Genetic and functional interaction between Ryh1 and Ypt3: two Rab GTPases that function in S. pombe secretory pathway

We have previously isolated ypt3-i5 mutant and showed that Ypt3 GTPase functions in the fission yeast secretory pathway. Here, the same genetic screen led to the isolation of ryh1-i6, a mutant allele of the ryh1+ gene encoding a homolog of Rab6. The ryh1-i6 mutant showed phenotypes that support its role in retrograde traffic from endosome to the Golgi. Interestingly, ryh1+ gene deletion was synthetically lethal with ypt3-i5 mutation. Consistently, the over-expression of the GDP-conformational mutant, Ryh1T25 N, inhibited the growth of ypt3-i5 mutant but had no effect on that of wild-type cells. Furthermore, the over-expression of the Ryh1T25N mutant inhibited the acid phosphatase glycosylation and exacerbated the cell wall integrity of ypt3-i5 mutant, but had no effect on those of wild-type cells. GFP-Ryh1 and GFP-Ypt3 both localized at the Golgi/endosome, but showed distinct subcellular localizations. The localization of GFP-Ryh1 in ypt3-i5 mutant and that of GFP-Ypt3 in ryh1-i6 mutant were distinct from those in wild-type cells. In addition, Ryh1 as well as Ypt3 were shown to be involved in acid phosphatase secretion. These results suggest that Ryh1 is involved in the secretory pathway and may have a potential overlapping function with Ypt3 in addition to its role in recycling.     

Biallelic loss‐of‐function variants in TBC1D2B cause a neurodevelopmental disorder with seizures and gingival overgrowth

The family of Tre2‐Bub2‐Cdc16 (TBC)‐domain containing GTPase activating proteins (RABGAPs) is not only known as key regulatorof RAB GTPase activity but also has GAP‐independent functions. Rab GTPases are implicated in membrane trafficking pathways, such as vesicular trafficking. We report biallelic loss‐of‐function variants in TBC1D2B, encoding a member of the TBC/RABGAP family with yet unknown function, as the underlying cause of cognitive impairment, seizures, and/or gingival overgrowth in three individuals from unrelated families. TBC1D2B messenger RNA amount was drastically reduced, and the protein was absent in fibroblasts of two patients. In immunofluorescence analysis, ectopically expressed TBC1D2B colocalized with vesicles positive for RAB5, a small GTPase orchestrating early endocytic vesicle trafficking. In two independent TBC1D2B CRISPR/Cas9 knockout HeLa cell lines that serve as cellular model of TBC1D2B deficiency, epidermal growth factor internalization was significantly reduced compared with the parental HeLa cell line suggesting a role of TBC1D2B in early endocytosis. Serum deprivation of TBC1D2B‐deficient HeLa cell lines caused a decrease in cell viability and an increase in apoptosis. Our data reveal that loss of TBC1D2B causes a neurodevelopmental disorder with gingival overgrowth, possibly by deficits in vesicle trafficking and/or cell survival.     

Chemical genetic analysis of FTY720‐ and Ca2+‐sensitive mutants reveals a functional connection between FTY720 and membrane trafficking

FTY720, a sphingosine‐1‐phosphate (S1P) analog, is used as an immune modulator to treat multiple sclerosis. Accumulating evidence has suggested the mode of action of FTY720 independent of an S1P modulator. In fission yeast, FTY720 induces an increase in intracellular Ca²⁺ and ROS levels. We have previously identified 49 genes of which deletion causes FTY720 sensitivity. Here, we characterized the FTY720‐sensitive mutants in terms of their relevance to the Ca²⁺ homeostasis and identified the 16 F TY720‐ and C a²⁺‐s ensitive mutants (fcs mutants). Most of the FTY720‐sensitive mutants showed elevated Ca²⁺ levels and exhibited Ca²⁺ dysregulation by FTY720 treatment. One of the functional categories among the genes whose deletion renders cells susceptible to FTY720 and Ca²⁺ include the Golgi/endosomal membrane trafficking. Notably, FTY720, but not phosphorylated FTY720 incapable of inducing Ca²⁺ increase, inhibited the secretion of acid phosphatase in the wild‐type cells. Importantly, secretory defects of the Golgi/endosomal trafficking mutants, Vps45, or Ryh1 deletion, were further exacerbated by FTY720. Our fcs mutant screen also identified the adenylyl cyclase‐associated protein Cap1 and a Rictor homolog Ste20, whose deletion markedly exacerbated FTY720‐sensitive secretory impairment. Collectively, our data may suggest a synergistic impact of FTY720 combined with secretion perturbation on proliferation and Ca²⁺ homeostasis.     

Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner

Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.     

Chlamydia pneumoniae inclusion membrane protein Cpn0585 interacts with multiple Rab GTPases

Chlamydiae are intracellular bacteria that develop within a membrane-bound vacuole called an inclusion. To ensure that the inclusion is a safe niche for chlamydial replication, chlamydiae exploit a number of host cell processes, including membrane-trafficking pathways. Recently, several Rab GTPases were found to associate with the inclusions of various chlamydial species. Here we report that Cpn0585, a Chlamydia pneumoniae inclusion membrane protein (Inc), interacts with multiple Rab GTPases. The results from yeast two-hybrid experiments revealed that an amino-terminally truncated form of Cpn0585 (Cpn0585_102-651) interacts with Rab1, Rab10, and Rab11 but not with Rab4 or Rab6. Cpn0585-Rab GTPase interactions are direct and GTP dependent as shown in glutathione S-transferase pull-down assays using native and recombinant Cpn0585. In C. pneumoniae-infected HEp-2 cells transfected with enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, the colocalization with Cpn0585 at the inclusion membrane was partial for EGFP-Rab1 and EGFP-Rab10, but extensive for wild-type EGFP-Rab11A and the constitutively active GTPase-deficient EGFP-Rab11AQ70L. Moreover, Cpn0585 colocalized with EGFP-Rab11AQ70L as early as 2 h postinfection. Upon delivery into live C. pneumoniae-infected cells, Cpn0585_628-651-specific antibodies bound to the inclusion membrane, demonstrating that the Rab GTPase-interacting domain of Cpn0585 faces the host cell cytosol. Finally, ectopic expression of Cpn0585_102-651 partially inhibited the development of C. pneumoniae inclusions in EGFP. but not in EGFP-Rab11AQ70L-expressing HEp-2 cells. Collectively, these data suggest that Cpn0585 is involved in the recruitment of Rab GTPases to the inclusion membrane and that interfering with this function may adversely impact the fitness of the C. pneumoniae inclusion for chlamydial replication.     

Rab GTPases as regulators of endocytosis,targets of disease and therapeutic opportunities

Agola JO, Jim PA, Ward HH, BasuRay S, Wandinger‐Ness A. Rab GTPases as regulators of endocytosis, targets of disease and therapeutic opportunities. Rab GTPases are well‐recognized targets in human disease, although are underexplored therapeutically. Elucidation of how mutant or dysregulated Rab GTPases and accessory proteins contribute to organ specific and systemic disease remains an area of intensive study and an essential foundation for effective drug targeting. Mutation of Rab GTPases or associated regulatory proteins causes numerous human genetic diseases. Cancer, neurodegeneration and diabetes represent examples of acquired human diseases resulting from the up‐ or downregulation or aberrant function of Rab GTPases. The broad range of physiologic processes and organ systems affected by altered Rab GTPase activity is based on pivotal roles in responding to cell signaling and metabolic demand through the coordinated regulation of membrane trafficking. The Rab‐regulated processes of cargo sorting, cytoskeletal translocation of vesicles and appropriate fusion with the target membranes control cell metabolism, viability, growth and differentiation. In this review, we focus on Rab GTPase roles in endocytosis to illustrate normal function and the consequences of dysregulation resulting in human disease. Selected examples are designed to illustrate how defects in Rab GTPase cascades alter endocytic trafficking that underlie neurologic, lipid storage, and metabolic bone disorders as well as cancer. Perspectives on potential therapeutic modulation of GTPase activity through small molecule interventions are provided.     

