Microtubule motors power plasma membrane tubulation in clathrin-independent endocytosis.

How the plasma membrane is bent to accommodate clathrin-independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin-independent carriers by binding and cross-linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin-induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B-subunit into surface-derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin-independent endocytosis.

Overexpression of caveolin-1 is sufficient to phenocopy the behavior of a disease-associated mutant.

Mutations and alterations in caveolin-1 expression levels have been linked to a number of human diseases. How misregulation of caveolin-1 contributes to disease is not fully understood, but has been proposed to involve the intracellular accumulation of mutant forms of the protein. To better understand the molecular basis for trafficking defects that trap caveolin-1 intracellularly, we compared the properties of a GFP-tagged version of caveolin-1 P132L, a mutant form of caveolin-1 previously linked to breast cancer, with wild-type caveolin-1. Unexpectedly, wild-type caveolin-1-GFP also accumulated intracellularly, leading us to examine the mechanisms underlying the abnormal localization of the wild type and mutant protein in more detail. We show that both the nature of the tag and cellular context impact the subcellular distribution of caveolin-1, demonstrate that even the wild-type form of caveolin-1 can function as a dominant negative under some conditions, and identify specific conformation changes associated with incorrectly targeted forms of the protein. In addition, we find intracellular caveolin-1 is phosphorylated on Tyr14, but phosphorylation is not required for mistrafficking of the protein. These findings identify novel properties of mistargeted forms of caveolin-1 and raise the possibility that common trafficking defects underlie diseases associated with overexpression and mutations in caveolin-1.

Lipid sorting by ceramide structure from plasma membrane to ER for the cholera toxin receptor ganglioside GM1.

The glycosphingolipid GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane lipid can specify trafficking in these pathways, we synthesized GM1 isoforms with alternate ceramide domains and imaged their trafficking in live cells. Only GM1 with unsaturated acyl chains sorted efficiently from PM to TGN and ER. Toxin binding, which effectively crosslinks GM1 lipids, was dispensable, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. The results implicate a protein-dependent mechanism of lipid sorting by ceramide structure and provide a molecular explanation for the diversity and specificity of retrograde trafficking by CT in host cells.

Coordinated regulation of caveolin-1 and Rab11a in apical recycling compartments of polarized epithelial cells.

Recent studies have identified caveolin-1, a protein best known for its functions in caveolae, in apical endocytic recycling compartments in polarized epithelial cells. However, very little is known about the regulation of caveolin-1 in the endocytic recycling pathway. To address this question, in the current study we compared the relationship between compartments enriched in sub-apical caveolin-1 and Rab11a, a well-defined marker of apical recycling endosomes, using polarized MDCK cells as a model. We show that caveolin-1-containing vesicles define a compartment that partially overlaps with Rab11a, and that the distribution of subapical caveolin-1 and Rab11a shows a similar dependence on microtubule disruption. Mutants of the Rab11a effector, Rab11-FIP2 also altered the localization of caveolin-1. These findings indicate that caveolin-1 is coordinately regulated with Rab11a within the apical recycling system of polarized epithelial cells, suggesting that the two proteins are components of the same pathway.

Nucleocytoplasmic distribution and dynamics of the autophagosome marker EGFP-LC3.

The process of autophagy involves the formation of autophagosomes, double-membrane structures that encapsulate cytosol. Microtubule-associated protein light chain 3 (LC3) was the first protein shown to specifically label autophagosomal membranes in mammalian cells, and subsequently EGFP-LC3 has become one of the most widely utilized reporters of autophagy. Although LC3 is currently thought to function primarily in the cytosol, the site of autophagosome formation, EGFP-LC3 often appears to be enriched in the nucleoplasm relative to the cytoplasm in published fluorescence images. However, the nuclear pool of EGFP-LC3 has not been specifically studied in previous reports, and mechanisms by which LC3 shuttles between the cytoplasm and nucleoplasm are currently unknown. In this study, we therefore investigated the regulation of the nucleo-cytoplasmic distribution of EGFP-LC3 in living cells. By quantitative fluorescence microscopy analysis, we demonstrate that soluble EGFP-LC3 is indeed enriched in the nucleus relative to the cytoplasm in two commonly studied cell lines, COS-7 and HeLa. Although LC3 contains a putative nuclear export signal (NES), inhibition of active nuclear export or mutation of the NES had no effect on the nucleo-cytoplasmic distribution of EGFP-LC3. Furthermore, FRAP analysis indicates that EGFP-LC3 undergoes limited passive nucleo-cytoplasmic transport under steady state conditions, and that the diffusional mobility of EGFP-LC3 was substantially slower in the nucleus and cytoplasm than predicted for a freely diffusing monomer. Induction of autophagy led to a visible decrease in levels of soluble EGFP-LC3 relative to autophagosome-bound protein, but had only modest effects on the nucleo-cytoplasmic ratio or diffusional mobility of the remaining soluble pools of EGFP-LC3. We conclude that the enrichment of soluble EGFP-LC3 in the nucleus is maintained independently of active nuclear export or induction of autophagy. Instead, incorporation of soluble EGFP-LC3 into large macromolecular complexes within both the cytoplasm and nucleus may prevent its rapid equilibrium between the two compartments.

A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes.

Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells.

Attenuated endocytosis and toxicity of a mutant cholera toxin with decreased ability to cluster ganglioside GM1 molecules.

Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM(1). The homopentomeric B-subunit of the toxin can bind up to five GM(1) molecules at once. Here, we examined the role of polyvalent binding of GM(1) in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM(1). The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM(1) when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM(1) molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM(1) molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway.

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.

Ras diffusion is sensitive to plasma membrane viscosity.

The cell surface contains a variety of barriers and obstacles that slow the lateral diffusion of glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins below the theoretical limit imposed by membrane viscosity. How the diffusion of proteins residing exclusively on the inner leaflet of the plasma membrane is regulated has been largely unexplored. We show here that the diffusion of the small GTPase Ras is sensitive to the viscosity of the plasma membrane. Using confocal fluorescence recovery after photobleaching, we examined the diffusion of green fluorescent protein (GFP)-tagged HRas, NRas, and KRas in COS-7 cells loaded with or depleted of cholesterol, a well-known modulator of membrane bilayer viscosity. In cells loaded with excess cholesterol, the diffusional mobilities of GFP-HRas, GFP-NRas, and GFP-KRas were significantly reduced, paralleling the behavior of the viscosity-sensitive lipid probes DiIC(16) and DiIC(18). However, the effects of cholesterol depletion on protein and lipid diffusion in cell membranes were highly dependent on the depletion method used. Cholesterol depletion with methyl-beta-cyclodextrin slowed Ras diffusion by a viscosity-independent mechanism, whereas overnight cholesterol depletion slightly increased both protein and lipid diffusion. The ability of Ras to sense membrane viscosity may represent a general feature of proteins residing on the cytoplasmic face of the plasma membrane.