Vesicular Transport Machinery Research Articles

ARF1 compartments direct cargo flow via maturation into recycling endosomes.

Cellular membrane homoeostasis is maintained via a tightly regulated membrane and cargo flow between organelles of the endocytic and secretory pathways. Adaptor protein complexes (APs), which are recruited to membranes by the small GTPase ARF1, facilitate cargo selection and incorporation into trafficking intermediates. According to the classical model, small vesicles would facilitate bi-directional long-range transport between the Golgi, endosomes and plasma membrane. Here we revisit the intracellular organization of the vesicular transport machinery using a combination of CRISPR-Cas9 gene editing, live-cell high temporal (fast confocal) or spatial (stimulated emission depletion) microscopy as well as correlative light and electron microscopy. We characterize tubulo-vesicular ARF1 compartments that harbour clathrin and different APs. Our findings reveal two functionally different classes of ARF1 compartments, each decorated by a different combination of APs. Perinuclear ARF1 compartments facilitate Golgi export of secretory cargo, while peripheral ARF1 compartments are involved in endocytic recycling downstream of early endosomes. Contrary to the classical model of long-range vesicle shuttling, we observe that ARF1 compartments shed ARF1 and mature into recycling endosomes. This maturation process is impaired in the absence of AP-1 and results in trafficking defects. Collectively, these data highlight a crucial role for ARF1 compartments in post-Golgi sorting.

WSSV early protein WSSV004 enhances viral replication by suppressing LDH activity

White spot syndrome virus (WSSV) is known to upregulate glycolysis to supply biomolecules and energy for the virus's replication. At the viral genome replication stage, lactate dehydrogenase (LDH), a glycolytic enzyme, shows increased activity without any increase in expression. In the present study, yeast 2-hybrid screening was used to identify WSSV proteins that interacted with LvLDH isoform 1 and 2, and these included the WSSV early protein WSSV004. The interaction between WSSV004 and LvLDH1/2 was confirmed by co-immunoprecipitation. Immunofluorescence showed that WSSV004 co-localized with LvLDH1/2 in the cytoplasm. dsRNA silencing experiments showed that WSSV004 was crucial for WSSV replication. However, although WSSV004 silencing led to the suppression of total LvLDH gene expression during the viral late stage, there was nevertheless a significant increase in LvLDH activity at this time. We also used affinity purification-mass spectrometry to identify cellular proteins that interact with WSSV004, and found a total of 108 host proteins and 3 WSSV proteins with which it potentially interacts. Bioinformatics analysis revealed that WSSV004 and its interacting proteins might be responsible for various biological pathways during infection, including vesicular transport machinery and RNA-related functions. Collectively, our study suggests that WSSV004 serves as a multifunctional modulator to facilitate WSSV replication.

Golgi Complex form and Function: A Potential Hub Role Also in Skeletal Muscle Pathologies?

A growing number of disorders has been associated with mutations in the components of the vesicular transport machinery. The early secretory pathway consists of Endoplasmic Reticulum, numerous vesicles, and the Golgi Complex (GC), which work together to modify and package proteins to deliver them to their destination. The GC is a hub organelle, crucial for organization of the other secretory pathway components. As a consequence, GC's form and function are key players in the pathogenesis of several disorders. Skeletal muscle (SKM) damage can be caused by defective protein modifications and traffic, as observed in some Limb girdle muscular dystrophies. Interestingly, in turn, muscle damage in Duchenne dystrophic SKM cells also includes the alteration of GC morphology. Based on the correlation between GC's form and function described in non-muscle diseases, we suggest a key role for this hub organelle also in the onset and progression of some SKM disorders. An altered GC could affect the secretory pathway via primary (e.g., mutation of a glycosylation enzyme), or secondary mechanisms (e.g., GC mis-localization in Duchenne muscles), which converge in SKM cell failure. This evidence induces considering the secretory pathway as a potential therapeutic target in the treatment of muscular dystrophies.

Probing the Peculiarity of EhRabX10, a pseudoRab GTPase, from the Enteric Parasite Entamoeba histolytica through In Silico Modeling and Docking Studies.

