Interactive Fly, Drosophila



Profilin - actin interaction

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.
The enhancement of nucleotide exchange by profilin is therefore not involved in its promotion of actin assembly, and the productive growth of filaments from profilin-actin complex requires the coupling of ATP hydrolysis to profilin-actin assembly, a condition fulfilled by Mg-actin, and not by Ca-actin (Perelroizen, 1996).

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).

Profilin and actin filament turnover; synergy with cofilin

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).

Profilin - lipid interaction

Profilin is an actin- and phosphatidylinositol 4,5-bisphosphate-binding protein that plays a role in the organization of the cytoskeleton and may be involved in growth factor signaling pathways. The subcellular localization of profilin was examined in the yeast S. cerevisiae. Profilin was localized in both the plasma membrane and cytosolic fractions of the cell. Actin is bound to the profilin localized in the cytosol. The association of profilin with the membrane is peripheral and mediated through interaction with phospholipid. The phospholipid dependence of profilin for membrane binding was examined in vitro using pure profilin and defined unilamellar phospholipid vesicles. The presence of phosphatidylinositol 4,5-bisphosphate in phospholipid vesicles is required for maximum profilin binding. Moreover, the binding of profilin to phospholipid vesicles is dependent on the surface concentration of phosphatidylinositol 4,5-bisphosphate. The subcellular localization of profilin was examined in vivo under growth conditions (i.e. inositol starvation of ino1 cells and glucose starvation of respiratory deficient cells) where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate are depleted. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate levels results in a translocation of profilin from the plasma membrane to the cytosolic fraction. Profilin translocated back to the membrane fraction from the cytosol under growth conditions where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate are replenished. These results suggest that phosphoinositide metabolism played a role in the localization of profilin (Ostrander, 1995).

Bovine profilin isoforms bind both the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) (the target of Phospholipase C - see Protein kinase C for more information) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) (see Enabled) and cyclase-associated protein (CAP). Compared with profilin II, profilin I has a higher affinity for PIP2. However, proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes on binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, profilin II was identified as the preferred ligand for VASP. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways (Lambrechts, 1997).

Actin-binding proteins such as profilin and gelsolin bind to phosphatidylinositol (PI) 4,5-bisphosphate (PI 4,5-P2) and regulate the concentration of monomeric actin. Profilin and gelsolin stimulate PI 3-kinase-mediated phosphorylation of PI 4,5-P2 (lipid kinase activity) in a concentration-dependent manner. This effect is specific to profilin and gelsolin because other cytoskeletal proteins such as tau or actin do not affect PI 3-kinase activity. In addition to lipid kinase activity, PI 3-kinase also carries out protein kinase activity: it phosphorylates proteins (p85 subunit of PI 3-kinase). However, the protein kinase activity of PI 3-kinase is not affected in the presence of profilin. Profilin may also affect PI 3-kinase activity by its direct association to the enzyme. However, PI 3-kinase does not affect the actin-sequestering ability of profilin indicating that actin and p85 do not share a common binding site on profilin. These studies suggest that profilin and gelsolin may control the generation of 3-OH phosphorylated phosphoinositides, which in turn may regulate actin polymerization (Singh, 1996).

Although profilin's interactions with its three known ligands (poly-L-proline (PLP), phosphatidylinositol 4,5-bisphosphate (PIP2), and actin monomers) have been well characterized in vitro, its precise role in cells remains largely unknown. By binding to clusters of PIP2, profilin is able to inhibit the hydrolysis of PIP2 by phospholipase C gamma 1 (PLC gamma 1). This ability is the result of profilin's affinity for PIP2, but the specific residues of profilin's amino acid sequence involved in the binding of PIP2 are not known. The following mutants of human profilin were used (named according to the wild-type amino acid altered, its position, and the amino acid substituted in its place): Y6F, D8A, L10R, K25Q, K53I, R74L, R88L, R88L/K90E, H119D, G121D, and K125Q. With the exception of L10R, all of the mutants were successfully expressed in Escherichia coli and purified by affinity chromatography on PLP-Sepharose. Only Y6F and K25Q demonstrate moderately less stringent binding to PLP, indicating that most of the mutations do not induce marked alterations of profilin's structure. When tested for their relative abilities to inhibit the hydrolysis of PIP2 by PLC gamma 1, most of the mutants are indistinguishable from wild-type profilin. Exceptions include D8A, which demonstrates increased inhibition of PLC gamma 1, and R88L, which demonstrates decreased inhibition of PLC gamma 1. To assess the importance of the region surrounding residue 88 of human profilin, three synthetic decapeptides selected to correspond to non-overlapping stretches of the human profilin sequence were tested for their abilities to inhibit PLC gamma 1. Only the decapeptide that matches the peptide stretch centered around residue 88 is able to inhibit PLC gamma 1 activity substantially and is able to do so at nearly wild-type profilin levels. Taken together with the finding that mutating residue 88 results in decreased inhibition of PLC gamma 1 activity, these data provide strong evidence that this region of human profilin represents an important binding site for PIP2 (Sohn, 1995).

Depolymerization of actin filaments by profilin: effects of profilin on capping protein function

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1-5 microM). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. The effects were examined of higher concentrations of profilin (10-100 microM) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, 40 microM), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin has no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration is independent of monomer sequestration. Furthermore, it was found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments has not been appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization (Bubb, 2003).

Profilin response to Rho family members and interaction with Diaphanous homologs

continued: see Evolutionary Homologs part 2/2

chickadee: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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