Carles Rentero

PALM imaging and cluster analysis of protein heterogeneity at the cell surface

The authors employed photoactivatable localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM) imaging and image analysis based on Ripleys K-function to quantify the distribution and heterogeneity of proteins at the cell plasma membrane. The membrane targeting sequence of the N-terminal region of the T cell receptor-pathway kinase Lck fused to the photo-convertible fluorescent protein tdEos (Lck(N10)-tdEos), clusters into sub-100 nm regions which cover approximately 7% of the cell surface. 2-channel PALM imaging of Lck(N10)-tdEos and the N-terminus of the kinase Src (Src(N15)-PS-CFP2) are demonstrated. Finally, T cell microclusters at the immune synapse are imaged at super-resolution using dSTORM, showing that conventional TIRF images contain unresolved, small clusters. These methods are generally applicable to other cell and fluorophore systems to quantify 2-D molecular clustering at nanometer scales.

Actin Dynamics Drive Membrane Reorganization and Scission in Clathrin-Independent Endocytosis

Nascent transport intermediates detach from donor membranes by scission. This process can take place in the absence of dynamin, notably in clathrin-independent endocytosis, by mechanisms that are yet poorly defined. We show here that in cells scission of Shiga toxin-induced tubular endocytic membrane invaginations is preceded by cholesterol-dependent membrane reorganization and correlates with the formation of membrane domains on model membranes, suggesting that domain boundary forces are driving tubule membrane constriction. Actin triggers scission by inducing such membrane reorganization process. Tubule occurrence is indeed increased upon cellular depletion of the actin nucleator component Arp2, and the formation of a cortical actin shell in liposomes is sufficient to trigger the scission of Shiga toxin-induced tubules in a cholesterol-dependent but dynamin-independent manner. Our study suggests that membranes in tubular Shiga toxin-induced invaginations are poised to undergo actin-triggered reorganization leading to scission by a physical mechanism that may function independently from or in synergy with pinchase activity.

Quantitative imaging of membrane lipid order in cells and organisms

It is now recognized that lipids and proteins in cellular membranes are not homogenously distributed. A high degree of membrane order is the biophysical hallmark of cholesterol-enriched lipid rafts, which may induce the lateral sorting of proteins within the membrane. Here we describe a quantitative fluorescence microscopy technique for imaging localized lipid environments and measuring membrane lipid order in live and fixed cells, as well as in intact tissues. The method is based on the spectral ratiometric imaging of the polarity-sensitive membrane dyes Laurdan and di-4-ANEPPDHQ. Laurdan typically requires multiphoton excitation, making it suitable for the imaging of tissues such as whole, living zebrafish embryos, whereas di-4-ANEPPDHQ imaging can be achieved with standard confocal microscopes. This approach, which takes around 4 h, directly examines the organization of cellular membranes and is distinct from alternative approaches that infer membrane order by measuring probe partitioning or dynamics.

Quantitative Microscopy: Protein Dynamics and Membrane Organisation

The mobility of membrane proteins is a critical determinant of their interaction capabilities and protein functions. The heterogeneity of cell membranes imparts different types of motion onto proteins; immobility, random Brownian motion, anomalous sub‐diffusion, ‘hop’ or confined diffusion, or directed flow. Quantifying the motion of proteins therefore enables insights into the lateral organisation of cell membranes, particularly membrane microdomains with high viscosity such as lipid rafts. In this review, we examine the hypotheses and findings of three main techniques for analysing protein dynamics: fluorescence recovery after photobleaching, single particle tracking and fluorescence correlation spectroscopy. These techniques, and the physical models employed in data analysis, have become increasingly sophisticated and provide unprecedented details of the biophysical properties of protein dynamics and membrane domains in cell membranes. Yet despite these advances, there remain significant unknowns in the relationships between cholesterol‐dependent lipid microdomains, protein‐protein interactions, and the effect of the underlying cytoskeleton. New multi‐dimensional microscopy approaches may afford greater temporal and spatial resolution resulting in more accurate quantification of protein and membrane dynamics in live cells.