Entamoeba histolytica (Eh) is a pathogenic eukaryote that often resides silently in humans under asymptomatic stages. Upon indeterminate stimulus, it develops into fulminant amoebiasis that causes severe hepatic abscesses with 50% mortality. This neglected tropical pathogen relies massively on membrane modulation to flourish and cause disease; these modulations range from the phagocytic mode for food acquisition to a complex trogocytosis mechanism for tissue invasion. Rab GTPases form the largest branch of the Ras-like small GTPases, with a diverse set of roles across the eukaryotic kingdom. Rab GTPases are vital for the orchestration of membrane transport and the secretory pathway responsible for transporting the pathogenic effectors, such as cysteine proteases (EhCPs) which help in tissue invasion. Rab GTPases thus play a crucial role in executing the cytolytic effect of E. histolytica. First, they interact with Gal/Nac lectins required for adhering to the host cells, and then, they assist in the secretion of EhCPs. Additionally, amoebic Rab GTPases are vital for encystation because substantial vesicular trafficking is required to create dormant amoebic cysts. These cysts are the infective agent and help to spread the disease. The absence of a “bonafide” vesicular transport machinery in Eh and the existence of a diverse repertoire of amoebic Rab GTPases (EhRab) hint at their contribution in supporting this atypical machinery. Here, we provide insights into a pseudoRab GTPase, EhRabX10, by performing physicochemical analysis, predictive 3D structure modeling, protein-protein interaction studies, and in silico molecular docking. Our group is the first one to classify EhRabX10 as a pseudoRab GTPase with four nonconserved G-motifs. It possesses the basic fold of the P-loop containing nucleotide hydrolases. Through this in silico study, we provide an introduction to the characterization of the atypical EhRabX10 and set the stage for future explorations into the mechanisms of nucleotide recognition, binding, and hydrolysis employed by the pseudoEhRab GTPase family.

Herpes Simplex Virus: The Hostile Guest That Takes Over Your Home.

Alpha (α)-herpesviruses (HSV-1 and HSV-2), like other viruses, are obligate intracellular parasites. They hijack the cellular machinery to survive and replicate through evading the defensive responses by the host. The viral genome of herpes simplex viruses (HSVs) contains viral genes, the products of which are destined to exploit the host apparatus for their own existence. Cellular modulations begin from the entry point itself. The two main gateways that the virus has to penetrate are the cell membrane and the nuclear membrane. Changes in the cell membrane are triggered when the glycoproteins of HSV interact with the surface receptors of the host cell, and from here, the components of the cytoskeleton take over. The rearrangement in the cytoskeleton components help the virus to enter as well as transport to the nucleus and back to the cell membrane to spread out to the other cells. The entire carriage process is also mediated by the motor proteins of the kinesin and dynein superfamily and is directed by the viral tegument proteins. Also, the virus captures the cell’s most efficient cargo carrying system, the endoplasmic reticulum (ER)–Golgi vesicular transport machinery for egress to the cell membrane. For these reasons, the host cell has its own checkpoints where the normal functions are halted once a danger is sensed. However, a cell may be prepared for the adversities from an invading virus, and it is simply commendable that the virus has the antidote to these cellular strategies as well. The HSV viral proteins are capable of limiting the use of the transcriptional and translational tools for the cell itself, so that its own transcription and translation pathways remain unhindered. HSV prefers to constrain any self-destruction process of the cell—be it autophagy in the lysosome or apoptosis by the mitochondria, so that it can continue to parasitize the cell for its own survival. This review gives a detailed account of the significance of compartmentalization during HSV pathogenesis. It also highlights the undiscovered areas in the HSV cell biology research which demand attention for devising improved therapeutics against the infection.

Vesicular Transport Machinery in Brain Endothelial Cells: What We Know and What We Do not.

The vesicular transport machinery regulates numerous essential functions in cells such as cell polarity, signaling pathways, and the transport of receptors and their cargoes. From a pharmaceutical perspective, vesicular transport offers avenues to facilitate the uptake of therapeutic agents into cells and across cellular barriers. In order to improve receptor-mediated transcytosis of biologics across the blood-brain barrier and into the diseased brain, a detailed understanding of intracellular transport mechanisms is essential. The vesicular transport machinery is a highly complex network and involves an array of protein complexes, cytosolic adaptor proteins, and the subcellular structures of the endo-lysosomal system. The endo-lysosomal system includes several types of vesicular entities such as early, late, and recycling endosomes, exosomes, ectosomes, retromer-coated vesicles, lysosomes, trans-endothelial channels, and tubules. While extensive research has been done on the trafficking system in many cell types, little is known about vesicular trafficking in brain endothelial cells. Consequently, assumptions on the transport system in endothelial cells are based on findings in polarised epithelial cells, although recent studies have highlighted differences in the endothelial system. This review highlights aspects of the vesicular trafficking machinery in brain endothelial cells, including recent findings, limitations, and opportunities for further studies.

