Richard E. Pagano

Pathways of clathrin-independent endocytosis

There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. In addition to the classical clathrin-dependent mechanism of endocytosis, several pathways that do not use a clathrin coat are emerging. These pathways transport a diverse array of cargoes and are sometimes hijacked by bacteria and viruses to gain access to the host cell. Here, we review our current understanding of various clathrin-independent mechanisms of endocytosis and propose a classification scheme to help organize the data in this complex and evolving field.

Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles

Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1null (Cav1−/−) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1−/− MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveolae and to identify noncaveolar endocytic vehicles. In WT MEFs, a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2% per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.

Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells

We recently showed that human skin fibroblasts internalize fluorescent analogues of the glycosphingolipids lactosylceramide and globoside almost exclusively by a clathrin-independent mechanism involving caveolae. In contrast, a sphingomyelin analogue is internalized approximately equally via clathrin-dependent and caveolar routes. Here, we further characterized the caveolar pathway for glycosphingolipids, showing that Golgi targeting of sphingolipids internalized via caveolae required microtubules and phosphoinositol 3-kinases and was inhibited in cells expressing dominant-negative Rab7 and Rab9 constructs. In addition, overexpression of wild-type Rab7 or Rab9 (but not Rab11) in Niemann-Pick type C (NP-C) lipid storage disease fibroblasts resulted in correction of lipid trafficking defects, including restoration of Golgi targeting of fluorescent lactosylceramide and endogenous GM(1) ganglioside, and a dramatic reduction in intracellular cholesterol stores. Our results demonstrate a role for Rab7 and Rab9 in the Golgi targeting of glycosphingolipids and suggest a new therapeutic approach for restoring normal lipid trafficking in NP-C cells.

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.

Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases.

Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases

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.

Transforming Growth Factor-β Activation of Phosphatidylinositol 3-Kinase Is Independent of Smad2 and Smad3 and Regulates Fibroblast Responses via p21-Activated Kinase-2

Transforming growth factor-beta (TGF-beta) stimulates cellular proliferation and transformation to a myofibroblast phenotype in vivo and in a subset of fibroblast cell lines. As the Smad pathway is activated by TGF-beta in essentially all cell types, it is unlikely to be the sole mediator of cell type-specific outcomes to TGF-beta stimulation. In the current study, we determined that TGF-beta receptor signaling activates phosphatidylinositol 3-kinase (PI3K) in several fibroblast but not epithelial cultures independently of Smad2 and Smad3. PI3K activation occurs in the presence of dominant-negative dynamin and is required for p21-activated kinase-2 kinase activity and the increased proliferation and morphologic change induced by TGF-beta in vitro.

Regulation of Endocytic Recycling of KCNQ1/KCNE1 Potassium Channels

Stress-dependent regulation of cardiac action potential duration is mediated by the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. It is accompanied by an increased magnitude of the slow outward potassium ion current, IKs. KCNQ1 and KCNE1 subunits coassemble to form the IKs channel. Mutations in either subunit cause long QT syndrome, an inherited cardiac arrhythmia associated with an increased risk of sudden cardiac death. Here we demonstrate that exocytosis of KCNQ1 proteins to the plasma membrane requires the small GTPase RAB11, whereas endocytosis is dependent on RAB5. We further demonstrate that RAB-dependent KCNQ1/KCNE1 exocytosis is enhanced by the serum- and glucocorticoid-inducible kinase 1, and requires phosphorylation and activation of phosphoinositide 3-phosphate 5-kinase and the generation of PI(3,5)P2. Identification of KCNQ1/KCNE1 recycling and its modulation by serum- and glucocorticoid-inducible kinase 1-phosphoinositide 3-phosphate 5-kinase -PI(3,5)P2 provides a mechanistic insight into stress-induced acceleration of cardiac repolarization.

Pneumocystis carinii cell wall β-glucan induces release of macrophage inflammatory protein-2 from alveolar epithelial cells via a lactosylceramide-mediated mechanism

Infiltration of the lungs with neutrophils promotes respiratory failure during severePneumocystis carinii (PC) pneumonia. Recent studies have shown that alveolar epithelial cells (AECs), in addition to promoting PC attachment, also participate in lung inflammation by the release of cytokines and chemokines. Herein, we demonstrate that a PC β-glucan rich cell wall isolate (PCBG) stimulates the release of macrophage inflammatory protein-2 (MIP-2) from isolated AECs through a lactosylceramide-dependent mechanism. The results demonstrate that MIP-2 mRNA and protein production is significantly increased at both early and late time points after PCBG challenge. Although CD11b/CD18 (Mac-1, CR3) is the most widely studied β-glucan receptor, we demonstrate that CD11b/CD18 is not present on AECs. This study instead demonstrates that preincubation of AECs with an antibody directed against the membrane glycosphingolipid lactosylceramide (CDw17) results in a significant decrease in MIP-2 secretion. Preincubation of the anti-CDw17 antibody with solubilized lactosylceramide reverses this effect. Furthermore, incubation of AECs with inhibitors of glycosphingolipid biosynthesis, includingN-butyldeoxyno jirimycin andd-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-HCl, also results in a significant decrease in AEC MIP-2 production following challenge with PCBG. These data demonstrate that PC β-glucan induces significant production of MIP-2 from AECs and that CDw17 participates in the glucan-induced inflammatory signaling in lung epithelial cells during PC infection.

Broad screening test for sphingolipid-storage diseases.

BACKGROUND Lipid-storage diseases are collectively important because they cause substantial morbidity and mortality, and because they may present as dementia, major psychiatric illness, developmental delay, or cerebral palsy. At present, no single assay can be used as an initial general screen for lipid-storage diseases. METHODS We used a fluorescent analogue of lactosylceramide, called N-[5-(5,7-dimethylborondipyrromethenedifluoride)-1-pentanoyl]D- lactosylsphingosine (BODIPY-LacCer), the emission of which changes from green to red wavelengths with increasing concentrations in membranes, to examine the intracellular distribution of the lipid within living cells. FINDINGS During a brief pulse-chase experiment, the fluorescent lipid accumulated in the lysosomes of fibroblasts from patients with Fabrys disease, GM1 gangliosidosis, GM2 gangliosidosis (Tay-Sachs and Sandhoff forms), metachromatic leucodystrophy, mucolipidosis type IV, Niemann-Pick disease (types A, B, and C), and sphingolipid-activator-protein-precursor (prosaposin) deficiency. In control cells, the lipid was mainly confined to the Golgi complex. In a masked study, replicate samples of 25 of 26 unique cell lines representing ten different lipid-storage diseases, and 18 of 20 unique cell lines representing controls were correctly identified; the sensitivity was 96.2% (95% CI 80.4-99.9) and the specificity 90.0% (68.3-98.8). INTERPRETATION This method may be useful as an initial general screen for lipid-storage diseases, and, with modification, could be used for large-scale automated screening of drugs to abrogate lysosomal storage in various lipidoses. The unexpected accumulation of BODIPY-LacCer in several biochemically distinct diseases raises important questions about common mechanisms of cellular dysfunction in these disorders.