Multivesicular Body Formation Research Articles

Molecular Genetics of Frontotemporal Dementia Elucidated by Drosophila Models—Defects in Endosomal–Lysosomal Pathway

Frontotemporal dementia (FTD) is the second most common senile neurodegenerative disease. FTD is a heterogeneous disease that can be classified into several subtypes. A mutation in CHMP2B locus (CHMP2Bintron5), which encodes a component of endosomal sorting complex required for transport-III (ESCRT-III), is associated with a rare hereditary subtype of FTD linked to chromosome 3 (FTD-3). ESCRT is involved in critical cellular processes such as multivesicular body (MVB) formation during endosomal–lysosomal pathway and autophagy. ESCRT mutants causes diverse physiological defects primarily due to accumulation of endosomes and defective MVBs resulting in misregulation of signaling pathways. Charged multivesicular body protein 2B (CHMP2B) is important for neuronal physiology which especially rely on precise regulation of protein homeostasis due to their post-mitotic status. Drosophila has proven to be an excellent model for charaterization of mechanistic underpinning of neurodegenerative disorders including FTD. In this review, current understanding of various FTD-related mutations is discussed with a focus on Drosophila models of CHMP2Bintron5-associated FTD.

SMPD3 deficiency perturbs neuronal proteostasis and causes progressive cognitive impairment

Neutral sphingomyelinase smpd3 is most abundantly expressed in neurons of brain. The function of SMPD3 has remained elusive. Here, we report a pathogenetic nexus between absence of SMPD3 in the Golgi compartment (GC) of neurons of the smpd3-/- mouse brain, inhibition of Golgi vesicular protein transport and progressive cognitive impairment. Absence of SMPD3 activity in the Golgi sphingomyelin cycle impedes remodeling of the lipid bilayer, essential for budding and multivesicular body formation. Importantly, we show that inhibition of the Golgi vesicular protein transport causes accumulation of neurotoxic proteins APP, Aβ and phosphorylated Tau, dysproteostasis, unfolded protein response, and apoptosis, which ultimately manifests in progressive cognitive decline, similar to the pathognomonic signatures of familial and sporadic forms of Alzheimer´s disease. This discovery might contribute to the search for other primary pathogenic mechanisms, which link perturbed lipid bilayer structures and protein processing and transport in the neuronal Golgi compartment and neurodegeneration and cognitive deficits.

Leptin Modulates Exosome Biogenesis in Breast Cancer Cells: an Additional Mechanism in Cell‐to‐Cell Communication

Exosomes are small membrane vesicles secreted by both normal and malignant cells upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane, which play an important role in cell‐to‐cell communication. Several reports have highlighted the involvement of exosomes in many aspects of breast cancer development and progression, thus mounting interest in the potential exploitation of these vesicles as cancer biomarkers, drug delivery systems, and for the development of novel therapies. It has been extensively demonstrated the involvement of the obesity hormone leptin in all steps of breast tumorigenesis, but up to now its role in modulating breast cancer exosome generation have not been investigated.Here, we studied the effect of leptin on exosome biogenesis and secretion using as experimental model both estrogen receptor α (ERα)‐positive MCF‐7 and triple negative MDA‐MB‐231 breast cancer cells. First, we evaluated, by Transmission Electron Microscopy (TEM) MVB formation in cells treated with leptin. TEM analysis revealed that the number of MVBs in the cytoplasm of leptin‐treated MCF‐7 and MDA‐MB‐231 breast cancer cells was significantly increased compared to control untreated cells. Next, we characterized size distribution, particle number and protein cargo of exosomes, isolated by ultracentrifugation method, from conditioned media (CM) of cells treated or not with leptin. Nanoparticle Tracking Analysis revealed that the concentration of exosomes in the leptin treated MCF‐7‐ and MDA‐MB‐231‐CM was significantly higher compared to untreated samples. Furthermore, exosomes quantification by Acetylcholinesterase activity showed that the full leptin receptor antagonist, peptide LDFI, abrogated leptin‐induced exosome secretion. Exosomes from leptin treated cells showed an increased expression of the leptin target gene Heat shock protein 90 (Hsp90) and its client protein HER2, along with activated leptin signaling effectors such as pJAK2, pSTAT3, and pMAPK42/44 compared to exosomes from untreated cells. Mechanistically, our results demonstrated that, among protein involved in exosome biogenesis, leptin significantly increased protein expression of the well‐known exosomal luminal marker Tumor susceptibility gene 101 (Tsg101), without affecting its mRNA levels. Co‐immunoprecipitation studies revealed a specific interaction between the chaperone protein Hsp90 and Tsg101 in basal condition that was further increased after leptin exposure. Accordingly, leptin‐induced Tsg101 protein levels were completely abrogated in the presence of specific Hsp90 inhibitor, 17‐allylamino‐17‐demethoxygeldanamicin.In conclusion, our results demonstrate for the first time that leptin was able to increase exosomes release in breast cancer cells, through an up‐regulation of Tsg101 expression at posttranslational level. These findings, providing additional insights into the molecular mechanism governing exosome generation in breast cancer cells, might open new avenues for therapeutic intervention in breast carcinoma.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TP53 is required for BECN1- and ATG5-dependent cell death induced by sphingosine kinase 1 inhibition

