Measuring ligand-receptor binding kinetics and dynamics using k-space image correlation spectroscopy.
Accurate measurements of kinetic rate constants for interacting biomolecules are crucial for understanding the mechanisms underlying intracellular signalling pathways. The magnitude of binding rates plays a very important molecular regulatory role which can lead to very different cellular physiological responses under different conditions. Here, we extend the k-space image correlation spectroscopy (kICS) technique to study the kinetic binding rates of systems wherein: (a) fluorescently labelled, free ligands in solution interact with unlabelled, diffusing receptors in the plasma membrane and (b) systems where labelled, diffusing receptors are allowed to bind/unbind and interconvert between two different diffusing states on the plasma membrane. We develop the necessary mathematical framework for the kICS analysis and demonstrate how to extract the relevant kinetic binding parameters of the underlying molecular system from fluorescence video-microscopy image time-series. Finally, by examining real data for two model experimental systems, we demonstrate how kICS can be a powerful tool to measure molecular transport coefficients and binding kinetics.
Department of Physics, McGill University, Montréal, Québec H3A 2T8, Canada.
Hugo B
HB
Brandão
Department of Physics, McGill University, Montréal, Québec H3A 2T8, Canada.
Hussain
H
Sangji
Department of Physics, McGill University, Montréal, Québec H3A 2T8, Canada.
Elvis
E
Pandžić
Department of Biology, McGill University, Montréal, Québec H3A 1B1, Canada.
Susanne
S
Bechstedt
Department of Biology, McGill University, Montréal, Québec H3A 1B1, Canada.
Gary J
GJ
Brouhard
Department of Physics, McGill University, Montréal, Québec H3A 2T8, Canada; Department of Chemistry, McGill University, Montréal, Québec H3A 2K6, Canada. Electronic address: paul.wiseman@mcgill.ca.
Paul W
PW
Wiseman
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002772:Cholera Toxin / Q000737:chemistry; D000089622:Doublecortin Domain Proteins; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D062105:Molecular Docking Simulation; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000089622:Doublecortin Domain Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D008869:Microtubule-Associated Proteins; D009479:Neuropeptides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002772:Cholera Toxin
Chemical kinetics; Cholera toxin; Fluctuation spectroscopy; Fluorescence microscopy; Image correlation spectroscopy; Microtubules