The Rab6 effector Bicaudal D1 associates with Chlamydia trachomatis inclusions in a biovar-specific manner

Chlamydia species are obligate intracellular bacteria that replicate within a membrane-bound vacuole, the inclusion, which is trafficked to the peri-Golgi region by processes that are dependent on early chlamydial gene expression. Although neither the host nor the chlamydial proteins that regulate the intracellular trafficking have been clearly defined, several enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, including Rab6, are recruited to Chlamydia trachomatis inclusions. To further characterize the association of Rab6 with C. trachomatis inclusions, we examined the intracellular localization of guanine nucleotide-binding mutants of Rab6 and demonstrated that only active GTP-bound and not inactive GDP-bound EGFP-Rab6 mutants were recruited to the inclusion, suggesting that EGFP-Rab6 interacts with the inclusion via a host Rab6 effector or a chlamydial protein that mimics a Rab6 effector. Using EGFP-tagged fusion proteins, we also demonstrated that the Rab6 effector Bicaudal D1 (BICD1) localized to C. trachomatis inclusions in a biovar-specific manner. In addition, we demonstrated that EGFP-Rab6 and its effector EGFP-BICD1 are recruited to the inclusion in a microtubule- and Golgi apparatus-independent but chlamydial gene expression-dependent mechanism. Finally, in contrast to the Rab6-dependent Golgi apparatus localization of endogenous BICD1, EGFP-BICD1 was recruited to the inclusion by a Rab6-independent mechanism. Collectively, these data demonstrate that neither Rab6 nor BICD1 is trafficked to the inclusion via a Golgi apparatus-localized intermediate, suggesting that each protein is trafficked to the C. trachomatis serovar L2 inclusion by a unique, but as-yet-undefined, mechanism.     

Role of Rab GTPases in membrane traffic and cell physiology

Intracellular membrane traffic defines a complex network of pathways that connects many of the membrane-bound organelles of eukaryotic cells. Although each pathway is governed by its own set of factors, they all contain Rab GTPases that serve as master regulators. In this review, we discuss how Rabs can regulate virtually all steps of membrane traffic from the formation of the transport vesicle at the donor membrane to its fusion at the target membrane. Some of the many regulatory functions performed by Rabs include interacting with diverse effector proteins that select cargo, promoting vesicle movement, and verifying the correct site of fusion. We describe cascade mechanisms that may define directionality in traffic and ensure that different Rabs do not overlap in the pathways that they regulate. Throughout this review we highlight how Rab dysfunction leads to a variety of disease states ranging from infectious diseases to cancer.     

Expression profiling of Rab GTPases reveals the involvement of Rab20 and Rab32 in acute brain inflammation in mice

Rab GTPases have emerged as central regulators of vesicle trafficking and are essential for cytokine production during the pathogenesis of neuroinflammation. To characterize the roles of different Rab proteins in brain inflammation, we used quantitative PCR (qPCR) to examine the expression profiles of all members of the Rab family in an experimental model of brain inflammation in mice. We found that Rab20 and Rab32 were substantially up-regulated during the acute phase of inflammation. The increased expression of Rab20 was also confirmed by immunostaining of inflamed brains at different timepoints. The concomitant overexpression of Rabs (Rab20 and Rab32) and early response proinflammatory cytokines (TNF-α and IL-1β) suggested that these Rabs may be important for subsequent inflammatory responses in brain. Furthermore, we found that the expression of certain Rabs was dramatically reduced in cultured primary microglia, which was not observed in the in vivo profiling. In N9, a microglial cell line, however, there was no increase in the expression of Rab20 or Rab32, but Rab3c was significantly overexpressed. These results collectively indicate that Rabs may participate in inflammatory response in microglia during brain inflammation. The differential regulation of individual Rabs in different experimental systems is a caveat for the analysis of Rab functions.     

