During cell division, many animal cells transform into a spherical shape and assemble a contractile ring composed of actin filaments and myosin motors at the equator to separate the cell body into two. Although actomyosin regulatory proteins are spatio-temporally controlled during cytokinesis, the direct contribution of cell shape and actomyosin activity to the contractile ring assembly remains unclear. Here, we demonstrated in vitro that actin polymerization inside cell-sized spherical droplets induced the spontaneous formation of single ring-shaped actin bundles in the presence of bundling factors. Despite a lack of spatial regulatory signals, the rings always assembled at the equator to minimize the elastic energy of the bundles. Myosin promoted ring formation by the dynamic remodelling of actin networks, and an increase in the effective concentration of myosin triggered ring contraction. These results will help us understand how animal cells coordinate cell shape and actomyosin activities to direct cytokinesis.

In adult T-cell leukemia (ATL) cells, a defective human T-cell leukemia virus type 1 (HTLV-1) provirus lacking the 5 long terminal repeat (LTR), designated type 2 defective provirus, is frequently observed. To investigate the mechanism underlying the generation of the defective provirus, we sequenced HTLV-1 provirus integration sites from cases of ATL. In HTLV-1 proviruses retaining both LTRs, 6-bp repeat sequences were adjacent to the 5 and 3 LTRs. In 8 of 12 cases with type 2 defective provirus, 6-bp repeats were identified at both ends. In five of these cases, a short repeat was bound to CA dinucleotides of the pol and env genes at the 5 end, suggesting that these type 2 defective proviruses were formed before integration. In four cases lacking the 6-bp repeat, short (6-to 26-bp) deletions in the host genome were identified, indicating that these defective proviruses were generated after integration. Quantification indicated frequencies of type 2 defective provirus of less than 3.9% for two carriers, which are much lower than those seen for ATL cases (27.8%). In type 2 defective proviruses, the second exons of the tax, rex, and p30 genes were frequently deleted, leaving Tax unable to activate NF-B and CREB pathways. The HTLV-1 bZIP factor gene, located on the minus strand, is expressed in ATL cells with this defective provirus, and its coding sequences are intact, suggesting its significance in oncogenesis.

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.

Formins are force-sensing proteins that regulate actin polymerization dynamics. Here, we applied stretching tension to individual actin filaments under the regulation of formin mDia1 to investigate the mechanical responses in actin polymerization dynamics. We found that the elongation of an actin filament was accelerated to a greater degree by stretching tension for ADP-G-actin than that for ATP-G-actin. An apparent decrease in the critical concentration of G-actin was observed, especially in ADP-G-actin. These results on two types of G-actin were reproduced by a simple kinetic model, assuming the rapid equilibrium between pre- and posttranslocated states of the formin homology domain two dimer. In addition, profilin concentration dramatically altered the force-dependent acceleration of actin filament elongation, which ranged from twofold to an all-or-none response. Even under conditions in which actin depolymerization occurred, applications of a several-piconewton stretching tension triggered rapid actin filament elongation. This extremely high force-sensing mechanism of mDia1 and profilin could be explained by the force-dependent coordination of the biphasic effect of profilin; i.e., an acceleration effect masked by a depolymerization effect became dominant under stretching tension, negating the latter to rapidly enhance the elongation rate. Our findings demonstrate that the biphasic effect of profilin is controlled by mechanical force, thus expanding the function of mDia1 as a mechanosensitive regulator of actin polymerization.

Collective behaviors of motile units through hydrodynamic interactions induce directed fluid flow on a larger length scale than individual units. In cells, active cytoskeletal systems composed of polar filaments and molecular motors drive fluid flow, a process known as cytoplasmic streaming. The motor-driven elongation of microtubule bundles generates turbulent-like flow in purified systems; however, it remains unclear whether and how microtubule bundles induce large-scale directed flow like the cytoplasmic streaming observed in cells. Here, we adopted egg extracts as a model system of the cytoplasm and found that microtubule bundle elongation induces directed flow for which the length scale and timescale depend on the existence of geometrical constraints. At the lower activity of dynein, kinesins bundle and slide microtubules, organizing extensile microtubule bundles. In bulk extracts, the extensile bundles connected with each other and formed a random network, and vortex flows with a length scale comparable to the bundle length continually emerged and persisted for 1 min at multiple places. When the extracts were encapsulated in droplets, the extensile bundles pushed the droplet boundary. This pushing force initiated symmetry breaking of the randomly oriented bundle network, leading to bundles aligning into a rotating vortex structure. This vortex induced rotational cytoplasmic flows on the length scale and timescale that were 10- to 100-fold longer than the vortex flows emerging in bulk extracts. Our results suggest that microtubule systems use not only hydrodynamic interactions but also mechanical interactions to induce large-scale temporally stable cytoplasmic flow.