ABSTRACTThe bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the enzyme that produces it, SPHK1 (sphingosine kinase 1), regulate many processes important for the etiology of cancer. It has been suggested that SPHK1 levels are regulated by the tumor suppressor protein TP53, a key regulator of cell cycle arrest, apoptosis, and macroautophagy/autophagy. However, little is still known of the relationship between TP53 and SPHK1 activity in the regulation of these processes. To explore this link, we examined the effects of inhibiting SPHK1 in wild-type and TP53 null cancer cell lines. SK1-I, an analog of sphingosine and isozyme-specific SPHK1 inhibitor, suppressed cancer cell growth and clonogenic survival in a TP53-dependent manner. It also more strongly enhanced intrinsic apoptosis in wild-type TP53 cells than in isogenic TP53 null cells. Intriguingly, SK1-I induced phosphorylation of TP53 on Ser15, which increases its transcriptional activity. Consequently, levels of TP53 downstream targets such as pro-apoptotic members of the BCL2 family, including BAX, BAK1, and BID were increased in wild-type but not in TP53 null cells. Inhibition of SPHK1 also increased the formation of autophagic and multivesicular bodies, and increased processing of LC3 and its localization within acidic compartments in a TP53-dependent manner. SK1-I also induced massive accumulation of vacuoles, enhanced autophagy, and increased cell death in an SPHK1-dependent manner that also required TP53 expression. Importantly, downregulation of the key regulators of autophagic flux, BECN1 and ATG5, dramatically decreased the cytotoxicity of SK1-I only in cells with TP53 expression. Hence, our results reveal that TP53 plays an important role in vacuole-associated cell death induced by SPHK1 inhibition in cancer cells.

Targeting Transmembrane BAX Inhibitor Motif Containing 1 Alleviates Pathological Cardiac Hypertrophy.

Cardiac hypertrophy and its resultant heart failure are among the most common causes of mortality worldwide. Abnormal protein degradation, especially the impaired lysosomal degradation of large organelles and membrane proteins, is involved in the progression of cardiac hypertrophy. However, the underlying mechanisms have not been fully elucidated. We investigated cardiac transmembrane BAX inhibitor motif containing 1 (TMBIM1) mRNA and protein expression levels in samples from patients with heart failure and mice with aortic banding (AB)-induced cardiac hypertrophy. We generated cardiac-specific Tmbim1 knockout mice and cardiac-specific Tmbim1-overexpressing transgenic mice and then challenged them with AB surgery. We used microarray, confocal image, and coimmunoprecipitation analyses to identify the downstream targets of TMBIM1 in cardiac hypertrophy. Tmbim1/Tlr4 double-knockout mice were generated to investigate whether the effects of TMBIM1 on cardiac hypertrophy were Toll-like receptor 4 (TLR4) dependent. Finally, lentivirus-mediated TMBIM1 overexpression in a monkey AB model was performed to evaluate the therapeutic potential of TMBIM1. TMBIM1 expression was significantly downregulated on hypertrophic stimuli in both human and mice heart samples. Silencing cardiac Tmbim1 aggravated AB-induced cardiac hypertrophy. This effect was blunted by Tmbim1 overexpression. Transcriptome profiling revealed that the TLR4 signaling pathway was disrupted dramatically by manipulation of Tmbim1. The effects of TMBIM1 on cardiac hypertrophy were shown to be dependent on TLR4 in double-knockout mice. Fluorescent staining indicated that TMBIM1 promoted the lysosome-mediated degradation of activated TLR4. Coimmunoprecipitation assays confirmed that TMBIM1 directly interacted with tumor susceptibility gene 101 via a PTAP motif and accelerated the formation of multivesicular bodies that delivered TLR4 to the lysosomes. Finally, lentivirus-mediated TMBIM1 overexpression reversed AB-induced cardiac hypertrophy in monkeys. TMBIM1 protects against pathological cardiac hypertrophy through promoting the lysosomal degradation of activated TLR4. Our findings reveal the central role of TMBIM1 as a multivesicular body regulator in the progression of pathological cardiac hypertrophy, as well as the role of vesicle trafficking in signaling regulation during cardiac hypertrophy. Moreover, targeting TMBIM1 could be a novel therapeutic strategy for treating cardiac hypertrophy and heart failure.

SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events.

Trafficking within eukaryotic cells is a complex and highly regulated process; events such as recycling of plasma membrane receptors, formation of multivesicular bodies, regulated release of hormones and delivery of proteins to membranes all require directionality and specificity. The underpinning processes, including cargo selection, membrane fusion, trafficking flow and timing, are controlled by a variety of molecular mechanisms and engage multiple families of lipids and proteins. Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. We will describe how these proteins are controlled in a range of regulated trafficking events, with particular emphasis on the insulin-stimulated delivery of glucose transporters to the surface of adipose and muscle cells. Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion.

Effect of starvation and refeeding on the ultrastructure of the perigastric organ (hepatopancreas) in the whiteleg shrimp Litopenaeus vannamei (Boone, 1931) (Decapoda: Caridea: Penaeidae)

Farmed caridean shrimps can experience starvation periods attributable to disease outbreaks or adverse environmental conditions. The hepatopancreas, or perigastric organ, of decapods, being the principal organ for storage of nutrients that can be mobilized during non-feeding periods, plays a fundamental role during starvation. We studied the ultrastructural changes in the perigastric organ of whiteleg shrimp Litopenaeus vannamei (Boone, 1931), a commonly farmed species, during starvation and refeeding. Starvation induced a progressive increase of cellular immunity (haemocytic infiltration, both hyaline and granular and/or semigranular haemocytes), necrosis (swelling of organelles and cytolysis), and autophagy (formation of phagophores, autophagosomes, autolysosomes, and multivesicular and residual bodies). The complete depletion of lipid droplets and a few signs of apoptosis (chromatin condensation and nuclear fragmentation) were also observed during starvation. Refeeding resulted in a partial recovery of the perigastric organ. Findings demonstrate the capacity of the perigastric organ to recover when refed for ten days after five days of starvation. Longer starvation periods severely affect the perigastric organ, causing potential economic losses to famers.

Mutant Huntingtin Inhibits αB-Crystallin Expression and Impairs Exosome Secretion from Astrocytes.

In the brain, astrocytes secrete diverse substances that regulate neuronal function and viability. Exosomes, which are vesicles produced through the formation of multivesicular bodies and their subsequent fusion with the plasma membrane, are also released from astrocytes via exocytotic secretion. Astrocytic exosomes carry heat shock proteins that can reduce the cellular toxicity of misfolded proteins and prevent neurodegeneration. Although mutant huntingtin (mHtt) affects multiple functions of astrocytes, it remains unknown whether mHtt impairs the production of exosomes from astrocytes. We found that mHtt is not present in astrocytic exosomes, but can decrease exosome secretion from astrocytes in HD140Q knock-in (KI) mice. N-terminal mHtt accumulates in the nuclei and forms aggregates, causing decreased secretion of exosomes from cultured astrocytes. Consistently, there is a significant decrease in secreted exosomes in both female and male HD KI mouse striatum in which abundant nuclear mHtt aggregates are present. Conversely, injection of astrocytic exosomes into the striatum of HD140Q KI mice reduces the density of mHtt aggregates. Further, mHtt in astrocytes decreased the expression of αB-crystallin, a small heat shock protein that is enriched in astrocytes and mediates exosome secretion, by reducing the association of Sp1 with the enhancer of the αB-crystallin gene. Importantly, overexpression of αB-crystallin rescues defective exosome release from HD astrocytes as well as mHtt aggregates in the striatum of HD140Q KI mice. Our results demonstrate that mHtt reduces the expression of αB-crystallin in astrocytes to decrease exosome secretion in the HD brains, contributing to non-cell-autonomous neurotoxicity in HD.SIGNIFICANCE STATEMENT Huntington's disease (HD) is characterized by selective neurodegeneration that preferentially occurs in the striatal medium spiny neurons. Recent studies in different HD mouse models demonstrated that dysfunction of astrocytes, a major type of glial cell, leads to neuronal vulnerability. Emerging evidence shows that exosomes secreted from astrocytes contain neuroprotective cargoes that could support the survival of neighboring neurons. We found that mHtt in astrocytes impairs exosome secretion by decreasing αB-crystallin, a protein that is expressed mainly in glial cells and mediates exosome secretion. Overexpression of αB-crystallin could alleviate the deficient exosome release and neuropathology in HD mice. Our results revealed a new pathological pathway that affects the critical support of glial cells to neurons in the HD brain.

