Expression Of Rab7 Research Articles

Nuclear Targeting of an Endosomal E3 Ubiquitin Ligase

Ring finger protein 13 (RNF13) is an E3 ubiquitin ligase embedded in endosome membranes. The protein undergoes constitutive post-translational proteolysis, making its detection difficult unless cells are incubated with a proteasome inhibitor to allow biosynthetic forms to accumulate. When cells were treated with phorbol 12-myristate 13-acetate (PMA), RNF13 avoided proteolysis. A similar stabilization was seen on ionomycin treatment of cells. Drug treatment stabilized both the full-length protein and a membrane-embedded C-terminal fragment generated following ectodomain shedding. Immunofluorescence staining revealed that PMA treatment caused the protein to accumulate in recycling endosomes, where it colocalized with transferrin receptor, and on the inner nuclear membrane, where it colocalized with lamin B. Expression of dominant-negative Rab11 inhibited nuclear localization, suggesting RNF13 was targeted to the inner nuclear membrane through recycling endosomes. New protein synthesis was necessary for this targeting. Nuclear localization was confirmed by immunoelectron microscopy and by purification of the inner nuclear membrane. Stress-induced transport of an endosomal protein to the inner nuclear membrane is a novel mechanism for introduction of regulatory proteins to the DNA environment. RNF13, with its ubiquitin ligase-active RING domain, has the potential to turn over key nuclear proteins in response to signals received at the plasma membrane.

Mycobacterium tuberculosis H37Rv induces ectosome release in human polymorphonuclear neutrophils

Ectocytosis, the cellular process by which ectosomes (Ects) are released, is an important phenomenon by which eukaryotic cells exchange molecular information. Ects released from N-formylmethionyl-leucyl-phenylalanine (fMLP)-activated human polymorphonuclear neutrophils (PMNs) have recently been characterized. Molecules such as CD35 and phosphatidylserine (PS), and enzymes such as myeloperoxidase and elastase were found in these vesicles, suggesting that Ects from PMNs could function as ecto-organelles with anti-microbial activity. Here we show for the first time that human PMNs release ectosomes in response to Mycobacterium tuberculosis H37Rv infection. We found that the release of ectosomes was not associated exclusively with mycobacterial infection since infection with other microorganisms (e.g., Leishmania mexicana, Staphylococcus aureus, and Escherichia coli or activation with phorbol myristate acetate (PMA)) also induced ectocytosis. Ects release started as early as 10min after infection or activation. Expression of CD35, PS, Rab5, Rab7 and gp91(Phox), a subunit of Cyt b555 was demonstrated on the Ects membrane. Based on our observations we conclude that Ects are released from human neutrophils in response to cell activation and that this process is not related to apoptosis.

Loss of Rab25 promotes the development of intestinal neoplasia in mice and is associated with human colorectal adenocarcinomas

Transformation of epithelial cells is associated with loss of cell polarity, which includes alterations in cell morphology as well as changes in the complement of plasma membrane proteins. Rab proteins regulate polarized trafficking to the cell membrane and therefore represent potential regulators of this neoplastic transition. Here we have demonstrated a tumor suppressor function for Rab25 in intestinal neoplasia in both mice and humans. Human colorectal adenocarcinomas exhibited reductions in Rab25 expression independent of stage, with lower Rab25 expression levels correlating with substantially shorter patient survival. In wild-type mice, Rab25 was strongly expressed in cells luminal to the proliferating cells of intestinal crypts. While Rab25-deficient mice did not exhibit gross pathology, ApcMin/+ mice crossed onto a Rab25-deficient background showed a 4-fold increase in intestinal polyps and a 2-fold increase in colonic tumors compared with parental ApcMin/+ mice. Rab25-deficient mice had decreased beta1 integrin staining in the lateral membranes of villus cells, and this pattern was accentuated in Rab25-deficient mice crossed onto the ApcMin/+ background. Additionally, Smad3+/- mice crossed onto a Rab25-deficient background demonstrated a marked increase in colonic tumor formation. Taken together, these results suggest that Rab25 may function as a tumor suppressor in intestinal epithelial cells through regulation of protein trafficking to the cell surface.

