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Cell Sep 2020The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular...
The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular functions, from cell division to lysosome degradation and autophagy. How it breaks membranes, though, remains unknown. Here, we characterize a sequential polymerization of ESCRT-III subunits that, driven by a recruitment cascade and by continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of Vps24 for Did2 induces a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filament to drive the formation of membrane protrusions, and ends with the formation of a highly constricted Did2-Ist1 co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of stepwise changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to catalyze ESCRT-III activity.
Topics: Adenosine Triphosphatases; Cell Line, Tumor; Cell Membrane; Endosomal Sorting Complexes Required for Transport; Endosomes; HeLa Cells; Humans; Membrane Fusion; Polymerization; Protein Transport
PubMed: 32814015
DOI: 10.1016/j.cell.2020.07.021 -
Nature Communications Oct 2023The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for autophagosome-lysosome fusion in mammals, yet...
The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for autophagosome-lysosome fusion in mammals, yet reconstituting the mammalian HOPS complex remains a challenge. Here we propose a "hook-up" model for mammalian HOPS complex assembly, which requires two HOPS sub-complexes docking on membranes via membrane-associated Rabs. We identify Rab39A as a key small GTPase that recruits HOPS onto autophagic vesicles. Proper pairing with Rab2 and Rab39A enables HOPS complex assembly between proteoliposomes for its tethering function, facilitating efficient membrane fusion. GTP loading of Rab39A is important for the recruitment of HOPS to autophagic membranes. Activation of Rab39A is catalyzed by C9orf72, a guanine exchange factor associated with amyotrophic lateral sclerosis and familial frontotemporal dementia. Constitutive activation of Rab39A can rescue autophagy defects caused by C9orf72 depletion. These results therefore reveal a crucial role for the C9orf72-Rab39A-HOPS axis in autophagosome-lysosome fusion.
Topics: Animals; Autophagy; C9orf72 Protein; Catalysis; Guanosine Triphosphate; Mammals; Membrane Fusion; Vacuoles
PubMed: 37821429
DOI: 10.1038/s41467-023-42003-0 -
Cell Calcium Jan 2022In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where...
In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca-dependent exocytosis regulated by distinct Casources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.
Topics: Animals; Cell Membrane; Cholesterol; Exocytosis; Lactotrophs; Membrane Fusion; Rats; Rats, Wistar; Secretory Vesicles
PubMed: 34844123
DOI: 10.1016/j.ceca.2021.102503 -
Biochemical Society Transactions Aug 2022Visualization of cellular dynamics using fluorescent light microscopy has become a reliable and indispensable source of experimental evidence for biological studies.... (Review)
Review
Visualization of cellular dynamics using fluorescent light microscopy has become a reliable and indispensable source of experimental evidence for biological studies. Over the past two decades, the development of super-resolution microscopy platforms coupled with innovations in protein and molecule labeling led to significant biological findings that were previously unobservable due to the barrier of the diffraction limit. As a result, the ability to image the dynamics of cellular processes is vastly enhanced. These imaging tools are extremely useful in cellular physiology for the study of vesicle fusion and endocytosis. In this review, we will explore the power of stimulated emission depletion (STED) and confocal microscopy in combination with various labeling techniques in real-time observation of the membrane transformation of fusion and endocytosis, as well as their underlying mechanisms. We will review how STED and confocal imaging are used to reveal fusion and endocytic membrane transformation processes in live cells, including hemi-fusion; hemi-fission; hemi-to-full fusion; fusion pore opening, expansion, constriction and closure; shrinking or enlargement of the Ω-shape membrane structure after vesicle fusion; sequential compound fusion; and the sequential endocytic membrane transformation from flat- to O-shape via the intermediate Λ- and Ω-shape transition. We will also discuss how the recent development of imaging techniques would impact future studies in the field.
Topics: Cell Membrane; Endocytosis; Exocytosis; Membrane Fusion; Microscopy, Confocal; Secretory Vesicles
PubMed: 35960003
DOI: 10.1042/BST20210263 -
PLoS Pathogens Mar 2023Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As...
Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.
Topics: Glucosylceramides; Membrane Fusion; Glycoproteins; Lactosylceramides; RNA Viruses
PubMed: 36920967
DOI: 10.1371/journal.ppat.1011232 -
Traffic (Copenhagen, Denmark) Oct 2018A cell is able to sense the biomechanical properties of the environment such as the rigidity of the extracellular matrix and adapt its tension via regulation of plasma... (Review)
Review
A cell is able to sense the biomechanical properties of the environment such as the rigidity of the extracellular matrix and adapt its tension via regulation of plasma membrane and underlying actomyosin meshwork properties. The cell's ability to adapt to the changing biomechanical environment is important for cellular homeostasis and also cell dynamics such as cell growth and motility. Membrane trafficking has emerged as an important mechanism to regulate cell biomechanics. In this review, we summarize the current understanding of the role of cell mechanics in exocytosis, and reciprocally, the role of exocytosis in regulating cell mechanics. We also discuss how cell mechanics and membrane trafficking, particularly exocytosis, can work together to regulate cell polarity and motility.
