Role In Vesicle Transport Research Articles

Calix[6]arene dismantles extracellular vesicle biogenesis and metalloproteinases that support pancreatic cancer hallmarks

Many challenges are faced in pancreatic cancer treatment due to late diagnosis and poor prognosis because of high recurrence and metastasis. Extracellular vesicles (EVs) and matrix metalloproteinases (MMPs), besides acting in intercellular communication, are key players in the cancer cell plasticity responsible for initiating metastasis. Therefore, these entities provide valuable targets for the development of better treatments. In this context, this study aimed to evaluate the potential of calix[6]arene to disturb the release of EVs and the activity of MMPs in pancreatic cancer cells. We found a correlation between the endocytic-associated mediators and the prognosis of pancreatic cancer patients. We observed a more active EV machinery in the pancreatic cancer cell line PANC-1, which was reduced three-fold by treatment with calix[6]arene at subtoxic concentration (5 μM; p 〈0,001). We observed the modulation of 186 microRNAs (164 miRNAs upregulated and 22 miRNAs downregulated) upon calix[6]arene treatment. Interestingly, some of them as miR-4443 and miR-3909, regulates genes HIF1A e KIF13A that are well known to play a role in transport of vesicles. Furthermore, Calix[6]arene downmodulated matrix metalloproteinases (MMPs) -2 and − 9 and disturbed the viability of pancreatic organoids which recapitulate the cellular heterogeneity, structure, and functions of primary tissues. Our findings shed new insights on calix[6]arene's antitumor mechanism, including its intracellular effects on vesicle production and trafficking, as well as MMP activity, which may harm the tumor microenvironment and contribute to a reduction in cancer cell dissemination, which is one of the challenges associated with high mortality in pancreatic cancer.

Clinico-radiological characteristics of two patients with geroderma osteodysplasticum caused by new nucleotide variants in the GORAB gene

Geroderma osteodysplasticum (OMIM #231070) is a rare autosomal recessive disorder characterized by congenital wrinkled skin on the dorsal surface of the limbs and abdomen; marked osteoporosis in childhood. Pathogenic variants of the nucleotide sequence in the GORAB gene, which encodes the GORAB protein located in the Golgi apparatus and plays a crucial role in vesicle transport in the Golgi complex, lead to the disease. This is the first report of 2 patients with this disease in Russia. A detailed description of the clinical-radiological and genetic characteristics of these patients with new nucleotide variants in the GORAB gene in a compound heterozygous state is presented: c.170C>G (p.Ser57Ter) and c.790G>C (p.Ala264Pro); c.295C>T (p.Gln99Ter) and heterozygous deletion on chromosome 1 (chr1:g.170531967–170539494del), affecting exons 1 and 2 of the gene. The main clinical manifestations of the syndrome were congenital wrinkled skin, sagging cheeks, hypoplasia of the cheekbones, prognathism, muscle hypotonia and joint hypermobility. Radiological signs included congenital bilateral dislocation of the hip, compression fractures of the vertebral bodies, and disruption of the continuity of the growth plate of the distal femur and proximal tibia, referred to as “insertion.” The rarity of the syndrome, as well as the similarity of clinical manifestations with a heterogeneous group of hereditary diseases accompanied by decreased skin elasticity, leads to a late correct diagnosis. This necessitates the description of the clinical-genetic characteristics of the disease, the study of the dynamics of the formation of its phenotypic manifestations, and the methods of molecular-genetic diagnostics.

Palmitate-mediated disruption of the endoplasmic reticulum decreases intracellular vesicle motility

Essential cellular processes such as metabolism, protein synthesis, and autophagy require the intracellular transport of membrane-bound vesicles. The importance of the cytoskeleton and associated molecular motors for transport is well documented. Recent research has suggested that the endoplasmic reticulum (ER) may also play a role in vesicle transport through a tethering of vesicles to the ER. We use single-particle tracking fluorescence microscopy and a Bayesian change-point algorithm to characterize vesicle motility in response to the disruption of the ER, actin, and microtubules. This high-throughput change-point algorithm allows us to efficiently analyze thousands of trajectory segments. We find that palmitate-mediated disruption of the ER leads to a significant decrease in vesicle motility. A comparison with the disruption of actin and microtubules shows that disruption of the ER has a significant impact on vesicle motility, greater than the disruption of actin. Vesicle motility was dependent on cellular region, with greater motility in the cell periphery than the perinuclear region, possibly due to regional differences in actin and the ER. Overall, these results suggest that the ER is an important factor in vesicle transport.