The Connecdenn DENN Domain: A GEF for Rab35 Mediating Cargo-Specific Exit from Early Endosomes

The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn/DENND1A DENN domain functions as a guanine nucleotide exchange factor (GEF) for Rab35 to regulate endosomal membrane trafficking. Loss of Rab35 activity causes an enlargement of early endosomes and inhibits MHC class I recycling. Moreover, it prevents early endosomal recruitment of EHD1, a common component of tubules involved in endosomal cargo recycling. Our data reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.

Endocytic Rab proteins are required for hepatitis C virus replication complex formation

During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 and 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 and Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation.

Identification of Rab5 as a Gene Involved in Chronic Myeloid Leukemia (CML) Progression.

Abstract 3470Poster Board III-358The role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, however, the mechanisms leading to CML progression remain poorly understood.By using our model of transgenic Drosophila Melanogaster (Dm) for human Bcr-Abl driven CML we have identified Rab5 as a gene involved in the regulation of CML progression. The Rab5 is a member of gene family small GTPases which are involved in the regulation of vesicular transport. Lately several important reports have linked some members of the Rab family to invesivness and migration of cancer cells. Rab5 is associate with alpha-integrin subunits and modulates their endosomal traffic and subcellular localization. We have observed that a loss of function of Rab5 gene have induced a worsening of the CML phenotype generated by hBcr-Abl expression. In contrast, Rab gain of function rescued Bcr-Abl phenotype. The aim of the study was to evaluate the expression of Rab5 in CML cells to better understand if a potential correlation with progression, which has been observed in the model, could be confirmed in patients. MethodsRab5 gene expression was measured by Real Time PCR in 90 samples from 80 CML patients (32 PB and 58 BM). Among those, 53 are collected at diagnosis (19 of 53 patients have been enrolled in TOPS study). In addition, 9 samples from in CP patients have been collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC. In 14 patients, genes expression was analyzed during remission as, well. In parallel, 21 healthy donors (10 PB and 11 BM) have been evaluated. Rab5 protein expression was investigated by Western Blot and Immunofluorescence. We have also utilized K562 transfected with Rab5 plasmid, which we have generated to gain insight about the effects of Rab5 on cell proliferation and apoptosis. ResultsRab5 transfection and overexpression in K562 significantly reduced proliferation and affected apoptosis. We found that in CML patients Rab5 expression levels were significantly decreased in either BM or PB (p<0.001 and p<0.0001) as compared to healthy subjects. Furthermore, in blast crisis samples we have found Rab5 transcripts levels to be further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Our preliminary analysis of samples from TOPS trial have shown a trend that Rab5 levels are lower among those patients achieving MMR by 12 months, when compared to the group of patients non achieving MMR on 400 mg, but that difference was not statistically significant (p=0.2). Among those randomized to receive imatinib 800 mg the difference was statistically significant with a median value among those achieving MMR of 1.27 vs 2.14 in the group without MMR (p=0.04). The protein levels have been analyzed by Western Blot and immunofluorescence and allow us to show detectable levels of Rab5 in samples collected at remission, but undetectable levels in course of active CML disease. Although preliminary, our results show a significant decrease of Rab5 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Rab5 in slowing down or suppressing a progression. Surprisingly, among CP CML patients the responders to TKI therapy have been detected to express a lower level of Rab5 than non responders. We are conducting further studies to better explain these data, which we find intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network DisclosuresNo relevant conflicts of interest to declare.