TMED2 is Required in both the Chorion and Placenta for Placental Labyrinth Layer Development

The transmembrane emp24 domain/p24 (TMED) family are essential components of the vesicular transport machinery. TMED family members serve as cargo receptors important for selection and packaging of endoplasmic reticulum (ER) luminal proteins into coatomer (COP) II coated vesicles for anterograde transport to the Golgi. Until recently the role of TMED proteins in development and disease remained a mystery. We postulated that members of the TMED family regulate transport of cargo proteins essential for normal development and homeostasis. Tmed2, the sole member of the vertebrate Tmed β‐subfamily, shows tissue and temporal specific patterns of expression during embryogenesis, but is ubiquitously expressed in all adult organs. We previously showed that mouse embryos homozygous mutant for a single point mutation, the 99J mutation, in the signal sequence of Tmed2 fail to form a functional placenta, and that heterozygous mice carrying this mutation developed non‐alcoholic liver disease. Using explants of chorion and allantois, the precursors of the developing placenta, we identified several secreted and membrane‐bound proteins that were abnormally expressed in Tmed2 mutant tissues, indicating a role for TMED2 in transport of secreted and membrane bound cargoes. In addition, explants between wild type chorion and Tmed2 homozygous mutant allantois, and vice‐versa, revealed both cell‐non‐autonomous and cell‐autonomous requirements for TMED2 during placental development. Our studies indicate that TMED2 is required for organogenesis of the placenta, which, is essential for survival of the growing fetus.Support or Funding InformationNatural Sciences and Engineering Research CouncilThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Non-alcoholic fatty liver disease in mice with heterozygous mutation in TMED2.

The transmembrane emp24 domain/p24 (TMED) family are essential components of the vesicular transport machinery. Members of the TMED family serve as cargo receptors implicated in selection and packaging of endoplasmic reticulum (ER) luminal proteins into coatomer (COP) II coated vesicles for anterograde transport to the Golgi. Deletion or mutations of Tmed genes in yeast and Drosophila results in ER-stress and activation of the unfolded protein response (UPR). The UPR leads to expression of genes and proteins important for expanding the folding capacity of the ER, degrading misfolded proteins, and reducing the load of new proteins entering the ER. The UPR is activated in non-alcoholic fatty liver disease (NAFLD) in human and mouse and may contribute to the development and the progression of NAFLD. Tmed2, the sole member of the vertebrate Tmed β subfamily, exhibits tissue and temporal specific patterns of expression in embryos and developing placenta but is ubiquitously expressed in all adult organs. We previously identified a single point mutation, the 99J mutation, in the signal sequence of Tmed2 in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Histological and molecular analysis of livers from heterozygous mice carrying the 99J mutation, Tmed299J/+, revealed a requirement for TMED2 in liver health. We show that Tmed299J/+ mice had decreased levels of TMED2 and TMED10, dilated endoplasmic reticulum membrane, and increased phosphorylation of eIF2α, indicating ER-stress and activation of the UPR. Increased expression of Srebp1a and 2 at the newborn stage and increased incidence of NAFLD were also found in Tmed299J/+ mice. Our data establishes Tmed299J/+ mice as a novel mouse model for NAFLD and supports a role for TMED2 in liver health.

A microRNA negative feedback loop downregulates vesicle transport and inhibits fear memory.

The SNARE-mediated vesicular transport pathway plays major roles in synaptic remodeling associated with formation of long-term memories, but the mechanisms that regulate this pathway during memory acquisition are not fully understood. Here we identify miRNAs that are up-regulated in the rodent hippocampus upon contextual fear-conditioning and identify the vesicular transport and synaptogenesis pathways as the major targets of the fear-induced miRNAs. We demonstrate that miR-153, a member of this group, inhibits the expression of key components of the vesicular transport machinery, and down-regulates Glutamate receptor A1 trafficking and neurotransmitter release. MiR-153 expression is specifically induced during LTP induction in hippocampal slices and its knockdown in the hippocampus of adult mice results in enhanced fear memory. Our results suggest that miR-153, and possibly other fear-induced miRNAs, act as components of a negative feedback loop that blocks neuronal hyperactivity at least partly through the inhibition of the vesicular transport pathway.

