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Molecular Biology of the Cell Aug 2023As a prelude to fusion, the R-SNARE on one membrane zippers with Qa-, Qb-, and Qc-SNAREs from its apposed fusion partner, forming a four-helical bundle that draws the...
As a prelude to fusion, the R-SNARE on one membrane zippers with Qa-, Qb-, and Qc-SNAREs from its apposed fusion partner, forming a four-helical bundle that draws the two membranes together. Because Qa- and Qb-SNAREs are anchored to the same membrane and are adjacent in the 4-SNARE bundle, their two anchors might be redundant. Using the recombinant pure protein catalysts of yeast vacuole fusion, we now report that the specific distribution of transmembrane (TM) anchors on the Q-SNAREs is critical for efficient fusion. A TM anchor on the Qa-SNARE supports rapid fusion even when the other two Q-SNAREs are unanchored, while a TM anchor on the Qb-SNARE is dispensable and is insufficient for rapid fusion as the sole Q-SNARE anchor. This does not depend on which specific TM domain is attached to the Qa-SNARE but rather is due to the Qa-SNARE being anchored per se. The need for Qa-SNARE anchoring is even seen when the motypic fusion and vacuole rotein orting protein (HOPS), the physiological catalyst of tethering and SNARE assembly, is replaced by an artificial tether. The need for a Qa TM anchor is thus a fundamental property of vacuolar SNARE zippering-induced fusion and may reflect the need for the Qa juxtamembrane (JxQa) region to be anchored between its SNARE and TM domains. This requirement for Qa-SNARE anchoring and correct JxQa position is bypassed by Sec17/Sec18, exploiting a platform of partially zippered SNAREs. Because Qa is the only synaptic Q-SNARE with a TM anchor, the need for Qa-specific anchoring may reflect a general requirement for SNARE-mediated fusion.
Topics: Vacuoles; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; SNARE Proteins; Qa-SNARE Proteins; Q-SNARE Proteins; Recombinant Proteins
PubMed: 37314849
DOI: 10.1091/mbc.E23-02-0052 -
Autophagy Dec 2023I have been surprised at the number of papers I edit where the schematic of macroautophagy/autophagy is wrong, and it is wrong in the same way. How could this happen?...
I have been surprised at the number of papers I edit where the schematic of macroautophagy/autophagy is wrong, and it is wrong in the same way. How could this happen? Did people get together and agree to misrepresent the morphological intermediates of autophagy? Not exactly.
Topics: Humans; Autophagy; Autophagosomes; Macroautophagy; Lysosomes
PubMed: 37850823
DOI: 10.1080/15548627.2023.2266977 -
Molecular Cell Jan 2024Membraneless organelles formed by phase separation of proteins and nucleic acids play diverse cellular functions. Whether and, if yes, how membraneless organelles in...
Membraneless organelles formed by phase separation of proteins and nucleic acids play diverse cellular functions. Whether and, if yes, how membraneless organelles in ways analogous to membrane-based organelles also undergo regulated fusion and fission is unknown. Here, using a partially reconstituted mammalian postsynaptic density (PSD) condensate as a paradigm, we show that membraneless organelles can undergo phosphorylation-dependent fusion and fission. Without phosphorylation of the SAPAP guanylate kinase domain-binding repeats, the upper and lower layers of PSD protein mixtures form two immiscible sub-compartments in a phase-in-phase organization. Phosphorylation of SAPAP leads to fusion of the two sub-compartments into one condensate accompanied with an increased Stargazin density in the condensate. Dephosphorylation of SAPAP can reverse this event. Preventing SAPAP phosphorylation in vivo leads to increased separation of proteins from the lower and upper layers of PSD sub-compartments. Thus, analogous to membrane-based organelles, membraneless organelles can also undergo regulated fusion and fission.
Topics: Animals; Biomolecular Condensates; Phosphorylation; Post-Synaptic Density; Cell Physiological Phenomena; Protein Binding; Organelles; Mammals
PubMed: 38096828
DOI: 10.1016/j.molcel.2023.11.011 -
Results and Problems in Cell... 2024The Drosophila trachea is an interconnected network of epithelial tubes, which delivers gases throughout the entire organism. It is the premier model to study the... (Review)
Review
The Drosophila trachea is an interconnected network of epithelial tubes, which delivers gases throughout the entire organism. It is the premier model to study the development of tubular organs, such as the human lung, kidney, and blood vessels. The Drosophila embryonic trachea derives from a series of segmentally repeated clusters. The tracheal precursor cells in each cluster migrate out in a stereotyped pattern to form primary branches. Thereafter, the neighboring branches need to fuse to form an interconnected tubular network. The connection between neighboring branches is orchestrated by specialized cells, called fusion cells. These cells fuse with their counterparts to form a tube with a contiguous lumen. Branch fusion is a multi-step process that includes cell migration, cell adhesion, cytoskeleton track formation, vesicle trafficking, membrane fusion, and lumen formation. This review summarizes the current knowledge on fusion process in the Drosophila trachea. These mechanisms will greatly contribute to our understanding of branch fusion in mammalian systems.
