Single-Molecule Imaging of Signaling Molecules in Living Cells

Abstract

Single-molecule imaging is an ideal technology to study molecular mechanisms of biological reactions in vitro. Recently, we extended this technology to real-time observation of complexes of fluorescent dye-labeled epidermal growth factor (EGF) and its receptor (EGFR). Detection of single molecules was confirmed by single-step photobleaching. Using the single-molecule technique, the process of dimerization, a key step of EGFR signal transduction was observed. In addition to EGFR, singlemolecules of fluorescently labeled nerve growth factor, GFP fusion forms of small G proteins and Raf1 kinase, and a fluorescent analogue of cAMP were observed in living cells.Single-molecule imaging enables us to observe localization, movement, oligomerization and turnover of individual signaling molecules in the plasma membrane of living cells.

Modeling the Mechanosensitivity of Neutrophils Passing through a Narrow Channel

“…In the experiment (9), t e z 15 s for the slow filtration rate and 2 s for the fast filtration rate. These are comparable to the reciprocal of the kinetic rate of the Rho GTPases (14)(15)(16). These findings offer further evidence for a Rho-GTPasesbased pathway for neutrophil activation after fluidization.…”

Modeling the Mechanosensitivity of Neutrophils Passing through a Narrow Channel

“…In the experiment (9), t e z 15 s for the slow filtration rate and 2 s for the fast filtration rate. These are comparable to the reciprocal of the kinetic rate of the Rho GTPases (14)(15)(16). These findings offer further evidence for a Rho-GTPasesbased pathway for neutrophil activation after fluidization.…”

Modelling Cell Polarization Driven by Synthetic Spatially Graded Rac Activation

“…We denote the concentration of a given GTPase by in its active membrane form and , in the inactive membrane bound and cytosolic forms respectively. We make the biologically reasonable assumptions that each Rho protein has a constant total amount, , over the timescale of the experiments and that membrane cycling dynamics are much faster than activation/inactivation dynamics [22] . The latter hypothesis is a convenient simplification, that is not critical for model dynamics.…”

A Density-Dependent Switch Drives Stochastic Clustering and Polarization of Signaling Molecules

“…Second, on the timescale of cell polarization, the amount of Cdc42 can be considered to be roughly constant. Third, the inactive cytosolic pool can be considered well mixed assuming: (i) the amount of GDI in the cell is not a limiting factor [37] , [38] ; and (ii) switching between membrane and cytosolic states is rapid [39] . Then, we can use the fast exchange (rapid equilibrium approximation) between the membrane and cytosolic GDP-bound forms to derive a net “effective diffusion coefficient” [40] for the inactive forms.…”

Modeling the Mechanosensitivity of Neutrophils Passing through a Narrow Channel

“…In the experiment (9), t e z 15 s for the slow filtration rate and 2 s for the fast filtration rate. These are comparable to the reciprocal of the kinetic rate of the Rho GTPases (14)(15)(16). These findings offer further evidence for a Rho-GTPasesbased pathway for neutrophil activation after fluidization.…”

Modelling Cell Polarization Driven by Synthetic Spatially Graded Rac Activation

“…We denote the concentration of a given GTPase by in its active membrane form and , in the inactive membrane bound and cytosolic forms respectively. We make the biologically reasonable assumptions that each Rho protein has a constant total amount, , over the timescale of the experiments and that membrane cycling dynamics are much faster than activation/inactivation dynamics [22] . The latter hypothesis is a convenient simplification, that is not critical for model dynamics.…”

A Density-Dependent Switch Drives Stochastic Clustering and Polarization of Signaling Molecules

“…Second, on the timescale of cell polarization, the amount of Cdc42 can be considered to be roughly constant. Third, the inactive cytosolic pool can be considered well mixed assuming: (i) the amount of GDI in the cell is not a limiting factor [37] , [38] ; and (ii) switching between membrane and cytosolic states is rapid [39] . Then, we can use the fast exchange (rapid equilibrium approximation) between the membrane and cytosolic GDP-bound forms to derive a net “effective diffusion coefficient” [40] for the inactive forms.…”

Modeling the Mechanosensitivity of Neutrophils Passing through a Narrow Channel

“…In the experiment (9), t e z 15 s for the slow filtration rate and 2 s for the fast filtration rate. These are comparable to the reciprocal of the kinetic rate of the Rho GTPases (14)(15)(16). These findings offer further evidence for a Rho-GTPasesbased pathway for neutrophil activation after fluidization.…”

Modelling Cell Polarization Driven by Synthetic Spatially Graded Rac Activation

“…We denote the concentration of a given GTPase by in its active membrane form and , in the inactive membrane bound and cytosolic forms respectively. We make the biologically reasonable assumptions that each Rho protein has a constant total amount, , over the timescale of the experiments and that membrane cycling dynamics are much faster than activation/inactivation dynamics [22] . The latter hypothesis is a convenient simplification, that is not critical for model dynamics.…”

A Density-Dependent Switch Drives Stochastic Clustering and Polarization of Signaling Molecules

“…Second, on the timescale of cell polarization, the amount of Cdc42 can be considered to be roughly constant. Third, the inactive cytosolic pool can be considered well mixed assuming: (i) the amount of GDI in the cell is not a limiting factor [37] , [38] ; and (ii) switching between membrane and cytosolic states is rapid [39] . Then, we can use the fast exchange (rapid equilibrium approximation) between the membrane and cytosolic GDP-bound forms to derive a net “effective diffusion coefficient” [40] for the inactive forms.…”