Involvement of Rab6 in the Regulation of Phagocytosis against Virus

Aug 28, 2012 - Phagocytosis, which is of fundamental importance for innate and adaptive immunity in animals, is driven by organization of the actin cy...
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Involvement of Rab6 in the Regulation of Phagocytosis against Virus Infection in Invertebrates Ting Ye, Wen Tang, and Xiaobo Zhang* Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education, Key Laboratory of Animal Virology of Ministry of Agriculture and College of Life Sciences, Zhejiang University, Hangzhou 310058, The People’s Republic of China ABSTRACT: Phagocytosis, which is of fundamental importance for innate and adaptive immunity in animals, is driven by organization of the actin cytoskeleton. To date, however, the molecular events involved in the regulation of phagocytosis through reorganization of actin by small G proteins remains to be elucidated. To address this issue, the molecular mechanism of Rab6 in phagocytosis against virus infection in invertebrates was characterized in this study. The results showed that the Rab6 obtained from shrimp could interact with actin to regulate shrimp hemocyte phagocytosis through induction of the rearrangement of actin to protect against white spot syndrome virus (WSSV) infection. The Rab6 protein in Drosophila melanogaster shared the same mechanism of action as that of Rab6 in shrimp, indicating that the function of Rab6 in phagocytosis was conserved in invertebrates. By comparison with the early marker (Rab5) and late marker (LAMP1) of phagosomes, Rab6 was critically involved in the regulation of actin organization throughout the entire phagocytosis process. The presence of the evolutionarily conserved amino acid sequences of Rab6 in invertebrates and vertebrates indicated a conserved mechanism of Rab6 function in phagocytosis of animals. Therefore, our findings presented novel molecular events in the regulation of phagocytosis by small G proteins. KEYWORDS: ativiral phagocytosis, actin, Rab6, invertebrate

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INTRODUCTION Rab proteins (Rab GTPases), constituting the largest subset of the Ras family (Ras, Rho/Rac/Cdc42, Ran, Sar/Arf and Rab), are evolutionarily conserved in all eukaryotes.1−4 To date, more than 60 members of the Rab family have been indentified.5−7 Among these, Rab5, Rab6, Rab7 and Rab11 have been shown to be involved in phagocytosis,8−10 although information on the mechanism of Rab protein regulation of phagocytosis is limited.8−10 Rab proteins act as molecular switches, being active in the GTP-bound state and inactive in the GDP-bound state. They regulate key steps in the formation, motility and docking of vesicles in specific trafficking pathways by recruiting specific effector proteins to different membrane compartments.11−13 Some studies indicate that Rab proteins are also implicated in phagosome formation and maturation.14,15 Phagocytosis, an actin-dependent process, is a crucial part of the immune responses in multicellular organisms,8,16,17which mediates rapid engulfment of pathogens and apoptotic cells by specialized phagocytes. Phagocytosis consists of multiple stages, including the binding of particles to cell receptors, activation of a signaling pathway that leads to temporal and spatial regulation of F-actin formation and actin polymerization to engulf particles and removal of the actin coat from a newly formed phagosome to generate a mature phagolysosome. In this phagocytic process, Rab is known to be a key regulator of the early endocytic pathway and maturation of the phagosome.18−22 © 2012 American Chemical Society

The small GTPase Rab5 integrates the targeting, tethering and fusion of early endosomes and also appears to be involved in the dynamics of early phagosomes.8 In C57BL/6 mice, the depletion of Gapex-5, a guanine nucleotide exchange factor of Rab5, leads to inhibition of Rab5 activation during phagocytosis.8,9,23 In mammalian cells, Rab11 has been proposed to be involved in the formation of the phagocytic cup.24 On the basis of studies in Dictyostelium, Rab7 appears to direct a subset of lysosomal proteins to newly formed phagosomes.25,26 This process is coupled to internalization of particles, suggesting that Rab7 regulates phagosome maturation. Recently, it has been demonstrated that Rab6 is involved in phagocytosis in shrimp.10,27,28 However, the biological systems of phagocytosis are extremely complicated and the molecular events that form this process are, as of yet, unknown. In this study, Rab6 was characterized in invertebrates including shrimp and fruit fly to further the regulatory mechanism of phagocytosis mediated by Rab protein. The results showed that the Rab6 plays an important role in the regulation of phagocytosis by manipulation of actin in both shrimp and fruit fly, leading to significant changes in host antiviral activity. Therefore, our study demonstrated a universal role of Rab6 in the regulation of phagocytosis in invertebrates. Received: March 20, 2012 Published: August 28, 2012 4834