Topics: Animals; Cytoskeleton; Drosophila; Drosophila melanogaster; Drosophila Proteins; Mammals; Microtubules; Morphogenesis; Trachea
PubMed: 37996674
DOI: 10.1007/978-3-031-37936-9_5 -
Nanoscale Sep 2023Gene delivery has great potential in modulating protein expression in specific cells to treat diseases. Such therapeutic gene delivery demands sufficient cellular...
Gene delivery has great potential in modulating protein expression in specific cells to treat diseases. Such therapeutic gene delivery demands sufficient cellular internalization and endosomal escape. Of various nonviral nucleic acid delivery systems, lipid nanoparticles (LNPs) are the most advanced, but still, are very inefficient as the majority are unable to escape from endosomes/lysosomes. Here, we develop a highly efficient gene delivery system using fusogenic coiled-coil peptides. We modified LNPs, carrying EGFP-mRNA, and cells with complementary coiled-coil lipopeptides. Coiled-coil formation between these lipopeptides induced fast nucleic acid uptake and enhanced GFP expression. The cellular uptake of coiled-coil modified LNPs is likely driven by membrane fusion thereby omitting typical endocytosis pathways. This direct cytosolic delivery circumvents the problems commonly observed with the limited endosomal escape of mRNA. Therefore fusogenic coiled-coil peptide modification of existing LNP formulations to enhance nucleic acid delivery efficiency could be beneficial for several gene therapy applications.
PubMed: 37671560
DOI: 10.1039/d3nr02175k -
Molecular Biology of the Cell Mar 2024Insulin secretion depends on the Ca-regulated fusion of granules with the plasma membrane. A recent model of Ca-triggered exocytosis in secretory cells proposes that...
Insulin secretion depends on the Ca-regulated fusion of granules with the plasma membrane. A recent model of Ca-triggered exocytosis in secretory cells proposes that lipids in the plasma membrane couple the calcium sensor Syt1 to the membrane fusion machinery (Kiessling , 2018). Specifically, Ca-mediated binding of Syt1's C2 domains to the cell membrane shifts the membrane-anchored SNARE syntaxin-1a to a more fusogenic conformation, straightening its juxtamembrane linker. To test this model in live cells and extend it to insulin secretion, we enriched INS1 cells with a panel of lipids with different acyl chain compositions. Fluorescence lifetime measurements demonstrate that cells with more disordered membranes show an increase in fusion efficiency, and vice versa. Experiments with granules purified from INS1 cells and recombinant SNARE proteins reconstituted in supported membranes confirmed that lipid acyl chain composition determines SNARE conformation and that lipid disordering correlates with increased fusion. Addition of Syt1's C2AB domains significantly decreased lipid order in target membranes and increased SNARE-mediated fusion probability. Strikingly, Syt's action on both fusion and lipid order could be partially bypassed by artificially increasing unsaturated phosphatidylserines in the target membrane. Thus, plasma membrane lipids actively participate in coupling Ca/synaptotagmin-sensing to the SNARE fusion machinery in cells.
Topics: Membrane Fusion; Membrane Lipids; SNARE Proteins; Insulin-Secreting Cells; Cell Membrane; Synaptotagmin I; Exocytosis; Recombinant Proteins; Calcium
PubMed: 38117594
DOI: 10.1091/mbc.E23-06-0225 -
International Journal of Molecular... Nov 2023Membrane-spanning portions of proteins' polypeptide chains are commonly known as their transmembrane domains (TMDs). The structural organisation and dynamic behaviour of... (Review)
Review
Membrane-spanning portions of proteins' polypeptide chains are commonly known as their transmembrane domains (TMDs). The structural organisation and dynamic behaviour of TMDs from proteins of various families, be that receptors, ion channels, enzymes etc., have been under scrutiny on the part of the scientific community for the last few decades. The reason for such attention is that, apart from their obvious role as an "anchor" in ensuring the correct orientation of the protein's extra-membrane domains (in most cases functionally important), TMDs often actively and directly contribute to the operation of "the protein machine". They are capable of transmitting signals across the membrane, interacting with adjacent TMDs and membrane-proximal domains, as well as with various ligands, etc. Structural data on TMD arrangement are still fragmentary at best due to their complex molecular organisation as, most commonly, dynamic oligomers, as well as due to the challenges related to experimental studies thereof. Inter alia, this is especially true for viral fusion proteins, which have been the focus of numerous studies for quite some time, but have provoked unprecedented interest in view of the SARS-CoV-2 pandemic. However, despite numerous structure-centred studies of the spike (S) protein effectuating target cell entry in coronaviruses, structural data on the TMD as part of the entire spike protein are still incomplete, whereas this segment is known to be crucial to the spike's fusogenic activity. Therefore, in attempting to bring together currently available data on the structure and dynamics of spike proteins' TMDs, the present review aims to tackle a highly pertinent task and contribute to a better understanding of the molecular mechanisms underlying virus-mediated fusion, also offering a rationale for the design of novel efficacious methods for the treatment of infectious diseases caused by SARS-CoV-2 and related viruses.
