Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin ... more Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin (HA) and neuraminidase (NA), and is a major determinant of virus infectivity. To translocate a virus particle on the cell surface, HA molecules exchange viral receptors and NA molecules accelerate the receptor exchange of HA. This type of virus motility was recently identified in influenza A virus (IAV). To determine if other influenza virus types have a similar receptor exchange mechanism-driven motility, we investigated influenza C virus (ICV) motility on a receptor-fixed glass surface. This system excludes receptor mobility, which makes it more desirable than a cell surface for demonstrating virus motility by receptor exchange. Like IAV, ICV was observed to move across the receptor-fixed surface. However, in contrast to the random movement of IAV, a filamentous ICV strain, Ann Arbor/1/50 (AA), moved in a straight line, in a directed manner, and at a constant rate, whereas a spherical ...
Japan General IncorporatedAssociation unctear. Recently, we showed that the cellular rcdox {reduc... more Japan General IncorporatedAssociation unctear. Recently, we showed that the cellular rcdox {reduction-oxidation) peise deterrnines the phototactic sign/ ce]ls shew positive phototaxis after tTeutment with reactive oxygen species (ROS), whereas they show negative phototaxis after treatment with ROS quenchers. This finding raised two questions of 1) how cellular redox poise changes in vivo and 2) what is the motecutar basis for the redox-regulated switching ofthe phototactic sign. Regarding ]), we foeused on state transitions of photosynthesis, because the ROS generation has been suggested to greatly change depending on the state of photosynthesis. Lowtemperuture fluoreseence spectroseopy revea]ed that cetls shewing positive phototaxis are in State 1, whereas those showing negative phDtotaxis are in State 2. We propose a rnode] that changes in the intrace]lular ROS amount. caused by state transitions. function as a slgna] for the phototactic sign switching. Regarding 2), we found that two previous]y isolated phototaxis-deficient mutants have defects in redox sensing. We also isolated a new mutant that responds to the treatment with redox reagents in an opposite manner to that of wilcl type. We will present the resu]ts ofphenotypic analyses.
to be heterogeneous. Such a spatial hcteTogeneity of signaling molecules could affect the cAMP rc... more to be heterogeneous. Such a spatial hcteTogeneity of signaling molecules could affect the cAMP rcsponsc. !n the present study, we first show the transicnt response of Ptdlns(3,4,5)P3 by the step increase of cAMP eoncentrations, and found that the respense is spatially localized against the small increase of cAMP concentration.
... Sato Masayuki J. Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka... more ... Sato Masayuki J. Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka Univ. Egmond Wouter N. van; Dept of Molecular Cell ... NII論文ID(NAID) : 110007013715. NII書誌ID(NCID) : AN00129693. 本文言語コード : ENG. ISSN : 05824052. 収録DB : NII-ELS. ...
Dynamics maintaining diversity of cell types in a multi-cellular system are studied in relationsh... more Dynamics maintaining diversity of cell types in a multi-cellular system are studied in relationship with the plasticity of cellular states. First, we introduce a new theoretical framework, reaction-diffusion system on `chemical species space' to model intra-cellular chemical reaction dynamics. Then, by considering the cell division and death of such cells, developmental process from a single cell is studied. Cell differentiation process is found to occur through instability in transient dynamics and cell-cell interaction. In a long time behavior, extinction of multiple cells is repeated, which leads to itinerancy over successive quasi-stable multi-cellular states consisting of different types of cells. By defining the plasticity of a cellular state, it is shown that the plasticity of cells decreases before the large extinction, from which diversity and plasticity are recovered. After this switching, decrease of plasticity again occurs, leading to the next extinction of multiple...