Abstract 5554: FoxO1 Stimulates Autophagic Flux Through Upregulation of the Small GTP Binding Protein Rab7

We have shown previously that FoxO1, a member of the FoxO transcription factor family, is upregulated by nutrient starvation and cardiac unloading. FoxO1 in turn promotes autophagy, thereby mediating survival and regression of hypertrophy in cardiac myocytes. The current investigation addressed the molecular mechanism by which FoxO1 promotes autophagy in cardiac myocytes. In order to evaluate formation of both autophagosomes and autolysosomes, cultured cardiac myocytes were transduced with adenovirus harboring mRFP-GFP-LC3. Adenoviral mediated overexpression of FoxO1 increased accumulation of both GFP-LC3 dots (1.5 fold vs Ad-LacZ) and mRFP-LC3 dots (2 fold vs Ad-LacZ, p&lt;0.01), which was further enhanced in the presence of protease inhibitors(PI) (1.5 fold vs Ad-LacZ+PI, p&lt;0.01). FoxO1 overexpression caused greater increases in mRFP-LC3 dots than GFP-LC3 dots, suggesting that FoxO1 enhances autolysosome formation more strongly than autophagosome formation, thereby stimulating autophagic flux. Rab7 is a small GTP binding protein which plays an important role in mediating maturation of late autophagic vacuoles. mRNA expression of Rab7 is strongly upregulated in cardiac myocytes (4.7 fold, p&lt;0.05) and hearts (2.5 fold) with FoxO1 overexpression. Overexpression of Rab7 was sufficient to stimulate autolysosome formation and autophagic flux in cultured cardiac myocytes, as evidenced by accumulation of LC3-II (2.8 fold, p&lt;0.05) and mRFP-LC3 dots (2.3 fold, p&lt;0.05), and decreased p62 (−40%, p&lt;0.05). Lentivirus mediated knockdown of Rab7 (LV-sh-Rab7) attenuated FoxO1- induced autophagy, as indicated by LC3-II accumulation (Ad-Foxo1WT 1.9, Ad-Foxo1WT + LV-sh-Rab7 0.11 vs Ad-LacZ, p&lt;0.05) and p62 expression levels (Ad-Foxo1WT 0.49, Ad-Foxo1WT + LV-sh-Rab7 0.84 vs Ad-LacZ, p&lt;0.05), suggesting that Rab7 mediates FoxO1-induced autophagy in cardiac myocytes. In summary, FoxO1 stimulates autophagosome/autolysosome formation and autophagic flux in cardiac mycoytes, accompanied by upregulation of Rab7. Rab7 plays an essential role in mediating FoxO1-induced stimulation of autophagy, possibly by stimulating autophagosome -lysosomal fusion in cardiac myocytes.

Ultrastructural characterization of giant endosomes induced by GTPase-deficient Rab5

The small GTPase Rab5 controls the fusogenic properties of early endosomes through GTP-dependent recruitment and activation of effector proteins. Expression of a GTPase-defective mutant, Rab5(Q79L), is known to cause formation of enlarged early endosomes. The ability of Rab5-GTP to recruit multiple effectors raises the question whether the Rab5(Q79L)-induced giant endosomes simply represent enlarged early endosomes or whether they have a more complex phenotype. In this report, we have addressed this issue by generating a HEp2 cell line with inducible expression of Rab5(Q79L) and performing ultrastructural analysis of Rab5(Q79L)-induced endosomes. We find that Rab5(Q79L) not only induces formation of enlarged early endosomes but also causes enlargement of later endocytic profiles. Most strikingly, Rab5(Q79L) causes formation of enlarged multivesicular endosomes with a large number of intraluminal vesicles, and endosomes that contain both early and late endocytic markers are frequently observed. In addition, we observe defects in the sorting of the EGF receptor and the transferrin receptor through this compartment.

