Nobuhisa Umeki

The significance of membrane fluidity of feeder cell-derived substrates for maintenance of iPS cell stemness

The biological activity of cell-derived substrates to maintain undifferentiated murine-induced pluripotent stem (iPS) cells was correlated to membrane fluidity as a new parameter of cell culture substrates. Murine embryonic fibroblasts (MEFs) were employed as feeder cells and their membrane fluidity was tuned by chemical fixation using formaldehyde (FA). Membrane fluidity was evaluated by real-time single-molecule observations of green fluorescent protein-labeled epidermal growth factor receptors on chemically fixed MEFs. Biological activity was monitored by colony formation of iPS cells. Treatment with a low concentration of FA sustained the membrane fluidity and biological activity, which were comparable to those of mitomycin C-treated MEFs. The biological activity was further confirmed by sustained expression of alkaline phosphatase, SSEA-1, and other pluripotency markers in iPS cells after 3–5 days of culture on FA-fixed MEFs. Chemical fixation of feeder cells has several advantages such as providing ready-to-use culture substrates without contamination by proliferating feeder cells. Therefore, our results provide an important basis for the development of chemically fixed culture substrates for pluripotent stem cell culture as an alternative to conventional treatment by mitomycin C or x-ray irradiation.

Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein.

To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive.

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility

Background: The roles of conformational changes of actin in myosin motility are unclear. Results: A G146V mutation in actin, which perturbed its conformation, impaired force generation by myosin II, but not by myosin V. Conclusion: Conformational changes of actin involving Gly-146 have critical roles in motility of myosin II, but not of myosin V. Significance: The mechanism of motility may be different between myosin types. The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.

Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral

RalGDS is one of the Ras effectors and functions as a guanine nucleotide exchange factor for the small G-protein, Ral, which regulates membrane trafficking and cytoskeletal remodeling. The translocation of RalGDS from the cytoplasm to the plasma membrane is required for Ral activation. In this study, to understand the mechanism of Ras–Ral signaling we performed a single-molecule fluorescence analysis of RalGDS and its functional domains (RBD and REMCDC) on the plasma membranes of living HeLa cells. Increased molecular density of RalGDS and RBD, but not REMCDC, was observed on the plasma membrane after EGF stimulation of the cells to induce Ras activation, suggesting that the translocation of RalGDS involves an interaction between the GTP-bound active form of Ras and the RBD of RalGDS. Whereas the RBD played an important role in increasing the association rate constant between RalGDS and the plasma membrane, the REMCDC domain affected the dissociation rate constant from the membrane, which decreased after Ras activation or the hyperexpression of Ral. The Y64 residue of Ras and clusters of RalGDS molecules were involved in this reduction. From these findings, we infer that Ras activation not merely increases the cell-surface density of RalGDS, but actively stimulates the RalGDS–Ral interaction through a structural change in RalGDS and/or the accumulation of Ral, as well as the GTP–Ras/RalGDS clusters, to induce the full activation of Ral.

Mutation-Specific Mechanisms of Hyperactivation of Noonan Syndrome SOS Molecules Detected with Single-molecule Imaging in Living Cells

Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases.

Ca2+ Independent and Tail Dependent Regulation of the Motor Activity of Myosin X

Myosin X is involved in the actin cytoskeletal reorganization and protrusion of filopodia. Here we studied the molecular mechanism of regulation of myosin X. The actin-activated ATPase activity of M10Full was Ca2+ independent and significantly lower than that of M10HMM. The tail domain significantly inhibited the actin-activated ATPase activity of M10HMM regardless of Ca2+. The inhibition showed significant dependence on salt concentration, suggesting that the inhibition is dependent on ionic interaction between the tail domain and the head/neck domain of myosin X. The in vitro actin gliding velocity was markedly inhibited (4 fold) by the tail. These results suggest that the tail domain functions as an intra-molecular inhibitor of the myosin X motor function. The deletion of FERM domain abolished the inhibitory activity of the tail. On the other hand, deletion of the N-terminal PEST domain did not affect the inhibitory activity. Further truncation of the PH domain abolished the inhibitory activity of the tail. These results suggest that both the PH and FERM domains of the tail are required for the inhibition. On the other hand, the elimination of both IQ domains and the SAH/coiled-coil domain showed no effect on the tail induced inhibition. Furthermore, M10IQo co-immunoprecipitated with M10PH-FERM. The result indicated that the tail domain (PH-FERM) directly interacts with the motor domain to inhibit the motor activity. Electron microscopy revealed that the full-length myosin X molecules were monomeric, showing the wider molecules in low salt with ATP, while narrow molecules, similar head shapes to the M10HMM, in high salt. Our observation suggested that the tail domain folds backward to the head, such that it appeared to interact with the motor domain, and thus inhibits the motor activity of myosin X.(Supported by NIH).

Characterization of the Plant-Specific Rice Kinesins

Kinesin is a motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics and signal transduction. Kinesin converts chemical energy from ATP into mechanical force. Kinesin family is classified into some subfamilies. Some species of kinesin derived from vertebrate have been well studied. However, not so many studies for kinesins of plants have been done yet. Recently, the genome sequences of rice were completed. Bioinformatical analyses revealed that at least 41 kinesin-related proteins were encoded on the rice genome. In this study, we focused on the two rice kinesins; 1. O12 that has a calponin homology domain, 2. K23 that belongs to At1 subfamily in kinesin-7. The cDNAs of the kinesin motor domain was subcloned into expression vector pET and transformed into E. coli BL21 (DE3). kinesin motor domains were expressed and purified by Co-NTA column. The biochemical characterizations of the two rice kinesins were studied. The microtubule-dependent ATPase activity of the two rice kinesins motor domains were 30∼60-fold lower than that of conventional kinesin. Kinetic analyses using stopped-flow demonstrated that ATP binding to O12 in the absence of microtubule was extremely slow compared with that of conventional kinesin. While, ATP binding to K23 was not accelerated by microtubule. Furthermore, interestingly ATPase activity of O12 in the absence microtubule regulated by actin. The O12-tail fused with GFP was observed to localize in the actin filament in the onion cell. The two plant specific rice kinesin O12 and K23 were shown to have unique enzymatic properties.