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.     

Characterization of a Rab11-like GTPase,EhRab11, of Entamoeba histolytica

The Entamoeba histolytica Rab11 family of small molecular weight GTPases consists of three members, EhRab11, EhRab11B, and EhRab11C. The functions of these Rabs in Entamoeba have not been determined. Therefore, as an approach to elucidate the role of the Rab11 family of GTPases in Entamoeba, immunofluorescence microscopy was undertaken to define the subcellular localization of one member of this family, EhRab11. Under conditions of growth, EhRab11 displayed a punctate pattern in the cytoplasm of trophozoites. EhRab11 did not colocalize with markers for the Golgi apparatus, endoplasmic reticulum, pinosomes, phagosomes, or compartments formed by receptor-mediated endocytosis, suggesting that this Rab may not play a role in vesicle trafficking between these organelles. Under conditions of iron and serum starvation, EhRab11 was translocated to the periphery of the cell. The altered cellular localization was accompanied by multinucleation of the cells as well as the acquisition of detergent resistance by the cells, features that are characteristic of Entamoeba cysts. The translocation of EhRab11 to the periphery of the cell during iron and serum starvation was specific as the subcellular localizations of two other Rab GTPases, EhRab7 and EhRabA, were not altered under the same conditions. In addition, the formation of multinucleated cells by inhibition of cytokinesis was not sufficient to induce the translocation of EhRab11 to the cell periphery. Taken together, the data suggest that iron and serum starvation may induce encystation in E. histolytica and that EhRab11 may play a role in this process. Moreover, these studies are the first to describe a putative role for a Rab GTPase in encystation in Entamoeba sp.     

Cell-cycle and developmental regulation of TbRAB31 localisation, a GTP-locked Rab protein from Trypanosoma brucei

Rab proteins are small GTPases that control the direction and timing of vesicle fusion during intracellular trafficking between membraneous compartments. Genome sequencing and EST analysis of Trypanosoma brucei indicates that the trypanosome Rab (TbRAB) gene family, and hence complexity of intracellular transport pathways, is intermediate between Saccharomyces cerevisiae and mammals. TbRAB31 is a constitutively expressed T. brucei Rab protein (formerly Trab7p) and is the product of one of two closely linked TbRAB genes, the other being TbRAB2 (TbRab2p, in: Field H, Ali BRS, Sherwin T, Gull K, Croft SL, Field MC. TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells. J Cell Sci 1999; 112:147-156), involved in ER to Golgi transport. TbRAB31 has high homology to members of the Sec4/Ypt1 subfamily of Rab proteins from S. cerevisiae and to Rab13 and Rab11 from higher eukaryotes. Recombinant TbRAB31 binds GTP but, unusually for a Rab protein, has undetectable GTPase activity resulting in a constitutively GTP-bound protein. Antibodies against TbRAB31 recognise a discrete structure located between the kinetoplast and nucleus in interphase procyclic cells; by contrast the structure is morphologically more complex in bloodstream form (BSF) parasites, consisting of at least two foci. TbRAB31 behaviour was also studied during the cell cycle; TbRAB31 always localised to a discrete structure that duplicated very early in mitosis and relocated to daughter cells in a coordinate manner with the basal body and kinetoplast, suggesting the involvement of microtubules. Additional evidence suggests that TbRAB31 localises to the trypanosome Golgi complex. Firstly, the interphase position of TbRAB31 is consistent with a Golgi location. Secondly, the TbRAB31 structure is also recognised by cross-reacting antibodies to mammalian beta-coatomer protein (beta-COP), which localises to the Golgi in mammalian cells. Thirdly, the fluorescent ceramide analogue, BODIPY-TR-ceramide, a reliable marker of the mammalian Golgi apparatus, exhibited overlapping distribution with TbRAB31. The location of BODIPY-TR-ceramide was confirmed at the trypanosome Golgi by histochemistry with diaminobenzidine and electron microscopy.     

