Raman Deep Singh

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent (“caveolar-like”) mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a “vital marker” for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

Glycosphingolipids Internalized via Caveolar-related Endocytosis Rapidly Merge with the Clathrin Pathway in Early Endosomes and Form Microdomains for Recycling

We have previously demonstrated that glycosphingolipids are internalized from the plasma membrane of human skin fibroblasts by a clathrin-independent, caveolar-related mechanism and are subsequently transported to the Golgi apparatus by a process that is dependent on microtubules, phosphatidylinositol 3-kinase, Rab7, and Rab9. Here we characterized the early steps of intracellular transport of a fluorescent glycosphingolipid analog, BODIPY-lactosylceramide (LacCer), and compared this to fluorescent transferrin (Tfn), a well established marker for the clathrin pathway. Although these two markers were initially internalized into separate vesicles by distinct mechanisms, they became co-localized in early endosomes within 5 min. These results demonstrate that glycosphingolipid-containing vesicles derived from caveolar-related endocytosis fuse with the classical endosomal system. However, in contrast to Tfn, internalization and trafficking of LacCer was independent of Rab5a, a key regulator of transport to early endosomes. By taking advantage of the monomer/excimer properties of the fluorescent lipid analog, we were also able to visualize LacCer segregation into distinct microdomains of high (red emission) and low (green emission) concentrations in the early endosomes of living cells. Interestingly, the high concentration “red” microdomains co-localized with fluorescent Tfn upon exit from early endosomes and passed through Rab11-positive “recycling endosomes” prior to being transported back to the plasma membrane. These results together with our previous studies suggest that glycosphingolipids internalized by caveolar endocytosis are rapidly delivered to early endosomes where they are fractionated into two major pools, one that is transported via late endosomes to the Golgi apparatus and the other that is returned to the plasma membrane via the recycling compartment.

Membrane microdomains, caveolae, and caveolar endocytosis of sphingolipids (Review)

Caveolae are flask-shape membrane invaginations of the plasma membrane that have been implicated in endocytosis, transcytosis, and cell signaling. Recent years have witnessed the resurgence of studies on caveolae because they have been found to be involved in the uptake of some membrane components such as glycosphingolipids and integrins, as well as viruses, bacteria, and bacterial toxins. Accumulating evidence shows that endocytosis mediated by caveolae requires unique structural and signaling machinery (caveolin-1, src kinase), which indicates that caveolar endocytosis occurs through a mechanism which is distinct from other forms of lipid microdomain-associated, clathrin-independent endocytosis. Furthermore, a balance of glycosphingolipids, cholesterol, and caveolin-1 has been shown to be important in regulating caveolae endocytosis.

Type II Transforming Growth Factor-β Receptor Recycling Is Dependent upon the Clathrin Adaptor Protein Dab2

Transforming growth factor-β receptor recycling is regulated by the clathrin adaptor Dab2 protein. In the absence of Dab2, receptors localize in a perinuclear locale because they are unable to transit from the early endosomal antigen 1-positive early endosome to the Rab11-positive endosomal recycling compartment.

Inhibition of caveolar uptake, SV40 infection, and β1-integrin signaling by a nonnatural glycosphingolipid stereoisomer

Caveolar endocytosis is an important mechanism for the uptake of certain pathogens and toxins and also plays a role in the internalization of some plasma membrane (PM) lipids and proteins. However, the regulation of caveolar endocytosis is not well understood. We previously demonstrated that caveolar endocytosis and β1-integrin signaling are stimulated by exogenous glycosphingolipids (GSLs). In this study, we show that a synthetic GSL with nonnatural stereochemistry, β-d-lactosyl-N-octanoyl-l-threo-sphingosine, (1) selectively inhibits caveolar endocytosis and SV40 virus infection, (2) blocks the clustering of lipids and proteins into GSLs and cholesterol-enriched microdomains (rafts) at the PM, and (3) inhibits β1-integrin activation and downstream signaling. Finally, we show that small interfering RNA knockdown of β1 integrin in human skin fibroblasts blocks caveolar endocytosis and the stimulation of signaling by a GSL with natural stereochemistry. These experiments identify a new compound that can interfere with biological processes by inhibiting microdomain formation and also identify β1 integrin as a potential mediator of signaling by GSLs.

Caveolar endocytosis and microdomain association of a glycosphingolipid analog is dependent on its sphingosine stereochemistry

We have previously shown that glycosphingolipid analogs are internalized primarily via caveolae in various cell types. This selective internalization was not dependent on particular carbohydrate headgroups or sphingosine chain length. Here, we examine the role of sphingosine structure in the endocytosis of BODIPY™-tagged lactosylceramide (LacCer) analogs via caveolae. We found that whereas the LacCer analog with the natural (d-erythro) sphingosine stereochemistry is internalized mainly via caveolae, the non-natural (l-threo) LacCer analog is taken up via clathrin-, RhoA-, and Cdc42-dependent mechanisms and largely excluded from uptake via caveolae. Unlike the d-erythro-LacCer analog, the l-threo analog did not cluster in membrane microdomains when added at higher concentrations (5–20 μm). In vitro studies using small unilamellar vesicles and giant unilamellar vesicles demonstrated that l-threo-LacCer did not undergo a concentration-dependent excimer shift in fluorescence emission such as that seen with BODIPY™-sphingolipids with natural stereochemistry. Molecular modeling studies suggest that in d-erythro-LacCer, the disaccharide moiety extends above and in the same plane as the sphingosine hydrocarbon chain, while in l-threo-LacCer the carbohydrate group is nearly perpendicular to the hydrocarbon chain. Together, these results suggest that the altered stereochemistry of the sphingosine group in l-threo-LacCer results in a perturbed structure, which is unable to pack closely with natural membrane lipids, leading to a reduced inclusion in plasma membrane microdomains and decreased uptake by caveolar endocytosis. These findings demonstrate the importance of the sphingolipid stereochemistry in the formation of membrane microdomains.

