Disrupting microtubule network immobilizes amoeboid chemotactic receptor in the plasma membrane

Abstract

Signaling cascades are initiated in the plasma membrane via activation of one molecule by another. The interaction depends on the mutual availability of the molecules to each other and this is determined by their localization and lateral diffusion in the cell membrane. The cytoskeleton plays a very important role in this process by enhancing or restricting the possibility of the signaling partners to meet in the plasma membrane. In this study we explored the mode of diffusion of the cAMP receptor, cAR1, in the plasma membrane of Dictyostelium discoideum cells and how this is regulated by the cytoskeleton. Single-particle tracking of fluorescently labeled cAR1 using total internal reflection microscopy showed that 70% of the cAR1 molecules were mobile. These receptors showed directed motion and we demonstrate that this is not because of tracking along the actin cytoskeleton. Instead, destabilization of the microtubules abolished cAR1 mobility in the plasma membrane and this was confirmed by fluorescence recovery after photobleaching. As a result of microtubule stabilization, one of the first downstream signaling events, the jump of the PH domain of CRAC, was decreased. These results suggest a role for microtubules in cAR1 dynamics and in the ability of cAR1 molecules to interact with their signaling partners.

Cytoskeletal Control of CD36 Diffusion Promotes Its Receptor and Signaling Function

“…The radial arrangement of the linear trajectories around the nucleus suggested that MTs, closely apposed to the cell cortex in macrophages (Figure S6A), could play a role. Previous studies have implicated MTs in regulating receptor dynamics (Bouzigues et al, 2007; de Keijzer et al, 2011; Serge et al, 2003), although generally resulting in directed movement and not “1D diffusion” as observed for CD36.…”

“…While receptors were initially thought to cluster upon binding multivalent ligands (Heldin, 1995), there is increasing evidence that receptors can also exist in pre-formed clusters that get reorganized and activated upon ligand binding (Cambi et al, 2006; Chung et al, 2010; Iino et al, 2001; Livnah et al, 1999; Sako et al, 2000; Schamel et al, 2005; Varma and Mayor, 1998). Membrane microdomains enriched in cholesterol and sphingolipids (Foster et al, 2003; Friedrichson and Kurzchalia, 1998; Hess et al, 2007; Pike, 2003; Sharma et al, 2004), interactions between transmembrane proteins and the cytoskeleton (Andrews et al, 2008; Bouzigues et al, 2007; de Keijzer et al, 2011; Goswami et al, 2008; Kaizuka et al, 2009; Plowman et al, 2005; Serge et al, 2003; Suzuki et al, 2007), and interactions between proteins within the membrane (Douglass and Vale, 2005; Espenel et al, 2008) have been implicated in regulating receptor dynamics and clustering. Very little is known about these emerging mechanisms of unligated receptor clustering and their roles in controlling the signaling competence of receptors.…”

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules

“…A previous study showed that disrupting the actin cytoskeleton increases the mobility of cAR1 at the plasma membrane, whereas microtubule disruption inhibits cAR1’s mobility and its mediated production of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P 3 ] in response to cAMP stimulation [45]. These observations suggested that the potential anchoring of cAR1 to microtubules is important for proper cAR1 signaling.…”

Single Molecule Imaging Deciphers the Relation between Mobility and Signaling of a Prototypical G Protein-coupled Receptor in Living Cells

“…GPCRs can interact directly or indirectly with the cytoskeleton of the cell (14,44,45). Here we probed the influence of the cytoskeleton on NK1R diffusion using cytoskeleton-depolymerizing toxins.…”