Benzyl-N-acetyl-alpha-D-galactosaminide induces a storage disease-like phenotype by perturbing the endocytic pathway

The sugar analog O-benzyl-N-acetyl-alpha-d-galactosaminide (BG) is an inhibitor of glycan chain elongation and inhibits alpha2,3-sialylation in mucus-secreting HT-29 cells. Long-term exposure of these cells to BG is associated with the accumulation of apical glycoproteins in cytoplasmic vesicles. The mechanisms involved therein and the nature of the vesicles have not been elucidated. In these cells, a massive amount of BG metabolites is synthesized. Because sialic acid is mainly distributed apically in epithelial cells, it has been proposed that the BG-induced undersialylation of apical membrane glycoproteins is responsible for their intracellular accumulation due to a defect in anterograde traffic and that sialic acid may constitute an apical targeting signal. In this work, we demonstrate that the intracellular accumulation of membrane glycoproteins does not result mainly from defects in anterograde traffic. By contrast, in BG-treated cells, endocytosed membrane proteins were retained intracellularly for longer periods of time than in control cells and colocalized with accumulated MUC1 and beta(1) integrin in Rab7/lysobisphosphatidic acid(+) vesicles displaying features of late endosomes. The phenotype of BG-treated cells is reminiscent of that observed in lysosomal storage disorders. Sucrose induced a BG-like, lysosomal storage disease-like phenotype without affecting sialylation, indicating that undersialylation is not a requisite for the intracellular accumulation of membrane glycoproteins. Our findings strongly support the notion that the effects observed in BG-treated cells result from the accumulation of BG-derived metabolites and from defects in the endosomal pathway. We propose that abnormal subcellular distribution of membrane glycoproteins involved in cellular communication and/or signaling may also take place in lysosomal storage disorders and may contribute to their pathogenesis.     

C. elegans tubby regulates life span and fat storage by two independent mechanisms

In C. elegans, similar to in mammals, mutations in the tubby homolog, tub-1, promote increased fat deposition. Here, we show that mutation in tub-1 also leads to life span extension dependent on daf-16/FOXO. Interestingly, function of tub-1 in fat storage is independent of daf-16. A yeast two-hybrid screen identified a novel TUB-1 interaction partner (RBG-3); a RabGTPase-activating protein. Both TUB-1 and RBG-3 localize to overlapping neurons. Importantly, RNAi of rbg-3 decreases fat deposition in tub-1 mutants but does not affect life span. We demonstrate that TUB-1 is expressed in ciliated neurons and undergoes both dendritic and ciliary transport. Additionally, tub-1 mutants are chemotaxis defective. Thus, tub-1 may regulate fat storage either by modulating transport, sensing, or responding to signals in ciliated neurons. Taken together, we define a role for tub-1 in regulation of life span and show that tub-1 regulates life span and fat storage by two independent mechanisms.     

Akt pathway as a target for therapeutic intervention in HNSCC

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common form of cancer worldwide. One frequent alteration found in this type of cancer is overactivation of the PI3K/PTEN/mTOR pathway, of which protein kinase B (PKB)/Akt is a central key element, controlling important cellular processes such as metabolism, cell size, proliferation and apoptosis, ultimately regulating cell growth and survival. Thus, drugs that target Akt directly or elements of the pathway are plausible candidates for cancer treatment. Accordingly, numerous clinical trials in various phases are being performed for these drugs. In this review, we discuss the tumorigenic capacity of Akt and focus on its role in HNSCC, paying special attention to the current efforts in treating this cancer in a more specific, Akt-targeted way, based on its primordial role in this type of cancer.     

Immunoliposomes with different acid sensitivities as probes for the cellular endocytic pathway

By combining dioleoylphosphatidylethanolamine (DOPE) with oleic acid (OA), palmitoylhomocysteine (PHC) or dipalmitoylsuccinylglycerol (DPSG) we have prepared pH-sensitive liposomes with different acid sensitivities. DOPE/OA liposomes are the most acid sensitive, while DOPE/DPSG liposomes are the least acid sensitive. Incubation of DOPE/OA liposomes with mouse L929 cells reduces the pH-sensitivity of these liposomes by altering the lipid composition. Using diphtheria toxin fragment A as a marker for cytoplasmic delivery, we find that the delivery kinetics of pH-sensitive immunoliposomes closely correlates with the modified acid sensitivities of the liposomes. Immunoliposomes encounter pH 6-6.2 with a t1/2 of 5-15 min after internalization. By contrast, acidification of the endosomes to pH 5.0 takes longer (t1/2 approximately 25 min). We also used a whole cell null point technique (Yamishiro and Maxfield (1987) J. Cell Biol. 105, 2713-2721) to directly determine the average pH encountered by the endocytosed immunoliposomes. We find that acidification determined by the null point method proceeds less rapidly than that estimated from DTA delivery data. This is likely due to the fact that the measured DTA delivery is done by those liposomes which first arrive at the endosomes with sufficient acidity. Our data suggests that DOPE/PHC immunoliposomes deliver at the early endosome while DOPE/DPSG immunoliposomes deliver at the late endosomes. The DOPE/OA immunoliposomes, with the altered composition and acid sensitivity, deliver with a kinetics intermediate between the other two immunoliposomes. Thus, pH-sensitive liposomes represent useful probes for studying the kinetics of endosome acidification.     

The endocytic pathway: a mosaic of domains

Organelles in the endocytic pathway are composed of a mosaic of structural and functional regions. These regions consist, at least in part, of specialized protein-lipid domains within the plane of the membrane, or of protein complexes associated with specific membrane lipids. Whereas some of these molecular assemblies can be found in more than one compartment, a given combination seems to be unique to each compartment, indicating that membrane organization might be modular.     

Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

PTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.Subject terms: Cancer, Oncogenes     

A non-exchangeable fluorescent phospholipid analog as a membrane traffic marker of the endocytic pathway

The fluorescent phospholipid analog N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) was inserted into the plasma membrane of Baby hamster kidney cells at low temperature (2 degrees C). The mobility characteristics of the analog--as revealed by fluorescence photobleaching recovery--were very similar to those of membrane-inserted 1-acyl-2[6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]caproyl] phosphatidylcholine (C6-NBD-PC). Upon warming to 37 degrees C, followed by a 1-h incubation, all N-Rh-PE was located intracellularly. By contrast, after the same time interval, approximately 10% of the cell-associated PC-derivative was found intracellularly. Furthermore, the analogs moved to different intracellular sites, as N-Rh-PE associates with perinuclear and peri-Golgi structures, whereas C6-NBD-PC appears mainly in the Golgi complex. Colocalization with organelle-specific probes and Percoll gradient analysis identified the N-Rh-PE-labeled structures as lysosomes. Temperature and energy-dependent experiments supported the endocytic pathway as the mechanism of N-Rh-PE internalization. The mechanism of N-Rh-PE internalization appears to differ from that of C6-NBD-PC. In conjunction with a difference in the efficiency of removal of the lipid derivatives from the plasma membrane, the results suggest that N-Rh-PE is selectively internalized, implying that sorting of the lipid analogs already occurs at the level of the plasma membrane. The distinct difference in physical appearance of the probes after membrane insertion, i.e., N-Rh-PE being present as small clusters and C6-NBD-PC as monomers, could explain the selective sorting and internalization of N-Rh-PE. The results demonstrate that N-Rh-PE may serve as a useful marker for studying membrane traffic during endocytosis.     

The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.     

C. elegans fat storage and metabolic regulation

C. elegans has long been used as an experimentally tractable organism for discovery of fundamental mechanisms that underlie metazoan cellular function, development, neurobiology, and behavior. C. elegans has more recently been exploited to study the interplay of environment and genetics on lipid storage pathways. As an experimental platform, C. elegans is amenable to an extensive array of forward and reverse genetic, a variety of “omics” and anatomical approaches that together allow dissection of complex physiological pathways. This is particularly relevant to the study of fat biology, as energy balance is ultimately an organismal process that involves behavior, nutrient digestion, uptake and transport, as well as a variety of cellular activities that determine the balance between lipid storage and utilization. C. elegans offers the opportunity to dissect these pathways and various cellular and organismal homeostatic mechanisms in the context of a genetically tractable, intact organism.     

Macropinocytosis: an endocytic pathway for internalising large gulps

Macropinocytosis is a regulated form of endocytosis that mediates the non-selective uptake of solute molecules, nutrients and antigens. It is an actin-dependent process initiated from surface membrane ruffles that give rise to large endocytic vacuoles called macropinosomes. Macropinocytosis is important in a range of physiological processes; it is highly active in macrophages and dendritic cells where it is a major pathway for the capture of antigens, it is relevant to cell migration and tumour metastasis and it represents a portal of cell entry exploited by a range of pathogens. The molecular basis for the formation and maturation of macropinosomes has only recently begun to be defined. Here, we review the general characteristics of macropinocytosis, describe some of the regulators of this pathway, which have been identified to date and highlight strategies to explore the relevance of this endocytosis pathway in vivo.     

Angiogenetic pathway as a therapeutic target in renal cell carcinoma

For several decades, metastatic renal cell carcinoma (mRCC) was considered resistant to therapy and thus linked to poor prognosis. Recent years have seen a complete revolution of the therapeutic landscape, with the introduction of new target therapies that have radically changed treatment strategies in this setting. These new agents have proved to be effective if directed mainly against the angiogenic pathway. The vascular endothelial growth factor family is, in fact, the main target of a number of new drugs, such as monoclonal antibodies and different generations of tyrosine-kinase inhibitors that are currently approved or in different stages of clinical trial for the treatment of mRCC patients.     

Evidence for a clathrin-independent endocytic pathway for APP internalization in the neuronal somatodendritic compartment

1. Download : Download high-res image (267KB)
2. Download : Download full-size image

     

A cell-free analysis of the endocytic pathway

The molecular control of the endocytic pathway is poorly understood. To obtain this information requires the use of cell-free systems which faithfully recreate the various endocytic events as they occur in the intact cell. Here I describe our approach to elucidating the mechanism which controls the fusion between different vesicles on the pathway.     

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae, and is not inhibited by overexpression of GTPase-null dynamin mutants. This mechanism, however, has been defined largely in these negative terms. We applied a ferro-fluid-based purification of endosomes to identify endosomal proteins. One of the proteins identified in this way was flotillin-1 (also called reggie-2). Here, we show that flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both glycosylphosphatidylinositol (GPI)-linked proteins and cholera toxin B subunit. Endocytosis in flotillin-1-containing intermediates is clathrin-independent. Total internal reflection microscopy and immuno-electron microscopy revealed that flotillin-1-containing regions of the plasma membrane seem to bud into the cell, and are distinct from clathrin-coated pits and caveolin-1-positive caveolae. Flotillin-1 small interfering RNA (siRNA) inhibited both clathrin-independent uptake of cholera toxin and endocytosis of a GPI-linked protein. We propose that flotillin-1 is one determinant of a clathrin-independent endocytic pathway in mammalian cells.