Structural templating of J-aggregates: Visualizing bis(monoacylglycero)phosphate domains in live cells

Identifying the key structural and dynamical determinants that drive the association of biomolecules, whether in solution, or perhaps more importantly in a membrane environment, has critical implications for our understanding of cellular dynamics, processes, and signaling. With recent advances in high-resolution imaging techniques, from the development of new molecular labels to technical advances in imaging methodologies and platforms, researchers are now reaping the benefits of being able to directly characterize and quantify local dynamics, structures, and conformations in live cells and tissues. These capabilities are providing unique insights into association stoichiometries, interactions, and structures on sub-micron length scales. We previously examined the role of lipid headgroup chemistry and phase state in guiding the formation of pseudoisocyanine (PIC) dye J-aggregates on supported planar bilayers [Langmuir, 25, 10719]. We describe here how these same J-aggregates can report on the in situ formation of organellar membrane domains in live cells. Live cell hyperspectral confocal microscopy using GFP-conjugated GTPase markers of early (Rab5) and late (Rab7) endosomes revealed that the PIC J-aggregates were confined to domains on either the limiting membrane or intralumenal vesicles (ILV) of late endosomes, known to be enriched in the anionic lipid bis(monoacylglycero)phosphate (BMP). Correlated confocal fluorescence - atomic force microscopy performed on endosomal membrane-mimetic supported planar lipid bilayers confirmed BMP-specific templating of the PIC J-aggregates. These data provide strong evidence for the formation of BMP-rich lipid domains during multivesicular body formation and portend the application of structured dye aggregates as markers of cellular membrane domain structure, size, and formation.

Adverse Effect of Superovulation Treatment on Maturation, Function and Ultrastructural Integrity of Murine Oocytes.

Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve ultrastructural integrity and developmental competence of oocytes following oocyte activation and embryo culture. A randomized study using mouse model was designed and in vitro development (experiment 1), ultrastructural morphology (experiment 2) and functional integrity of the oocytes (experiment 3) retrieved after PMSG/hCG injection (superovulation group) or not (natural ovulation; control group) were evaluated. In experiment 1, more oocytes were retrieved following superovulation than following natural ovulation, but natural ovulation yielded higher (p < 0.0563) maturation rate than superovulation. The capacity of mature oocytes to form pronucleus and to develop into blastocysts in vitro was similar. In experiment 2, a notable (p < 0.0186) increase in mitochondrial deformity, characterized by the formation of vacuolated mitochondria, was detected in the superovulation group. Multivesicular body formation was also increased, whereas early endosome formation was significantly decreased. No obvious changes in other microorganelles, however, were detected, which included the formation and distribution of mitochondria, cortical granules, microvilli, and smooth and rough endoplasmic reticulum. In experiment 3, significant decreases in mitochondrial activity, ATP production and dextran uptake were detected in the superovulation group. In conclusion, superovulation treatment may change both maturational status and functional and ultrastuctural integrity of oocytes. Superovulation effect on preimplantation development can be discussed.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools.

Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca2+ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes.

Post-translational control of T cell development by the ESCRT protein CHMP5.