Prion Aggregates Are Recruited to the Insoluble Protein Deposit (IPOD) via Myosin 2-Based Vesicular Transport

Aggregation of amyloidogenic proteins is associated with several neurodegenerative diseases. Sequestration of misfolded and aggregated proteins into specialized deposition sites may reduce their potentially detrimental properties. Yeast exhibits a distinct deposition site for amyloid aggregates termed “Insoluble PrOtein Deposit (IPOD)”, but nothing is known about the mechanism of substrate recruitment to this site. The IPOD is located directly adjacent to the Phagophore Assembly Site (PAS) where the cell initiates autophagy and the Cytoplasm-to-Vacuole Targeting (CVT) pathway destined for delivery of precursor peptidases to the vacuole. Recruitment of CVT substrates to the PAS was proposed to occur via vesicular transport on Atg9 vesicles and requires an intact actin cytoskeleton and “SNAP (Soluble NSF Attachment Protein) Receptor Proteins (SNARE)” protein function. It is, however, unknown how this vesicular transport machinery is linked to the actin cytoskeleton. We demonstrate that recruitment of model amyloid PrD-GFP and the CVT substrate precursor-aminopeptidase 1 (preApe1) to the IPOD or PAS, respectively, is disturbed after genetic impairment of Myo2-based actin cable transport and SNARE protein function. Rather than accumulating at the respective deposition sites, both substrates reversibly accumulated often together in the same punctate structures. Components of the CVT vesicular transport machinery including Atg8 and Atg9 as well as Myo2 partially co-localized with the joint accumulations. Thus we propose a model where vesicles, loaded with preApe1 or PrD-GFP, are recruited to tropomyosin coated actin cables via the Myo2 motor protein for delivery to the PAS and IPOD, respectively. We discuss that deposition at the IPOD is not an integrated mandatory part of the degradation pathway for amyloid aggregates, but more likely stores excess aggregates until downstream degradation pathways have the capacity to turn them over after liberation by the Hsp104 disaggregation machinery.

Role of Intermediate Filaments in Vesicular Traffic.

Intermediate filaments are an important component of the cellular cytoskeleton. The first established role attributed to intermediate filaments was the mechanical support to cells. However, it is now clear that intermediate filaments have many different roles affecting a variety of other biological functions, such as the organization of microtubules and microfilaments, the regulation of nuclear structure and activity, the control of cell cycle and the regulation of signal transduction pathways. Furthermore, a number of intermediate filament proteins have been involved in the acquisition of tumorigenic properties. Over the last years, a strong involvement of intermediate filament proteins in the regulation of several aspects of intracellular trafficking has strongly emerged. Here, we review the functions of intermediate filaments proteins focusing mainly on the recent knowledge gained from the discovery that intermediate filaments associate with key proteins of the vesicular membrane transport machinery. In particular, we analyze the current understanding of the contribution of intermediate filaments to the endocytic pathway.

The ESCRT complex: from endosomal transport to the development of multicellular organisms

Since its discovery more than 50 years ago, the endo-lysosomal system has emerged as a central integrator of different cellular activities. This vesicular trafficking apparatus governs processes as diverse as the transduction of stimuli by growth factor receptors, the recycling and secretion of signaling molecules and the regulation of cellular homeostasis through autophagy. Accordingly, dysfunctions of the vesicular transport machinery have been linked to a growing number of pathologies. In this review we take the "Endosomal Sorting Complex Required for Transport" (ESCRT) as an example to illustrate the multiple functions of an evolutionarily conserved endosomal transport machinery. We describe the major concepts that have emerged from the study of this machinery at the level of the development and the physiology of multi-cellular organisms. In particular, we highlight the essential contributions of ESCRT proteins on the regulation of three biological processes: the endocytic regulation of cell signaling, autophagy and its role in neuronal morphogenesis and finally the biogenesis and function of extracellular vesicles.

Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis

Cilia formation is a multi-step process that starts with the docking of a vesicle at the distal part of the mother centriole. This step marks the conversion of the mother centriole into the basal body, from which axonemal microtubules extend to form the ciliary compartment. How vesicles are stably attached to the mother centriole to initiate ciliary membrane biogenesis is unknown. Here, we investigate the molecular role of the mother centriolar component Cep164 in ciliogenesis. We show that Cep164 was indispensable for the docking of vesicles at the mother centriole. Using biochemical and functional assays, we identified the components of the vesicular transport machinery, the GEF Rabin8 and the GTPase Rab8, as interacting partners of Cep164. We propose that Cep164 is targeted to the apical domain of the mother centriole to provide the molecular link between the mother centriole and the membrane biogenesis machinery that initiates cilia formation.