Therapeutic Targeting of Rab GTPases: Relevance for Alzheimer's Disease.

Rab GTPases (Rabs) are small proteins that play crucial roles in vesicle transport and membrane trafficking. Owing to their widespread functions in several steps of vesicle trafficking, Rabs have been implicated in the pathogenesis of several disorders, including cancer, diabetes, and multiple neurodegenerative diseases. As treatments for neurodegenerative conditions are currently rather limited, the identification and validation of novel therapeutic targets, such as Rabs, is of great importance. This review summarises proof-of-concept studies, demonstrating that modulation of Rab GTPases in the context of Alzheimer’s disease (AD) can ameliorate disease-related phenotypes, and provides an overview of the current state of the art for the pharmacological targeting of Rabs. Finally, we also discuss the barriers and challenges of therapeutically targeting these small proteins in humans, especially in the context of AD.

Decreased Podocyte Vesicle Transcytosis and Albuminuria in APC C-Terminal Deficiency Mice with Puromycin-Induced Nephrotic Syndrome.

In minimal change nephrotic syndrome, podocyte vesicle transport is enhanced. Adenomatous polyposis coli (APC) anchors microtubules to cell membranes and plays an important role in vesicle transport. To clarify the role of APC in vesicle transport in podocytes, nephrotic syndrome was induced by puromycin amino nucleoside (PAN) injection in mice expressing APC1638T lacking the C-terminal of microtubule-binding site (APC1638T mouse); this was examined in renal tissue changes. The kidney size and glomerular area of APC1638T mice were reduced (p = 0.014); however, the number of podocytes was same between wild-type (WT) mice and APC1638T mice. The ultrastructure of podocyte foot process was normal by electron microscopy. When nephrotic syndrome was induced, the kidneys of WT+PAN mice became swollen with many hyaline casts, whereas these changes were inhibited in the kidneys of APC1638T+PAN mice. Electron microscopy showed foot process effacement in both groups; however, APC1638T+PAN mice had fewer vesicles in the basal area of podocytes than WT+PAN mice. Cytoplasmic dynein-1, a motor protein for vesicle transport, and α-tubulin were significantly reduced in APC1638T+PAN mice associated with suppressed urinary albumin excretion compared to WT+PAN mice. In conclusion, APC1638T mice showed reduced albuminuria associated with suppressed podocyte vesicle transport when minimal change nephrotic syndrome was induced.

Loss of Profilin3 Impairs Spermiogenesis by Affecting Acrosome Biogenesis, Autophagy, Manchette Development and Mitochondrial Organization.

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis, and metastasis of tumor cells. The testes-specific PFN3 is localized in the acroplaxome–manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3–/– males are subfertile, displaying a type II globozoospermia. We revealed that Pfn3–/– sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3–/– sperm showed reduced sperm motility resulting from flagellum deformities. We show that acrosome biogenesis is impaired starting from the Golgi phase, and mature sperm seems to suffer from a cytoplasm removal defect. An RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed, resulting in the inhibition of autophagy. This dysregulation of AMPK/mTOR affected the autophagic flux, which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3–/– testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3–ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3–/– testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicate this protein as a candidate for male factor infertility in humans.

On the role of vesicle transport in neurite growth: Modeling and experiments

The processes that determine the establishment of the complex morphology of neurons during development are still poorly understood. Here, we focus on the question how a difference in the length of neurites affects vesicle transport. We performed live imaging experiments and present a lattice-based model to gain a deeper theoretical understanding of intracellular transport in neurons. After a motivation and appropriate scaling of the model we present numerical simulations showing that initial differences in neurite length result in phenomena of biological relevance, i.e. a positive feedback that enhances transport into the longer neurite and oscillation of vesicles concentrations that can be interpreted as cycles of extension and retraction observed in experiments. Thus, our model is a first step towards a better understanding of the interplay between the transport of vesicles and the spatial organization of cells.

ShNPSN11, a vesicle-transport-related gene, confers disease resistance in tomato to Oidium neolycopersici.