Both of the Rab5 subfamily small GTPases of Trypanosoma brucei are essential and required for endocytosis

Endocytosis is an essential process in Trypanosoma brucei and all evidence suggests it is exclusively clathrin-mediated. The trypanosome genome encodes two Rab5 proteins, small GTPases that play a role in very early stages of endocytosis. In the mammalian bloodstream stage TbRAB5A localises to compartments containing internalised antibody, variant surface glycoprotein (VSG) and transferrin, whilst TbRAB5B localises to compartments containing the transmembrane protein ISG(100). Dominant-active forms of TbRAB5A stimulate endocytosis in procyclic forms and alter the kinetics of anti-VSG antibody and transferrin turnover in bloodstream stages. Similar mutants of TbRAB5B increase fluid phase uptake in procyclic cells but do not significantly affect endocytosis in bloodstream forms. Here, we use RNA interference to evaluate the relative importance of TbRAB5A and TbRAB5B and show that both GTPases are essential in the bloodstream form. Depletion of either TbRAB5A or TbRAB5B results in morphological abnormalities, including enlargement of the flagellar pocket, consistent with a potent block to endocytosis. Also, RNAi compromises transferrin accumulation in both cases but induces distinct patterns of mislocalisation of endosomal markers. Finally, RNAi of either TbRAB5A or TbRAB5B results in a decrease in levels of clathrin. Taken together, these data indicate that both TbRAB5A and TbRAB5B are required for endocytosis in trypanosomes and demonstrate that there are multiple essential endocytic routes in this organism.     

The clustered Fcgamma receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner

Phosphorylation of clustered Fcgamma receptor II (FcgammaRII) by Src family tyrosine kinases is the earliest event in the receptor signaling cascade. However, the molecular mechanisms for the interaction between FcgammaRII and these kinases are not elucidated. To asses this problem we isolated high molecular weight complexes of cross-linked FcgammaRII from non-ionic detergent lysates of U937 monocytic cells. CD55, a glycosylphosphatidylinositol-anchored protein, a ganglioside GM1 and Lyn, a Src family tyrosine kinase, were also located in these complexes. Gradient centrifugation demonstrated that the complexes containing cross-linked FcgammaRII displayed a low buoyant density. The FcgammaRII present in the complexes underwent tyrosine phosphorylation. Cross-linked FcgammaRII and Lyn occupied common 100-200 nm detergent-resistant membrane fragments, as demonstrated by immunoprecipitation and microscopy studies. Pretreatment of the cells with beta-cyclodextrin, a cholesterol acceptor, depleted membrane cholesterol and released CD55, GM1 and Lyn from the detergent-resistant complexes. In parallel, the association of Lyn with cross-linked FcgammaRII was disrupted and phosphorylation of the receptor inhibited. Reincorporation of cholesterol evoked the relocation of Lyn into the detergent-resistant membrane fraction and restored both Lyn association with cross-linked FcgammaRII and tyrosine phosphorylation of the receptor. Our data demonstrate that cholesterol-enriched membrane rafts can facilitate tyrosine phosphorylation of clustered FcgammaRII by Lyn kinase.     

Early and late endosomal compartments of Entamoeba histolytica are enriched in cysteine proteases, acid phosphatase and several Ras-related Rab GTPases.

Pure populations of early and late endosomes of Entamoeba histolytica were isolated by magnetic fractionation and characterized. It was shown that these vesicles were enriched in acid phosphatase and cysteine protease activities. An important virulence factor, a 27-kDa cysteine protease, was also enriched in early and late endosomes of E. histolytica. These data suggest that E. histolytica hydrolases reside in compartments that are part of or communicate with the endosomal pathway. To begin to identify the role of Rab GTPases in E. histolytica, an oligonucleotide approach was employed to screen an E. histolytica cDNA library for genes encoding Rab-like proteins. cDNAs encoding a Rab11-like protein (EhRab11) and a novel Rab protein (EhRabA) were isolated and characterized. The EhRab11 cDNA predicts a polypeptide of at least 206 amino acids with a molecular mass of at least 23.2 kDa. Phylogenetic analysis and alignment of EhRab11 with other Rab proteins demonstrated that EhRab11 shared significant homology at the amino acid level with Rab11-like proteins from a number of other eukaryotes, suggesting that EhRab11 is a Rab11 homolog for E. histolytica. The EhRabA clone predicts a polypeptide of 219 amino acids with a molecular mass of at least 24.5 kDa. EhRabA shared only limited homology at the amino acid level with other Rab proteins, suggesting that it is a novel member of this family of GTP-binding proteins. Finally, Western blot analysis demonstrated that EhRab11 and a previously described Rab7-like GTPase from E. histolytica was enriched in magnetically purified endosomal compartments of this organism.     