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MATERIALS AND METHODS

as control. Cells were transfected or cotransfected with the recombinant plasmids using Cellfectin transfection reagent (Invitrogen) according to the instructions provided by the manufacturer. Three days later, the cells were collected and lysed in 0.4 mL of lysis buffer containing protease inhibitors.10 The supernatant of the cell lysate was obtained by centrifuge (15 000× g, 4 °C) for 15 min. For CoIP, the supernatant was mixed with anti-His-actin for 2 h at 4 °C, followed by incubation with protein A-Sepharose (Bio-Rad) for 1 h at 4 °C. After washing with lysis buffer, the coupled proteins were subjected to SDS-PAGE and Western blot analysis with APconjugated anti-V5 antibody (Invitrogen). S2 cells (Invitrogen, USA) were propagated at 27 °C in Drosophila medium (Invitrogen) supplemented with 10% heatinactivated fetal bovine serum (FBS). The Drosophila megalogaster Rab6 and β-actin genes were cloned into the BamHI-XhoI sites of the pAc5.1/V5-His plasmid (Invitrogen). GFP-pAc5.1/V5-His plasmid was used as control. S2 cells at confluence of approximately 70% were transfected with plasmids. Transfection or cotransfections of plasmids was conducted with Cellfectin II transfection reagent (Invitrogen). Three days later, the cells were collected. CoIP was performed as described above using the specific anti-His-actin IgG. After washing with lysis buffer, the coupled proteins were subjected to SDS-PAGE and Western blot analysis with AP-conjugated anti-V5 antibody (Invitrogen).

Shrimp Culture and Shrimp Infection by WSSV

Groups of 30 Marsupenaeus japonicus shrimp (approximately 10 g and 10−12 cm each) were maintained in 80 L aquariums at 25 °C. PCR of shrimp gill tissues was conducted with WSSVspecific primers to experiments to confirm that they were WSSV-free.29 Infection of shrimp infected with WSSV was carried out by intramuscular injection of virus particles. The WSSV inoculum was obtained from virus-infected Marsupenaeus japonicus shrimp by homogenizing infected tissues in TN buffer (0.1 g/mL).10 After centrifugation at 2000× g for 10 min, the supernatant was diluted (1:100) with 0.9% NaCl and filtered (0.45 μm). Subsequently, 0.1 mL of filtrate (105 WSSV copies/ mL) was intramuscularly injected into virus-free shrimp in the lateral area of the fourth abdominal segment. Virus-infected specimens were collected and immediately stored at −70 °C. Recombinant Expressions of Genes in E. coli and Antibody Preparations

The shrimp β-actin and Rab6 genes were expressed in E. coli BL21 (DE3) as fusion proteins with a 6× His tag using the following primers incorporating restriction enzyme sites (italic): Actin 5′-AAGGATCCATGTGTGACGACGAAGTAGC3′ (BamHI) 5′-CCGCTCGAGTTAGAAGCACTTGCGGTG-3′ (XhoI) Rab6 5′-AAGGATCCATGTCGGGCGAATTCGG- 3′ (BamHI) 5′-AACTCGAGCGAAGGTTAAGCAAGCACATCC −3′ (XhoI) The fruit fly Rab6 and β-actin genes were also expressed in E.coli BL21 using the following primers: Rab6: 5′-CGGAATTCATGTCATCCGGAGATTTTGG-3′ (EcoRI) 5′-AAACTCGAGCGGCAGGCGCAGCCGCCCT3′(XhoI) Actin: 5′-AAGGATCCATG TGTGACGACGAAGTAGC-3′ (BamHI) 5′-AACTCGAGCGGAAGCACTTCC TGTGAAC-3′ (XhoI) Gene expression and protein purification were conducted according to the instructions provided by manufacturers (Amersham Biosciences, USA). Purified recombinant fusion proteins were used as antigens to immunize mice for antibody generation as previously described.10 The titers of antisera were approximately 1:10 000 as determined by enzyme-linked immunosorbent assay (ELISA). The immunoglobulin (IgG) fraction was purified by protein A-Sepharose (Bio-Rad, USA) and stored at −70 °C.