Ano1, a Ca2+‐activated Cl− channel, coordinates contractility in mouse intestine by Ca2+ transient coordination between interstitial cells of Cajal

Ano1, a Ca2+‐activated Cl− channel, is expressed in interstitial cells of Cajal (ICC) throughout the gut. We report here that it is required to maintain coordinated Ca2+ transients within myenteric ICC of mouse small intestine. Ca2+ transients in Ano1 WT mice were rhythmic and coordinated whereas uncoordinated Ca2+ transients were seen in knockout mice. Ca2+ transients were un‐coordinated following pharmacological block of Ano1 in WT mice using niflumic acid, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid and 4,4′‐diisothiocyanato‐2,2′‐stilbenedisulfonic acid disodium salt. Transient knockdown of Ano1 in organotypic cultures with short hairpin RNA to Ano1 in WT tissues also caused loss of coordinated Ca2+ transients. Contractility of Ano1 knockout mouse intestinal segments in organ bath experiments was significantly decreased, less coordinated and non‐rhythmic. Spatiotemporal maps from knockout mouse small intestine also showed loss of phasic contractile activity. This study provides important information on the basic mechanisms driving coordinated contractile activity in the gastrointestinal tract.

Switching the targeting pathways of a therapeutic antibody by nanodesign.

Application of nonmaterials in medicine is an emerging field with potentials to positively impact human health care [1]. Nanoconjugates of monoclonal antibodies including EGFR (epidermal growth factor receptor) antibody cetuximab (C225), herceptin, are potential candidates for various biomedical applications including targeted delivery, detection/diagnosis and imaging [2][3]. However, mechanisms by which C225 and its nanoconjugates transport to the cell and molecular machineries involved for intracellular delivery are not well understood. A better understanding of the regulatory components involved in endocytosis of these nanoconjugates will aide us for successful nanodesign to achieve specific intracellular targeting. Exploiting strong affinity of gold nanoparticles (AuNPs) to bind to –SH and –NH2 moieties present in the cysteine and lysine residues of C225, we design two Au-C225 conjugates having variable number of antibody to demonstrate that the mechanism of endocytosis of C225 in PANC-1 cells can be tailored by precise design of the nanoconjugates [4]. Using pharmacological inhibitors (Supplementary Figure 1), genetic approaches and Cdc42 null cells we demonstrate that both C225 and gold-conjugated C225 with complete surface coverage (Au-C225-C; ∼3 C225/particle) requires clustering in the GSL (Glycosphingolipid) domains at the plasma membrane followed by dynamin-2 (dyn-2) dependent caveolar endocytosis. However, partially covered nanoconjugate (Au-C225-P; ∼1 C225/particle) is internalized via dyn-2 independent Cdc42 dependent pinocytosis/phagocytosis that requires actin polymerization. Regulating the endocytosis of cetuximab between caveolar and pinocytic internalization by appropriate nanodesign may be useful to target specific intracellular pathways for better therapeutic intervention with reduced side effects.

An association between type Iγ PI4P 5-kinase and Exo70 directs E-cadherin clustering and epithelial polarization

Type Iγ phosphatidylinositol-4-phosphate 5-kinase and Exo70 cooperate in the directed targeting of E-cadherin on the plasma membrane to newly formed adherens junctions. This promotes the regional accumulation of E-cadherin, expansion and maturation of adherens junctions, and differentiation of the lateral membrane domain.

Co-regulation of caveolar and Cdc42-dependent fluid phase endocytosis by phosphocaveolin-1.

Several clathrin-independent endocytosis mechanisms have been identified that can be distinguished by specific requirements for certain proteins, such as caveolin-1 (Cav1) and the Rho GTPases, RhoA and Cdc42, as well as by specific cargo. Some endocytic pathways may be co-regulated such that disruption of one pathway leads to the up-regulation of another; however, the underlying mechanisms for this are unclear. Cav1 has been reported to function as a guanine nucleotide dissociation inhibitor (GDI), which inhibits Cdc42 activation. We tested the hypothesis that Cav1 can regulate Cdc42-dependent, fluid phase endocytosis. We demonstrate that Cav1 overexpression decreases fluid phase endocytosis, whereas silencing of Cav1 enhances this pathway. Enhancement of Cav1 phosphorylation using a phosphatase inhibitor reduces Cdc42-regulated pinocytosis while stimulating caveolar endocytosis. Fluid phase endocytosis was inhibited by expression of a putative phosphomimetic mutant, Cav1-Y14E, but not by the phospho-deficient mutant, Cav1-Y14F. Overexpression of Cav2, or a Cav1 mutant in which the GDI region was altered to the corresponding sequence in Cav2, did not suppress fluid phase endocytosis. These results suggest that the Cav1 expression level and phosphorylation state regulates fluid phase endocytosis via the interaction between the Cav1 GDI region and Cdc42. These data define a novel molecular mechanism for co-regulation of two distinct clathrin-independent endocytic pathways.