Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.

Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.     

Glycans in autophagy,endocytosis and lysosomal functions

Glycoconjugate Journal - Glycans have been shown to function as versatile molecular signals in cells. This prompted us to look at their roles in endocytosis, endolysosomal system and autophagy. We...     

Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins

The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery.     

Molecular interplay of autophagy and endocytosis in human health and diseases

Autophagy, an evolutionarily conserved process for maintaining the physio‐metabolic equilibrium of cells, shares many common effector proteins with endocytosis. For example, tethering proteins involved in fusion like Ras‐like GTPases (Rabs), soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs), lysosomal‐associated membrane protein (LAMP), and endosomal sorting complex required for transport (ESCRT) have a dual role in endocytosis and autophagy, and the trafficking routes of these processes converge at lysosomes. These common effectors indicate an association between budding and fusion of membrane‐bound vesicles that may have a substantial role in autophagic lysosome reformation, by sensing cellular stress levels. Therefore, autophagy–endocytosis crosstalk may be significant and implicates a novel endocytic regulatory pathway of autophagy. Moreover, endocytosis has a pivotal role in the intake of signalling molecules, which in turn activates cascades that can result in pathophysiological conditions. This review discusses the basic mechanisms of this crosstalk and its implications in order to identify potential novel therapeutic targets for various human diseases.     

Interaction of the endocytic scaffold protein Pan1 with the type I myosins contributes to the late stages of endocytosis

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1DeltaPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5-Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.     

Powering membrane traffic in endocytosis and recycling

Early in evolution, the diversification of membrane-bound compartments that characterize eukaryotic cells was accompanied by the elaboration of molecular machineries that mediate intercompartmental communication and deliver materials to specific destinations. Molecular motors that move on tracks of actin filaments or microtubules mediate the movement of organelles and transport between compartments. The subjects of this review are the motors that power the transport steps along the endocytic and recycling pathways, their modes of attachment to cargo and their regulation.     

Interaction of Lassa virus fusion and membrane proximal peptides with late endosomal membranes

Mammarenaviruses include many significant worldwide-widespread human pathogens, among them Lassa virus (LASV), having a dramatic morbidity and mortality rate. They are a potential high-risk menace to the worldwide public health since there are no treatments and there is a high possibility of animal-to-human and human-to-human viral transmission. These viruses enter into the cells by endocytosis fusing its membrane envelope with the late endosomal membrane thanks to the glycoprotein GP2, a membrane fusion protein of class I. This protein contains different domains, among them the N-terminal fusion peptide (NFP), the internal fusion loop (IFL), the membrane proximal external region (MPER) and the transmembrane domain (TMD). All these domains are implicated in the membrane fusion process. In this work, we have used an all-atom molecular dynamics study to know the binding of these protein domains with a complex membrane mimicking the late endosome one. We show that the NFP/IFL domain is capable of spontaneously inserting into the membrane without a significant change of secondary structure, the MPER domain locates at the bilayer interface with an orientation parallel to the membrane surface and tends to interact with other MPER domains, and the TMD domain tilts inside the bilayer. Moreover, they predominantly interact with negatively charged phospholipids. Overall, these membrane-interacting domains would characterise a target that would make possible to find effective antiviral molecules against LASV in particular and Mammarenaviruses in general.     

Expanding into new markets--VCP/p97 in endocytosis and autophagy

The AAA-ATPase p97 (also called VCP for Valosin-containing protein) is essential for a number of cellular processes as diverse as ER-associated degradation, DNA damage response, and cell cycle control. Mechanistically, p97 cooperates with its cofactor Ufd1-Npl4 in these processes to segregate polyubiquitinated misfolded or regulatory client proteins from intracellular structures for subsequent degradation by the proteasome. Recent work now connects p97, independently of Ufd1-Npl4, to endosomal trafficking and autophagy. Interestingly, these pathways also deliver proteins for degradation, albeit by the lysosome. While monoubiquitination and alternative p97-cofactors, including UBXD1, have been associated with these activities, the underlying molecular mechanism(s) are still unclear or controversial. In this review, we aim to summarize the available data and discuss mechanistic models.     

Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

Background

Dendritic cells (DCs) are among the first cells to encounter HIV-1 and play important roles in viral transmission and pathogenesis. Immature DCs allow productive HIV-1 replication and long-term viral dissemination. The pro-inflammatory factor lipopolysaccharide (LPS) induces DC maturation and enhances the efficiency of DC-mediated HIV-1 transmission. Type I interferon (IFN) partially inhibits HIV-1 replication and cell-cell transmission in CD4⁺ T cells and macrophages. Tetherin is a type I IFN-inducible restriction factor that blocks HIV-1 release and modulates CD4⁺ T cell-mediated cell-to-cell transmission of HIV-1. However, the role of type I IFN and tetherin in HIV-1 infection of DCs and DC-mediated viral transmission remains unknown.