C. elegansRab GTPase activating protein TBC-2 promotes cell corpse degradation by regulating the small GTPase RAB-5

During apoptosis, dying cells are quickly internalized by neighboring cells or phagocytes, and are enclosed in phagosomes that undergo a maturation process to generate the phagoslysosome, in which cell corpses are eventually degraded. It is not well understood how apoptotic cell degradation is regulated. Here we report the identification and characterization of the C. elegans tbc-2 gene, which is required for the efficient degradation of cell corpses. tbc-2 encodes a Rab GTPase activating protein (GAP) and its loss of function affects several events of phagosome maturation, including RAB-5 release, phosphatidylinositol 3-phosphate dynamics, phagosomal acidification, RAB-7 recruitment and lysosome incorporation, which leads to many persistent cell corpses at various developmental stages. Intriguingly, the persistent cell corpse phenotype of tbc-2 mutants can be suppressed by reducing gene expression of rab-5, and overexpression of a GTP-locked RAB-5 caused similar defects in phagosome maturation and cell corpse degradation. We propose that TBC-2 functions as a GAP to cycle RAB-5 from an active GTP-bound to an inactive GDP-bound state, which is required for maintaining RAB-5 dynamics on phagosomes and serves as a switch for the progression of phagosome maturation.

A Dominant-Negative Mutant of rab5 Inhibits Infection of Cells by Foot-and-Mouth Disease Virus: Implications for Virus Entry

Foot-and-mouth disease virus (FMDV) can use a number of different integrins (alphavbeta1, alphavbeta3, alphavbeta6, and alphavbeta8) as receptors to initiate infection. Infection mediated by alphavbeta6 is known to occur by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes. On internalization, virus is detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomal compartments. Due to the extreme sensitivity of FMDV to acidic pH, it is thought that EE can provide a pH low enough for infection to occur; however, definitive proof that infection takes place from within these compartments is still lacking. Here we have investigated the intracellular transport steps required for FMDV infection of IBRS-2 cells, which express alphavbeta8 as their FMDV receptor. These experiments confirmed that FMDV infection mediated by alphavbeta8 is also dependent on clathrin-mediate endocytosis and an acidic pH within endosomes. Also, the effect on FMDV infection of dominant-negative (DN) mutants of cellular rab proteins that regulate endosomal traffic was examined. Expression of DN rab5 reduced the number of FMDV-infected cells by 80%, while expression of DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent ( approximately 35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE.

The Expression Analysis of Innate Immune-related Genes in Kuruma Shrimp Penaeus japonicus after DNA Vaccination against Penaeid Rod-shaped DNA Virus

In this study, we have developed a DNA vaccine encoding viral envelope protein VP28 of penaeid rod-shaped DNA virus (PRDV, = WSSV). Protective efficacy of the DNA vaccine against PRDV was confirmed at 7 days post vaccination in kuruma shrimp Penaeus japonicus. The VP28 transcript derived from the vaccine was detected in various tissues at 1, 3 and 7 days post vaccination. Moreover, notable up-regulated expression of Rab7, penaeidin, lysozyme and crustin genes was observed upon DNA vaccination. These results suggest that the DNA vaccine significantly increased the protection and innate immune responses in kuruma shrimp against PRDV.

Syndecan-1 Ectodomain Shedding Is Regulated by the Small GTPase Rab5

The ectodomain shedding of syndecan-1, a major cell surface heparan sulfate proteoglycan, modulates molecular and cellular processes central to the pathogenesis of inflammatory diseases. Syndecan-1 shedding is a highly regulated process in which outside-in signaling accelerates the proteolytic cleavage of syndecan-1 ectodomains at the cell surface. Several extracellular agonists that induce syndecan-1 shedding and metalloproteinases that cleave syndecan-1 ectodomains have been identified, but the intracellular mechanisms that regulate syndecan-1 shedding are largely unknown. Here we examined the role of the syndecan-1 cytoplasmic domain in the regulation of agonist-induced syndecan-1 shedding. Our results showed that the syndecan-1 cytoplasmic domain is essential because mutation of invariant cytoplasmic Tyr residues abrogates ectodomain shedding, but not because it is Tyr phosphorylated upon shedding stimulation. Instead, our data showed that the syndecan-1 cytoplasmic domain binds to Rab5, a small GTPase that regulates intracellular trafficking and signaling events, and this interaction controls the onset of syndecan-1 shedding. Syndecan-1 cytoplasmic domain bound specifically to Rab5 and preferentially to inactive GDP-Rab5 over active GTP-Rab5, and shedding stimulation induced the dissociation of Rab5 from the syndecan-1 cytoplasmic domain. Moreover, the expression of dominant-negative Rab5, unable to exchange GDP for GTP, interfered with the agonist-induced dissociation of Rab5 from the syndecan-1 cytoplasmic domain and significantly inhibited syndecan-1 shedding induced by several distinct agonists. Based on these data, we propose that Rab5 is a critical regulator of syndecan-1 shedding that serves as an on-off molecular switch through its alternation between the GDP-bound and GTP-bound forms.

