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Masahiro Ueda

Publications and source records attributed to Masahiro Ueda.

8 recordsLinked to original sources
PubMed2006

Input-output relationship in galvanotactic response of Dictyostelium cells.

Under a direct current electric field, Dictyostelium cells exhibit migration towards the cathode. To determine the input-output relationship of the cell's galvanotactic response, we developed an experimental instrument in which electric signals applied to the cells are highly reproducible and the motile response are analyzed quantitatively. With no electric field, the cells moved randomly in all directions. Upon applying an electric field, cell migration speeds became about 1.3 times faster than those in the absence of an electric field. Such kinetic effects of electric fields on the migration were observed for cells stimulated between 0.25 and 10 V/cm of the field strength. The directions of cell migrations were biased toward the cathode in a positive manner with field strength, showing galvanotactic response in a dose-dependent manner. Quantitative analysis of the relationship between field strengths and directional movements revealed that the biased movements of the cells depend on the square of electric field strength, which can be described by one simple phenomenological equation. The threshold strength for the galvanotaxis was between 0.25 and 1 V/cm. Galvanotactic efficiency reached to half-maximum at 2.6 V/cm, which corresponds to an approximate 8 mV voltage difference between the cathode and anode direction of 10 microm wide, round cells. Based on these results, possible mechanisms of galvanotaxis in Dictyostelium cells were discussed. This development of experimental system, together with its good microscopic accessibility for intracellular signaling molecules, makes Dictyostelium cells attractive as a model organism for elucidating stochastic processes in the signaling systems responsible for cell motility and its regulations.

Animals↗
PubMed2006

Stochastic signal inputs for chemotactic response in Dictyostelium cells revealed by single molecule imaging techniques.

Chemotactic cells can exhibit extreme sensitivity to chemical gradients. Theoretical estimations of the signal inputs required for chemotaxis suggest that the response can be achieved under the strong influence of stochastic input noise generated by the receptors during the transmembrane signaling. This arises a fundamental question regarding the mechanisms for directional sensing: how do cells obtain reliable information regarding gradient direction by using stochastically operating receptors and the downstream molecules? To address this question, we have developed single molecule imaging techniques to visualize signaling molecules responsible for chemotaxis in living Dictyostelium cells, allowing us to monitor the stochastic signaling processes directly. Single molecule imaging of a chemoattractant bound to a receptor demonstrates that signal inputs fluctuate with time and space. Downstream signaling molecules, such as PTEN and a PH domain-containing protein that are constituent parts of chemotactic signaling system, can also be followed at single molecule level in living cells, illuminating the stochastic nature of chemotactic signaling processes. In this report, we start with a brief introduction of chemotactic response of the eukaryotic cells, followed by an explanation for single molecule imaging techniques, and finally discuss these applications to chemotactic signaling system of Dictyostelium cells.

Animals↗
PubMed2006

Brownian motion, fluctuation and life.

The measurements of dynamic behaviors of biomolecules in relation to their functions have been allowed using single molecule measurements. Thermal Brownian motion causes random step motion of motor proteins and structural fluctuation of protein molecules between multiple states. In hierarchic structure of life, the fluctuation is modulated. Random fluctuation is biased to directional motion and reactions as a result of interaction of proteins. The fluctuation of kinetic state of signaling proteins results in polarization and localization of cells. A recognition process in brain is also explained by the equation analogous to biochemical reaction at the molecular level. Thus dynamic processes originated from thermal motion may play an important role in activation processes in life.

Actins↗
PubMed2006

Tumor suppressor PTEN acts through dynamic interaction with the plasma membrane.

The tumor suppressor function of PTEN is strongly linked to its ability to dephosphorylate phosphatidylinositol-3,4,5 trisphosphate and, thereby, control cell growth, survival, and migration. However, the mechanism of action of PTEN in living cells is largely unexplored. Here we use single-molecule TIRF microscopy in living cells to reveal that the enzyme binds to the membrane for a few hundred milliseconds, sufficient to degrade several phosphatidylinositol-3,4,5 trisphosphate molecules. Deletion of an N-terminal lipid-binding motif completely abrogates membrane interaction and in vivo function. Several mechanisms, including C-terminal tail phosphorylations, appear to hold PTEN in a constrained conformation that limits its rate of association with the membrane. The steady-state level of bound PTEN is highest at sites of retracting membrane, including the rear of highly polarized cells. The dynamic membrane association could be modulated temporally or spatially to alter PTEN activity in specific physiological situations and could have important implications for tumor suppressor function.