Results

We demonstrated that IFN-alpha (IFNα)-induced mature DCs restricted HIV-1 replication and trans-infection of CD4⁺ T cells. Tetherin expression in monocyte-derived immature DCs was undetectable or very low. High levels of tetherin were transiently expressed in LPS- and IFNα-induced mature DCs, while HIV-1 localized into distinct patches in these DCs. Knockdown of induced tetherin in LPS- or IFNα-matured DCs modestly enhanced HIV-1 transmission to CD4⁺ T cells, but had no significant effect on wild-type HIV-1 replication in mature DCs. Intriguingly, we found that HIV-1 replication in immature DCs induced significant tetherin expression in a Nef-dependent manner.

Conclusions

The restriction of HIV-1 replication and transmission in IFNα-induced mature DCs indicates a potent anti-HIV-1 response; however, high levels of tetherin induced in mature DCs cannot significantly restrict wild-type HIV-1 release and DC-mediated HIV-1 transmission. Nef-dependent tetherin induction in HIV-1-infected immature DCs suggests an innate immune response of DCs to HIV-1 infection.     

Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis

Endophilin/SH3p4 is a protein highly enriched in nerve terminals that binds the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin, two proteins implicated in synaptic vesicle endocytosis. We show here that antibody-mediated disruption of endophilin function in a tonically stimulated synapse leads to a block in the invagination of clathrin-coated pits adjacent to the active zone and therefore to a block of synaptic vesicle recycling. We also show that in a cell-free system, endophilin is not associated with clathrin coats and is a functional partner of dynamin. Our findings suggest that endophilin is part of a biochemical machinery that acts in trans to the clathrin coat from early stages to vesicle fission.     

Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis

Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.     

Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts

Cell–cell fusion is an evolutionarily conserved process that leads to the formation of multinucleated myofibers, syncytiotrophoblasts and osteoclasts, allowing their respective functions. Although cell–cell fusion requires the presence of fusogenic membrane proteins and actin-dependent cytoskeletal reorganization, the precise machinery allowing cells to fuse is still poorly understood. Using an inducible knockout mouse model to generate dynamin 1– and 2–deficient primary osteoclast precursors and myoblasts, we found that fusion of both cell types requires dynamin. Osteoclast and myoblast cell–cell fusion involves the formation of actin-rich protrusions closely associated with clathrin-mediated endocytosis in the apposed cell. Furthermore, impairing endocytosis independently of dynamin also prevented cell–cell fusion. Since dynamin is involved in both the formation of actin-rich structures and in endocytosis, our results indicate that dynamin function is central to the osteoclast precursors and myoblasts fusion process, and point to an important role of endocytosis in cell–cell fusion.     

Flow and shuttle of plasma membrane during endocytosis

A striking feature of endocytosis is the large amount of surface membrane that is brought into the cells through the formation of endocytic vesicles. Little is known about the fate of this membrane material. It is implausible that it would be destroyed in lysosomes, as the rate of turnover of the constituents of plasma membrane is much too low with respect to the rate of endocytosis in all cells studied so far. Conversely, plasma membrane fragments, internalized by endocytosis cannot merely be incorporated in lysosomes, as these organelles have been shown to maintain their size, despite continuous and active endocytosis. We present evidence that plasma membrane antigens, detected by means of specific antibodies, are internalized during endocytosis and reach lysosomes. They are thereafter returned back to cell surface. These results indicate the existence of a shuttle of membrane elements between the cell surface and lysosomes.     

In vitro fusion of endosomes following receptor-mediated endocytosis

Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment via the macrophage mannose receptor and (ii) that mutually recognize each other. Dinitrophenol-derivatized beta-glucuronidase (DNP-beta-glucuronidase), a ligand for the mannose receptor, was endocytosed by one population of J774 E clone cells, and mannose-derivatized monoclonal anti-DNP IgG (Man-IgG) was internalized by a second set of cells. Both ligands were localized in endosomes as determined by fractionation on Percoll gradients. Incubation of vesicles prepared from the two set of cells resulted in vesicle fusion as indicated by the formation of DNP-beta-glucuronidase-Man-IgG complexes. Under standard conditions, fusion was time-, ATP-, and temperature-dependent. KCl was required for fusion. Fusion required both cytosolic- and membrane-associated proteins. N-Ethylmaleimide treatment of cytosol inhibited fusion. Proton ionophores and amines had no effect on the fusion reaction. ATP-dependent fusion was only observed between early endocytic compartments. While in the presence of a Ca2+ chelator fusion was ATP-dependent, in its absence fusion was also observed in an ATP-independent fashion.