Caspase 8 Promotes Peripheral Localization and Activation of Rab5

Caspase 8 is a cysteine protease that initiates apoptotic signaling via the extrinsic pathway in a manner dependent upon association with early endosomes. Previously, we identified caspase 8 as an effector of migration, promoting motility in a manner dependent upon phosphorylation on Tyr-380 by Src family kinases and its subsequent association with Src homology 2 domain-containing proteins. Here we demonstrate the regulation of the small GTPase Rab5, which mediates early endosome formation, homotypic fusion, and maturation by caspase 8. Regulation requires the Tyr-380 phosphorylation site but not caspase proteolytic activity. Tyr-380 is essential for interaction with the Src homology 2 domains of p85α, a multifunctional adaptor for phosphatidylinositol 3-kinase, that possesses Rab-GAP activity. Interaction between caspase 8 and p85α promotes Rab5 GTP loading, alters endosomal trafficking, and results in the accumulation of Rab5-positive endosomes at the edge of the cell. Conversely, caspase 8-dependent GTP loading of Rab5 is overcome by increased expression of p85α in a Rab-GAP-dependent manner. Thus, we demonstrate a novel function for caspase 8 as a modulator of p85α Rab-GAP activity and endosomal trafficking.

Developmental expression of Rab11, a small GTP‐binding protein in Drosophila epithelia

Intracellular membrane trafficking regulates a wide variety of developmental processes, including cell and tissue morphogenesis. Here we report developmental expression of Drosophila Rab11, a small GTP-binding protein, required for both endocytic recycling and exocytosis. Rab11 is expressed in the epithelial cell types of diverse lineages at all developmental stages, beginning from the cellular blastoderm in early embryos to adult primordia and adult tissues, like the columnar epithelia lining male ejaculatory bulb. A robust expression of Rab11 is seen both in the amnioserosa and in the lateral epidermis during embryonic dorsal closure, a morphogenetic event that involves spreading and fusion of the contra-lateral sides of epidermis. Rab11 mutant embryos fail to display the characteristic morphological changes in these two epithelial tissues during dorsal closure, providing a strong basis to dissect the role of Rab11 in coordinated epithelial sheet movements. genesis 47:32-39, 2009. (c) 2008 Wiley-Liss, Inc.

Regulation of Histone Deacetylase in Waldenström Macroglobulinemia

Background: Waldenström's Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Histone deacetylase (HDAC) inhibitors represent promising new treatment strategy in B cell malignancies. We therefore investigated the in vitro effect of the novel hydroxamic acid derivative HDAC inhibitor LBH589 in WM.Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTS assay and thymidine uptake assay. Cell signaling and apoptotic pathways were determined by Western Blot and immunofluorescence.Results: LBH589 induced a significant decrease of proliferation and triggered cytotoxicity in all cell lines tested and primary CD19+ WM cells (IC50 of 20–40nM), even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, LBH589 did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells. LBH589 induced both intrinsic and extrinsic apoptotic pathways, with caspase-9, caspase-8, caspase-3, and PARP cleavage in a dose-dependent manner. We also demonstrated significant upregulation of the proapoptotic transcription factor p53 and down-regulation of the anti-apoptotic proteins BclxL, Mcl-1 and c-myc. We then demonstrated that LBH589 induced apoptosis in WM cells in a caspase-independent manner through induction of autophagy, as shown by upregulation of LC3B and Rab7 expression. We further determined the mechanism of action of LBH589 in WM, investigating the effect of LBH589 on histone acetylation and NF-kB pathways. We found that LBH589 induced a dose-dependent increase in histone H3-H4 acetylation; and inhibited both canonical and non-canonical pathways of NF-κB, as shown by western blot and immunofluorescence. In addition, LBH589 augmented rituximab, fludarabine, bortezomib, and perifosine-induced cyotoxicity in WM cells.Conclusion: LBH589 has significant antitumor activity in WM in vitro, providing the framework for clinical trials evaluating LBH589 as a new therapeutic agent in patients with WM.

