Membrane Fusion Events Research Articles

A Novel Homozygous Abnormal Splice Variant in the Myoferlin Gene Leading to Floppy Infant Syndrome in a Saudi Family

Myoferlin (MYOF) (OMIM#604603) is a type II membrane protein that belongs to the ferlin family, which is expressed in cardiac and skeletal muscles. This protein has seven C2 domains that mediate calcium-dependent membrane fusion events and membrane trafficking, while MYOF dysfunction is associated with muscular disorder. We are reporting a case from Saudi Arabia of an 18-month-old male patient with generalized hypotonia, which might be a floppy infant syndrome. In this study, whole exome sequencing (WES) was done, and a novel homozygous abnormal splice variant c.4982+1G>T, p.Val1661fs was identified in the MYOF gene. The results of WES were further validated by using Sanger sequencing; the proband showed homozygous mutation while both parents were heterozygous at this position. Implementing WES improves the screening and detection of novel and causative genetic variants and comprehends patient management. The results of this study, therefore, will add to the literature on the role of MYOF gene and any pathogenic variants that might lead to muscular dysfunction. Furthermore, this will establish a disease database, providing a groundwork for understanding the critical genomic regions.

Proteome analysis of Ehrlichia chaffeensis containing phagosome membranes revealed the presence of numerous bacterial and host proteins.

Tick-transmitted Ehrlichia chaffeensis, the causative agent for human monocytic ehrlichiosis, resides and multiplies within a host cell phagosome. Infection progression of E. chaffeensis includes internalization into a host cell by host cell membrane fusion events following engulfment leading to the formation of E. chaffeensis containing vacuole (ECV). Revealing the molecular composition of ECV is important in understanding the host cellular processes, evasion of host defense pathways and in defining host-pathogen interactions. ECVs purified from infected host cells were analyzed to define both host and bacterial proteomes associated with the phagosome membranes. About 160 bacterial proteins and 2,683 host proteins were identified in the ECV membranes. The host proteins included predominantly known phagosome proteins involved in phagocytic trafficking, fusion of vesicles, protein transport, Ras signaling pathway and pathogenic infection. Many highly expressed proteins were similar to the previously documented proteins of phagosome vacuole membranes containing other obligate pathogenic bacteria. The finding of many bacterial membrane proteins is novel; they included multiple outer membrane proteins, such as the p28-Omps, the 120 kDa protein, preprotein translocases, lipoproteins, metal binding proteins, and chaperonins, although the presence of ankyrin repeat proteins, several Type I and IV secretion system proteins is anticipated. This study demonstrates that ECV membrane is extensively modified by the pathogen. This study represents the first and the most comprehensive description of ECV membrane proteome. The identity of many host and Ehrlichia proteins in the ECV membrane will be a valuable to define pathogenic mechanisms critical for the replication of the pathogen within macrophages.

An imaging-based RNA interference screen for modulators of the Rab6-mediated Golgi-to-ER pathway in mammalian cells.

In mammalian cells, membrane traffic pathways play a critical role in connecting the various compartments of the endomembrane system. Each of these pathways is highly regulated, requiring specific machinery to ensure their fidelity. In the early secretory pathway, transport between the endoplasmic reticulum (ER) and Golgi apparatus is largely regulated via cytoplasmic coat protein complexes that play a role in identifying cargo and forming the transport carriers. The secretory pathway is counterbalanced by the retrograde pathway, which is essential for the recycling of molecules from the Golgi back to the ER. It is believed that there are at least two mechanisms to achieve this - one using the cytoplasmic COPI coat complex, and another, poorly characterised pathway, regulated by the small GTPase Rab6. In this work, we describe a systematic RNA interference screen targeting proteins associated with membrane fusion, in order to identify the machinery responsible for the fusion of Golgi-derived Rab6 carriers at the ER. We not only assess the delivery of Rab6 to the ER, but also one of its cargo molecules, the Shiga-like toxin B-chain. These screens reveal that three proteins, VAMP4, STX5, and SCFD1/SLY1, are all important for the fusion of Rab6 carriers at the ER. Live cell imaging experiments also show that the depletion of SCFD1/SLY1 prevents the membrane fusion event, suggesting that this molecule is an essential regulator of this pathway.