Topics: Animals; Biomechanical Phenomena; Cell Membrane; Cell Movement; Cell Polarity; Exocytosis; Humans; Membrane Fusion; Protein Transport
PubMed: 29943478
DOI: 10.1111/tra.12584 -
Annual Review of Biophysics May 2022Cellular membranes self-assemble from and interact with various molecular species. Each molecule locally shapes the lipid bilayer, the soft elastic core of cellular... (Review)
Review
Cellular membranes self-assemble from and interact with various molecular species. Each molecule locally shapes the lipid bilayer, the soft elastic core of cellular membranes. The dynamic architecture of intracellular membrane systems is based on elastic transformations and lateral redistribution of these elementary shapes, driven by chemical and curvature stress gradients. The minimization of the total elastic stress by such redistribution composes the most basic, primordial mechanism of membrane curvature-composition coupling (CCC). Although CCC is generally considered in the context of dynamic compositional heterogeneity of cellular membrane systems, in this article we discuss a broader involvement of CCC in controlling membrane deformations. We focus specifically on the mesoscale membrane transformations in open, reservoir-governed systems, such as membrane budding, tubulation, and the emergence of highly curved sites of membrane fusion and fission. We reveal that the reshuffling of molecular shapes constitutes an independent deformation mode with complex rheological properties.This mode controls effective elasticity of local deformations as well as stationary elastic stress, thus emerging as a major regulator of intracellular membrane remodeling.
Topics: Cell Membrane; Elasticity; Lipid Bilayers; Membrane Fusion
PubMed: 35239417
DOI: 10.1146/annurev-biophys-011422-100054 -
Biochimica Et Biophysica Acta.... Dec 2021Autophagy is an essential process in cell self-repair and survival. The centre of the autophagic event is the generation of the so-called autophagosome (AP), a vesicle... (Review)
Review
Autophagy is an essential process in cell self-repair and survival. The centre of the autophagic event is the generation of the so-called autophagosome (AP), a vesicle surrounded by a double membrane (two bilayers). The AP delivers its cargo to a lysosome, for degradation and re-use of the hydrolysis products as new building blocks. AP formation is a very complex event, requiring dozens of specific proteins, and involving numerous instances of membrane biogenesis and architecture, including membrane fusion and fission. Many stages of AP generation can be rationalised in terms of curvature, both the molecular geometry of lipids interpreted in terms of 'intrinsic curvature', and the overall mesoscopic curvature of the whole membrane, as observed with microscopy techniques. The present contribution intends to bring together the worlds of biophysics and cell biology of autophagy, in the hope that the resulting cross-pollination will generate abundant fruit.
Topics: Autophagosomes; Autophagy; Biophysics; Cell Communication; Lipid Bilayers; Lysosomes; Membrane Fusion; Phagocytosis
PubMed: 34419487
DOI: 10.1016/j.bbamem.2021.183731 -
Protein Science : a Publication of the... Aug 2018Research for three decades and major recent advances have provided crucial insights into how neurotransmitters are released by Ca -triggered synaptic vesicle exocytosis,... (Review)
Review
Research for three decades and major recent advances have provided crucial insights into how neurotransmitters are released by Ca -triggered synaptic vesicle exocytosis, leading to reconstitution of basic steps that underlie Ca -dependent membrane fusion and yielding a model that assigns defined functions for central components of the release machinery. The soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) syntaxin-1, SNAP-25, and synaptobrevin-2 form a tight SNARE complex that brings the vesicle and plasma membranes together and is key for membrane fusion. N-ethyl maleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble the SNARE complex to recycle the SNAREs for another round of fusion. Munc18-1 and Munc13-1 orchestrate SNARE complex formation in an NSF-SNAP-resistant manner by a mechanism whereby Munc18-1 binds to synaptobrevin and to a self-inhibited "closed" conformation of syntaxin-1, thus forming a template to assemble the SNARE complex, and Munc13-1 facilitates assembly by bridging the vesicle and plasma membranes and catalyzing opening of syntaxin-1. Synaptotagmin-1 functions as the major Ca sensor that triggers release by binding to membrane phospholipids and to the SNAREs, in a tight interplay with complexins that accelerates membrane fusion. Many of these proteins act as both inhibitors and activators of exocytosis, which is critical for the exquisite regulation of neurotransmitter release. It is still unclear how the actions of these various proteins and multiple other components that control release are integrated and, in particular, how they induce membrane fusion, but it can be expected that these fundamental questions can be answered in the near future, building on the extensive knowledge already available.
Topics: Animals; Humans; Membrane Fusion; Mice; Neurotransmitter Agents; SNARE Proteins; Synaptic Transmission
PubMed: 29893445
DOI: 10.1002/pro.3445 -
Langmuir : the ACS Journal of Surfaces... Nov 2021Fusion events in living cells are intricate phenomena that require the coordinate action of multicomponent protein complexes. However, simpler synthetic tools to control...
Fusion events in living cells are intricate phenomena that require the coordinate action of multicomponent protein complexes. However, simpler synthetic tools to control membrane fusion in artificial cells are highly desirable. Native membrane fusion machinery mediates fusion, driving a delicate balance of membrane curvature and tension between two closely apposed membranes. Here, we show that silica nanoparticles (SiO NPs) at a size close to the cross-over between tension-driven and curvature-driven interaction regimes initiate efficient fusion of biomimetic model membranes. Fusion efficiency and mechanisms are studied by Förster resonance energy transfer and confocal fluorescence microscopy. SiO NPs induce a slight increase in lipid packing likely to increase the lateral tension of the membrane. We observe a connection between membrane tension and fusion efficiency. Finally, real-time confocal fluorescence microscopy reveals three distinct mechanistic pathways for membrane fusion. SiO NPs show significant potential for inclusion in the synthetic biology toolkit for membrane remodeling and fusion in artificial cells.
Topics: Biomimetics; Membrane Fusion; Membranes; Nanoparticles; Silicon Dioxide
PubMed: 34788054
DOI: 10.1021/acs.langmuir.1c02492