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.