SNARE Protein DdVam7 of the Nematode-Trapping Fungus Drechslerella dactyloides Regulates Vegetative Growth, Conidiation, and the Predatory Process via Vacuole Assembly.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play conserved roles in membrane fusion events in eukaryotes and have been documented to be involved in fungal growth and pathogenesis. However, little is known about the roles of SNAREs in trap morphogenesis in nematode-trapping fungi (NTF). Drechslerella dactyloides, one of the constricting ring-forming NTF, captures free-living nematodes via rapid ring cell inflation. Here, we characterized DdVam7 of D. dactyloides, a homolog of the yeast SNARE protein Vam7p. Deletion of DdVam7 significantly suppressed vegetative growth and conidiation. The mutation significantly impaired trap formation and ring cell inflation, resulting in a markedly decreased nematode-trapping ability. A large vacuole could develop in ring cells within ~2.5 s after instant inflation in D. dactyloides. In the ΔDdVam7 mutant, the vacuoles were small and fragmented in hyphae and uninflated ring cells, and the large vacuole failed to form in inflated ring cells. The localization of DdVam7 in vacuoles suggests its involvement in vacuole fusion. In summary, our results suggest that DdVam7 regulates vegetative growth, conidiation, and the predatory process by mediating vacuole assembly in D. dactyloides, and this provides a basis for studying mechanisms of SNAREs in NTF and ring cell rapid inflation. IMPORTANCE D. dactyloides is a nematode-trapping fungus that can capture nematodes through a constricting ring, the most sophisticated trapping device. It is amazing that constricting ring cells can inflate to triple their size within seconds to capture a nematode. A large centrally located vacuole is a unique signature associated with inflated ring cells. However, the mechanism underpinning trap morphogenesis, especially vacuole dynamics during ring cell inflation, remains unclear. Here, we documented the dynamics of vacuole assembly during ring cell inflation via time-lapse imaging for the first time. We characterized a SNARE protein in D. dactyloides (DdVam7) that was involved in vacuole assembly in hyphae and ring cells and played important roles in vegetative growth, conidiation, trap morphogenesis, and ring cell inflation. Overall, this study expands our understanding of biological functions of the SNARE proteins and vacuole assembly in NTF trap morphogenesis and provides a foundation for further study of ring cell rapid inflation mechanisms.

Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibits the infection of SARS-CoV-2 variants and syncytium formation

The continuous emergence of SARS-CoV-2 variants prolongs COVID-19 pandemic. Although SARS-CoV-2 vaccines and therapeutics are currently available, there is still a need for development of safe and effective drugs against SARS-CoV-2 and also for preparedness for the next pandemic. Here, we discover that astersaponin I (AI), a triterpenoid saponin in Aster koraiensis inhibits SARS-CoV-2 entry pathways at the plasma membrane and within the endosomal compartments mainly by increasing cholesterol content in the plasma membrane and interfering with the fusion of SARS-CoV-2 envelope with the host cell membrane. Moreover, we find that this functional property of AI as a fusion blocker enables it to inhibit the infection with SARS-CoV-2 variants including the Alpha, Beta, Delta, and Omicron with a similar efficacy, and the formation of syncytium, a multinucleated cells driven by SARS-CoV-2 spike protein-mediated cell-to-cell fusion. Finally, we claim that the triterpene backbone as well as the attached hydrophilic sugar moieties of AI are structurally important for its inhibitory activity against the membrane fusion event. Overall, this study demonstrates that AI is a natural viral fusion inhibitor and proposes that it can be a broad-spectrum antiviral agent against current COVID-19 pandemic and future outbreaks of novel viral pathogens.