Western Blot Analysis

The proteins, separated in a 12% SDS-PAGE gel, were transferred onto a nitrocellulose membrane (Bio-Rad, America). The membrane was immersed in phosphate buffered saline (PBS) containing 5% nonfat dried milk at 4 °C overnight. Subsequently, the membrane was incubated with the primary antibody, followed by incubation with AP-conjugated secondary antibody. Two hours later, proteins were detected with NBT and BCIP solutions (BBI, Canada). Construction of the Phylogenetic Tree

A phylogenetic tree of Rab6 was constructed using MEGA 5.05 (National Institutes of Health, USA). All Rab6 genes were from National Center for Biotechnology Information (NCBI) and their accession numbers were as follows: Marsupenaeus japonicus ABC68472.1, Homo sapiens CAG46781.1, Canis lupus XP_850271.1, Rattus norvegicus NP_445818.1, Mus musculus NP_077249.1, Bos taurus XP_001252030.1, Taeniopygia guttata XP_002189206.1, Xenopus tropicalis NP_989315.1, Danio rerio AAI57380.1, Salmo salar NP_001133408.1, Anas platyrhynchos ABC42924.1), Monodelphis domestica XP_001363218.1, Anolis carolinensis XP_003218408.1, Ciona intestinalis XP_002121682.1, Saccoglossus kowalevskii XP_002737729.1, Daphnia pulex EFX82462.1, Acyrthosiphon pisum NP_001155439.1), Apis mellifera XP_392533.1, Bombyx mori NP_001040270.1, Ixodes scapularis XP_002404303.1, Bombus terrestris XP_003397813.1, Camponotus f loridanus EFN73950.1, Nasonia vitripennis XP_001604718.2, Pediculus humanus corporis XP_002423803.1, Tribolium castaneum XP_972453.2, Drosophila melanogaster NP_477172.1, Anopheles gambiae XP_001238010.2 and Aedes aegypti XP_001657423.1. The Rab6 contained 208 amino acids in Human and Drosophila and 209 amino acids in shrimp.

Cell Culture, Transfection and Coimmunoprecipitation (CoIP)

High Five (Hi 5) insect cells were cultured following standard procedures (Invitrogen, USA). Cells were grown at 27 °C in GIBCO insect culture medium (Invitrogen) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Thermo, USA). Rab6 gene deletion mutants, the full-length Rab6 gene and the full-length β-actin gene of shrimp were cloned into the BamHIXhoI sites of the pIZ/V5-His plasmid. The constructs were confirmed by sequencing. GFP-pIZ/V5-His plasmid was used 4835

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Synthesis of siRNAs

0.5% Triton for 5 min. Subsequently, slides were stained with rhodamine-phalloidin and DAPI, air-dried and mounted with antifade solution (Invitrogen). Cellular morphology was observed using a Confocal Laser Scanning Microscope. The shrimp hemocytes without the Rab6-siRNA injection were used as a control. To evaluate the influence of Rab6 gene silencing on phagocytosis, shrimp hemocytes were collected at 48 h after the Rab6-siRNA injection and subjected to phagocytosis assays analyzed by flow cytometry. All above assays were repeated on three occasions.

