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

Abstract

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.

Regulation of the Total Cell Surface Area in Dividing Dictyostelium Cells

“…We examined the contribution of CME on the surface area of dividing Dictyostelium cells by observing cells expressing GFP-clathrin under a total internal reflection fluorescence (TIRF) microscope. Many fluorescent dots, representing coated pits, were observed in the cell cortex of cells at the interphase and mitotic stages ( Figure 3A), which confirmed previous observations (Macro et al, 2012;Fujimoto et al, 2019). Figure 3B shows a typical time course of individual dots that appeared and then disappeared in the cortex.…”

“…Therefore, CME is not a key contributor to the suppression of the total membrane uptake. Incidentally, we could not find any gene homologous to caveolin in the Dictyostelium genome that would account for caveola-mediated endocytosis (Fujimoto et al, 2019). Presumably, other endocytosis mechanisms, such as macro-pinocytosis, may also be suppressed.…”

“…In Dictyostelium cells, we have not observed clathrin at the spindle. In Dictyostelium cells, clathrin evenly localizes in the cell cortex, whereas dynamin is known to localize at the cleavage furrow (Masud Rana et al, 2013;Fujimoto et al, 2019).…”

“…Expression vectors containing GFP-ABD (actin-binding domain of filamin), GFP-alpha-tubulin, GFP-clathrin light chain (Fujimoto et al, 2019), or GFP-histone were transformed into AX2 cells by electroporation or laserporation as described previously (Yumura et al, 1995;Yumura, 2016). Positive cells were selected using 10 µg/mL G418 (Wako, Osaka, Japan).…”

“…The fluorescence intensity of the stained cells was measured by a fluorescence spectrophotometer. To synchronize the cells, they were cultured at 10 • C for 16 h and then arrested at the metaphase by treating with 100 µM thiabendazole (TB), a microtubule depolymerizer, at 22 • C for 3.5 h (Fujimoto et al, 2019). After the removal of TB by centrifugation, most of the cells divided within 20 min.…”