Dysferlin facilitates the de novo biogenesis of tubular membranes in hypertrophic remodelling

Abstract Introduction Ferlins are transmembrane proteins with multiple C2 domains that may mediate Ca2+ dependent membrane fusion and repair events. In ventricles, Dysferlin expression is dominant, and patient mutations have been associated with cardiomyopathies. Hence, we hypothesized that Dysferlin is essential for the functional integrity of the tubular endomembrane system in ventricular myocytes (VM), and may facilitate the cellular hypertrophic remodelling in pressure-overload. Results In 30 weeks old Dysferlin knockout (KO) mice, echocardiography revealed dilative cardiomyopathy with reduced left-ventricular ejection fractions (difference between means KO vs. wildtype (WT) −13.45±2.17%, p<0.001, n=16/20 mice). Asking for the subcellular relevance of Dysferlin, we investigated VM nanodomains by immunolabelling in STimulated Emission Depletion (STED) nanoscopy and visualized punctual Dysferlin clusters decorating the T-tubule system of mouse and human VM. Precisely, Dysferlin clusters localized to junctions of the tubular system and the sarcoplasmic reticulum (SR) in nanometric proximity to RyR2 Ca2+ release units. Complexome profiling (LC-MS/MS) also suggested interactions of Dysferlin with RyR2 and the SR membrane-tethering protein Junctophilin-2 (JP2) in high molecular weight complexes. In JP2-overexpressing mice, live-cell STED membrane imaging captured polyadic junctional membrane structures in VM, specifically surrounded by dense Dysferlin clusters, which proposes Dysferlin as stabilizing protein of tubule-SR junctions. Further, we studied the ultrastructural remodelling of the tubular system in a mouse model of cardiac hypertrophy and heart failure induced by transaortic constriction (TAC). Interestingly, mortality four weeks post TAC was not increased in KO mice. However, echocardiography uncovered that Dysf-KO prevents left-ventricular hypertrophy in response to pressure-overload, which was confirmed by single cell measurements (VM area: WT 3559±120 μm2 vs. KO 2671±87 μm2, n=163/158 VM, p<0.001). Thus, we assumed Dysferlin to mediate tubular network proliferation during hypertrophic remodelling. Indeed, WT VM showed an augmented Dysferlin expression post TAC (158±0,125%, p=0,005, n=7/7 mice), and immunofluorescence STED nanoscopy detected an increased Dysferlin network complexity. In detail, we visualized highly abundant Dysferlin clusters decorating newly shaped axial tubules, which were quantified by component-specific tubule network analysis. Conclusion Nanoscale imaging proposes Dysferlin as stabilizer of tubule-SR junctions, thereby promoting functional excitation-contraction coupling, and demonstrates the crucial importance of Dysferlin in pressure-overload induced hypertrophic remodelling, facilitating the de novo biogenesis of axial tubule membranes and tubule-SR junctions necessary for VM growth. Thus, Dysferlin may emerge as potential novel therapeutic rationale to control subcellular membrane remodelling in cardiac diseases. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Multiscale Bioimaging Cluster of Excellence: From Molecular Machines to Networks of Excitable Cells;German Research Foundation (DFG) - Collaborative Research Centre 1002 (SFB 1002): Modulatory Units in Heart Failure

The intramembrane proteases SPPL2a and SPPL2b regulate the homeostasis of selected SNARE proteins.

Signal peptide peptidase (SPP) and SPP-like (SPPL) aspartyl intramembrane proteases are known to contribute to sequential processing of type II-oriented membrane proteins referred to as regulated intramembrane proteolysis. The ER-resident family members SPP and SPPL2c were shown to also cleave tail-anchored proteins, including selected SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitating membrane fusion events. Here, we analysed whether the related SPPL2a and SPPL2b proteases, which localise to the endocytic or late secretory pathway, are also able to process SNARE proteins. Therefore, we screened 18 SNARE proteins for cleavage by SPPL2a and SPPL2b based on cellular co-expression assays, of which the proteins VAMP1, VAMP2, VAMP3 and VAMP4 were processed by SPPL2a/b demonstrating the capability of these two proteases to proteolyse tail-anchored proteins. Cleavage of the four SNARE proteins was scrutinised at the endogenous level upon SPPL2a/b inhibition in different cell lines as well as by analysing VAMP1-4 levels in tissues and primary cells of SPPL2a/b double-deficient (dKO) mice. Loss of SPPL2a/b activity resulted in an accumulation of VAMP1-4 in a cell type- and tissue-dependent manner, identifying these proteins as SPPL2a/b substrates validated in vivo. Therefore, we propose that SPPL2a/b control cellular levels of VAMP1-4 by initiating the degradation of these proteins, which might impact cellular trafficking.