Rab 23 is expressed in the glomerulus and plays a role in the development of focal segmental glomerulosclerosis

Rab23, a member of the Rab family of small GTPase, has a function in antagonizing sonic hedgehog signal transduction. Both Rab-family and hedgehog-related proteins are involved in sclerosis and fibrosis in certain pathological states, but their roles in focal segmental glomerulosclerosis (FSGS) remain unclear. The FSGS model was established in Balb/c mice by a single injection of adriamycin. Serum, urine and mice kidneys were collected on Days 0, 7, 15 and 20. Western blot analysis was performed to detect the levels of Rab23 in the samples. Immunohistochemistry was used to examine the expressional profiles of Rab23 in kidneys. The expressions of transcripts of Rab23, extracellular matrix (ECM) proteins, and various hedgehog signalling pathway genes in kidneys or mesangial cells were evaluated by real-time RT-PCR. The effect of Rab23 on ECM protein expressions was evaluated by the knockdown or overexpression of Rab23 in mesangial cells. Our results show that elevations of Rab23 were observed in the urine, but not in the serum, of the FSGS mice. Rab23 and hedgehog signalling pathway genes were constitutively expressed in normal kidneys and were significantly up-regulated in the kidneys of FSGS mice. The basal expression of Rab23 was identified in glomeruli, and mesangial cells displayed obvious elevation of Rab23 in the FSGS state. The knockdown or overexpression of Rab23 affected the collagen expression in cultured mesangial cells. An autocrine loop of hedgehog signalling could be activated in mesangial cells in the FSGS state, and Rab23 may be elevated to suppress hedgehog signalling and/or influence collagen synthesis. Importantly, Rab23 could serve as a biomarker that indicates the severity of FSGS.

Membrane Ruffles Capture C3bi-opsonized Particles in Activated Macrophages

A widespread belief in phagocyte biology is that FcgammaR-mediated phagocytosis utilizes membrane pseudopods, whereas Mac-1-mediated phagocytosis does not involve elaborate plasma membrane extensions. Here we report that dynamic membrane ruffles in activated macrophages promote binding of C3bi-opsonized particles. We identify these ruffles as components of the macropinocytosis machinery in both PMA- and LPS-stimulated macrophages. C3bi-particle capture is facilitated by enrichment of high-affinity Mac-1 and the integrin-regulating protein talin in membrane ruffles. Membrane ruffle formation and C3bi-particle binding are cytoskeleton dependent events, having a strong requirement for F-actin and microtubules (MTs). MT disruption blunts ruffle formation and PMA- and LPS-induced up-regulation of surface Mac-1 expression. Furthermore, the MT motor, kinesin participates in ruffle formation implicating a requirement for intracellular membrane delivery to active membrane regions during Mac-1-mediated phagocytosis. We observed colocalization of Rab11-positive vesicles with CLIP-170, a MT plus-end binding protein, at sites of particle adherence using TIRF imaging. Rab11 has been implicated in recycling endosome dynamics and mutant Rab11 expression inhibits both membrane ruffle formation and C3bi-sRBC adherence to macrophages. Collectively these findings represent a novel membrane ruffle "capture" mechanism for C3bi-particle binding during Mac-1-mediated phagocytosis. Importantly, this work also demonstrates a strong functional link between integrin activation, macropinocytosis and phagocytosis in macrophages.

