Accumulation Of Vesicles Research Articles

Deletion of the Plasmodium falciparum exported protein PTP7 leads to Maurer's clefts vesiculation, host cell remodeling defects, and loss of surface presentation of EMP1.

Presentation of the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (EMP1), at knob-like protrusions on the surface of infected red blood cells, underpins the parasite's pathogenicity. Here we describe a protein PF3D7_0301700 (PTP7), that functions at the nexus between the intermediate trafficking organelle, the Maurer's cleft, and the infected red blood cell surface. Genetic disruption of PTP7 leads to accumulation of vesicles at the Maurer's clefts, grossly aberrant knob morphology, and failure to deliver EMP1 to the red blood cell surface. We show that an expanded low complexity sequence in the C-terminal region of PTP7, identified only in the Laverania clade of Plasmodium, is critical for efficient virulence protein trafficking.

Plant cytokinesis and the construction of new cell wall.

Cytokinesis in plants is fundamentally different from that in animals and fungi. In plant cells, a cell plate forms through the fusion of cytokinetic vesicles and then develops into the new cell wall, partitioning the cytoplasm of the dividing cell. The formation of the cell plate entails multiple stages that involve highly orchestrated vesicle accumulation, fusion and membrane maturation, which occur concurrently with the timely deposition of polysaccharides such as callose, cellulose and cross-linking glycans. This review summarizes the major stages in cytokinesis, endomembrane components involved in cell plate assembly and its transition to a new cell wall. An animation that can be widely used for educational purposes further summarizes the process.

Production of cholesterol-like molecules impacts Escherichia coli robustness, production capacity, and vesicle trafficking

The economic viability of bioprocesses is constrained by the limited range of operating conditions that can be tolerated by the cell factory. Engineering of the microbial cell membrane is one strategy that can increase robustness and thus alter this range. In this work, we targeted cellular components that contribute to maintenance of appropriate membrane function, such as: flotillin-like proteins, membrane structural proteins, and membrane lipids. Specifically, we exploited the promiscuity of squalene hopene cyclase (SHC) to produce polycyclic terpenoids with properties analogous to cholesterol. Strains producing these cholesterol-like molecules were visualized by AFM and height features were observed. Production of these cholesterol-like molecules was associated with increased tolerance towards a diversity of chemicals, particularly alcohols, and membrane trafficking processes such as lipid droplet accumulation and production of extracellular vesicles. This engineering approach improved the production titers for wax-esters and ethanol by 80- and 10-fold, respectively. Expression of SHC resulted in the production of steroids. Strains engineered to also express truncated squalene synthase (tERG9) produced diplopterol and generally did not perform as well. Increased expression of several membrane-associated proteins, such as YqiK, was observed to impact vesicle trafficking and further improve tolerance relative to SHC alone, but did not improve bio-production. Deletion of YbbJ increased lipid droplet accumulation as well as production of intracellular wax esters. This work serves as a proof of concept for engineering strategies targeting membrane physiology and trafficking to expand the production capacity of microbial cell factories.

Rab11FIP1-deficient mice develop spontaneous inflammation and show increased susceptibility to colon damage.