Tomato powdery mildew, caused by Oidium neolycopersici, is a fungal disease that results in severe yield loss in infected plants. Herein, we describe the function of a class of proteins, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which play a role in vesicle transport during defense signaling. To date, there have been no reports describing the function of tomato SNAREs during resistance signaling to powdery mildew. Using a combination of classical plant pathology-, genetics-, and cell biology-based approaches, we evaluate the role of ShNPSN11 in resistance to the powdery mildew pathogen O. neolycopersici. Quantitative RT-PCR analysis of tomato SNAREs revealed that ShNPSN11 mRNA accumulation in disease-resistant varieties was significantly increased following pathogen, compared with susceptible varieties, suggesting a role during induced defense signaling. Using in planta subcellular localization, we demonstrate that ShNPSN11 was primarily localized at the plasma membrane, consistent with the localization of SNARE proteins and their role in defense signaling and trafficking. Silencing of ShNPSN11 resulted in increased susceptibility to O. neolycopersici, with pathogen-induced levels of H2O2 and cell death elicitation in ShNPSN11-silenced lines showing a marked reduction. Transient expression of ShNPSN11 did not result in the induction of a hypersensitive cell death response or suppress cell death induced by BAX. Taken together, these data demonstrate that ShNPSNl11 plays an important role in defense activation and host resistance to O. neolycopersici in tomato LA1777.

Intracellular Transport of Ribosome-Inactivating Proteins Depends on Annexin 13.

In the present study, we assessed the role of annexin 13 membrane-binding protein (ANXA13) in the intracellular transport of vesicles containing type II ribosome-inactivating proteins (RIP-IIs). A modified human intestinal epithelial cell line HT29 was used, in which the expression of ANXA13 was significantly reduced. The cytotoxic effect of ricin and viscumin was evaluated by modification of 28S ribosome RNA. The observed differences in the activity of toxins on the parental and modified HT29 lines indicate that ANXA13 plays a different role in the intracellular transport of vesicles containing the RIP-IIs.

Rab11a regulates MMP2 expression by activating the PI3K/AKT pathway in human hepatocellular carcinoma cells

As a member of the Rab GTPase family, Rab11a plays an important role in vesicle transport and tumor progression. However, it is not clear whether it can also be used as an oncoprotein in hepatocellular carcinoma (HCC). In this study, database and immunohistochemical analyses showed that Rab11a was highly expressed in HCC tissues, and associated with poor clinical prognosis. Rab11a overexpression promoted the proliferation, migration, invasion, and anti-apoptosis of human HCC cell lines, MHCC-97H and HCC-LM3, whereas the downregulation of Rab11a inhibited these biological tumor activities. Nude mice xenograft demonstrated that Rab11a had a positive effect on the growth of hepatocellular carcinoma cells in vivo. Further studies found that the PI3K/AKT pathway and matrix metalloproteinase 2 (MMP2) upregulation can be activated by over-expression of Rab11a. However, MMP2 upregulation induced by Rab11a can be inhibited by the PI3K/AKT pathway inhibitor, LY294002. Altogether, our study established for the first time that Rab11a can play a pro-cancer role in HCC, as a novel oncoprotein, by activating the PI3K/AKT pathway to regulate MMP2 expression.

Mouse TMCO5 is localized to the manchette microtubules involved in vesicle transfer in the elongating spermatids.

As a result of a high-throughput in situ hybridization screening for adult mouse testes, we found that the mRNA for Tmco5 is expressed in round and elongating spermatids. Tmco5 belongs to the Tmco (Transmembrane and coiled-coil domains) gene family and has a coiled-coil domain in the N-terminal and a transmembrane domain in the C-terminal region. A monoclonal antibody raised against recombinant TMCO5 revealed that the protein is expressed exclusively in the elongating spermatids of step 9 to 12 and is localized to the manchette, a transiently emerging construction, which predominantly consists of cytoskeleton microtubules and actin filaments. This structure serves in the transport of Golgi-derived non-acrosomal vesicles. Moreover, induced expression of TMCO5 in CHO cells resulted in the co-localization of TMCO5 with β-tubulin besides the reorganization of the Golgi apparatus. Judging from the results and considering the domain structure of TMCO5, we assume that Tmco5 may have a role in vesicle transport along the manchette.

Myosin Va and spermine synthase: partners in exosome transport.