Lafora disease proteins malin and laforin are recruited to aggresomes in response to proteasomal impairment

Lafora disease (LD), an autosomal recessive neurodegenerative disorder, is characterized by the presence of cytoplasmic polyglucosan inclusions known as Lafora bodies in several tissues including the brain. Laforin, a protein phosphatase, and malin, an ubiquitin ligase, are two of the proteins that are known to be defective in LD. Malin interacts with laforin and promotes its polyubiquitination and degradation. Here we show that malin and laforin co-localize in endoplasmic reticulum (ER) and that they form centrosomal aggregates when treated with proteasomal inhibitors in both neuronal and non-neuronal cells. Laforin/malin aggregates co-localize with gamma-tubulin and cause redistribution of alpha-tubulin. These aggregates are also immunoreactive to ubiquitin, ubiquitin-conjugating enzyme, ER chaperone and proteasome subunits, demonstrating their aggresome-like properties. Furthermore, we show that the centrosomal aggregation of laforin and malin is dependent on the functional microtubule network. Laforin and malin form aggresome when expressed together or otherwise, suggesting that the two proteins are recruited to the centrosome independent of each other. Taken together, our results suggest that the centrosomal accumulation of malin, possibly with the help of laforin, may enhance the ubiquitination of its substrates and facilitate their efficient degradation by proteasome. Defects in malin or laforin may thus lead to increased levels of misfolded and/or target proteins, which may eventually affect the physiological processes of the neuron. Thus, defects in protein degradation and clearance are likely to be the primary trigger in the physiopathology of LD.     

Molecular cloning and characterization of human RAB23, a member of the group of Rab GTPases

The RAB small G protein family is composed of approximately 40 members. Many of them are ubiquitous and are expressed and participate in transport processes, such as endocytosis and exocytosis, whereas others are expressed only within a specific cell group carrying out specific functions. In the current study, we present the molecular characterisation and chromosomal location of the human RAB23 gene, a new member of the RAB family. This gene, expressed in retina, is composed of 7 exons spanning 34 kb of genomic DNA and located in the pericentromeric region of chromosome 6 between microsatellite markers D6S257 and D6S1695, within the critical region of RP25. Since proteins belonging to the Rab family have already been related to retinal degeneration we considered RAB23 an interesting candidate for the RP25 locus. However the absence of pathogenic variations after molecular analysis of the coding sequence in the index patients of RP25 linked families would be consistent with the exclusion of RAB23 as responsible for RP25 phenotype.     

Two checkpoint complexes are independently recruited to sites of DNA damage in vivo

The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by "sensing" DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1-GFP or Ddc2-GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2-GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.     

Caenorhabditis elegans Rab escort protein (REP‐1) differently regulates each Rab protein function and localization in a tissue‐dependent manner

Rab proteins play a critical role in intracellular vesicle trafficking and require post‐translational modification by adding lipids at the C‐terminus for proper functions. This modification is preceded by the formation of a trimeric protein complex with the Rab escort protein (REP) and the Rab geranylgeranyltransferase (RabGGTase). However, the genetic hierarchy among these proteins and the tissue‐specificity of each protein function are not yet clearly understood. Here we identified the Caenorhabditis elegans rep‐1 gene and found that a rep‐1 mutant showed a mild defect in synaptic transmission and defecation behaviors. Genetic analyses using the exocytic Rab mutants rab‐3 or rab‐27 suggested that rep‐1 functions only in the RAB‐27 pathway, and not in the RAB‐3 pathway, for synaptic transmission at neuromuscular junctions. However, the disruption of REP‐1 did not cause defecation defects compared to severe defects in either RAB‐27 or RabGGTase disruption, suggesting that REP‐1 is not essential for RAB‐27 signaling in defection. Some Rab proteins did not physically interact with REP‐1, and localization of these Rab proteins was not severely affected by REP‐1 disruption. These findings suggest that REP‐1 functions are required in specific Rab pathways and in specific tissues, and that some Rab proteins are functionally prenylated without REP‐1.     

