A spatially restricted increase in receptor mobility is involved in directional sensing duringDictyostelium discoideumchemotaxis

Abstract

The directed cell migration towards a chemotactic source, chemotaxis, involves three complex and interrelated processes: directional sensing, cell polarization and motility. Directional sensing allows migrating eukaryotic cells to chemotax in extremely shallow gradients (<2% across the cell body) of the chemoattractant. Although directional sensing has been observed as spatially restricted responses along the plasma membrane, our understanding of the `compass' of the cell that controls the gradient-induced translocation of proteins during chemotactic movements is still largely lacking. Until now, the dynamical behaviour and mobility of the chemoattractant-receptor molecule has been neglected in models describing the directional sensing mechanisms. Here, we show by single-molecule microscopy an agonist-induced increase in the mobile fraction of cAMP-receptor at the leading edge of chemotacting Dictyostelium discoideum cells. The onset of receptor mobility was correlated to the uncoupling and activation of the Gα2-protein. A finite-element simulation showed that the increase in mobile fraction of the activated receptor enabled the amplified generation of activated Gβγ-dimers at the leading edge of the cell, faithfully representing a primary linear amplification step in directional sensing. We propose here that modulation of the receptor mobility is directly involved in directional sensing and provides a new mechanistic basis for the primary amplification step in current theoretical models that describe directional sensing.

Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging

“…We compared two different strategies for achieving low fluorescent protein concentration. One approach uses high-intensity light to bleach cells expressing moderate or high amounts of fluorescent proteins down to concentrations that are compatible with single-molecule imaging (de Keijzer et al, 2008;van Hemert et al, 2010). When we analyzed XTC cells expressing low levels of the p40 subunit of the Arp2/3 complex under both high-and low-intensity light (supplementary material Fig.…”

Androgen receptor complexes probe DNA for recognition sequences by short random interactions

“…Using a laserbased fluorescence microscopy setup equipped with a highsensitivity and high-speed charge-coupled device (CCD) camera (Schmidt et al, 1996), SMM has successfully been applied to proteins fused to autofluorescent proteins, like GFP, providing insight into the mobility patterns of several proteins at a time resolution of ,5 ms and a positional accuracy of ,40 nm (de Keijzer et al, 2008;Harms et al, 1999;Iino et al, 2001;Lommerse et al, 2005). Initially, these studies mostly focused on membrane proteins, but in recent years, data on the threedimensional (3D) mobility of fluorescently labeled proteins in the nuclei of living cells have been extracted using this approach.…”

Mobility of G proteins is heterogeneous and polarized during chemotaxis

“…Approximately 7.7ϫ10 4 G-YFP were expressed, which is at the lower end of the expression level of reported endogenous G molecules of 8ϫ10 4 -40ϫ10 4 molecules (Jin et al, 2000). It was reported earlier that 4ϫ10 4 receptors were expressed in wild-type and in transformed cells (Van Haastert et al, 1996;de Keijzer et al, 2008), the active fraction of which, 2ϫ10 4 (~50% of 4ϫ10 4 ) (de Keijzer et al, 2008) corresponds very well to the number of slow G molecules, 2.5ϫ10 4 (~32% of 7.7ϫ10 4 ).…”