A recent paper in Bioscience Reports (BSR20182189) describes the discovery of an interaction between the motor protein myosin Va and the metabolic enzyme spermine synthase. Myosin Va is a molecular motor which plays a key role in vesicle transport. Mutations in the gene which encodes this protein are associated with Griscelli syndrome type 1 and the ‘dilute’ phenotype in animals. Spermine synthase catalyzes the conversion of spermidine to spermine. This largely cytoplasmic enzyme can also be localized to the soluble fraction in exosomes. Mutations in the spermine synthase gene are associated with Snyder Robinson mental retardation syndrome. The interaction between the two proteins was detected using the yeast two hybrid method and verified by microscale thermophoresis of recombinant proteins. Knockdown of the MYO5A gene reduced the expression of mRNA coding for spermine synthase. The amount of this transcript was also reduced in cells derived from a patient with Griscelli syndrome type 1. This suggests that, in addition to a direct physical interaction between the two proteins, myosin Va also modulates the transcription of the spermine synthase gene. The mechanism for this modulation is currently unknown. These findings have implications for Griscelli syndrome type 1 and Snyder Robinson mental retardation syndrome. They also suggest that interactions between myosin Va and soluble exosome proteins such as spermine synthase may be important in the mechanism of exosome transport.

Investigating a Role for TCL/RhoJ in Endocytic Pathways Using Live Cell Imaging

TCL (or RhoJ) is a plasma membrane localized Rho‐GTPase bearing an additional eighteen N‐terminal amino acids in comparison to its close homologue Cdc42. Previous experiments in the lab have demonstrated that deleting the N‐terminal extension shifts TCL localization to vesicular membranes. To determine how this deletion may influence membrane trafficking of TCL, HeLa cells were transfected with plasmids encoding YFP‐tagged TCL and an N‐terminal deletion mutant (ΔN), and live‐cell imaging was performed using an FV10i fluorescence microscope. The live‐cell imaging demonstrated that wild‐type YFP‐TCL localization was present at the plasma membrane, and it also revealed an organized movement of YFP‐TCL labeled intracellular vesicles. Cells expressing the YFP‐TCL ΔN deletion mutant displayed more of a vesicular localization of the fusion protein, and vesicular movement within the cell was more erratic and random. Previous research has used fluorescent‐labeled transferrin as a marker to investigate a role for TCL in endocytic pathways; therefore, cells were exposed to Alexa 647‐labeled holo‐transferrin to determine if TCL colocalizes with vesicles involved with transferrin receptor uptake and recycling. Colocalization of the two fluorescent labels in time‐lapse images was measured using CellSens software, and initial results show significant colocalization of YFP‐TCL ΔN with transferrin‐labeled vesicles in comparison to wild‐type TCL. However, it is unclear if the alterations in TCL localization are influencing transferrin transport kinetics or if YFP‐TCL ΔN is inappropriately localized to transferrin‐labeled vesicles. Together, these results show TCL has a potential role in vesicle transport, and future experiments will be aimed at determining if it influences vesicle transport kinetics.Support or Funding InformationSupport was provided by the Nielson Foundation, Bemidji, MN and a grant from Regenerative Medicine Minnesota (RMM‐2017‐EP‐04)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Power Stroke Distance of Human Cytoplasmic Dynein

Cytoplasmic dynein is a motor protein moving along microtubules, and plays important roles in vesicle transport and mitosis. To understand the molecular mechanism of dynein motility, we measured the efficiency of FRET from dynein ring-BFP to linker-GFP, and the displacement driven by single-headed dynein interacting with microtubules by optical tweezers. The efficiency and the apparent stroke displacement depend on ATP concentration. The low efficiency and displacement at low ATP concentration indicate no conformational change of dynein at no ATP binding (apo) state that is predominant at low ATP concentration. With increasing ATP concentration, population of apo state decreases and that of the pre-power stroke state such as dynein ADP-Pi should increase. Dynein at the pre-power stroke state will generate the power stroke at binding to microtubule. Therefore, the displacement increased with ATP concentration. This is the first obtained interesting result and has not been reported for myosin and kinesin power strokes. High FRET efficiency and distance (8-9 nm) at saturated concentration of ATP indicate that the 8-9 nm power stroke distance is generated by swing of linker. This is supported by the result that the dynein deleted at the loops interacted with the linker did not generate the power stroke. The power stroke driven by the conformational change of dynein linker will be the fundamental mechanism of dynein motility.

