Abstract
We introduce a new generalized theoretical framework for image correlation spectroscopy (ICS). Using this framework, we extend the ICS method in time–frequency (ν, nu) space to map molecular flow of fluorescently tagged proteins in individual living cells. Even in the presence of a dominant immobile population of fluorescent molecules, nu-space ICS (nICS) provides an unbiased velocity measurement, as well as the diffusion coefficient of the flow, without requiring filtering. We also develop and characterize a tunable frequency-filter for spatio-temporal ICS (STICS) that allows quantification of the density, the diffusion coefficient and the velocity of biased diffusion. We show that the techniques are accurate over a wide range of parameter space in computer simulation. We then characterize the retrograde flow of adhesion proteins (α6- and αLβ2-GFP integrins and mCherry-paxillin) in CHO.B2 cells plated on laminin and intercellular adhesion molecule 1 (ICAM-1) ligands respectively. STICS with a tunable frequency filter, in conjunction with nICS, measures two new transport parameters, the density and transport bias coefficient (a measure of the diffusive character of a flow/biased diffusion), showing that molecular flow in this cell system has a significant diffusive component. Our results suggest that the integrin–ligand interaction, along with the internal myosin-motor generated force, varies for different integrin–ligand pairs, consistent with previous results.
Highlights
Protein transport and trafficking play an important role in the function and organization of the cell
We established a generalized theoretical framework for image correlation spectroscopy (ICS) that serves as a basis for connecting and comparing variants of these methods
STICS with the IIR filter emerged as a robust technique to measure molecular flow velocities in single cells, densities and transport bias coefficients
Summary
Protein transport and trafficking play an important role in the function and organization of the cell. Spatio-temporal image correlation spectroscopy (STICS, [4]) can create vector maps of the transport of fluorescently-tagged proteins at normal protein expression levels. It has been used in multiple systems to measure flow of adhesion proteins [4, 5], movement of actin during cytokinesis [6], transport of vesicles in growing pollen tubes [7]. After characterizing the ν-space methods in simulation, we use them to measure new properties of retrograde flow of adhesion proteins (α6- and αLβ2-GFP integrins and mCherry-paxillin) in living CHO.B2 cells. We present an overview of different image correlation methodologies in figure 1
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