In silico prediction, characterization, docking studies and molecular dynamics simulation of human p97 in complex with p37 cofactor

BackgroundThe AAA + ATPase p97 is an essential unfoldase/segragase involved in a multitude of cellular processes. It functions as a molecular machine critical for protein homeostasis, homotypic membrane fusion events and organelle biogenesis during mitosis in which it acts in concert with cofactors p47 and p37. Cofactors assist p97 in extracting and unfolding protein substrates through ATP hydrolysis. In contrast to other p97ʼs cofactors, p37 uniquely increases the ATPase activity of p97. Disease-causing mutations in p97, including mutations that cause neurodegenerative diseases, increase cofactor association with its N-domain, ATPase activity and improper substrate processing. Upregulation of p97 has also been observed in various cancers. This study aims towards the characterization of the protein–protein interaction between p97 and p37 at the atomic level. We defined the interacting residues in p97 and p37. The knowledge will facilitate the design of unique small molecules inhibiting this interaction with insights into cancer therapy and drug design.ResultsThe homology model of human p37 UBX domain was built from the X-ray crystal structure of p47 C-terminus from rat (PDB code:1S3S, G) as a template and assessed by model validation analysis. According to the HDOCK, HAWKDOCK, MM-GBSA binding free energy calculations and Arpeggio, we found that there are several hydrophobic and two hydrogen-bonding interactions between p37 UBX and p97 N-D1 domain. Residues of p37 UBX predicted to be involved in the interactions with p97 N-D1 domain interface are highly conserved among UBX cofactors.ConclusionThis study provides a reliable structural insight into the p37-p97 complex binding sites at the atomic level though molecular docking coupled with molecular dynamics simulation. This can guide the rational design of small molecule drugs for inhibiting mutant p97 activity.

Potential roles of synaptotagmin family members in cancers: Recent advances and prospects.

With the continuous development of bioinformatics and public database, more and more genes that play a role in cancers have been discovered. Synaptotagmins (SYTs) are abundant, evolutionarily conserved integral membrane proteins composed of a short N-terminus, a variable linker domain, a single transmembrane domain, and two C2 domains, and they constitute a family of 17 isoforms. The synaptotagmin family members are known to regulate calcium-dependent membrane fusion events. Some SYTs play roles in hormone secretion or neurotransmitter release or both, and much evidence supports SYTs as Ca2+ sensors of exocytosis. Since 5 years ago, an increasing number of studies have found that SYTs also played important roles in the occurrence and development of lung cancer, gastric cancer, colon cancer, and other cancers. Down-regulation of SYTs inhibited cell proliferation, migration, and invasion of cancer cells, but promoted cell apoptosis. Growth of peritoneal nodules is inhibited and survival is prolonged in mice administrated with siSYTs intraperitoneally. Therefore, most studies have found SYTs serve as an oncogene after overexpression and may become potential prognostic biomarkers for multiple cancers. This article provides an overview of recent studies that focus on SYT family members’ roles in cancers and highlights the advances that have been achieved.

The Sperm Olfactory Receptor OLFR601 is Dispensable for Mouse Fertilization.

Fertilization involves the fusion of two gametes by means of yet unknown membrane binding and fusion events. Over the last years, many sperm proteins have been uncovered to play essential roles in sperm-egg fusion in mammals, but their precise role in fertilization remains unknown, being unclear how these proteins interact with each other or with other yet unknown sperm proteins. The aim of this study has been to identify possible sperm proteins interacting with TMEM95, a protein essential for fertilization located in the sperm membrane. A list of 41 sperm proteins that were pulled down with TMEM95 and identified by mass spectrometry did not include other sperm proteins known to play a role in fertilization, suggesting an independent role of TMEM95 in fertilization. Between these lists, OLFR601 is allocated to the acrosomal region and may mediate affinity for an odorant involved in fertilization. However, Olfr601 disruption did not impair the sperm fertilization ability, suggesting that its function may be redundant with that of other sperm proteins.

Unlocking the gate to GABARAPL2.