The siRNAs targeting the shrimp Rab6 gene (Rab-siRNA, 5′AATTCGGAAACC CGTTGAG-3′) or the fruit fly Rab6 gene (Rab6-specific-siRNA, 5′-CCTCGCTGAT TACACGATT-3′) were synthesized using a commercial kit (TaKaRa, Japan) according to a previously described procedure.30 The siRNAs consisted of 21-nucleotide double-stranded RNAs, each strand of which contained a 19-nucleotide target sequence and a twouracil (U) overhang at the 3′ end. The sequence of the shrimp Rab6 siRNA was randomly mutated at one nucleotide, yielding the corresponding Rab6-mutation-siRNA (5′-AATGCGGAAACCCGTTGA G-3′). The fruit fly Rab6-specific-siRNA was also randomly mutated at two nucleotides, generating mutation-siRNA (5′-CCTCGCAGA TTACACCATT-3′). The formation of double-stranded RNAs was monitored by determining the size shift in agarose gel electrophoresis. The synthesized siRNAs were dissolved in siRNA buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl) and quantified by spectrophotometry.

Evaluation of Fruit Fly Rab6 Protein in Antiviral Phagocytosis in S2 Cells

The fruit fly Rab6 gene was cloned into the EcoRI-XhoI sites of the pAc5.1/V5- His plasmid (Invitrogen). In this construct, the Rab6 gene was mutated at two nucleotides (position 78 A→C and position 90 C→A) to prevent recognition by the fruit fly Rab6-specific-siRNA. This construct was confirmed by sequencing. S2 cells were cultured for 24 h to 70% confluence prior to transfection. Then 5 μg of Rab6-specfic-siRNA, mutationsiRNA or plasmids containing the mutated fruit fly Rab6 gene, or the Rab6-specific-siRNA+plasmid containing the mutated Rab6 (5 μg each) was transfected into the S2 cells using Lip2000. At different times after transfection, the S2 cells were collected and subjected to analysis by real-time PCR, Western blot, phagocytosis and multiplicity of infection (MOI) assays. For confocal imaging, S2 cells at 36 h after transfection were mounted onto poly-L-lysine-coated glass slides and incubated for 30 min at 4 °C. After washing in HL3 buffer,31,32 the slides were fixed for 10 min with 10% paraformaldehyde in HL3 buffer. Cells were then processed as described above. To evaluate the effects of Rab6 on phagocytosis by imaging at 36 h after transfection, inactivated FITC-labeled DCV virions were added into the cells and incubated at 28 °C for different times (0, 5, 15, 25, and 35 min). Subsequently, cells were mounted onto glass slides and stained with rhodamine-phalloidin and DAPI. Images were obtained by confocal microscopy. All assays were repeated on three occasions.

Flow Cytometry Analysis of Shrimp Hemocytic Phagocytosis of WSSV

Purified WSSV virions (105 copies/mL) were treated with 1% formaldehyde overnight. After being washed with 0.1 M NaHCO3 (pH 9.0), the virions were incubated in 0.1 M NaHCO3 containing 1 mg/mL FITC isomer 1 (Sigma) for 1 h at 25 °C with gentle stirring. Then the FITC-labeled WSSV virions were rinsed with PBS until the supernatant was free of visible FITC. Shrimp hemocytes were resuspended in PBS at a density of 1 × 106 cells/mL, followed by incubation with the FITC-labeled WSSV for 30 min at 28 °C. After washing three times in PBS, the fluorescence of nonphagocytosed WSSV was quenched with the trypan blue solution (2 mg/mL PBS) (Amresco, USA). Thirty minutes later, after five washes with PBS, the hemocytic phagocytosis was examined by flow cytometry using the CELLQuest program (Becton Dickinson, USA). Phagocytosis was quantified as the percentage of fluorescence-positive cells within gate M3. For each sample, 5000 to 10 000 cells were counted. The phagocytic percentage (PP) was calculated as: PP = (number of cells ingesting virus/ number of cells observed) × 100%. All of the assays were repeated on three occasions. Data were analyzed using one-way analysis of variance (ANOVA).