Global cellular responses induced by epidermal growth factor (EGF) receptor (EGFR) occur immediat... more Global cellular responses induced by epidermal growth factor (EGF) receptor (EGFR) occur immediately with a less than 1% occupancy among tens of thousands of EGFR molecules on single cell surface. Activation of EGFR requires the formation of a signaling dimer of EGFR bound with a single ligand to each molecule. How sufficient numbers of signaling dimers are formed at such low occupancy rate is still not known. Here, we have analyzed the kinetics of EGF binding and the formation of the signaling dimer using single-molecule imaging and mathematical modeling. A small number of EGFR on the cell surface formed dimeric binding sites, which bound EGF two orders of magnitude faster than the monomeric binding sites. There was a positive cooperative binding of EGF to the dimeric binding sites through a newly discovered kinetic intermediate. These two mechanisms facilitate the formation of signaling dimers of EGF/EGFR complexes.
Proceedings of the National Academy of Sciences, 2007
Phosphorylation of the cytoplasmic tyrosine residues of the epidermal growth factor receptor (EGF... more Phosphorylation of the cytoplasmic tyrosine residues of the epidermal growth factor receptor (EGFR) upon binding of EGF induces recognition of various intracellular signaling molecules, including Grb2. Here, the reaction kinetics between EGFR and Grb2 was analyzed by visualizing single molecules of Grb2 conjugated to the fluorophore Cy3 (Cy3-Grb2). The plasma membrane fraction was purified from human epithelial carcinoma A431 cells after stimulation with EGF and attached to coverslips. Unitary events of association and dissociation of Cy3-Grb2 on the EGFR in the membrane fraction were observed at different concentrations of Grb2 (0.1-100 nM). The dissociation kinetics could be explained by using a multiple-exponential function with a major (>90%) dissociation rate of 8 s ؊1 and a few minor components, suggesting the presence of multiple bound states. In contrast, the association kinetics could be described by a stretched exponential function, suggesting the presence of multiple reaction channels from many unbound substates. Transitions between the unbound substates were also suggested. Unexpectedly, the rate of association was not proportional to the Grb2 concentration: an increase in Cy3-Grb2 concentration by a factor of 10 induced an increase in the reaction frequency approximately by a factor of three. This effect can compensate for fluctuation of the signal transduction from EGFR to Grb2 caused by variations in the expression level of Grb2 in living cells.
Under a direct current electric field, Dictyostelium cells exhibit migration towards the cathode.... more 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.
Quantitative relationships between inputs and outputs of signaling systems are fundamental inform... more Quantitative relationships between inputs and outputs of signaling systems are fundamental information for the understanding of the mechanism of signal transduction. Here we report the correlation between the number of epidermal growth factor (EGF) bindings and the response probability of intracellular calcium elevation. Binding of EGF molecules and changes of intracellular calcium concentration were measured for identical HeLa human epithelial cells. It was found that 300 molecules of EGF were enough to induce calcium response in half of the cells. This number is quite small compared to the number of EGF receptors (EGFR) expressed on the cell surface (50,000). There was a sigmoidal correlation between the response probability and the number of EGF bindings, meaning an ultrasensitive reaction. Analysis of the cluster size distribution of EGF demonstrated that dimerization of EGFR contributes to this switch-like ultrasensitive response. Single-molecule analysis revealed that EGF bound faster to clusters of EGFR than to monomers. This property should be important for effective formation of signaling dimers of EGFR under very small numbers of EGF bindings and suggests that the expression of excess amounts of EGFR on the cell surface is required to prepare predimers of EGFR with a large association rate constant to EGF.
Mechanochemical coupling was studied for two different types of myosin motors in cells: myosin V,... more Mechanochemical coupling was studied for two different types of myosin motors in cells: myosin V, which carries cargo over long distances by as a single molecule; and myosin II, which generates a contracting force in cooperation with other myosin II molecules. Both mean and variance of myosin V velocity at various [ATP] obeyed Michaelis-Menten mechanics, consistent with tight mechanochemical coupling. Myosin II, working in an ensemble, however, was explained by a loose coupling mechanism, generating variable step sizes depending on the ATP concentration and realizing a much larger step (200 nm) per ATP hydrolysis than myosin V through its cooperative nature at zero load. These different mechanics are ideal for the respective myosin's physiological functions.
Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin ... more Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin (HA) and neuraminidase (NA), and is a major determinant of virus infectivity. To translocate a virus particle on the cell surface, HA molecules exchange viral receptors and NA molecules accelerate the receptor exchange of HA. This type of virus motility was recently identified in influenza A virus (IAV). To determine if other influenza virus types have a similar receptor exchange mechanism-driven motility, we investigated influenza C virus (ICV) motility on a receptor-fixed glass surface. This system excludes receptor mobility, which makes it more desirable than a cell surface for demonstrating virus motility by receptor exchange. Like IAV, ICV was observed to move across the receptor-fixed surface. However, in contrast to the random movement of IAV, a filamentous ICV strain, Ann Arbor/1/50 (AA), moved in a straight line, in a directed manner, and at a constant rate, whereas a spherical ...
Japan General IncorporatedAssociation unctear. Recently, we showed that the cellular rcdox {reduc... more Japan General IncorporatedAssociation unctear. Recently, we showed that the cellular rcdox {reduction-oxidation) peise deterrnines the phototactic sign/ ce]ls shew positive phototaxis after tTeutment with reactive oxygen species (ROS), whereas they show negative phototaxis after treatment with ROS quenchers. This finding raised two questions of 1) how cellular redox poise changes in vivo and 2) what is the motecutar basis for the redox-regulated switching ofthe phototactic sign. Regarding ]), we foeused on state transitions of photosynthesis, because the ROS generation has been suggested to greatly change depending on the state of photosynthesis. Lowtemperuture fluoreseence spectroseopy revea]ed that cetls shewing positive phototaxis are in State 1, whereas those showing negative phDtotaxis are in State 2. We propose a rnode] that changes in the intrace]lular ROS amount. caused by state transitions. function as a slgna] for the phototactic sign switching. Regarding 2), we found that two previous]y isolated phototaxis-deficient mutants have defects in redox sensing. We also isolated a new mutant that responds to the treatment with redox reagents in an opposite manner to that of wilcl type. We will present the resu]ts ofphenotypic analyses.
to be heterogeneous. Such a spatial hcteTogeneity of signaling molecules could affect the cAMP rc... more to be heterogeneous. Such a spatial hcteTogeneity of signaling molecules could affect the cAMP rcsponsc. !n the present study, we first show the transicnt response of Ptdlns(3,4,5)P3 by the step increase of cAMP eoncentrations, and found that the respense is spatially localized against the small increase of cAMP concentration.
... Sato Masayuki J. Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka... more ... Sato Masayuki J. Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka Univ. Egmond Wouter N. van; Dept of Molecular Cell ... NII論文ID(NAID) : 110007013715. NII書誌ID(NCID) : AN00129693. 本文言語コード : ENG. ISSN : 05824052. 収録DB : NII-ELS. ...
Dynamics maintaining diversity of cell types in a multi-cellular system are studied in relationsh... more Dynamics maintaining diversity of cell types in a multi-cellular system are studied in relationship with the plasticity of cellular states. First, we introduce a new theoretical framework, reaction-diffusion system on `chemical species space' to model intra-cellular chemical reaction dynamics. Then, by considering the cell division and death of such cells, developmental process from a single cell is studied. Cell differentiation process is found to occur through instability in transient dynamics and cell-cell interaction. In a long time behavior, extinction of multiple cells is repeated, which leads to itinerancy over successive quasi-stable multi-cellular states consisting of different types of cells. By defining the plasticity of a cellular state, it is shown that the plasticity of cells decreases before the large extinction, from which diversity and plasticity are recovered. After this switching, decrease of plasticity again occurs, leading to the next extinction of multiple...