GABARAPL2 was initially characterized for its involvement in protein transport and membrane fusion events, but has since gained notoriety for its role in autophagy. GABARAPL2 is frequently studied alongside its GABARAP subfamily members, GABARAP and GABARAPL1. Although functional redundancy exists among the subfamily members, a complex network of molecular interactions, physiological processes and pathologies can be primarily related to GABARAPL2. GABARAPL2 has a multifaceted role, ranging from cellular differentiation to intracellular degradation. Much of what we know about GABARAPL2 is gained through identifying its interacting partners-a list that is constantly growing. In this article, we review both the autophagy-dependent and autophagy-independent roles of GABARAPL2, and emphasize their implications for both health and disease.

A genetically targeted sensor reveals spatial and temporal dynamics of acrosomal calcium and sperm acrosome exocytosis

Secretion of the acrosome, a single vesicle located rostrally in the head of a mammalian sperm, through a process known as “acrosome exocytosis” (AE), is essential for fertilization. However, the mechanisms leading to and regulating this complex process are controversial. In particular, poor understanding of Ca2+ dynamics between sperm subcellular compartments and regulation of membrane fusion mechanisms have led to competing models of AE. Here, we developed a transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE) to investigate the spatial and temporal Ca2+ dynamics in AE in live sperm. AcroSensE combines a genetically encoded Ca2+ indicator (GCaMP) fused with an mCherry indicator to spatiotemporally resolve acrosomal Ca2+ rise (ACR) and membrane fusion events, enabling real-time study of AE. We found that ACR is dependent on extracellular Ca2+ and that ACR precedes AE. In addition, we show that there are intermediate steps in ACR and that AE correlates better with the ACR rate rather than absolute Ca2+ amount. Finally, we demonstrate that ACR and membrane fusion progression kinetics and spatial patterns differ with different stimuli and that sites of initiation of ACR and sites of membrane fusion do not always correspond. These findings support a model involving functionally redundant pathways that enable a highly regulated, multistep AE in heterogeneous sperm populations, unlike the previously proposed “acrosome reaction” model.

Phylogeography and evolutionary dynamics analysis of porcine delta-coronavirus with host expansion to humans.

From 2003 onwards, three pandemics have been caused by coronaviruses: severe acute respiratory syndrome coronavirus (SARS-CoV); middle east respiratory syndrome coronavirus (MERS-CoV); and, most recently, SARS-CoV-2. Notably, all three were transmitted from animals to humans. This would suggest that animals are potential sources of epidemics for humans. The emerging porcine delta-coronavirus was reported to infect children. This is a red flag that marks the ability of PDCoV to break barriers of cross-species transmission to humans. Therefore, we conducted molecular genetic analysis of global clade PDCoV to characterize spatiotemporal patterns of viral diffusion and genetic diversity. PDCoV was classified into three major lineages, according to distribution and phylogenetic analysis of PDCoV. It can be inferred based on the analysis results of the currently known PDCoV strains that PDCoV might originate in Asia. We also selected six special spike amino acid sequences to align and analyze to find seven significant mutation sites. The accumulation of these mutations may enhance dynamic movements, accelerating spike protein membrane fusion events and transmission. Altogether, our study offers a novel insight into the diversification, evolution, and interspecies transmission and origin of PDCoV and emphasizes the need to study the zoonotic potential of the PDCoV and comprehensive surveillance and enhanced biosecurity precautions for PDCoV.

Genome-Wide Identification and Expression Analysis of SNARE Genes in Brassica napus.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components that drive membrane fusion events during exocytosis and endocytosis and play important roles in different biological processes of plants. In this study, we identified 237 genes encoding SNARE family proteins in B. napus in silico at the whole-genome level. Phylogenetic analysis showed that BnaSNAREs could be classified into five groups (Q (a-, b-, c-, bc-) and R) like other plant SNAREs and clustered into twenty-five subclades. The gene structure and protein domain of each subclade were found to be highly conserved. In many subclades, BnaSNAREs are significantly expanded compared with the orthologous genes in Arabidopsis thaliana. BnaSNARE genes are expressed differentially in the leaves and roots of B. napus. RNA-seq data and RT-qPCR proved that some of the BnaSNAREs are involved in the plant response to S. sclerotiorum infection as well as treatments with toxin oxalic acid (OA) (a virulence factor often secreted by S. sclerotiorum) or abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA), which individually promote resistance to S. sclerotiorum. Moreover, the interacted proteins of BnaSNAREs contain some defense response-related proteins, which increases the evidence that BnaSNAREs are involved in plant immunity. We also found the co-expression of BnaSYP121/2s, BnaSNAPs, and BnaVAMP722/3s in B. napus due to S. sclerotiorum infection as well as the probable interaction among them.

