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.

Cytokinesis D is known as the midwife mechanism in which neighboring cells facilitate cell division by crossing the cleavage furrow of dividing cells. Cytokinesis D is thought to be mediated by chemotaxis, where midwife cells migrate toward dividing cells by sensing an unknown chemoattractant secreted from the cleavage furrow. In this study, to validate this chemotaxis model, we aspirated the fluid from the vicinity of the cleavage furrow of a dividing Dictyostelium cell and discharged it onto a neighboring cell using a microcapillary. However, the neighboring cells did not show any chemotaxis toward the fluid. In addition, the cells did not manifest an increase in the levels of intracellular Ca²⁺, cAMP, or cGMP, which are expected to rise in chemotaxing cells. From several lines of our experiments, including these findings, we concluded that chemotaxis does not contribute to cytokinesis D. As an alternative, we propose a cortical-flow model, where a migrating cell attaches to a dividing cell by chance and is guided toward the furrow by the cortical flow on the dividing cell, and then physically assists the separation of the daughter cells.

In the field of cell and tissue engineering, there is an increasing demand for techniques to spatially control the adhesion of cells to substrates of desired sizes and shapes. Here, we describe two novel methods for fabricating a substrate for adhesion of cells to a defined area. In the first method, the surface of the coverslip or plastic dish was coated with Lipidure, a non-adhesive coating material, and air plasma was applied through a mask with holes, to confer adhesiveness to the surface. In the second method, after the surface of the coverslip was coated with gold by sputtering and then with Lipidure; the Lipidure coat was locally removed using a novel scanning laser ablation method. These methods efficiently confined cells within the adhesive area and enabled us to follow individual cells for a longer duration, compared to the currently available commercial substrates. By following single cells within the confined area, we were able to observe several new aspects of cell behavior in terms of cell division, cell–cell collisions, and cell collision with the boundary between adhesive and non-adhesive areas.