Global cellular responses induced by epidermal growth factor (EGF) receptor (EGFR) occur immediat... more Global cellular responses induced by epidermal growth factor (EGF) receptor (EGFR) occur immediately with a less than 1% occupancy among tens of thousands of EGFR molecules on single cell surface. Activation of EGFR requires the formation of a signaling dimer of EGFR bound with a single ligand to each molecule. How sufficient numbers of signaling dimers are formed at such low occupancy rate is still not known. Here, we have analyzed the kinetics of EGF binding and the formation of the signaling dimer using single-molecule imaging and mathematical modeling. A small number of EGFR on the cell surface formed dimeric binding sites, which bound EGF two orders of magnitude faster than the monomeric binding sites. There was a positive cooperative binding of EGF to the dimeric binding sites through a newly discovered kinetic intermediate. These two mechanisms facilitate the formation of signaling dimers of EGF/EGFR complexes.
Proceedings of the National Academy of Sciences, 2007
Phosphorylation of the cytoplasmic tyrosine residues of the epidermal growth factor receptor (EGF... more Phosphorylation of the cytoplasmic tyrosine residues of the epidermal growth factor receptor (EGFR) upon binding of EGF induces recognition of various intracellular signaling molecules, including Grb2. Here, the reaction kinetics between EGFR and Grb2 was analyzed by visualizing single molecules of Grb2 conjugated to the fluorophore Cy3 (Cy3-Grb2). The plasma membrane fraction was purified from human epithelial carcinoma A431 cells after stimulation with EGF and attached to coverslips. Unitary events of association and dissociation of Cy3-Grb2 on the EGFR in the membrane fraction were observed at different concentrations of Grb2 (0.1-100 nM). The dissociation kinetics could be explained by using a multiple-exponential function with a major (>90%) dissociation rate of 8 s ؊1 and a few minor components, suggesting the presence of multiple bound states. In contrast, the association kinetics could be described by a stretched exponential function, suggesting the presence of multiple reaction channels from many unbound substates. Transitions between the unbound substates were also suggested. Unexpectedly, the rate of association was not proportional to the Grb2 concentration: an increase in Cy3-Grb2 concentration by a factor of 10 induced an increase in the reaction frequency approximately by a factor of three. This effect can compensate for fluctuation of the signal transduction from EGFR to Grb2 caused by variations in the expression level of Grb2 in living cells.
Under a direct current electric field, Dictyostelium cells exhibit migration towards the cathode.... more 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.
Quantitative relationships between inputs and outputs of signaling systems are fundamental inform... more Quantitative relationships between inputs and outputs of signaling systems are fundamental information for the understanding of the mechanism of signal transduction. Here we report the correlation between the number of epidermal growth factor (EGF) bindings and the response probability of intracellular calcium elevation. Binding of EGF molecules and changes of intracellular calcium concentration were measured for identical HeLa human epithelial cells. It was found that 300 molecules of EGF were enough to induce calcium response in half of the cells. This number is quite small compared to the number of EGF receptors (EGFR) expressed on the cell surface (50,000). There was a sigmoidal correlation between the response probability and the number of EGF bindings, meaning an ultrasensitive reaction. Analysis of the cluster size distribution of EGF demonstrated that dimerization of EGFR contributes to this switch-like ultrasensitive response. Single-molecule analysis revealed that EGF bound faster to clusters of EGFR than to monomers. This property should be important for effective formation of signaling dimers of EGFR under very small numbers of EGF bindings and suggests that the expression of excess amounts of EGFR on the cell surface is required to prepare predimers of EGFR with a large association rate constant to EGF.
Mechanochemical coupling was studied for two different types of myosin motors in cells: myosin V,... more Mechanochemical coupling was studied for two different types of myosin motors in cells: myosin V, which carries cargo over long distances by as a single molecule; and myosin II, which generates a contracting force in cooperation with other myosin II molecules. Both mean and variance of myosin V velocity at various [ATP] obeyed Michaelis-Menten mechanics, consistent with tight mechanochemical coupling. Myosin II, working in an ensemble, however, was explained by a loose coupling mechanism, generating variable step sizes depending on the ATP concentration and realizing a much larger step (200 nm) per ATP hydrolysis than myosin V through its cooperative nature at zero load. These different mechanics are ideal for the respective myosin's physiological functions.
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Papers by Hiroaki Takagi