Amino Acid Motifs↗
PubMed2006

Single-molecule analysis of chemoattractant-stimulated membrane recruitment of a PH-domain-containing protein.

Molecular mechanisms of chemotactic response are highly conserved among many eukaryotic cells including human leukocytes and Dictyostelium discoideum cells. The cells can sense the differences in chemoattractant concentration across the cell body and respond by extending pseudopods from the cell side facing to a higher concentration. Pseudopod formation is regulated by binding of pleckstrin homology (PH)-domain-containing proteins to phosphatidylinositol 3,4,5-trisphosphates [PtdIns(3,4,5)P3] localized at the leading edge of chemotaxing cells. However, molecular mechanisms underlying dynamic features of a pseudopod have not been fully explained by the known properties of PH-domain-containing proteins. To investigate the mechanisms, we visualized single molecules of green fluorescent protein tagged to Crac (Crac-GFP), a PH-domain-containing protein in D. discoideum cells. Whereas populations of Crac molecules exhibited a stable steady-state localization at pseudopods, individual molecules bound transiently to PtdIns(3,4,5)P3 for approximately 120 milliseconds, indicating dynamic properties of the PH-domain-containing protein. Receptor stimulation did not alter the binding stability but regulated the number of bound PH-domain molecules by metabolism of PtdIns(3,4,5)P3. These results demonstrate that the steady-state localization of PH-domain-containing proteins at the leading edge of chemotaxing cells is dynamically maintained by rapid recycling of individual PH-domain-containing proteins. The short interaction between PH domains and PtdIns(3,4,5)P3 contributes to accurate and sensitive chemotactic movements through the dynamic redistributions. These dynamic properties might be a common feature of signaling components involved in chemotaxis.

Animals↗
PubMed2005

Total internal reflection fluorescence microscopy in single molecule nanobioscience.

Recent development in total internal reflection fluorescence microscopy (TIRFM) has made it possible to directly monitor the behaviors of biomolecules at the single-molecule level both in vitro and in living cells. This technique has opened a new era in bioscience, so-called single-molecule nanobioscience. In this review, we will summarize the theoretical basis of TIRFM, explain the instruments of TIRFM for single-molecules imaging, and discuss some novel applications of TIRFM to single-molecule nanobioscience.

Equipment Design↗
PubMed2002

Efficacy of a B virus gD DNA vaccine for induction of humoral and cellular immune responses in Japanese macaques.

It is desirable to prevent dissemination of B virus (BV) in macaque colonies because transmission of BV to humans causes deadly encephalomyelitis. Vaccination of monkeys is one method that could confine spread of BV within macaque colonies. Availability of a BV DNA vaccine for use in macaques would eliminate the risk of working with infectious BV. Toward this end, we constructed a plasmid expressing the BV glycoprotein D (gD). Immunogenicity of this construct as a DNA vaccine was assessed in adult Japanese macaques by four intracutaneous injections at a dose of 500 microg per head. Results of enzyme-linked immunosorbent assay (ELISA) using a recombinant herpes simplex virus type 1 (HSV1) gD, a homologue of BV gD, showed that significant levels of antibody was induced in all vaccinated animals following each booster injection. Western blot of sera from vaccinated macaques confirmed the specific recognition of authentic BV gD. Immune sera were also demonstrated to contain neutralizing activity against infectious BV. Weak lymphoproliferative responses were also observed in vaccinated macaques using recombinant HSV1 gD as a stimulating antigen and flow cytometry analysis of one individual revealed the presence of HSV1 gD-responsive effector T cells. Thus, the BV gD DNA vaccine was demonstrated to induce both humoral and cellular immune responses in macaques which recognized BV gD.

Animals↗