The small GTPase, Rab11a, regulates vesicle trafficking and cell polarity in epithelial cells through interaction with Rab11 family-interacting proteins (Rab11-FIPs). We hypothesized that deficiency of Rab11-FIP1 would affect mucosal integrity in the intestine. Global Rab11FIP1 knockout (KO) mice were generated by deletion of the second exon. Pathology of intestinal tissues was analyzed by immunostaining of colonic sections and RNA-sequencing of isolated colonic epithelial cells. A low concentration of dextran sodium sulfate (DSS, 2%) was added to drinking water for 5 days, and injury score was compared between Rab11FIP1 KO, Rab11FIP2 KO, and heterozygous littermates. Rab11FIP1 KO mice showed normal fertility and body weight gain. More frequent lymphoid patches and infiltration of macrophages and neutrophils were identified in Rab11FIP1 KO mice before the development of rectal prolapse compared with control mice. The population of trefoil factor 3 (TFF3)-positive goblet cells was significantly lower, and the ratio of proliferative to nonproliferative cells was higher in Rab11FIP1 KO colons. Transcription signatures indicated that Rab11FIP1 deletion downregulated genes that mediate stress tolerance response, whereas genes mediating the response to infection were significantly upregulated, consistent with the inflammatory responses in the steady state. Lack of Rab11FIP1 also resulted in abnormal accumulation of subapical vesicles in colonocytes and the internalization of transmembrane mucin, MUC13, with Rab14. After DSS treatment, Rab11FIP1 KO mice showed greater body weight loss and more severe mucosal damage than those in heterozygous littermates. These findings suggest that Rab11FIP1 is important for cytoprotection mechanisms and for the maintenance of colonic mucosal integrity.NEW & NOTEWORTHY Although Rab11FIP1 is important in membrane trafficking in epithelial cells, the gastrointestinal phenotype of Rab11FIP1 knockout (KO) mice had never been reported. This study demonstrated that Rab11FIP1 loss induces mistrafficking of Rab14 and MUC13 and decreases in colonic goblet cells, resulting in impaired mucosal integrity.

Abstract 3002: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

Abstract Autophagy is a dynamic, multi-step process that cells use to degrade damaged, abnormal, and potentially harmful cellular substances. While autophagy is maintained at a basal level in all cells, it is activated at a higher level in many cancer cells and promotes tumor growth, anti-tumor immune response, and resistance to cancer therapy. As a result, autophagy is increasingly being recognized to have an important role in cancer progression and emerging as a potential target for cancer therapy. We recently discovered that small GTPase Rab27B, a well-known regulator of vesicle trafficking and exosome secretion, also regulates autophagy process. Depletion of Rab27B in colorectal cancer (CRC) cells by CRISPR-Cas9 and siRNA resulted in an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62 indicating a defect in the autophagy flux. To characterize this autophagy defect, we used EGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining and identified that the degradation step of the autophagy flux is inhibited when Rab27B is silenced. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that autophagy defect resulting from Rab27B deletion will cause reduced stress response and tumor growth. Indeed, Rab27B knockout resulted in a 60% reduction in cell viability in response to starvation and a 94% reduction in colony formation in soft agar assay. Rab27B deletion also prevented spheroid formation in vitro. Finally, to analyze the effect of Rab27B deletion in tumor formation in vivo, we performed a xenograft study with wildtype and Rab27B knockout CRC cells HCT116 and observed that Rab27B deletion resulted in a significant reduction of tumor size (p<0.0001). Next, to analyze the therapeutic potential of Rab27B inhibition in CRC, we sought to identify small-molecule inhibitors of Rab27B GTPase activity in vitro through high-throughput drug screening from a panel of ∼2,100 compounds contained within the KU-HTSL chemical library. This screening resulted in the identification of three compounds (BTB00809, BTB03584, and KM01532) that demonstrated Rab27B GTPase activity inhibition. Interestingly, inhibition of Rab27B in CRC cells with the compound km01532 resulted in a similar increase of LC3-II in CRC cells HCT116 and SW480 as seen in the Rab27B knockout. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on investigating the mechanism of how Rab27B functions in the autophagy process, and whether Rab27B can be targeted as a potential therapeutic strategy for CRC using the small molecule compound km01532. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan Dixon. Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3002.

Autophagy protein LC3C binding to phospholipid and interaction with lipid membranes

Autophagy is a process in which parts of the eukaryotic cell are selectively degraded in the lysosome. The materials to be catabolized are first surrounded by a double-membrane structure, the autophagosome. Autophagosome generation is a complex event, in which many proteins are involved. Among the latter, yeast Atg8 or its mammalian orthologues are essential in autophagosome membrane elongation, shaping and closure. A subfamily of the human Atg8 orthologues is formed by the proteins LC3A, LC3B, and LC3C. Previous studies suggest that, at variance with the other two, LC3C does not participate in cardiolipin-mediated mitophagy. The present study was devoted to exploring the binding of LC3C to lipid vesicles, bilayers and monolayers, and the ensuing protein-dependent perturbing effects, in the absence of the mitochondrial lipid cardiolipin. All Atg8 orthologues are covalently bound to a phospholipid prior to their involvement in autophagosome elongation. In our case, a mutant in the C-terminal amino acid, LC3C G126C, together with the use of a maleimide-derivatized phosphatidyl ethanolamine, ensured LC3C lipidation, up to 100% under certain conditions. Ultracentrifugation, surface pressure measurements, spectroscopic and cryo-electron microscopic techniques revealed that lipidated LC3C induced vesicle aggregation (5-fold faster in sonicated than in large unilamellar vesicles) and inter-vesicular lipid mixing (up to 82%), including inner-monolayer lipid mixing (up to 32%), consistent with in vitro partial vesicle fusion. LC3C was also able to cause the release of 80–90% vesicular aqueous contents. The data support the idea that LC3C would be able to help in autophagosome elongation/fusion in autophagy phenomena.