Oral mucosal progenitor cells are potently immunosuppressive in a dose-independent manner

Oral mucosal lamina propria progenitor cells (OMLP-PCs) are a novel, clonally derived PC population of neural crest origin with the potential to differentiate down both mesenchymal and neuronal cell lineages. In this study we aimed to determine the immunological properties of OMLP-PCs and to establish whether they would be suitable candidates for allogeneic tissue engineering and in the treatment of immune-related diseases. OMLP-PCs demonstrated no inherent immunogenicity with insignificant expression of costimulatory molecules (CD40, CD80, CD86, CD154, and CD178) or human leukocyte antigen (HLA) class II. OMLP-PCs required 7 days of stimulation with interferon-γ (IFN-γ) to induce cell surface expression of HLA II. Mixed lymphocyte cultures and mitogen stimulation demonstrated the potent immunosuppressive capability of OMLP-PCs in a contact-independent manner. Complete inhibition of lymphocyte proliferation was seen at doses as low as 0.001% OMLP-PCs to responder lymphocytes, while annexin V staining confirmed that this immunosuppressive effect was not due to the induction of lymphocyte apoptosis. These data demonstrate, for the first time, that OMLP-PC immunomodulation, unlike that for mesenchymal stem cells, occurs via a dose- and HLA II-independent mechanism by the release of immunosuppressive soluble factors and suggests these cells may have wide ranging potential in future immune-related therapies.     

Resident alveolar macrophages are replaced by recruited monocytes in response to endotoxin-induced lung inflammation

In the acute respiratory distress syndrome, recruitment of peripheral blood monocytes results in expansion of the total pool of resident alveolar macrophages. The fate of resident macrophages, or whether recruited monocytes are selectively eliminated from the alveolar airspace or differentiate into resident alveolar macrophages during the resolving phase of inflammation, has not been determined. Here, we analyzed the kinetics of resident and recruited macrophage turnover within the alveolar airspace of untreated and LPS-challenged mice. Using bone marrow chimeric CD45.2 mice that were generated by lethal irradiation of CD45.2 alloantigen-expressing recipient mice and bone marrow transplantation from CD45.1 alloantigen-expressing donor mice, we employed a flow cytometric approach to distinguish recipient from donor-type macrophages in bronchoalveolar lavage fluids. Our data show that resident alveolar macrophages of untreated chimeric CD45.2 mice are very slowly replaced by constitutively immigrating CD45.1 positive monocytes, resulting in a replacement rate of approximately 40% by 1 yr. In contrast, more than 85% of the resident CD45.2 positive alveolar and lung homogenate macrophages were exchanged by donor CD45.1-expressing macrophages within 2 mo after treatment with Escherichia coli endotoxin (LPS). Importantly, fluorescence-activated cell sorter analysis of increased annexin V binding to both recipient and donor-type macrophages revealed increased apoptotic events to underlie this endotoxin-driven inflammatory macrophage turnover. Collectively, the data show that under baseline conditions the alveolar macrophage turnover exhibits very slow kinetics, whereas acute lung inflammation in response to treatment with LPS triggers a brisk acceleration of recruitment of monocytes that replace the resident alveolar macrophage population.     

Automated postural responses are modified in a functional manner by instruction

The restoration of upright balance after a perturbation relies on highly automated and, to a large extent, stereotyped postural responses. Although these responses occur before voluntary control comes into play, previous research has shown that they can be functionally modulated on the basis of cognitive set (experience, advanced warning, instruction, etc.). It is still unknown, however, how the central nervous system deals with situations in which the postural response is not necessarily helpful in the execution of a task. In the present study, the effects of instruction on automated postural responses in neck, trunk, shoulder, and leg muscles were investigated when people were either instructed to recover balance after being released from an inclined standing posture [balance recovery (BR) trials], or not to recover at all and fall onto a safety mattress in the most comfortable way [fall (F) trials], in both backward and leftward directions. Participants were highly successful in following the instructions, consistently exhibiting stepping responses for balance recovery in BR trials, and suppressing stepping in the F trials. Yet EMG recordings revealed similar postural responses with onset latencies between 70 and 130 ms in both BR and F trials, with slightly delayed responses in F trials. In contrast, very pronounced and early differences were observed between BR and F trials in response amplitudes, which were generally much higher in BR than in F trials, but with clear differentiation between muscles and perturbation directions. These results indicate that a balance perturbation always elicits a postural response, irrespective of the task demands. However, when a specific balance recovery response is not desired after a perturbation, postural responses can be selectively downregulated and integrated into the motor output in a functional and goal-oriented way.