Fluorescence Lifetime and Cross-correlation Spectroscopy for Observing Membrane Fusion of Liposome Models Containing Synaptic Proteins.

Watching events of membrane fusion in real time and distinguishing between intermediate steps of these events is useful for mechanistic insights but at the same time a challenging task. In this chapter, we describe how to use fluorescence cross-correlation spectroscopy and Förster-resonance energy transfer to resolve the tethering and fusion of membranes by SNARE proteins (syntaxin-1, SNAP-25, and synaptobrevin-2) as an example. The given protocols can easily be adapted to other membrane proteins to investigate their ability to tether or even fuse vesicular membrane.

In Vitro Reconstitution Studies of SNAREs and Their Regulators Mediating GLUT4 Vesicle Fusion.

The GLUT4 vesicle fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and a variety of regulatory proteins. For example, synip and tomosyn negatively regulate GLUT4 SNARE-mediated membrane fusion. Here we describe in vitro reconstituted assays to determine the molecular mechanisms of SNAREs, synip, and tomosyn. These methods can also be extended to the studies of other types of membrane fusion events.

Monitoring the Recruitment and Fusion of Autophagosomes to Phagosomes During the Clearance of Apoptotic Cells in the Nematode Caenorhabditis elegans.

During an animal's development, a large number of cells undergo apoptosis, a suicidal form of death. These cells are promptly phagocytosed by other cells and degraded inside phagosomes. The recognition, engulfment, and degradation of apoptotic cells is an evolutionarily conserved process occurring in all metazoans. Recently, we discovered a novel event in the nematode Caenorhabditis elegans: the double-membrane autophagosomes are recruited to the surface of phagosomes; subsequently, the outer membrane of an autophagosome fuses with the phagosomal membrane, allowing the inner vesicle to enter the phagosomal lumen and accumulate there over time. This event facilitates the degradation of the apoptotic cell inside the phagosome. During this study, we developed a real-time imaging protocol monitoring the recruitment and fusion of autophagosomes to phagosomes over two hours during embryonic development. This protocol uses a deconvolution-based microscopic imaging system with an optimized setting to minimize photodamage of the embryo during the recording period for high-resolution images. Furthermore, acid-resistant fluorescent reporters are chosen to label autophagosomes, allowing the inner vesicles of an autophagosome to remain visible after entering the acidic phagosomal lumen. The methods described here, which enable high sensitivity, quantitative measurement of each step of the dynamic incorporation in developing embryos, are novel since the incorporation of autophagosomes to phagosomes has not been reported previously. In addition to studying the degradation of apoptotic cells, this protocol can be applied to study the degradation of non-apoptotic cell cargos inside phagosomes, as well as the fusion between other types of intracellular organelles in living C. elegans embryos. Furthermore, its principle of detecting the membrane fusion event can be adapted to study the relationship between autophagosomes and phagosomes or other intracellular organelles in any biological system in which real-time imaging can be conducted. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.72466.

Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy.

The fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion events, respectively. Pore dynamics govern vesicle cargo release and the mode of vesicle recycling, but the mechanisms are poorly understood. This is partly due to a lack of reconstituted assays that combine single-pore sensitivity and high time resolution. Total internal reflection fluorescence (TIRF) microscopy offers unique advantages for characterizing single membrane fusion events, but signals depend on effects that are difficult to disentangle, including the polarization of the excitation electric field, vesicle size, photobleaching, orientation of the excitation dipoles of the fluorophores with respect to the membrane, and the evanescent field depth. Commercial TIRF microscopes do not allow control of excitation polarization, further complicating analysis. To overcome these challenges, we built a polarization-controlled total internal reflection fluorescence (pTIRF) microscope and monitored fusion of proteoliposomes with planar lipid bilayers with single molecule sensitivity and ∼15 ms temporal resolution. Using pTIRF microscopy, we detected docking and fusion of fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNARE proteins (vSUVs), with a supported bilayer containing the cognate t-SNAREs (tSBL). By varying the excitation polarization angle, we were able to identify a dye-dependent optimal polarization at which the fluorescence increase upon fusion was maximal, facilitating event detection and analysis of lipid transfer kinetics. An improved algorithm allowed us to estimate the size of the fusing vSUV and the fusion pore openness (the fraction of time the pore is open) for every event. For most events, lipid transfer was much slower than expected for diffusion through an open pore, suggesting that fusion pore flickering limits lipid release. We find a weak correlation between fusion pore openness and vesicle area. The approach can be used to study mechanisms governing fusion pore dynamics in a wide range of membrane fusion processes.

EhVps23: A Component of ESCRT-I That Participates in Vesicular Trafficking and Phagocytosis of Entamoeba histolytica

The endosomal sorting complex required for transport (ESCRT) is formed by ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III complexes, and accessory proteins. It conducts vesicular trafficking in eukaryotes through the formation of vesicles and membrane fission and fusion events. The trophozoites of Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents an active membrane movement in basal state that increases during phagocytosis and tissue invasion. ESCRT-III complex has a pivotal role during these events, but ESCRT-0, ESCRT-I and ESCRT-II have been poorly studied. Here, we unveiled the E. histolytica ESCRT-I complex and its implication in vesicular trafficking and phagocytosis, as well as the molecular relationships with other phagocytosis-involved molecules. We found a gene encoding for a putative EhVps23 protein with the ubiquitin-binding and Vps23 core domains. In basal state, it was in the plasma membrane, cytoplasmic vesicles and multivesicular bodies, whereas during phagocytosis it was extensively ubiquitinated and detected in phagosomes and connected vesicles. Docking analysis, immunoprecipitation assays and microscopy studies evidenced its interaction with EhUbiquitin, EhADH, EhVps32 proteins, and the lysobisphosphatidic acid phospholipid. The knocking down of the Ehvps23 gene resulted in lower rates of phagocytosis. Our results disclosed the concert of finely regulated molecules and vesicular structures participating in vesicular trafficking-related events with a pivotal role of EhVps23.

Miro1 functions as an inhibitory regulator of MFN at elevated mitochondrial Ca2+ levels.

Mitochondria function as an integrated network that moves along the microtubules within cells and changes the morphology through membrane fusion and fission events. Mitofusin (MFN) mediates membrane tethering and subsequent fusion of the mitochondrial outer membrane. Understanding the regulatory mechanisms of MFN function is critical to tackling the pathology related to mitochondrial network imbalance. Here, we reveala novel inhibitory mechanism of MFN-mediated fusion by mitochondrial Rho GTPase (Miro1) in response to elevated mitochondrial Ca2+ concentration ([Ca2+ ]m ). We showed that elevated [Ca2+ ]m prevents the fusion between mitochondria forming the outer membrane tether by ectopically expressing MFN. Lowering [Ca2+ ]m by treating cells with an inhibitor of mitochondrial calcium uniporter or knocking down Miro1/2 induces more fused networks. Miro1 interacts with MFN as supported by co-immunoprecipitation and protein association identified by proximity labeling proteomics. It suggests that Miro1 functions as a Ca2+ -sensor and inhibits MFN function at elevated [Ca2+ ]m. Miro1 EF-hand mutant has a compromised inhibitory effect, which reiterates Ca2+ -modulated regulation. Dysregulated Ca2+ -handling and mitochondrial network imbalance are highly relevant in the pathology of cancers, cardiovascular, and neurodegenerative diseases. Miro1 functions as a coordinated Ca2+ -responder by pausing mitochondrial transport while reducing network fusion and cooperating with Drp1-mediated fission. It likely prevents the detrimental effect of Ca2+m overload and facilitates mitophagy. Our finding reveals a novel regulation of mitochondrial network dynamics responding to [Ca2+ ]m through the interplay of Miro1 and MFN. Modulation of Miro1 and MFN interaction is a potential intervention to promote network homeostasis.