Impaired GATE16-mediated exocytosis in exocrine tissues causes Sjögren's syndrome-like exocrinopathy.

Sjögren's syndrome (SjS) is a chronic autoimmune disease characterized by immune cell infiltration of the exocrine glands, mainly the salivary and lacrimal glands. Despite recent advances in the clinical and mechanistic characterization of the disease, its etiology remains largely unknown. Here, we report that mice with a deficiency for either Atg7 or Atg3, which are enzymes involved in the ubiquitin modification pathway, in the salivary glands exhibit a SjS-like phenotype, characterized by immune cell infiltration with autoantibody detection, acinar cell death, and dry mouth. Prior to the onset of the SjS-like phenotype in these null mice, we detected an accumulation of secretory vesicles in the acinar cells of the salivary glands and found that GATE16, an uncharacterized autophagy-related molecule activated by ATG7 (E1-like enzyme) and ATG3 (E2-like enzyme), was highly expressed in these cells. Notably, GATE16 was activated by isoproterenol, an exocytosis inducer, and localized on the secretory vesicles in the acinar cells of the salivary glands. Failure to activate GATE16 was correlated with exocytosis defects in the acinar cells of the salivary glands in Atg7 and Atg3 cKO mice. Taken together, our results show that GATE16 activation regulated by the autophagic machinery is crucial for exocytosis and that defects in this pathway cause SjS.

Metabolic Reprogramming and Lipophagy Mediates Survival of Ascites Derived Metastatic Ovarian Cancer Cells.

Objective:The study was aimed at understanding the survival of metastatic ovarian cancer spheroids in the malignant ascites microenvironment. Methods:All the assays were performed using aseptically collected patient samples. The cells were characterized for the expression of ovarian and cancer stem cell markers using immunocytochemistry. The presence of lipid in the primary metastatic cancer spheroids were confirmed by neutral fat staining using Oil Red-O and transmission electron microscopy. The mRNA expression of autophagy and lipid metabolism genes was analyzed using RT-PCR. The lipid content was analyzed using lipidomics analysis. Etomoxir and chloroquine were used to study the effect of inhibition of autophagy in the metastatic cells. The data were analyzed using appropriate statistical tools and a p-value <0.05 was considered to be statistically significant.Results:Metastatic ovarian cancer spheroids exhibit cancer stem like properties and undergo a metabolic reprogramming when they disseminate from the primary tumor. We report here the accumulation of numerous cytoplasmic lipid droplets and lipophagic vesicles in the metastatic cells in contrast to their primary tumors. In addition we also report that these cells depend on lipophagy for the utilization of lipids rather than the conventional lipolytic pathway. The lipidomics analysis data reveals that the metastatic cells possess high levels of unsaturated fatty acids. We have also reported the occurrence of distinct accumulation of multiple nuclei in the patient derived metastatic cells. Inhibition of beta-oxidation and autophagic machinery using etomoxir and chloroquine resulted in cell death suggesting a potential mode to suppress metastatic cancer cells. Conclusion: Metabolic reprogramming is a characteristic feature of the metastatic ovarian cancer cells that are persisting in the malignant ascites. Targeting of the metastatic cells by gaining an insight into the various metabolic and molecular changes that occur in the metastatic niche provides a promising therapeutic approach in management of the disease.

Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target

Histone deacetylase (HDAC) targeting drugs have entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules, notably for HDAC6, and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-beta-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nM potency. MBLAC2 enzymatic inhibition and knock down led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Actomyosin activity-dependent apical targeting of Rab11 vesicles reinforces apical constriction.

During tissue morphogenesis, the changes in cell shape, resulting from cell-generated forces, often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. In this study, we identify an actomyosin-dependent mechanism involving Rab11-mediated trafficking in regulating apical constriction in the Drosophila embryo. During Drosophila mesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures ("Rab11 vesicles") by promoting a directional bias in dynein-mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network, and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding.

Elevated amyloid beta disrupts the nanoscale organization and function of synaptic vesicle pools in hippocampal neurons.

Alzheimer's disease is linked to increased levels of amyloid beta (Aβ) in the brain, but the mechanisms underlying neuronal dysfunction and neurodegeneration remain enigmatic. Here, we investigate whether organizational characteristics of functional presynaptic vesicle pools, key determinants of information transmission in the central nervous system, are targets for elevated Aβ. Using an optical readout method in cultured hippocampal neurons, we show that acute Aβ42 treatment significantly enlarges the fraction of functional vesicles at individual terminals. We observe the same effect in a chronically elevated Aβ transgenic model (APPSw,Ind) using an ultrastructure-function approach that provides detailed information on nanoscale vesicle pool positioning. Strikingly, elevated Aβ is correlated with excessive accumulation of recycled vesicles near putative endocytic sites, which is consistent with deficits in vesicle retrieval pathways. Using the glutamate reporter, iGluSnFR, we show that there are parallel functional consequences, where ongoing information signaling capacity is constrained. Treatment with levetiracetam, an antiepileptic that dampens synaptic hyperactivity, partially rescues these transmission defects. Our findings implicate organizational and dynamic features of functional vesicle pools as targets in Aβ-driven synaptic impairment, suggesting that interventions to relieve the overloading of vesicle retrieval pathways might have promising therapeutic value.

BECLIN1 Is Essential for Podocyte Secretory Pathways Mediating VEGF Secretion and Podocyte-Endothelial Crosstalk.

Vascular endothelial growth factor A (VEGFA) secretion from podocytes is crucial for maintaining endothelial integrity within the glomerular filtration barrier. However, until now, the molecular mechanisms underlying podocyte secretory function remained unclear. Through podocyte-specific deletion of BECLIN1 (ATG6 or Becn1), a key protein in autophagy initiation, we identified a major role for this molecule in anterograde Golgi trafficking. The Becn1-deficient podocytes displayed aberrant vesicle formation in the trans-Golgi network (TGN), leading to dramatic vesicle accumulation and complex disrupted patterns of intracellular vesicle trafficking and membrane dynamics. Phenotypically, podocyte-specific deletion of Becn1 resulted in early-onset glomerulosclerosis, which rapidly progressed and dramatically reduced mouse life span. Further, in vivo and in vitro studies clearly showed that VEGFA secretion, and thereby endothelial integrity, greatly depended on BECLIN1 availability and function. Being the first to demonstrate the importance of a secretory pathway for podocyte integrity and function, we identified BECLIN1 as a key component in this complex cellular process. Functionally, by promoting VEGFA secretion, a specific secretory pathway emerged as an essential component for the podocyte-endothelial crosstalk that maintains the glomerular filtration barrier.

Extracellular vesiculo-tubular structures associated with suberin deposition in plant cell walls

Suberin is a fundamental plant biopolymer, found in protective tissues, such as seed coats, exodermis and endodermis of roots. Suberin is deposited in most suberizing cells in the form of lamellae just outside of the plasma membrane, below the primary cell wall. How monomeric suberin precursors, thought to be synthesized at the endoplasmic reticulum, are transported outside of the cell, for polymerization into suberin lamellae has remained obscure. Using electron-microscopy, we observed large numbers of extracellular vesiculo-tubular structures (EVs) to accumulate specifically in suberizing cells, in both chemically and cryo-fixed samples. EV presence correlates perfectly with root suberization and we could block suberin deposition and vesicle accumulation by affecting early, as well as late steps in the secretory pathway. Whereas many previous reports have described EVs in the context of biotic interactions, our results suggest a developmental role for extracellular vesicles in the formation of a major cell wall polymer.

Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages invivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT.

An evidence of pores in phospholipid membrane induced by an antimicrobial peptide NK-2.

NK-2, a peptide derived from a cationic core region of NK-lysin, has emerged as a promising candidate for new antibiotics. In contrast to classical antibiotics, antimicrobial peptides target bacterial membranes and disintegrate the membrane by forming the transmembrane pores. However, complete understanding of the precise mechanisms of cellular apoptosis and molecular basis of membrane selectivity is still in dispute. In the present study, we have shown that NK-2 forms trans-membrane pores on negatively charged phospholipid membranes using phase contrast microscopy. As bacteria mimicking membranes, we have chosen large unilamellar vesicles (LUV) and giant unilamellar vesicles (GUV) composed of negatively charged phospholipid, dioleoyl phosphatidyl glycerol (DOPG) and neutral phospholipid, dioleoyl phophatidylcholine (DOPC). Leakage of internal fluid of giant unilamellar vesicles (GUV), leading to decrease in intensity in the halo region of phase contrast micrographs, suggests the formation of transmembrane pores. No such reduction of intensity in the halo region of DOPC was observed, indicating, neutral vesicles does not exhibit pores. Rate constant reckoned from the decaying intensity in the halo region was found to be 0.007s-1. Further, significant interaction of NK-2 with anionic membranes has been envisaged from zeta potential and dynamic light scattering. Binding free energy and other interaction parameters have been delineated using theoretical ansatz. A proliferation of average Size of anionic LUV on increasing NK-2 concentration indicates membrane-membrane interaction leading to peptide induced large aggregates of vesicles.

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles.

BackgroundThe cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle‐secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology.MethodsSporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS‐R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged‐matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT‐qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human‐derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls.ResultsAn accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy‐derived denervation‐naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome‐like vesicles and disruption of RNA‐processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA‐processing proteins. The RNA‐processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti‐CD63 immuno‐blocking of vesicle uptake.ConclusionsALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle‐mediated toxicity in ALS.

Deciphering Ethanol-Driven Swelling, Rupturing, Aggregation, and Fusion of Lipid Vesicles Using Coarse-Grained Molecular Dynamics Simulations.

Traditionally, liquid ethanol is known to enhance the permeability of lipid membranes and causes vesicle aggregation and fusion. However, how the amphiphilic ethanol molecules perturb the lipid vesicles to facilitate their aggregation or fusion has not been addressed at any level of molecular simulations. Herein, not only have we developed a coarse-grained (CG) model for liquid ethanol, its aqueous mixture, and hydrated lipid membranes for molecular dynamics (MD) simulations, but also utilized it to delineate the aggregation and fusion of lipid vesicles using CG-MD simulations with multimillion particles. We have systematically parametrized the force-field for pure ethanol and its interactions with hydrated POPC and POPE model lipid membranes. In this process, we have successfully reproduced the bulk ethanol structure and concentration-dependent density of aqueous ethanol. To quantify the interaction of ethanol with lipid membranes, we have reproduced the transfer free energy of the ethanol molecule across the hydrated bilayers, and the concentration-dependent distribution of ethanol molecules across the lipid bilayers. After having acceptable force-field parameters for ethanol-membrane interactions, we have checked the effect of ethanol toward the vesicles comprising POPC lipids. We observe a rapid increase in the size of the POPC lipid vesicles with increasing amounts of ethanol up to 30 mol %. We unambiguously observe swelling and decrease in the thickness of the POPC vesicles with increasing amounts of ethanol up to 30 mol %, beyond which the vesicles begin to lose their integrity and rupture at higher mol % of ethanol. The fusion study of two vesicles demonstrates that fused vesicles can be obtained from 20 to 30 mol % of ethanol provided that they are brought closer than a critical distance at a particular mol %. The multivesicle simulations show that along with the increase in the sizes of vesicles the propensity of vesicle aggregation increases as the mol % of ethanol increases.

