Bridging cytoskeletal intersections.
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Actins↗
Biomedical subjects
I Karakesisoglou
Publications and source records attributed to I Karakesisoglou.
An epidermal plakin that integrates actin and microtubule networks at cellular junctions.
Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.
Actin Cytoskeleton↗
Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants.
Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated 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.
Animals↗
Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMP-II homologue.
Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme-mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type-like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin.
Amino Acid Sequence↗
Plant profilins rescue the aberrant phenotype of profilin-deficient Dictyostelium cells.
To characterize the function of plant profilins in vivo, we expressed two pollen specific Zea mays (maize) profilin isoforms in profilin-minus Dictyostelium discoideum mutants. In maize, profilins exist as a multigene family containing 4 or more members which are highly similar to each other but substantially less similar to profilins from animals and lower eukaryotes. Previously we have shown that D. discoideum profilin-minus cells have an aberrant phenotype due to defects in cell shape, cytokinesis, and development. These defects could be rescued by introducing the pollen-specific profilins 1 or 2 from maize using a newly constructed expression vector. Expression of the heterologous profilins in Dictyostelium clones was assayed by affinity purification of the pollen profilins with poly-proline agarose and by immunoblotting with a polyclonal antiserum raised against maize pollen profilin. In contrast to the profilin-minus mutants, Dictyostelium cells expressing plant profilins showed normal cell shape, contained less F-actin, and were able to form fruiting bodies. These data provide genetic evidence that maize pollen profilins, even though they are specific for a distinct developmental stage, share functional properties with profilin from a lower eukaryote and apparently act as G-actin-sequestering proteins in this system.
Animals↗
Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.
In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.
Actins↗