Topics: Humans; Membrane Fusion; Protein Domains; Viral Fusion Proteins; Peptides; SARS-CoV-2
PubMed: 38003610
DOI: 10.3390/ijms242216421 -
Cell Discovery Jun 2024Membrane budding, which underlies fundamental processes like endocytosis, intracellular trafficking, and viral infection, is thought to involve membrane coat-forming...
Membrane budding, which underlies fundamental processes like endocytosis, intracellular trafficking, and viral infection, is thought to involve membrane coat-forming proteins, including the most observed clathrin, to form Ω-shape profiles and helix-forming proteins like dynamin to constrict Ω-profiles' pores and thus mediate fission. Challenging this fundamental concept, we report that polymerized clathrin is required for Ω-profiles' pore closure and that clathrin around Ω-profiles' base/pore region mediates pore constriction/closure in neuroendocrine chromaffin cells. Mathematical modeling suggests that clathrin polymerization at Ω-profiles' base/pore region generates forces from its intrinsically curved shape to constrict/close the pore. This new fission function may exert broader impacts than clathrin's well-known coat-forming function during clathrin (coat)-dependent endocytosis, because it underlies not only clathrin (coat)-dependent endocytosis, but also diverse endocytic modes, including ultrafast, fast, slow, bulk, and overshoot endocytosis previously considered clathrin (coat)-independent in chromaffin cells. It mediates kiss-and-run fusion (fusion pore closure) previously considered bona fide clathrin-independent, and limits the vesicular content release rate. Furthermore, analogous to results in chromaffin cells, we found that clathrin is essential for fast and slow endocytosis at hippocampal synapses where clathrin was previously considered dispensable, suggesting clathrin in mediating synaptic vesicle endocytosis and fission. These results suggest that clathrin and likely other intrinsically curved coat proteins are a new class of fission proteins underlying vesicle budding and fusion. The half-a-century concept and studies that attribute vesicle-coat contents' function to Ω-profile formation and classify budding as coat-protein (e.g., clathrin)-dependent or -independent may need to be re-defined and re-examined by considering clathrin's pivotal role in pore constriction/closure.
PubMed: 38862506
DOI: 10.1038/s41421-024-00677-w -
Biochimica Et Biophysica Acta.... Oct 2023Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as... (Review)
Review
Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.
Topics: Humans; Viral Envelope; Viral Envelope Proteins; Flavivirus; Zika Virus; Zika Virus Infection; Peptides; Dengue; Antiviral Agents; Phospholipids
PubMed: 37437754
DOI: 10.1016/j.bbamem.2023.184198 -
Antiviral Research Feb 2024The World Health Organization advices the use of a quadrivalent vaccine as prophylaxis against influenza, to prevent severe influenza-associated disease and -mortality,... (Review)
Review
The World Health Organization advices the use of a quadrivalent vaccine as prophylaxis against influenza, to prevent severe influenza-associated disease and -mortality, and to keep up with influenza antigenic diversity. Different small molecule antivirals to treat influenza have become available. However, emergence of drug resistant influenza viruses has been observed upon use of these antivirals. An appealing alternative approach to prevent or treat influenza is the use of antibody-based antivirals, such as conventional monoclonal antibodies and single-domain antibodies (sdAbs). The surface of the influenza A and B virion is decorated with hemagglutinin molecules, which act as receptor-binding and membrane fusion proteins and represent the main target of neutralizing antibodies. SdAbs that target influenza A and B hemagglutinin have been described. In addition, sdAbs directed against the influenza A virus neuraminidase have been reported, whereas no sdAbs targeting influenza B neuraminidase have been described to date. SdAbs directed against influenza A matrix protein 2 or its ectodomain have been reported, while no sdAbs have been described targeting the influenza B matrix protein 2. Known for their high specificity, ease of production and formatting, sdAb-based antivirals could be a major leap forward in influenza control.
Topics: Humans; Influenza, Human; Single-Domain Antibodies; Antibodies, Viral; Hemagglutinins; Neuraminidase; Influenza Vaccines; Orthomyxoviridae Infections; Antiviral Agents; Hemagglutinin Glycoproteins, Influenza Virus
PubMed: 38219914
DOI: 10.1016/j.antiviral.2024.105807