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\nmaintenance of normal cellular physiology. Site-directed mutagenesis of profilin II from\nDictyostelium discoideum by PCR resulted in the point mutations W3N and K114E, whereby\nthe W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight\nreduction in actin-binding, whereas the K114E profilin shows profound decrease in its ability\nto interact with actin but its affinity for poly-(L)-proline remained unaltered. The in vivo\nproperties of the point-mutated profilins were studied by expressing either W3N or K114E in\nthe profilin-minus D. discoideum mutants which have defects in the F-actin content,\ncytokinesis and development (Haugwitz et al., 1994). Expression resulted in normal cell\nphysiology, a reduction in the F-actin content, and a complete development. Interestingly,\nonly cells which overexpressed W3N could restore the aberrant phenotype, while the K114E\nprofilin with its fully functional poly-(L)-proline binding and its strongly reduced actinbinding\nactivities rescued the phenotype at low concentrations. Both the wild-type and pointmutated\nprofilins are enriched in phagocytic cups during uptake of yeast particles. These data\nsuggest a) that a functional poly-(L)-proline binding activity is more important for\nsuppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that\nthe enrichment of profilin in highly active phagocytic cups might be independent of either\npoly-(L)-proline or actin-binding activities.\nTo have a better understanding of the in vivo role of profilin, D. discoideum profilin II has\nbeen tagged at its C-terminus with the green fluorescent protein (GFP) with a 100-aa linker\nseparating profilin and GFP. This fusion construct was introduced in D. discoideum profilinminus\ncells and expression of the fusion protein could restore the aberrant phenotype\npartially. The partial rescue might be due to the uneven expression of the fusion protein\nleading to mixed populations even after repeated recloning. The profilin-GFP transformants\nshowed normal cell morphology, could be cultivated in shaking suspensions, and could\ndevelop fruiting bodies which closely resembled those of the wild-type. In vivo studies\nrevealed the distribution of the fusion protein in highly active regions of the cells such as\nphagocytic cups, macropinocytotic crowns, cell cortex and at the leading edges of locomoting\ncells. Thus profilin appears to play a significant role in the regulation of the dynamic actinbased\ncellular processes.\nA second actin-regulatory protein from D. discoideum namely, severin, a Ca2+-dependent Factin\nfragmenting 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\nseverin null D. discoideum mutant exhibits normal growth, cell motility, chemotaxis and\ndevelopment. Examination of the live dynamics of severin-GFP should clarify the in vivo role\nof severin and other functionally redundant cytoskeletal proteins. The 70 kDa severin-GFP\nfusion protein has been sufficiently expressed and partially purified from the severin null cells\nwhereby in vitro assays confirmed the ability of this fusion protein to sever F-actin only in the\npresence of Ca2+. Data from confocal microscopy showed that the fusion protein was\ntransiently detected in macropinocytotic crowns, phagocytic cups, membrane ruffles, at the\nleading edges of motile cells and cell-cell contacts of aggregating cells in directed motion.\nThese data suggest an in vivo role for severin in the remodulation of existing F-actin\nstructures as supported by the in vitro data.\nThe highly dynamic cytoskeleton also plays a significant part in the defence of the cells\nagainst pathogens. The behaviour of the actin cytoskeleton of cultured mammalian cells in\nresponse to Yersinia enterocolitica infection was examined by confocal microscopy with the\naid of GFP-tagged actin, cofilin and profilin II. The translocated Yersinia outer proteins\n(Yops) encoded by a virulence plasmid in the wild-type bacteria have been observed to\ndisrupt the actin microfilaments, resulting in diffuse actin staining which subsequently\ndisappeared completely upon prolonged bacterial infection. In addition, F-actin structures\nresembling phagocytic cups were found at the sites of bacterial adherence, suggesting the\nlikelihood of the involvement of the Rho family of small GTPases in the regulation of the\nactin cytoskeleton. The secreted Yops appeared to have no major effect on the distribution of\nGFP-profilin whereas the staining pattern of GFP-cofilin seemed to be modified by the Yops,\nresulting in a decrease in length of the actin-cofilin rods and a diffuse localization of cofilin.\nThe exact mechanisms of interaction between the Yops and their host targets remain to be\ndetermined. However, a clearer insight into the interaction between pathogens and the host\ncytoskeleton will certainly aid in the cellular defence and the prevention of pathogenesis.