Distinctive roles of Rac1 and Rab29 in LRRK2 mediated membrane trafficking and neurite outgrowth

Parkinson's disease (PD) associated leucine-rich repeat kinase 2 (LRRK2) mutants have shown pathogenic effects on a variety of subcellular processes. Two small GTPases Rac1 and Rab29 have been indicated as possible downstream effectors participating in LRRK2 signaling but their detail mechanisms remain unclear. In this study, we have used biochemical and cell biology approaches to address whether two GTPases interact with LRRK2 and hence function differently in LRRK2 mediated pathogenesis. Here we show that Rac1 and Rab29 specifically interact with LRRK2 with higher affinity for Rab29 and with different preference in functional domain binding. Mutant Rab29 but not Rac1 alters the endosome-to-TGN retrograde trafficking of a cargo protein cation-independent mannose-6- phosphate receptor (CI-M6PR) and its stability. On the other hand, overexpressed wild type Rab29 but not Rac1 rescued the altered retrograde membrane trafficking induced by the pathogenic mutant LRRK2G2019S. Furthermore, both Rac1 and Rab29 rescued neurite shortening in differentiated SH-SY5Y cells induced by LRRK2G2019S. Our study strongly suggests that Rac1 and Rab29 are involved in distinct functions as downstream effectors in LRRK2 signaling pathways.     

A unique Rab GTPase, EhRabA, of Entamoeba histolytica, localizes to the leading edge of motile cells

Entamoeba histolytica, an enteric protozoan parasite, infects 10% of the world's population leading to 50 million cases of invasive amoebiasis annually. Parasite vesicle trafficking and motility, which relies on vesicle trafficking to deliver membrane and membrane components to the leading edge, are important for virulence however little is known about the molecular mechanisms regulating these functions. Since Rab GTPases are known modulators of vesicle trafficking we have characterized a Rab GTPase of Entamoeba, EhRabA. Sequence analysis revealed that EhRabA shared limited homology with any known Rab suggesting that it is a novel member of this protein family. Immunofluorescence microscopy using EhRabA-specific antibodies demonstrated that EhRabA did not colocalize with markers for the Golgi apparatus, endoplasmic reticulum, pinosomes, or phagosomes. These data suggest that this Rab may not play a role in vesicle trafficking between these organelles. In quiescent Entamoeba cells, EhRabA localized to vesicles throughout the cytoplasm consistent with a role in vesicle trafficking, however, in motile cells this protein localized to small vesicles in the leading edge. In addition, when E. histolytica trophozoites were exposed to an N-formyl peptide (N-formylmethionylleucylphenylalanine) cell polarization, the formation of membrane extensions, and the translocation of EhRabA to these membrane extensions was observed. Taken together, these results suggest that EhRabA may function in the formation of membrane extensions perhaps by regulating the delivery of membrane and/or cell surface molecules to the plasma membrane.     

PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation

Phosphatidylinositol‐3‐kinase gamma (PI3Kγ) is a leukocyte‐specific lipid kinase with signaling function downstream of G protein‐coupled receptors to regulate cell trafficking, but its role in T cells remains unclear. To investigate the requirement of PI3Kγ kinase activity in T‐cell function, we studied T cells from PI3Kγ kinase‐dead knock‐in (PI3Kγ^KD/KD) mice expressing the kinase‐inactive PI3Kγ protein. We show that CD4⁺ and CD8⁺ T cells from PI3Kγ^KD/KD mice exhibit impaired TCR/CD28‐mediated activation that could not be rescued by exogenous IL‐2. The defects in proliferation and cytokine production were also evident in naïve and memory T cells. Analysis of signaling events in activated PI3Kγ^KD/KD T cells revealed a reduction in phosphorylation of protein kinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycle progression. Furthermore, PI3Kγ^KD/KD CD4⁺ T cells displayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells. PI3Kγ^KD/KD mice also exhibited an impaired response to immunization and a reduced delayed‐type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation, as well as for mounting an efficient T cell‐mediated immune response. The results suggest that PI3Kγ kinase inhibitors could be beneficial in reducing the undesirable immune response in autoimmune diseases.     

Systems biology of lupus: Mapping the impact of genomic and environmental factors on gene expression signatures,cellular signaling,metabolic pathways,hormonal and cytokine imbalance,and selecting targets for treatment

Systemic lupus erythematosus (SLE) is characterized by the dysfunction of T cells, B cells, and dendritic cells, the release of pro-inflammatory nuclear materials from necrotic cells, and the formation of antinuclear antibodies (ANA) and immune complexes of ANA with DNA, RNA, and nuclear proteins. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a key factor in abnormal activation of T and B cells in SLE. In T cells, increased production of nitric oxide and mitochondrial hyperpolarization (MHP) were identified as metabolic checkpoints upstream of mTOR activation. mTOR controls the expression T-cell receptor-associated signaling proteins CD4 and CD3ζ through increased expression of the endosome recycling regulator Rab5 and HRES-1/Rab4 genes, enhances Ca²⁺ fluxing and skews the expression of tyrosine kinases both in T and B cells, and blocks the expression of Foxp3 and the generation of regulatory T cells. MHP, increased activity of mTOR, Rab GTPases, and Syk kinases, and enhanced Ca²⁺ flux have emerged as common T and B cell biomarkers and targets for treatment in SLE.     

Differential cell surface recruitment of the superoxide‐producing NADPH oxidases Nox1, Nox2 and Nox5: The role of the small GTPase Sar1

Transmembrane glycoproteins, synthesized at the endoplasmic reticulum (ER), generally reach the Golgi apparatus in COPII‐coated vesicles en route to the cell surface. Here, we show that the bona fide nonglycoprotein Nox5, a transmembrane superoxide‐producing NADPH oxidase, is transported to the cell surface in a manner resistant to co‐expression of Sar1 (H79G), a GTP‐fixed mutant of the small GTPase Sar1, which blocks COPII vesicle fission from the ER. In contrast, Sar1 (H79G) effectively inhibits ER‐to‐Golgi transport of glycoproteins including the Nox5‐related oxidase Nox2. The trafficking of Nox2, but not that of Nox5, is highly sensitive to over‐expression of syntaxin 5 (Stx5), a t‐SNARE required for COPII ER‐to‐Golgi transport. Thus, Nox2 and Nox5 mainly traffic via the Sar1/Stx5‐dependent and ‐independent pathways, respectively. Both participate in Nox1 trafficking, as Nox1 advances to the cell surface in two differentially N‐glycosylated forms, one complex and one high mannose, in a Sar1/Stx5‐dependent and ‐independent manner, respectively. Nox2 and Nox5 also can use both pathways: a glycosylation‐defective mutant Nox2 is weakly recruited to the plasma membrane in a less Sar1‐dependent manner; N‐glycosylated Nox5 mutants reach the cell surface in part as the complex form Sar1‐dependently, albeit mainly as the high‐mannose form in a Sar1‐independent manner.     