Virus-producing cells determine the host protein profiles of HIV-1 virion cores

BackgroundUpon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized.ResultsWe examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells.ConclusionsProfiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN.

The expression of a naturally occurring, truncated allele of an α-SNAP gene suppresses plant parasitic nematode infection

Transcriptional mapping experiments of the major soybean cyst nematode resistance locus, rhg1, identified expression of the vesicular transport machinery component, α soluble NSF attachment protein (α-SNAP), occurring during defense. Sequencing the α-SNAP coding regions from the resistant genotypes G. max ([Peking/PI 548402]) and G. max ([PI 437654]) revealed they are identical, but differ from the susceptible G. max ([Williams 82/PI 518671]) by the presence of several single nucleotide polymorphisms. Using G. max ([Williams 82/PI 518671]) as a reference, a G→T(2,822) transversion in the genomic DNA sequence at a functional splice site of the α-SNAP([Peking/PI 548402]) allele produced an additional 17 nucleotides of mRNA sequence that contains an in-frame stop codon caused by a downstream G→A(2,832) transition. The G. max ([Peking/PI 548402]) genotype has cell wall appositions (CWAs), structures identified as forming as part of a defense response by the activity of the vesicular transport machinery. In contrast, the 17 nt α-SNAP([Peking/PI 548402]) mRNA motif is not found in G. max ([PI 88788]) that exhibits defense to H. glycines, but lack CWAs. The α-SNAP([PI 88788]) promoter contains sequence elements that are nearly identical to the α-SNAP([Peking/PI 548402]) allele, but differs from the G. max ([Williams 82/PI 518671]) ortholog. Overexpressing the α-SNAP([Peking/PI 548402]) allele in the susceptible G. max ([Williams 82/PI 518671]) genotype suppressed H. glycines infection. The experiments indicate a role for the vesicular transport machinery during infection of soybean by the soybean cyst nematode. However, increased GmEREBP1, PR1, PR2, PR5 gene activity but suppressed PR3 expression accompanied the overexpression of the α-SNAP([Peking/PI 548402]) allele prior to infection.

Rab7 is a critical mediator in vesicular transport of tyrosinase‐related protein 1 in melanocytes

How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase-related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild-type (Rab7WT), constitutively active (Rab7Q67L) or dominant-negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio-distribution of melanosomal proteins between Rab7WT-expressing cells and mutant Rab7-expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.

Expression of vesicular transport genes in avisicular cells of microsporidia Paranosema (Antonospora) locustae

The long-term adaptation of microsporidia, a large group of fungi-related protozoa, to the intracellular lifestyle has resulted in a drastic minimization of the parasite cell. An ultrastructural analysis shows that the Golgi complex of microsporidia Paranosema (Antonospora) grylli and P. locustae appears as branching networks of thin tubules. These tubular networks are connected with the endoplasmic reticulum, plasma membrane, and forming polar tube, but have no vesicles. Vesicles were not found, even with the use of ultrafast cryofixation and membrane fusion/uncoating inhibition techniques. However, it was found that microsporidia genome has a number of genes involved in vesicular transport. In this study we used RT-PCR to analyze the content of mRNA transcripts encoding β and β′-subunits of COPI coatomer complex, Sec13 and Sec31 subunits of COPI, SNARE-protein synaptobrevin and syntaxin-like member of SFT family in P. locustae at intracellular stages of development. The expression level of these genes was comparable to that of the gene that encodes alternative oxidase, an enzyme engaged in the core metabolism of microsporidia. Moreover, the application of polyclonal antibodies raised against the recombinant COPII subunit Sec13 expressed in E. coli revealed the association of protein with membranes and its accumulation in spores and at stages of intracellular development. The presence of components of vesicular transport machinery in avesicular microsporidia cells requires their functional analysis.