A palmitoylation switch mechanism regulates Rac1 function and membrane organization

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell‐cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol‐rich liquid‐ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post‐translational modification targets Rac1 for stabilization at actin cytoskeleton‐linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C‐terminal polybasic region and is regulated by the triproline‐rich motif. Non‐palmitoylated Rac1 shows decreased GTP loading and lower association with detergent‐resistant (liquid‐ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation‐deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1‐deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization.

Functional implications of plasma membrane condensation for T cell activation.

The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.

The biliary epithelium gives rise to liver progenitor cells

Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1β). However, HNF1β expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)‐positive hepatocytes. In order to investigate the role of HNF1β‐expressing cells we used a tamoxifen‐inducible Hnf1βCreER/R26RYfp/LacZ mouse to lineage‐trace Hnf1β+ biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1β+ cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1β+ cells to hepatocyte was detected. We next assessed the contribution of Hnf1β+‐derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl‐1,4‐dihydro‐collidin (DDC)‐diet and a choline‐deficient ethionine‐supplemented (CDE)‐diet. The contribution of Hnf1β+ cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC‐diet treatment, mice fed with a CDE‐diet showed a small population of hepatocytes derived from Hnf1β+ cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome‐wide expression profile of Hnf1β+‐derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways. Conclusion: HNF1β+ biliary duct cells are the origin of LPC. HNF1β+ cells do not contribute to hepatocyte turnover in the healthy liver, but after certain liver injury, they can differentiate to hepatocytes contributing to liver regeneration. (Hepatology 2014;60:1367–1377)

Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity

Annexin A6 is a calcium-dependent membrane-binding protein that interacts with signalling proteins, including the GTPase-activating protein p120GAP, one of the most important inactivators of Ras. Since we have demonstrated that annexin A6 inhibits EGF- and TPA-induced Ras signalling, we investigated whether modulation of Ras activity by annexin A6 was mediated via altered subcellular localization of p120GAP. First, we exploited our observation that high-density lipoproteins (HDL) can activate the Ras/MAP kinase pathway. Expression of annexin A6 caused a significant reduction in HDL-induced activation of Ras and Raf-1. Annexin A6 promoted membrane binding of p120GAP in vitro, and plasma membrane targeting of p120GAP in living cells, both in a Ca2+-dependent manner, which is consistent with annexin A6 promoting the Ca2+-dependent assembly of p120GAP-Ras at the plasma membrane. We then extended these studies to other cell types and stimuli. Expression of annexin A6 in A431 cells reduced, while RNAi-mediated suppression of annexin A6 in HeLa cells enhanced EGF-induced Ras and Erk activation. Importantly, the enhancement of Ras activation following RNAi-mediated reduction in p120GAP levels was more marked in annexin A6-expressing A431 cells than controls, indicating that the effect of annexin A6 on Ras was mediated via p120GAP. Finally, we demonstrated that annexin A6 promotes plasma membrane targeting of p120GAP in A431 cells in response to a variety of stimuli, resulting in colocalization with H-Ras. These findings demonstrate an important role for annexin A6 in regulating plasma membrane localization of p120GAP and hence Ras activity.

AMPK activation promotes lipid droplet dispersion on detyrosinated microtubules to increase mitochondrial fatty acid oxidation

Lipid droplets (LDs) are intracellular organelles that provide fatty acids (FAs) to cellular processes including synthesis of membranes and production of metabolic energy. While known to move bidirectionally along microtubules (MTs), the role of LD motion and whether it facilitates interaction with other organelles are unclear. Here we show that during nutrient starvation, LDs and mitochondria relocate on detyrosinated MT from the cell centre to adopt a dispersed distribution. In the cell periphery, LD–mitochondria interactions increase and LDs efficiently supply FAs for mitochondrial beta-oxidation. This cellular adaptation requires the activation of the energy sensor AMPK, which in response to starvation simultaneously increases LD motion, reorganizes the network of detyrosinated MTs and activates mitochondria. In conclusion, we describe the existence of a specialized cellular network connecting the cellular energetic status and MT dynamics to coordinate the functioning of LDs and mitochondria during nutrient scarcity.

Cholesterol Regulates Syntaxin 6 Trafficking at trans-Golgi Network Endosomal Boundaries

Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN). Here, using Chinese hamster ovary (CHO) Niemann-Pick type C1 (NPC1) mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6) accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs). This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.