The CNK1 scaffold binds cytohesins and promotes insulin pathway signaling

Protein scaffolds play an important role in signal transduction, regulating the localization of signaling components and mediating key protein interactions. Here, we report that the major binding partners of the Connector Enhancer of KSR 1 (CNK1) scaffold are members of the cytohesin family of Arf guanine nucleotide exchange factors, and that the CNK1/cytohesin interaction is critical for activation of the PI3K/AKT cascade downstream from insulin and insulin-like growth factor 1 (IGF-1) receptors. We identified a domain located in the C-terminal region of CNK1 that interacts constitutively with the coiled-coil domain of the cytohesins, and found that CNK1 facilitates the membrane recruitment of cytohesin-2 following insulin stimulation. Moreover, through protein depletion and rescue experiments, we found that the CNK1/cytohesin interaction promotes signaling from plasma membrane-bound Arf GTPases to the phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) to generate a PIP₂-rich microenvironment that is critical for the membrane recruitment of insulin receptor substrate 1 (IRS1) and signal transmission to the PI3K/AKT cascade. These findings identify CNK1 as a new positive regulator of insulin signaling.     

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.     

Geranylgeranyltransferase Cwg2‐Rho4/Rho5 module is implicated in the Pmk1 MAP kinase‐mediated cell wall integrity pathway in fission yeast

Pmk1, a fission yeast homologue of mammalian ERK MAPK, regulates cell wall integrity, cytokinesis, RNA granule formation and ion homeostasis. Our screen for vic (viable in the presence of immunosuppressant and chloride ion) mutants identified regulators of the Pmk1 MAPK signaling, including Cpp1 and Rho2, based on the genetic interaction between calcineurin and Pmk1 MAPK. Here, we identified the vic2‐1 mutants carrying a mis‐sense mutation in the cwg2⁺ gene encoding a beta subunit of geranylgeranyltransferase I (GGTase I), which participates in the post‐translational C‐terminal modification of several small GTPases, allowing their targeting to the membrane. Analysis of the vic2‐1/cwg2‐v2 mutant strain showed that the localization of Rho1, Rho4, Rho5 and Cdc42, both at the plasma and vacuolar membranes, was impaired in the vic2‐1/cwg2‐v2 mutant cells. In addition, Rho4 and Rho5 deletion cells exhibited the vic phenotype and cell wall integrity defects, shared phenotypes among the components of the Pmk1 MAPK pathway. Consistently, the phosphorylation of Pmk1 MAPK on heat shock was decreased in the cwg2‐v2 mutants, and rho4‐ and rho5‐null cells. Moreover, Rho4 and Rho5 associate with Pck1/Pck2. Possible roles of Cwg2, Rho4 and Rho5 in the Pmk1 signaling will be discussed.     

Development of a Rab9 Transgenic Mouse and Its Ability to Increase the Lifespan of a Murine Model of Niemann-Pick Type C Disease

Niemann-Pick, type C (NP-C) disease is an autosomal recessive neurovisceral storage disorder in which cholesterol and sphingolipids accumulate. There is no specific treatment for this disease, which is characterized by progressive neurological deterioration, sometimes accompanied by hepatosplenomegaly. We and others have shown that overexpression of certain Rab GTPases corrects defective membrane trafficking and reduces lipid storage in cultured NP-C fibroblasts. Here, we tested the possibility that Rab protein overexpression might also have beneficial effects in vivo using a murine model of NP-C. We first generated several lines of transgenic mice that ubiquitously overexpress Rab9 up to ~30-fold more than endogenous levels and found that the transgene expression had no obvious effects on fertility, behavior, or lifespan in normal mice. These transgenic strains were then crossed with NP-C mutant mice to produce NP-C homozygous recessive mice with and without the Rab9 transgene. Life expectancy of the NPC1 homozygous recessive animals was extended up to 22% depending on gender and the transgenic strain that was used. Histological studies and lipid analysis of brain sections indicated that the NP-C mice carrying the Rab9 transgene had dramatically reduced storage of GM₂ and GM₃ gangliosides relative to NP-C animals lacking the transgene. These results demonstrate that Rab9 overexpression has the potential to reduce stored lipids and prolong lifespan in vivo.     

Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish

The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant-negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions.