An optogenetic model reveals cell shape regulation through FAK and fascin.

Cell shape regulation is important, but the mechanisms that govern shape are not fully understood, in part due to limited experimental models in which cell shape changes and underlying molecular processes can be rapidly and non-invasively monitored in real time. Here, we used an optogenetic tool to activate RhoA in the middle of mononucleated macrophages to induce contraction, resulting in a side with the nucleus that retained its shape and a non-nucleated side that was unable to maintain its shape and collapsed. In cells overexpressing focal adhesion kinase (FAK; also known as PTK2), the non-nucleated side exhibited a wide flat morphology and was similar in adhesion area to the nucleated side. In cells overexpressing fascin, an actin-bundling protein, the non-nucleated side assumed a spherical shape and was similar in height to the nucleated side. This effect of fascin was also observed in fibroblasts even without inducing furrow formation. Based on these results, we conclude that FAK and fascin work together to maintain cell shape by regulating adhesion area and height, respectively, in different cell types. This article has an associated First Person interview with the first author of the paper.

SRRF-Stream Imaging of Optogenetically Controlled Furrow Formation Shows Localized and Coordinated Endocytosis and Exocytosis Mediating Membrane Remodeling.

Cleavage furrow formation during cytokinesis involves extensive membrane remodeling. In the absence of methods to exert dynamic control over these processes, it has been a challenge to examine the basis of this remodeling. Here we used a subcellular optogenetic approach to induce this at will and found that furrow formation is mediated by actomyosin contractility, retrograde plasma membrane flow, localized decrease in membrane tension, and endocytosis. FRAP, 4-D imaging, and inhibition or upregulation of endocytosis or exocytosis show that ARF6 and Exo70 dependent localized exocytosis supports a potential model for intercellular bridge elongation. TIRF and Super Resolution Radial Fluctuation (SRRF) stream microscopy show localized VAMP2-mediated exocytosis and incorporation of membrane lipids from vesicles into the plasma membrane at the front edge of the nascent daughter cell. Thus, spatially separated but coordinated plasma membrane depletion and addition are likely contributors to membrane remodeling during cytokinetic processes.

Membrane Flow Drives an Adhesion-Independent Amoeboid Cell Migration Mode.

Cells migrate by applying rearward forces against extracellular media. It is unclear how this is achieved in amoeboid migration, which lacks adhesions typical of lamellipodia-driven mesenchymal migration. To address this question, we developed optogenetically controlled models of lamellipodia-driven and amoeboid migration. On a two-dimensional surface, migration speeds in both modes were similar. However, when suspended in liquid, only amoeboid cells exhibited rapid migration accompanied by rearward membrane flow. These cells exhibited increased endocytosis at the back and membrane trafficking from back to front. Genetic or pharmacological perturbation of this polarized trafficking inhibited migration. The ratio of cell migration and membrane flow speeds matched the predicted value from a model where viscous forces tangential to the cell-liquid interface propel the cell forward. Since this mechanism does not require specific molecular interactions with the surrounding medium, it can facilitate amoeboid migration observed in diverse microenvironments during immune function and cancer metastasis.

Role of Cysteine Residues in the Carboxyl-Terminus of the Follicle-Stimulating Hormone Receptor in Intracellular Traffic and Postendocytic Processing.

Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the absence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and ß-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these cysteine residues are S-palmitoylated, the data presented emphasize on this posttranslational modification as an important factor for both upward and downward trafficking of this receptor.

Carboxyl terminus-truncated α1D-adrenoceptors inhibit the ERK pathway.

Human α1D-adrenoceptors are G protein-coupled receptors that mediate adrenaline/noradrenaline actions. There is a growing interest in identifying regulatory domains in these receptors and determining how they function. In this work, we show that the absence of the human α1D-adrenoceptor carboxyl tail results in altered ERK (extracellular signal-regulated kinase) and p38 phosphorylation states. Amino terminus-truncated and both amino and carboxyl termini-truncated α1D-adrenoceptors were transfected into Rat-1, HEK293, and B103 cells, and changes in the phosphorylation state of extracellular signal-regulated kinase was assessed using biochemical and biophysical approaches. The phosphorylation state of other protein kinases (p38, MEK1, and Raf-1) was also studied. Noradrenaline-induced ERK phosphorylation in Rat-1 fibroblasts expressing amino termini-truncated α1D-adrenoceptors. However, in cells expressing receptors with both amino and carboxyl termini truncations, noradrenaline-induced activation was abrogated. Interestingly, ERK phosphorylation that normally occurs through activation of endogenous G protein-coupled receptors, EGF receptors, and protein kinase C, was also decreased, suggesting that downstream steps in the mitogen-activated protein kinase pathway were affected. A similar effect was observed in B103 cells but not in HEK 293 cells. Phosphorylation of Raf-1 and MEK1 was also diminished in Rat-1 fibroblasts expressing amino- and carboxyl-truncated α1D-adrenoceptors. Our data indicate that expression of carboxyl terminus-truncated α1D-adrenoceptors alters ERK and p38 phosphorylation state.

α1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).

Visualizing G protein-coupled receptors in action through confocal microscopy techniques.

G protein-coupled receptors constitute one of the most abundant entities in cellular communication. Elucidation of their structure and function as well as of their regulation began 30-40 years ago and the advance has markedly increased during the last 15 years. They participate in a plethora of cell functions such as regulation of metabolic fluxes, contraction, secretion, differentiation, or proliferation, and in essentially all activities of our organism; these receptors are targets of a large proportion of prescribed and illegal drugs. Fluorescence techniques have been used to study receptors for many years. The experimental result was usually a two-dimensional (2D) micrograph. Today, the result can be a spatiotemporal (four-dimensional, 4D) movie. Advances in microscopy, fluorescent protein design, and computer-assisted analysis have been of great importance to increase our knowledge on receptor regulation and function and create opportunities for future research. In this review we briefly depict the state of the art of the G protein-coupled receptor field and the methodologies used to study G protein-coupled receptor location, trafficking, dimerization, and other types of receptor-protein interaction. Fluorescence techniques now permit the capture of receptor images with high resolution and, together with a variety of fluorescent dyes that color organelles (such as the plasma membrane or the nucleus) or the cytoskeleton, allow researchers to obtain a much clearer idea of what is taking place at the cellular level. These developments are changing the way we explore cell communication and signal transduction, permitting deeper understanding of the physiological and pathophysiological processes.

Free fatty acids and protein kinase C activation induce GPR120 (free fatty acid receptor 4) phosphorylation.

GPR120, free fatty acid receptor 4, is a recently deorphanized G protein-coupled receptor that seems to play cardinal roles in the regulation of metabolism and in the pathophysiology of inflammatory and metabolic disorders. In the present work a GPR120-Venus fusion protein was expressed in HEK293 Flp-In T-REx cells and its function (increase in intracellular calcium) and phosphorylation were studied. It was observed that the fusion protein migrated in sodium dodecyl sulfate-polyacrylamide gels as a band with a mass of ≈70-75kDa, although other bands of higher apparent weight (>130kDa) were also detected. Cell stimulation with docosahexaenoic acid or α-linolenic acid induced concentration-dependent increases in intracellular calcium and GPR120 phosphorylation. Activation of protein kinase C with phorbol esters also induced a marked receptor phosphorylation but did not alter the ability of 1µM docosahexaenoic acid to increase the intracellular calcium concentration. Phorbol ester-induced GPR120 phosphorylation, but not that induced with docosahexaenoic acid, was blocked by protein kinase C inhibitors (bis-indolyl-maleimide I and Gö 6976) suggesting that conventional kinase isoforms mediate this action. The absence of effect of protein kinase C inhibitors on agonist-induced GPR120 phosphorylation indicates that this kinase does not play a major role in agonist-induced receptor phosphorylation. Docosahexaenoic acid action was associated with marked GPR120 internalization whereas that induced with phorbol esters was smaller at early times.

Sphingosine 1-phosphate-mediated α1B-adrenoceptor desensitization and phosphorylation. Direct and paracrine/autocrine actions.

Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation were studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appear to be functionally relevant in IGF-I- and EGF-induced α1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P1 receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor-α1B-adrenergic receptor crosstalk.