Interplay of Fusion, Leakage, and Electrostatic Lipid Clustering: Membrane Perturbations by a Hydrophobic Antimicrobial Polycation

Membrane active compounds are able to induce various types of membrane perturbations. Natural or biomimetic candidates for antimicrobial treatment or drug delivery scenarios are mostly designed and tested for their ability to induce membrane permeabilization, also termed leakage. Furthermore, the interaction of these usually cationic amphiphiles with negatively charged vesicles often causes colloidal instability leading to vesicle aggregation or/and vesicle fusion. We show the interplay of these modes of membrane perturbation in mixed phosphatidyl glycerol (PG)/phosphatidyl ethanolamine (PE) by the statistical copolymer MM:CO comprising, both, charged and hydrophobic subunits. MM:CO is a representative of partially hydrophobic, highly active, but less selective antimicrobial polycations. Cryo-electron microscopy indicates vesicle fusion rather than vesicle aggregation upon the addition of MM:CO to negatively charged PG/PE (1:1) vesicles. In a combination of fluorescence-based leakage and fusion assays, there is support for membrane permeabilization and pronounced vesicle fusion activity as distinct effects. To this end, membrane fusion and aggregation were prevented by including lipids with polyethylene glycol attached to their head groups (PEG-lipids). The leakage activity of MM:CO is very similar in the absence and presence of PEG-lipids. Vesicle aggregation and fusion however are largely suppressed. This strongly suggests that MM:CO induces leakage by asymmetric packing stress because of hydrophobically driven interactions which could lead to leakage. As a further membrane perturbation effect, MM:CO causes lipid clustering in model vesicles. We address potential artifacts and misinterpretations of experiments characterizing leakage and fusion. Additional to the leakage activity, the pronounced fusogenic activity of the polymer and potentially of many other similar compounds likely has implications for antimicrobial activity and beyond.

Exploring the focal role of LRRK2 kinase in Parkinson's disease.

The major breakthroughs in our knowledge of how biology plays a role in Parkinson's disease (PD) have opened up fresh avenues designed to know the pathogenesis of disease and identify possible therapeutic targets. Mitochondrial abnormal functioning is a key cellular feature in the pathogenesis of PD. An enzyme, leucine-rich repeat kinase 2 (LRRK2), involved in both the idiopathic and familial PD risk, is a therapeutic target. LRRK2 has a link to the endolysosomal activity. Enhanced activity of the LRRK2 kinase, endolysosomal abnormalities and aggregation of autophagic vesicles with imperfectly depleted substrates, such as α-synuclein, are all seen in the substantia nigra dopaminergic neurons in PD. Despite the fact that LRRK2 is involved in endolysosomal and autophagic activity, it is undefined if inhibiting LRRK2 kinase activity will prevent endolysosomal dysfunction or minimise the degeneration of dopaminergic neurons. The inhibitor's capability of LRRK2 kinase to inhibit endolysosomal and neuropathological alterations in human PD indicates that LRRK2 inhibitors could have significant therapeutic usefulness in PD. G2019S is perhaps the maximum common mutation in PD subjects. Even though LRRK2's well-defined structure has still not been established, numerous LRRK2 inhibitors have been discovered. This review summarises the role of LRRK2 kinase in Parkinson's disease.

Arabidopsis exocyst subunit SEC6 is involved in cell plate formation during Microgametogenesis

Cytokinesis during pollen mitosis I is critical for cell division and differentiation in the male gametophyte development, but the vesicle trafficking mechanisms in this process are largely unknown. Exocyst is an octameric tethering complex which plays multiple important roles in plant cell vesicle trafficking. Here we report the characterization of exocyst subunit SEC6 in the cytokinesis during pollen mitosis I. We found that significantly amount of pollen from two sec6/+ mutant alleles arrested at the transition from unicelluar stage microspore to bicellular stage. Further analysis showed that sec6 mutation impaired cell plate formation and led to vesicles accumulation in cytoplasm. The localization of KNOLLE on the cell plate was compromised. Consistently, SEC6 gene was expressed start from early pollen development stage and SEC6-GFP localized to the cell plate. These results indicated that SEC6 participated in the cell plate formation during pollen mitosis I, where it might help to tether the vesicles before fusion.