Northern Blot Analysis

Total RNA was extracted from shrimp hemolymph using 1 mL TRIzol reagent according to the instructions provide by the manufacturer (Promega, USA). Extracted RNA was treated with RNase-free DNase I (TaKaRa, Japan) and separated by electrophoresis on a 2% agarose gel in 1× TBE buffer. Subsequently, RNA was transferred to a nitrocellulose membrane (Amersham, USA). Blots were detected with a DIG-labeled probe. DIG labeling and detection were performed following the protocol of DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany).

Effects of Rab6 on the Actin Conformation and Phagocytosis of Shrimp

Shrimp were injected with the Rab6-siRNA to silence the Rab6 expression. The Rab6-mutation-siRNA was included in the injection as a control. At various time after the siRNA injection (0, 4, 8, 12, 24, 48, 72, and 96 h), shrimp hemocytes were collected and analyzed by Northern and Western blotting, detected with the Rab6-specific probe or antibody, respectively. Shrimp actin was used as control. At 48 h after the siRNA injection, shrimp hemocytes were collected and incubated with rhodamine-phalloidin (red) and DAPI (blue) to label actin and the nucleus, respectively. Hemocytes from shrimp injected with Rab6-mutation-siRNA were used as a control. To examine the time-course of phagocytosis against WSSV, shrimp hemocytes were collected at 48 h after the Rab6-siRNA injection and then incubated with inactivated FITC-labeled WSSV at 28 °C for 0, 5, 15, 25, or 35 min. Samples were smeared onto poly-L-lysine-coated glass slides and incubated for 30 min at 4 °C. Hemocytes were fixed with 4% paraformaldehyde for 10 min and permeabilized with

Real-time Quantitative PCR

Real-time quantitative PCR was performed using a TaqMan fluorogenic probe (5′-FAM-TGCTGCCGTCTCCAATAMRA-3′) and WSSV-specific primers: sense, 5′TTG GTTTCATGCCCGAGATT-3′; ant isense, 5′CCTTGGTCAGCCCCTTGA-3′. A plasmid containing a 1400 bp fragment from the WSSV genome was constructed as the internal standard.33 The plasmid was digested by EcoRI to form linearized template in order to minimize the effect of the structure on real-time PCR. The concentration of the digested plasmid was determined by Quibt (Invitrogen, USA). The reaction mixture (25 μL) consisted of WSSV genome, 200 4836

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Figure 1. Role of the Rab6-actin interaction in the antiviral phagocytosis of shrimp hemocytes. (a) Schematic diagram of Rab6 deletion mutants. Numbers indicated the positions of mutated amino acids in the Rab6 protein. (b) Expressions of Rab6 deletion mutants in the insect Hi 5 cells. The insect Hi5 cells were cotransfected with the full-length actin and the different Rab6 mutants cloned in the pIZ/V5-His plasmids, respectively. Cell lysates were detected by Western blots with the Rab6-specific antibody. The headings showed the plasmids. M, protein marker. (c) Interaction between Rab6 and actin. The insect Hi5 cells were cotransfected with the recombinant pIZ/V5-His plasmids as indicated, respectively. Cell lysates were immunoprecipitated with actin-specific antibodies, followed by Western blots with anti-V5 antibody. Lane headings indicated the plasmids used in cotransfections and numbers indicated mutated amino acids in the Rab6 protein. The GFP was used as a control. M, protein marker. (d) Evaluation of Rab6 RNAi assays. Shrimp were injected with the Rab6-siRNA to silence Rab6 expression. Rab6-mutation-siRNA was included in the injection as a control. At various times after the siRNA injection, shrimp hemocytes were collected and subjected to Northern blot (left) and Western blot (right) analyses. Lane headings indicated the time in hours after siRNA injection. Shrimp actin was used as a control. The probes or antibodies used were indicated on the right. (e) Influence of Rab6 depletion on actin depolymerization. Shrimp were injected with Rab6-siRNA. At 48 h after the siRNA injection, shrimp hemocytes were collected and incubated with rhodamine-phalloidin (red) and DAPI (blue) to label actin and the nucleus, respectively. Shrimp hemocytes injected with Rab6-mutation- siRNA were used as a control. The shrimp hemocytes were examined with an optical microscope (down; scale bar, 50 μm) or confocal microscope (up; scale bar, 20 μm). The enlarged hemocytes were on the top. (f) Timecourse of phagocytosis against WSSV. At 48 h after the Rab6-siRNA injection, shrimp hemocytes were collected and incubated with FITC-labeled WSSV (green), followed by incubation with rhodamine-phalloidin (red) and DAPI (blue). Shrimp hemocytes with the Rab6-mutation-siRNA injection were used as a control. Numbers indicated the time for phagocytosis against WSSV. Lane headings represented the labeling targets. Scale bar, 20 μm. (g) Effects of Rab6 on phagocytosis against WSSV. At 48 h after the Rab6-siRNA injection, shrimp hemocytes were collected and incubated with FITC-labeled WSSV for 35 min. The percentage of hemocytic phagocytosis was examined by flow cytometry. Hemocytes from shrimp injected with Rab6-mutation-siRNA were used as a control. All assays were repeated on three occasions. Each column indicated the mean of triplicate assays. Statistically significant difference between treatments was indicated by * (P < 0.05).

