The profile of profilins.

Profilins are small proteins involved in actin dynamics. In accordance with this function, they are found in all eukaryotes and are structurally highly conserved. However, their precise role in regulating actin-related functions is just beginning to emerge. This article recapitulates the wealth of information on structure, expression and functions accumulated on profilins from many different organisms in the 30 years after their discovery as actin-binding proteins. Emphasis is given to their interaction with a plethora of many different ligands in the cytoplasm as well as in the nucleus, which is considered the basis for their various activities and the significance of the tissue-specific expression of profilin isoforms.

Characterization of functional domains of mDia1, a link between the small GTPase Rho and the actin cytoskeleton.

The widely expressed diaphanous proteins, a subclass of formins, comprise links between the Rho GTPases and the actin-based cytoskeleton. They contain several functional domains that are thought to be responsible for interaction with different ligands: the FH1 domain for binding the actin-associated protein profilin; the RBD for targeting activated Rho; and the C-terminal CIID module for autoregulation of the overall diaphanous activity. Using deletion constructs of the murine mDia1, we have analyzed the functional properties of these three domains separately in in vitro assays and in transiently and stably transfected cell lines. We show that the proline-rich FH1 domain effectively binds to profilins in vitro as well as in cells, that the RBD complexes with the CIID in a species-restricted manner and that overexpression of RBD causes spontaneous ruffling and loss of stress fibers, together with loss of directional motility. Supertransfection of cells stably expressing the RBD with dominant negative Rac effectively suppresses ruffling. Our data contribute to the understanding of the function of these domains in linking the actin cytoskeleton with the Rho-signaling cascade. Furthermore, they suggest that inactivation of Rho by exogenous RBD causes upregulation of Rac activity in the transfected cells.

Functional characterization of green fluorescent protein-profilin fusion proteins.

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose. Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.

Accumulation of profilin II at the surface of Listeria is concomitant with the onset of motility and correlates with bacterial speed.

The spatial and temporal activity of the actin cytoskeleton is precisely regulated during cell motility by several microfilament-associated proteins of which profilin plays an essential role. We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Listeria-infected cells. Among the different GFP-profilin fusion proteins studied, only the construct in which the GFP moiety was fused to the carboxy terminus of profilin II (profilin II-GFP) was recruited by intracellular Listeria. The in vitro ligand-binding properties of this construct, e.g. the binding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisphosphate (PIP2), were unaffected by GFP. Profilin II-GFP co-localised with vinculin and Mena to the focal adhesions in REF-52 fibroblasts and was distributed as a thin line at the front of protruding lamellipodia in B16-F1 mouse melanoma cells. In Listeria-infected cells, profilin II-GFP was recruited, in an asymmetric fashion, to the surface of Listeria at the onset of motility whereas it was not detectable on non-motile bacteria. In contrast to the vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria. Moreover, the fluorescence intensity of profilin II-GFP directly correlated with the speed of the bacteria. Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility.

A role for polyproline motifs in the spinal muscular atrophy protein SMN. Profilins bind to and colocalize with smn in nuclear gems.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the loss of alpha-motoneurons in the spinal cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have been identified on human chromosome 5q. The corresponding SMN protein is expressed ubiquitously. It is coded by seven exons and contains conspicuous proline-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to bind to profilins (PFNs), small proteins engaged in the control of actin dynamics. We tested whether profilins interact with SMN via its polyproline stretches. Using the yeast two-hybrid system we show that profilins bind to SMN and that this binding depends on its proline-rich motifs. These results were confirmed by coimmunoprecipitation and by in vitro binding studies. Two PFN isoforms, I and II, are known, of which II is characteristic for central nervous system tissue. We show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons. In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN in the cytoplasm of the cell body and in nuclear gems. Likewise, SMN and PFN I colocalize in gems of HeLa cells. Although SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the SMN genes are expressed ubiquitously, our findings suggest that the interaction of PFN II with SMN may be involved in neuron-specific effects of SMN mutations.

Plant and animal profilins are functionally equivalent and stabilize microfilaments in living animal cells.

We have analyzed the degree of functional similarity between birth and mammalian profilins, two members of the profilin family which show only a moderate sequence homology (22%) in living animal cells. The plant profilin, derived from birch pollen, was stably expressed in BHK-21 cells. Plant and endogenous profilin synthesis and cellular distribution were monitored by specific monoclonal antibodies. Quantitation of profilin and actin on calibrated immunoblots showed that two stable clones contained in total 1.4 and 2.0 times as much profilin as the parental cells. Using double fluorescence and confocal laser scanning microscopy, it was seen that the endogenous and the plant profilin colocalized with dynamic microfilaments, in particular with F-actin-rich foci and cortical microfilament webs of spreading cells, with dynamic microfilament bundles induced by serum deprival, and with cytochalasin D- and latrunculin-induced transient F-actin aggregates. The increase in the overall profilin concentration correlated with a significantly higher resistance of actin filaments to these drugs. Our data indicate that even profilins of highly distant evolutionary origin can functionally substitute for each other and support the hypothesis that in animal cells, profilins are engaged in regulating either the stability or the kinetic properties of actin filaments.

The molecular architecture of focal adhesions.

This article outlines the present knowledge of the architecture, molecular composition, and dynamics of focal contacts of adhesive animal cells. These structures, developed at the plasma membrane at sites where cells touch their substratum, are essential for cellular attachment in tissue formation during embryogenesis and wound healing. In tissue culture, they are particularly prominent and thus amenable to detailed investigation. Focal contacts consist of a cytoplasmic face, comprising cytoskeletal elements, a transmembrane connecting region, and a extracellular face composed of proteins of the extracellular matrix. The molecular anatomy of the numerous proteins involved, the basis for classifying them as structural or regulatory components, and their in vitro interactions are described. Based on this information, current models on the dynamics of their assembly and of possible regulatory mechanisms involving a variety of signal transduction pathways are discussed.

Interaction of plant profilin with mammalian actin.

The mode of interaction of birch and bovine profilins with actin was compared using a number of techniques. Birch profilin was purified from pollen or as a recombinant protein from Escherichia coli, using poly(L-proline) affinity chromatography and a monoclonal antibody for the identification of the isolated product. On two-dimensional gels, the genuine and recombinant proteins were identical in molecular mass and isoelectric point and revealed that birch profilin, in contrast to the basic profilins found in mammals, is an acidic protein, analogous to maize profilins. Bovine profilin was obtained from calf thymus. In viscometric assays, the birch protein was seen to modulate actin filament formation analogous to animal profilin. Birch profilin increased the critical concentration required for muscle and brain actin polymerization in a concentration-dependent manner, supporting the notion of the formation of a heterologous complex between the plant protein and animal actin. The effect was Mg(2+)-sensitive, as had been described for homologous complexes. The dissociation constants obtained for the plant/vertebrate and the vertebrate/vertebrate system were both in the micromolar range. The affinity of birch profilin for muscle actin was slightly lower than that for nonmuscle (brain) actin. A binary complex of birch profilin and skeletal muscle actin could be isolated by gel chromatography. Cross-linking experiments with actin, birch profilin, the G-actin binding peptide thymosin beta 4 and gelsolin segment 1, the N-terminal fragment of an actin capping protein, showed that profilin competed with thymosin beta 4, but had no effect on segment 1 binding to actin. These data indicate that the actin-binding domains in plant and animal profilins are functionally highly conserved, although the overall sequence similarity is less than 25%.