one end of the actin filament, in a complementary fashion. ADF increases the flux of depolymerizing subunits from the pointed ends, while profilin increases the flux of assembly onto the barbed ends at steady state. Profilin optimizes the directionality of treadmilling in two ways as follows: (1) profilin drives the ADP-bound state of actin toward the ATP-bound state, thus preventing the reverse pathway (association of ADF-ADP-actin with filament ends); (2) profilin replaces ATP-G-actin, which can associate with the two ends of the filament, by profilin-ATP-G-actin, which associates only with the barbed ends. Even in the presence of profilin, the rate-limiting step in the treadmilling cycle is still the dissociation from the pointed ends. The ADF-induced increase in the rate constant for actin dissociation from the pointed ends therefore is certainly at least 5-fold higher than the 25-fold increase found earlier. The measured rate of depolymerization from the pointed end was probably tempered by the reverse reaction of ADF-ADP-actin association to the pointed ends. Taking into account the slight increase (27 ± 10%) in the number of filaments observed in the presence of ADF, the conclusion that emerges from this work is that ADF increases the rate constant of depolymerization from the pointed ends at least 50-fold (Didry, 1998).

The rapid dynamics of actin filaments is a fundamental process that powers a large number of cellular functions. However, the basic mechanisms that control and coordinate such dynamics remain a central question in cell biology. To reach beyond simply defining the inventory of molecules that control actin dynamics and to understand how these proteins act synergistically to modulate filament turnover, evanescent-wave microscopy was combined with a biomimetic system and the behavior of single actin filaments was followed in the presence of a physiologically relevant mixture of accessory proteins. This approach allows for the real-time visualization of actin polymerization and age-dependent filament severing. In the presence of actin-depolymerizing factor (ADF)/cofilin and profilin, actin filaments with a processive formin attached at their barbed ends were observed to oscillate between stochastic growth and shrinkage phases. Fragmentation of continuously growing actin filaments by ADF/cofilin is the key mechanism modulating the prominent and frequent shortening events. The net effect of continuous actin polymerization, driven by a processive formin that uses profilin-actin, and of ADF/cofilin-mediating severing that trims the aged ends of the growing filaments is an up to 155-fold increase in the rate of actin-filament turnover in vitro in comparison to that of actin alone. Lateral contact between actin filaments dampens the dynamics and favors actin-cable formation. A kinetic simulation accurately validates these observations. A proposed mechanism for the control of actin dynamics is dominated by ADF/cofilin-mediated filament severing that induces a stochastic behavior upon individual actin filaments. When combined with a selection process that stabilizes filaments in bundles, this mechanism could account for the emergence and extension of actin-based structures in cells (Michelot, 2007).