Dynasore, a Dynamin Inhibitor, Inhibits Trypanosoma cruzi Entry into Peritoneal Macrophages

Background Trypanosoma cruzi is an intracellular parasite that, like some other intracellular pathogens, targets specific proteins of the host cell vesicular transport machinery, leading to a modulation of host cell processes that results in the generation of unique phagosomes. In mammalian cells, several molecules have been identified that selectively regulate the formation of endocytic transport vesicles and the fusion of such vesicles with appropriate acceptor membranes. Among these, the GTPase dynamin plays an important role in clathrin-mediated endocytosis, and it was recently found that dynamin can participate in a phagocytic process.Methodology/Principal FindingsWe used a compound called dynasore that has the ability to block the GTPase activity of dynamin. Dynasore acts as a potent inhibitor of endocytic pathways by blocking coated vesicle formation within seconds of its addition. Here, we investigated whether dynamin is involved in the entry process of T. cruzi in phagocytic and non-phagocytic cells by using dynasore. In this aim, peritoneal macrophages and LLC-MK2 cells were treated with increasing concentrations of dynasore before interaction with trypomastigotes, amastigotes or epimastigotes. We observed that, in both cell lines, the parasite internalization was drastically diminished (by greater than 90% in LLC-MK2 cells and 70% in peritoneal macrophages) when we used 100 µM dynasore. The T. cruzi adhesion index, however, was unaffected in either cell line. Analyzing these interactions by scanning electron microscopy and comparing peritoneal macrophages to LLC-MK2 cells revealed differences in the stage at which cell entry was blocked. In LLC-MK2 cells, this blockade is observed earlier than it is in peritoneal macrophages. In LLC-MK2 cells, the parasites were only associated with cellular microvilli, whereas in peritoneal macrophages, trypomastigotes were not completely engulfed by a host cell plasma membrane.Conclusions/SignificanceTaken together our results demonstrate that dynamin is an essential molecule necessary for cell invasion and specifically parasitophorous vacuole formation by host cells during interaction with Trypanosoma cruzi.

Production of cecropin A in transgenic rice plants has an impact on host gene expression

Expression of the cecropin A gene in rice confers resistance to the rice blast fungus Magnaporthe oryzae. In this study, a polymerase chain reaction-based suppression subtractive hybridization approach was used to generate a cDNA macroarray from the elite japonica rice (Oryza sativa L.) cultivar 'Senia'. Gene expression studies revealed that the expression of components of the protein secretory and vesicular transport machinery is co-ordinately activated at the pre-invasive stage of infection of rice by the blast fungus. Comparisons of gene expression between wild-type and cecropin A plants revealed the over-expression of genes involved in protection against oxidative stress in transgenic plants in the absence of the pathogen, which correlated well with the tolerance of these plants to oxidative stress. A subcellular fractionation analysis suggested that cecropin A accumulates in the endoplasmic reticulum in cecropin A rice. Moreover, a large number of genes related to the processes of synthesis, folding and stabilization of proteins that enter into the secretory pathway are over-expressed in cecropin A rice, confirming that these plants constitutively express the unfolded protein response. Transgenic expression of cecropin A in rice has an effect on the transcriptional reprogramming that accompanies plant adaptation to fungal infection. Overall, this study provides evidence for transgene-induced changes in gene expression in cecropin A rice under both optimal growth conditions and stress conditions imposed by fungal infection. The data also indicate that resistance to blast in cecropin A rice may be the consequence of a combination of the antifungal activity of cecropin A and cecropin A-mediated over-expression of rice genes.

A Structural Model of the GDP Dissociation Inhibitor Rab Membrane Extraction Mechanism

Rab GDP dissociation inhibitors (GDI)-facilitated extraction of prenylated Rab proteins from membranes plays an important role in vesicular membrane trafficking. The investigated thermodynamic properties of yeast Rab.GDI and Rab.MRS6 complexes demonstrated differences in the Rab binding properties of the closely related Rab GDI and MRS6 proteins, consistent with their functional diversity. The importance of the Rab C terminus and its prenylation for GDI/MRS6 binding was demonstrated using both biochemical and structural data. The presented structures of the apo-form yeast Rab GDI and its two complexes with unprenylated Rab proteins, together with the earlier published structures of the prenylated Ypt1.GDI, provide evidence of allosteric regulation of the GDI lipid binding site opening, which plays a key role in the proposed mechanism of GDI-mediated Rab extraction. We suggest a model for the interaction of GDI with prenylated Rab proteins that incorporates a stepwise increase in affinity as the three different partial interactions are successively formed.