Functional studies of selected actin binding proteins by point mutations and GFP fusions

Profilin is an ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cellular physiology. Site-directed mutagenesis of profilin II from Dictyostelium discoideum by PCR resulted in the point mutations W3N and K114E, whereby the W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin-binding, whereas the K114E profilin shows profound decrease in its ability to interact with actin but its affinity for poly-(L)-proline remained unaltered. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in the profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., 1994). Expression resulted in normal cell physiology, a reduction in the F-actin content, and a complete development. Interestingly, only cells which overexpressed W3N could restore the aberrant phenotype, while the K114E profilin with its fully functional poly-(L)-proline binding and its strongly reduced actinbinding activities rescued the phenotype at low concentrations. Both the wild-type and pointmutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities. To have a better understanding of the in vivo role of profilin, D. discoideum profilin II has been tagged at its C-terminus with the green fluorescent protein (GFP) with a 100-aa linker separating profilin and GFP. This fusion construct was introduced in D. discoideum profilinminus cells and expression of the fusion protein could restore the aberrant phenotype partially. The partial rescue might be due to the uneven expression of the fusion protein leading to mixed populations even after repeated recloning. The profilin-GFP transformants showed normal cell morphology, could be cultivated in shaking suspensions, and could develop fruiting bodies which closely resembled those of the wild-type. In vivo studies revealed the distribution of the fusion protein in highly active regions of the cells such as phagocytic cups, macropinocytotic crowns, cell cortex and at the leading edges of locomoting cells. Thus profilin appears to play a significant role in the regulation of the dynamic actinbased cellular processes. A second actin-regulatory protein from D. discoideum namely, severin, a Ca2+-dependent Factin fragmenting and capping protein, was also investigated via fusion to GFP at its C-terminus. Although severin is a very active F-actin fragmenting protein in in vitro assays, the severin null D. discoideum mutant exhibits normal growth, cell motility, chemotaxis and development. Examination of the live dynamics of severin-GFP should clarify the in vivo role of severin and other functionally redundant cytoskeletal proteins. The 70 kDa severin-GFP fusion protein has been sufficiently expressed and partially purified from the severin null cells whereby in vitro assays confirmed the ability of this fusion protein to sever F-actin only in the presence of Ca2+. Data from confocal microscopy showed that the fusion protein was transiently detected in macropinocytotic crowns, phagocytic cups, membrane ruffles, at the leading edges of motile cells and cell-cell contacts of aggregating cells in directed motion. These data suggest an in vivo role for severin in the remodulation of existing F-actin structures as supported by the in vitro data. The highly dynamic cytoskeleton also plays a significant part in the defence of the cells against pathogens. The behaviour of the actin cytoskeleton of cultured mammalian cells in response to Yersinia enterocolitica infection was examined by confocal microscopy with the aid of GFP-tagged actin, cofilin and profilin II. The translocated Yersinia outer proteins (Yops) encoded by a virulence plasmid in the wild-type bacteria have been observed to disrupt the actin microfilaments, resulting in diffuse actin staining which subsequently disappeared completely upon prolonged bacterial infection. In addition, F-actin structures resembling phagocytic cups were found at the sites of bacterial adherence, suggesting the likelihood of the involvement of the Rho family of small GTPases in the regulation of the actin cytoskeleton. The secreted Yops appeared to have no major effect on the distribution of GFP-profilin whereas the staining pattern of GFP-cofilin seemed to be modified by the Yops, resulting in a decrease in length of the actin-cofilin rods and a diffuse localization of cofilin. The exact mechanisms of interaction between the Yops and their host targets remain to be determined. However, a clearer insight into the interaction between pathogens and the host cytoskeleton will certainly aid in the cellular defence and the prevention of pathogenesis.