S2 Phagocytosis of DCV

nM of each primer, 100 nM of each TaqMan probe and 1× PCR reaction buffer containing DNA polymerase. PCR amplification was performed for 4 min at 50 °C, followed by 45 cycles of 30 s at 94 °C, 30 s at 52 °C and 30 s at 72 °C. Gene-specific primers and TaqMan fluorogenic probes were used to detect fruit fly Rab5, Rab6 or LAMP1 mRNAs by realtime PCR. The fruit fly ribosome protein 49 (RP49) was used as the normalization control. The sequences of sense and antisense primers for the fruit fly Rab6 gene were 5′CGGGATTGATTTCCTATCG AAGAC-3′ (sense) and 5′GTCGCGTATGTACGAGGGTATC-3′ (antisense) and the TaqMan probe was 5′-FAMCGCTCCTGTCCCGCCGTATCCCA-TAMRA-3′. The primers for Rab5 and LAMP1 were 5′-CACTCAGCAGCAATCT TA-3′ (sense) and 5′-GATGGCAACTGATATTTGG-3′ (antisense), and 5′-CAACCATCCATTGG GGCT T-3′ (sense) and 5′-GCGTTGCTGGGGATGTTG-3′ (antisense), respectively. The TaqMan probes were 5′-FAM-TCTACGGACGGTTACTGCAACTC-TAMRA-3′ (Rab5) and 5′-FAMTCTTGTATTATGCTTCAAATGGCGGC-TAMRA-3′ (LAMP1). The primers for RP49 were 5′-CCGCTTCAAGGGACAGTATCTG-3′ and 5′-CACG TTGTGCACCAGGAACTT-3′. The TaqMan probe was 5′-FAM-GGCAGCATGTG GCGGGTGCGCTT-TAMRA-3′. Real-time PCR was performed as described above.

DCV-infected S2 cells were collected and homogenized in 500 mL TM buffer containing the protease inhibitor. Cells were then centrifuged at 10 000× g for 20 min at 4 °C. The supernatant was centrifuged at 110 000× g for 3 h at 4 °C. The virion pellet was resuspended in 10 mL TM buffer. The purity of virions was evaluated by negative-staining transmission electron microscopy (TEM). Purified DCV virions were incubated at 60 °C for 1 h followed labeled with FITC as described above. The collected S2 cells were resuspended in PBS at 1 × 106 cells/mL and incubated with the FITC-labeled DCV virions (approximately 106 copies/mL) for 30 min at 28 °C. The phagocytosis percentage was examined by flow cytometry (FCM). Three biological replicates were performed for all assays. Multiplicity of Infection (MOI)

S2 cells were infected with 10 μL of DCV virions (approximately 106 copies/ml). At different time postinfection (24, 36, and 48 h), the percentage of DCV-infected S2 cells was calculated. Subsequently the MOI was determined by the formula: MOI= -ln(P), where P represented the percentages of noninfected S2 cells. Three biological replicates were performed for all assays. RNAi Assays of Rab5 and LAMP1 in S2 Cells

