Dynamics of Actin Cytoskeleton and Their Signaling Pathways during Cellular Wound Repair.

The repair of wounded cell membranes is essential for cell survival. Upon wounding, actin transiently accumulates at the wound site. The loss of actin accumulation leads to cell death. The mechanism by which actin accumulates at the wound site, the types of actin-related proteins participating in the actin remodeling, and their signaling pathways are unclear. We firstly examined how actin accumulates at a wound site in Dictyostelium cells. Actin assembled de novo at the wound site, independent of cortical flow. Next, we searched for actin- and signal-related proteins targeting the wound site. Fourteen of the examined proteins transiently accumulated at different times. Thirdly, we performed functional analyses using gene knockout mutants or specific inhibitors. Rac, WASP, formin, the Arp2/3 complex, profilin, and coronin contribute to the actin dynamics. Finally, we found that multiple signaling pathways related to TORC2, the Elmo/Doc complex, PIP2-derived products, PLA2, and calmodulin are involved in the actin dynamics for wound repair.

Deletion of gmfA induces keratocyte-like migration in Dictyostelium.

Glia maturation factor (GMF) has been established as an inactivating factor of the actin-related protein 2/3 (Arp2/3) complex, which regulates actin assembly. Regulation of actin assembly and reorganization is crucial for various cellular events, such as cell migration, cell division, and development. Here, to examine the roles of ADF-H domain-containing protein (also known as glia maturation factor; GmfA), the product of a single GMF homologous gene in Dictyostelium, gmfA-null cells were generated. They had moderate defects in cell growth and cytokinesis. Interestingly, they showed a keratocyte-like fan shape with a broader pseudopod, where Arp3 accumulated at higher levels than in wild-type cells. They migrated with higher persistence, but their velocities were comparable to those of wild-type cells. The polar pseudopods during cell division were also broader than those in wild-type cells. However, GmfA did not localize at the pseudopods in migrating cells or the polar pseudopods in dividing cells. Adhesions of mutant cells to the substratum were much stronger than that of wild-type cells. Although the mutant cells showed chemotaxis comparable to that of wild-type cells, they formed disconnected streams during the aggregation stage; however, they finally formed normal fruiting bodies. These results suggest that GmfA plays a crucial role in cell migration.

Regulation of the Total Cell Surface Area in Dividing Dictyostelium Cells.

When a cell divides into two daughter cells, the total cell surface area should increase. There are two models for membrane supply to support cell division: (1) unfolding of small surface membrane reservoirs such as microvilli or wrinkles and (2) exocytosis of intracellular vesicles. Here, we precisely measured the total cell surface area in dividing Dictyostelium cells, flattened by the agar overlay that eliminated the complexity of unfolding surface membrane reservoirs. Because the cells divided normally under the agar overlay, unfolding of surface membrane reservoirs was not required for cell division. Under the agar overlay, the total cell surface area slightly decreased from the interphase to the metaphase and then increased about 20% during cytokinesis. Both endocytosis and exocytosis were suppressed in the early mitotic phase but recovered during cytokinesis. The imbalance of endocytosis and exocytosis could contribute to the changes observed in the cell surface area. Clathrin-dependent endocytosis was also substantially suppressed during cytokinesis, but contrary to previous reports in cultured animal cells, it did not significantly contribute to the regulation of the cell surface area. Furrowing during cytokinesis was indispensable for the cell membrane increase, and vice versa.

Ca2+-Calmodulin Dependent Wound Repair in Dictyostelium Cell Membrane.

Wound repair of cell membrane is a vital physiological phenomenon. We examined wound repair in Dictyostelium cells by using a laserporation, which we recently invented. We examined the influx of fluorescent dyes from the external medium and monitored the cytosolic Ca2+ after wounding. The influx of Ca2+ through the wound pore was essential for wound repair. Annexin and ESCRT components accumulated at the wound site upon wounding as previously described in animal cells, but these were not essential for wound repair in Dictyostelium cells. We discovered that calmodulin accumulated at the wound site upon wounding, which was essential for wound repair. The membrane accumulated at the wound site to plug the wound pore by two-steps, depending on Ca2+ influx and calmodulin. From several lines of evidence, the membrane plug was derived from de novo generated vesicles at the wound site. Actin filaments also accumulated at the wound site, depending on Ca2+ influx and calmodulin. Actin accumulation was essential for wound repair, but microtubules were not essential. A molecular mechanism of wound repair will be discussed.

Dynamin-Like Protein B of Dictyostelium Contributes to Cytokinesis Cooperatively with Other Dynamins.

Dynamin is a large GTPase responsible for diverse cellular processes, such as endocytosis, division of organelles, and cytokinesis. The social amoebozoan, Dictyostelium discoideum, has five dynamin-like proteins: dymA, dymB, dlpA, dlpB, and dlpC. DymA, dlpA, or dlpB-deficient cells exhibited defects in cytokinesis. DlpA and dlpB were found to colocalize at cleavage furrows from the early phase, and dymA localized at the intercellular bridge connecting the two daughter cells, indicating that these dynamins contribute to cytokinesis at distinct dividing stages. Total internal reflection fluorescence microscopy revealed that dlpA and dlpB colocalized at individual dots at the furrow cortex. However, dlpA and dlpB did not colocalize with clathrin, suggesting that they are not involved in clathrin-mediated endocytosis. The fact that dlpA did not localize at the furrow in dlpB null cells and vice versa, as well as other several lines of evidence, suggests that hetero-oligomerization of dlpA and dlpB is required for them to bind to the furrow. The hetero-oligomers directly or indirectly associate with actin filaments, stabilizing them in the contractile rings. Interestingly, dlpA, but not dlpB, accumulated at the phagocytic cups independently of dlpB. Our results suggest that the hetero-oligomers of dlpA and dlpB contribute to cytokinesis cooperatively with dymA.

A study of wound repair in Dictyostelium cells by using novel laserporation.

We examined the mechanism of cell membrane repair in Dictyostelium cells by using a novel laser-based cell poration method. The dynamics of wound pores opening and closing were characterized by live imaging of fluorescent cell membrane proteins, influx of fluorescent dye, and Ca2+ imaging. The wound closed within 2-4 sec, depending on the wound size. Cells could tolerate a wound size of less than 2.0 µm. In the absence of Ca2+ in the external medium, the wound pore did not close and cells ruptured. The release of Ca2+ from intracellular stores also contributed to the elevation of cytoplasmic Ca2+ but not to wound repair. Annexin C1 immediately accumulated at the wound site depending on the external Ca2+ concentration, and annexin C1 knockout cells had a defect in wound repair, but it was not essential. Dictyostelium cells were able to respond to multiple repeated wounds with the same time courses, in contrast to previous reports showing that the first wound accelerates the second wound repair in fibroblasts.