Mammalian Nonmuscle Myosin II Binds to Anionic Phospholipids with Concomitant Dissociation of the Regulatory Light Chain

Mammalian cells express three Class II nonmuscle myosins (NM): NM2A, NM2B, and NM2C. The three NM2s have well established essential roles in cell motility, adhesion, and cytokinesis and less well defined roles in vesicle transport and other processes that would require association of NM2s with cell membranes. Previous evidence for the mechanism of NM2-membrane association includes direct interaction of NM2s with membrane lipids and indirect interaction by association of NM2s with membrane-bound F-actin or peripheral membrane proteins. Direct binding of NM2s to phosphatidylserine-liposomes, but not to phosphatidylcholine-liposomes, has been reported, but the molecular basis of the interaction between NM2s and acidic phospholipids has not been previously investigated. We now show that filamentous, full-length NM2A, NM2B, and NM2C and monomeric, non-filamentous heavy meromyosin bind to liposomes containing one or more acidic phospholipids (phosphatidylserine, phosphatidylinositol 4,5-diphosphate, and phosphatidylinositol 3,4,5-triphosphate) but do not bind to 100% phosphatidylcholine-liposomes. Binding of NM2s to acidic liposomes occurs predominantly through interaction of the liposomes with the regulatory light chain (RLC) binding site in the myosin heavy chain with concomitant dissociation of the RLC. Phosphorylation of myosin-bound RLC by myosin light chain kinase substantially inhibits binding to liposomes of both filamentous NM2 and non-filamentous heavy meromyosin; the addition of excess unbound RLC, but not excess unbound essential light chain, competes with liposome binding. Consistent with the in vitro data, we show that endogenous and expressed NM2A associates with the plasma membrane of HeLa cells and fibrosarcoma cells independently of F-actin.

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport: A Potential Effector Pathway For Luminal Calcium

Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery.

Human stefin B role in cell's response to misfolded proteins and autophagy.

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

Interactomics of Qa-SNARE in Arabidopsis thaliana

Membrane trafficking in plants is involved in cellular development and the adaptation to various environmental changes. SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins mediate the fusion between vesicles and organelles to facilitate transport cargo proteins in cells. To characterize further the SNARE protein networks in cells, we carried out interactome analysis of SNARE proteins using 12 transgenic Arabidopsis thaliana plants expressing green fluorescent protein (GFP)-tagged Qa-SNAREs (SYP111, SYP121, SYP122, SYP123, SYP132, SYP21, SYP22, SYP31, SYP32, SYP41, SYP42 and SYP43). Microsomal fractions were prepared from each transgenic root, and subjected to immunoprecipitation (IP) using micromagnetic beads coupled to anti-GFP antibodies. To identify Qa-SNARE-interacting proteins, all immunoprecipitated products were then subjected to mass spectrometric (IP-MS) analysis. The IP-MS data revealed not only known interactions of SNARE proteins, but also unknown interactions. The IP-MS results were next categorized by gene ontology analysis. The data revealed that categories of cellular component organization, the cytoskeleton and endosome were enriched in the SYP2, SYP3 and SYP4 groups. In contrast, transporter activity was classified specifically in the SYP132 group. We also identified a novel interaction between SYP22 and VAMP711, which was validated using co-localization analysis with confocal microscopy and IP. Additional novel SNARE-interacting proteins play roles in vesicle transport and lignin biosynthesis, and were identified as membrane microdomain-related proteins. We propose that Qa-SNARE interactomics is useful for understanding SNARE interactions across the whole cell.

The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

Molecular characterization and functional significance of the Vti family of SNARE proteins in tick salivary glands

Exocytosis involves membrane fusion between secretory vesicles and the plasma membrane. The Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) interact to fuse vesicles with the membrane and trigger the release of their sialosecretome out of the tick salivary gland cells. In this study, we examined the functional significance of the Vti family of SNARE proteins of blood-feeding Amblyomma maculatum and Amblyomma americanum. Vti1A and Vti1B have been implicated in multiple functional roles in vesicle transport. QRT-PCR studies demonstrated that the highest transcriptional expression of vti1a and vti1b genes occurs in unfed salivary glands, suggesting that elevated secretory vesicle formation occurs prior to feeding but continues at low rates after blood feeding commences. Vti1A and Vti1B localize to the secretory vesicles in unfed tick salivary glands in immunofluorescence microscopy studies. Knockdown of vti1a and vti1b by RNA interference resulted in a significant decrease in the engorged tick weight compared to the control during prolonged blood-feeding on the host. RNA interference of vti1a or vti1b impaired oviposition and none of the ticks produced eggs masses. Surprisingly, the double knockdown did not produce a strong phenotype and ticks fed normally on the host and produced egg masses, suggesting a compensatory mechanism exists within the secretory system which may have been activated in the double knockdown. These results suggest an important functional role of the Vti family of SNARE proteins in tick blood feeding and ultimately oviposition. Understanding the basic functions of the Vti family of SNARE proteins in salivary glands may lead to better ways to prevent tick attachment and transmission of tick-borne diseases.