The acquisition of a protective vertebrate immune system hinges on the efficient generation of a diverse but self-tolerant repertoire of T cells by the thymus through mechanisms that remain incompletely resolved. Here we identified the endosomal-sorting-complex-required-for-transport (ESCRT) protein CHMP5, known to be required for the formation of multivesicular bodies, as a key sensor of thresholds for signaling via the T cell antigen receptor (TCR) that was essential for T cell development. CHMP5 enabled positive selection by promoting post-selection thymocyte survival in part through stabilization of the pro-survival protein Bcl-2. Accordingly, loss of CHMP5 in thymocyte precursor cells abolished T cell development, a phenotype that was 'rescued' by genetic deletion of the pro-apoptotic protein Bim or transgenic expression of Bcl-2. Mechanistically, positive selection resulted in the stabilization of CHMP5 by inducing its interaction with the deubiquitinase USP8. Our results thus identify CHMP5 as an essential component of the post-translational machinery required for T cell development.

Tmbim1 is a multivesicular body regulator that protects against non-alcoholic fatty liver disease in mice and monkeys by targeting the lysosomal degradation of Tlr4.

Non-alcoholic steatohepatitis (NASH) is an increasingly prevalent liver pathology that can progress from non-alcoholic fatty liver disease (NAFLD), and it is a leading cause of cirrhosis and hepatocellular carcinoma. There is currently no pharmacological therapy for NASH. Defective lysosome-mediated protein degradation is a key process that underlies steatohepatitis and a well-recognized drug target in a variety of diseases; however, whether it can serve as a therapeutic target for NAFLD and NASH remains unknown. Here we report that transmembrane BAX inhibitor motif-containing 1 (TMBIM1) is an effective suppressor of steatohepatitis and a previously unknown regulator of the multivesicular body (MVB)-lysosomal pathway. Tmbim1 expression in hepatocytes substantially inhibited high-fat diet-induced insulin resistance, hepatic steatosis and inflammation in mice. Mechanistically, Tmbim1 promoted the lysosomal degradation of toll-like receptor 4 by cooperating with the ESCRT endosomal sorting complex to facilitate MVB formation, and the ubiquitination of Tmbim1 by the E3 ubiquitin ligase Nedd4l was required for this process. We also found that overexpression of Tmbim1 in the liver effectively inhibited a severe form of NAFLD in mice and NASH progression in monkeys. Taken together, these findings could lead to the development of promising strategies to treat NASH by targeting MVB regulators to properly orchestrate the lysosome-mediated protein degradation of key mediators of the disease.

Exocytosis-Mediated Urinary Full-Length Megalin Excretion Is Linked With the Pathogenesis of Diabetic Nephropathy.

Efficient biomarkers for diabetic nephropathy (DN) have not been established. Using ELISA, we found previously that urinary levels of full-length megalin (C-megalin), a multiligand endocytic receptor in proximal tubules, was positively correlated with DN progression in patients with type 2 diabetes mellitus (T2DM). Here, we found that urinary extracellular vesicle (UEV) excretion and C-megalin content in UEVs or in their exosomal fraction increased along with the progression of the albuminuric stages in patients with T2DM. Cultured immortalized rat proximal tubule cells (IRPTCs) treated with fatty acid-free BSA or advanced glycation end product-modified BSA (AGE-BSA), endocytic ligands of megalin, increased EV excretion, and their C-megalin content. C-megalin excretion from IRPTCs via extracellular vesicles was significantly blocked by an exosome-specific inhibitor, GW4869, indicating that this excretion is mainly exocytosis-mediated. AGE-BSA treatment of IRPTCs caused apparent lysosomal dysfunction, which stimulated multivesicular body formation, resulting in increased exosomal C-megalin excretion. In a high-fat diet-induced, megalin-mediated kidney injury model in mice, urinary C-megalin excretion also increased via UEVs. Collectively, exocytosis-mediated urinary C-megalin excretion is associated with the development and progression of DN in patients with T2DM, particularly due to megalin-mediated lysosomal dysfunction in proximal tubules, and hence it could be a candidate biomarker linked with DN pathogenesis.

Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction.

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1 of lymphocytes was important for inducing the release of sequestered HIV-1 from epithelial cells and facilitating cell-to-cell spread of virus from epithelial cells to lymphocytes. This mechanism may serve as a pathway of HIV-1 mucosal transmission.

The formation of multivesicular bodies in activated blastocysts is influenced by autophagy and FGF signaling in mice

Dormant blastocysts during delayed implantation undergo autophagic activation, which is an adaptive response to prolonged survival in utero during less favorable environment. We observed that multivesicular bodies (MVBs) accumulate in the trophectoderm of dormant blastocysts upon activation for implantation. Since autophagosomes are shown to fuse with MVBs and efficient autophagic degradation requires functional MVBs, we examined if MVB formation in activated blastocysts are associated with protracted autophagic state during dormancy. We show here that autophagic activation during dormancy is one precondition for MVB formation in activated blastocysts. Furthermore, the blockade of FGF signaling with PD173074 partially interferes with MVB formation in these blastocysts, suggesting the involvement of FGFR signaling in this process. We believe that MVB formation in activated blastocysts after dormancy is a potential mechanism of clearing subcellular debris accumulated during prolonged autophagy.

Modulation of Membrane Rigidity by the ESCRT-III Complex

The ESCRT-III complexes is an evolutionary conserved membrane scission machinery and it is essential in many cellular processes such as cytokinesis, multivesicular bodies formation, HIV release and nuclear membrane repair. Membrane scission by ESCRT-III is accomplished by constricting the negatively curved membrane present inside the neck connecting two membrane-delimited compartments. In Homo Sapiens there are at least 12 ESCRT-III proteins, called CHMPs. However, only a subset of them, namely CHMP4B, CHMP2A/B and CHMP3 appear to be strictly required in all these processes, indicating that they might constitute the minimal scission machinery. Several CHMP2B mutations have been reported to cause neurological disfunctions; however the function of this protein is still unknown. We investigated the mechanical properties of CHMP2B polymer at the mesoscale and found that it can modulate membrane rigidity, providing a possible molecular mechanism explaining the pathogenic property of the mutated CHMP2B. Our results indicate that modulation of membrane rigidity is an important aspect of ESCRT-III function and assign a novel function for CHMP2B.

EGF Uptake and Degradation Assay to Determine the Effect of HTLV Regulatory Proteins on the ESCRT-Dependent MVB Pathway.

The endosomal sorting complex required for transport (ESCRT) pathway plays key roles in multivesicular bodies (MVBs) formation and lysosomal degradation of membrane receptors, viral budding, and midbody abscission during cytokinesis. The epidermal growth factor receptor (EGFR) is regarded as a prototypical cargo of the MVB/ESCRT pathway and following stimulation by epidermal growth factor (EGF) EGFR/EGF complexes are internalized, sorted into MVBs, and degraded by lysosomes or recycled back to the cell membrane. Here, we describe an assay to analyze the effect of human T-cell leukemia (HTLV) regulatory proteins on the functionality of ESCRT-dependent MVB/lysosomal trafficking of EGFR/EGF complexes. This is performed by direct visualization and quantification of the rate of EGF-Alexa595/EGFR internalization and degradation in HeLa cells expressing HTLV regulatory proteins by immunofluorescence and western blot.

PRNP/prion protein regulates the secretion of exosomes modulating CAV1/caveolin-1-suppressed autophagy

ABSTRACTPrion protein modulates many cellular functions including the secretion of trophic factors by astrocytes. Some of these factors are found in exosomes, which are formed within multivesicular bodies (MVBs) and secreted into the extracellular space to modulate cell-cell communication. The mechanisms underlying exosome biogenesis were not completely deciphered. Here, we demonstrate that primary cultures of astrocytes and fibroblasts from prnp-null mice secreted lower levels of exosomes than wild-type cells. Furthermore, prnp-null astrocytes exhibited reduced MVB formation and increased autophagosome formation. The reconstitution of PRNP expression at the cell membrane restored exosome secretion in PRNP-deficient astrocytes, whereas macroautophagy/autophagy inhibition via BECN1 depletion reestablished exosome release in these cells. Moreover, the PRNP octapeptide repeat domain was necessary to promote exosome secretion and to impair the formation of the CAV1-dependent ATG12–ATG5 cytoplasmic complex that drives autophagosome formation. Accordingly, higher levels of CAV1 were found in lipid raft domains instead of in the cytoplasm in prnp-null cells. Collectively, these findings demonstrate that PRNP supports CAV1-suppressed autophagy to protect MVBs from sequestration into phagophores, thus facilitating exosome secretion.