Integrative Genomics IdentifiesRAB23as an Invasion Mediator Gene in Diffuse-Type Gastric Cancer

Recurrent genomic amplifications and deletions are frequently observed in primary gastric cancers (GC). However, identifying specific oncogenes and tumor suppressor genes within these regions can be challenging, as they often cover tens to hundreds of genes. Here, we combined high-resolution array-based comparative genomic hybridization (aCGH) with gene expression profiling to target genes within focal high-level amplifications in GC cell lines, and identified RAB23 as an amplified and overexpressed Chr 6p11p12 gene in Hs746T cells. High RAB23 protein expression was also observed in some lines lacking RAB23 amplification, suggesting additional mechanisms for up-regulating RAB23 besides gene amplification. siRNA silencing of RAB23 significantly reduced cellular invasion and migration in Hs746T cells, whereas overexpression of RAB23 enhanced cellular invasion in AGS cells. RAB23 amplifications in primary gastric tumors were confirmed by both fluorescence in situ hybridization and genomic qPCR, and in two independent patient cohorts from Hong Kong and the United Kingdom RAB23 expression was significantly associated with diffuse-type GC (dGC) compared with intestinal-type GC (iGC). These results provide further evidence that dGC and iGC likely represent two molecularly distinct tumor types, and show that investigating focal chromosomal amplifications by combining high-resolution aCGH with expression profiling is a powerful strategy for identifying novel cancer genes in regions of recurrent chromosomal aberration.

Annexin A2 Interactions with Rab14 in Alveolar Type II Cells

Annexin A2, a calcium-dependent phospholipid-binding protein, is abundantly expressed in alveolar type II cells where it plays a role in lung surfactant secretion. Nevertheless, little is known about the details of its cellular pathways. To identify annexin A2-regulated or associated proteins, we silenced endogenous annexin A2 expression in rat alveolar type II cells by RNA interference and assessed the change of the cellular transcriptome by DNA microarray analysis. The loss of annexin A2 resulted in the change of 61 genes. Thirteen of the selected genes (11 down-regulated and 2 up-regulated genes) were validated by real time quantitative PCR. When the loss of rat annexin A2 was rescued by overexpressing EGFP-tagged human annexin A2, six of seven selected targets returned to their normal expression level, indicating that these genes are indeed annexin A2-associated targets. One of the targets, Rab14, co-immunoprecipitated with annexin A2. Rab14 also co-localized in part with annexin A2 and lamellar bodies in alveolar type II cells. The silencing of Rab14 resulted in a decrease in surfactant secretion, suggesting that Rab14 may play a role in surfactant secretion.

Rab23 Regulates Differentiation of ATDC5 Chondroprogenitor Cells

Insulin treatment of mouse ATDC5 chondroprogenitors induces these cells to differentiate into mature chondrocytes. To identify novel factors that are involved in this process, we carried out mutagenesis of ATDC5 cells through retroviral insertion and isolated two mutant clones incapable of differentiation. Inverse PCR analysis of these clones revealed that the retroviral DNA was inserted into the promoter region of the Rab23 gene, resulting in increased Rab23 expression. To investigate whether an elevated level of Rab23 protein led to inhibition of chondrogenic differentiation, we characterized ATDC5 cells that either overexpress endogenous Rab23 or stably express ectopic Rab23. Our results revealed that up-regulation of Rab23 can indeed inhibit chondrogenic differentiation with a concomitant down-regulation of matrix genes such as type II collagen and aggrecan. In addition, stable small interfering RNA knockdown of Rab23 also resulted in inhibition of chondrogenic differentiation as well as down-regulation of Sox9, a master regulator of chondrogenesis. Interestingly, Sox9 expression has recently been linked to Gli1, and we found that Rab23 knockdown decreased Gli1 expression in chondrocytes. Because the phenotypes of Rab23 mutations in mice and humans include defects in cartilage and bone development, our study suggests that Rab23 is involved in the control of Sox9 expression via Gli1 protein.