Quantitative fluorescence imaging of protein diffusion and interaction in living cells

Abstract

Diffusion processes and local dynamic equilibria inside cells lead to nonuniform spatial distributions of molecules, which are essential for processes such as nuclear organization and signaling in cell division, differentiation and migration. To understand these mechanisms, spatially resolved quantitative measurements of protein abundance, mobilities and interactions are needed, but current methods have limited capabilities to study dynamic parameters. Here we describe a microscope based on light-sheet illumination that allows massively parallel fluorescence correlation spectroscopy (FCS) measurements and use it to visualize the diffusion and interactions of proteins in mammalian cells and in isolated fly tissue. Imaging the mobility of heterochromatin protein HP1α (ref. 4) in cell nuclei we could provide high-resolution diffusion maps that reveal euchromatin areas with heterochromatin-like HP1α-chromatin interactions. We expect that FCS imaging will become a useful method for the precise characterization of cellular reaction-diffusion processes.

The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells

“…On the plot we show the effective viscosity corresponding to the D 1 in ldDNA, D 1 in hdDNA, D 2 in ldDNA, and D 2 in hdDNA. The heterogeneous structure of the nucleus is also represented by the data for HP1-GFP diffusing in the nucleus of 3T3 cells [60]. Both the slow and the fast component are comparable with results of Hinde et al [59].…”

“…Thus, the diffusion characteristics of EGFP in the nucleus was rather constant and did not depend on DNA density, however relative contribution of slow component was higher in the heterochromatin region. Yet slightly different results were found in the study by Capoulade et al [60], where a diffusional mapping in 3T3 cells was performed with the use of selective plane illumination microscope. They measured the diffusion coefficients for the heterochromatin protein 1 (HP1α-EGFP) in the nucleus of 3T3 fibroblasts.…”

Probing transcription factor diffusion dynamics in the living mammalian embryo with photoactivatable fluorescence correlation spectroscopy

“…Moreover, the short time windows needed to obtain multiple ACFs makes this method ideal for collecting data from samples that are difficult to maintain under observation. As paFCS does not require custom-made microscopy systems it should also be applicable with other FCS modalities [47][48][49] .…”

Morphogen transport

“…What does the environment through which morphogens move look like, and how does that environment affect dispersal? What are the nanometer to micrometer scale transport mechanisms that together lead to macroscopic gradient formation (Müller and Schier, 2011)? Answers to these questions might be found using quantitative imaging and modeling techniques, such as single-plane illumination microscopy FCS (SPIM-FCS) Wohland et al, 2010;Capoulade et al, 2011), single-particle tracking (Duchesne et al, 2012) and multi-scale modeling approaches (Kavousanakis et al, 2010;Sample and Shvartsman, 2010;Daniels et al, 2012), to analyze transport over different time and length scales (Grimm et al, 2010). Such approaches will help to reveal more clearly how morphogen gradients form during development.…”