The siRNAs targeting the Rab5 gene (Rab5-siRNA, 5′GCCTCACCAAACATTG TCA-3′) or the lysosome associated membrane protein 1 (LAMP1) gene (LAMP1- siRNA, 5′4838

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Figure 2. Interaction between the Rab6 and actin proteins in Drosophila melanogaster S2. (a) A phylogenetic tree was constructed with UPGMA method in MEGA 5.05 and a bootstrap analysis was performed using 1000 replicates to test the relative support for particular clades. Scale bar, 0.01. (b) Expressions of Rab6 and β- actin in S2 cells as detected by Western blot with the fruit fly Rab6-specific or β-actin-specific antibodies. (c) Interaction between the Rab6 and β-actin proteins of fruit fly. S2 cells were transfected with recombinant Rab6-pAc5.1-V5/His and GFP-pAc5.1-V5/ His plasmids or actin-pAc5.1-V5/His plasmids (as indicated at the top). Cell lysates were immunoprecipitated with the actin-specific antibody, followed by Western blot analysis with the anti-V5 antibody.

CTCGTGTTCAGAAGATAAA-3′) were synthesized according to the protocol described above. The sequences of siRNAs

were randomly mutated at one nucleotide, generating the corresponding Rab5-mutation-siRNA (5′-G CCTCACCAT 4839

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blot analysis indicated that the Rab6 expression was completely inhibited from 48 to 72 h after siRNA injection, whereas the control Rab6-mutation-siRNA had no effect on Rab6 expression (Figure 1d), showing that the Rab6-siRNA mediated highly specific silencing of the Rab6 gene expression only. The results presented that the number of hemocytes in the Rab6knocked-down shrimp by Rab6-siRNA was identical to that of hemocytes in the control shrimp treated with Rab6-mutationsiRNA at a density of 1 × 105 hemocytes/mL (Figure 1e, down), indicating that there was no toxicity effect with the Rab6 knockdown on shrimp hemocytes. In the hemocytes of control shrimp treated with Rab6-mutation- siRNA, actin fibers were distributed across the cells (Figure 1e, up). However, examination of actin stress fibers following Rab6 gene silencing revealed that actin fibers were distributed against the cells in hemocytes of shrimp injected with Rab6-siRNA (Figure 1e, up), suggesting that Rab6 was required to maintain the correct conformation of actin. To further evaluate the effects of Rab6 gene silencing on the antiviral phagocytosis, the time-course of phagocytosis of WSSV was examined using shrimp hemocytes collected at 48 h after the Rab6-siRNA injection. It was observed that the antiviral phagocytic activity of the hemocytes treated with Rab6-siRNA was decreased by comparison with that of the control (Figure 1f). Pagocytosis analysis by flow cytometry, showed that the phagocytic percentage of hemocytes treated with Rab6-siRNA was significantly reduced (P < 0.05) compared with that of the control (Figure 1g), indicating that the Rab6 played an important role in antiviral phagocytosis. It could be concluded from these data that the Rab6 protein was essential for the correct conformation of actin, thus indicating a critical role in phagocytosis against virus infection.

ACATTGTCA-3′) or LAMP1-mutation-siRNA (5′CTCGTGTTCACAAGATAAA-3′). S2 cells were cultured for 24 h to 70% confluence prior to transfection. Transfections of siRNAs into S2 cells were routinely carried out in 6-well plates using 10 mL of each 20 mM siRNA per well. After 4 h, the transfection mixture was removed and cells were overlaid with 1 mL Drosophila medium containing 10% FBS. At 0 and 24 h after transfection, the S2 cells were collected and subjected to real-time PCR using Rab5- or LAMP1-specific primers and TaqMan probes, Western blot analysis with Rab5- or LAMP1specific antibodies (Abcam, USA), S2 phagocytosis against DCV and the pHrodo dye-based phagocytosis assays. The pHrodo Dye-based Phagocytosis Assay in S2 Cells

DCV was labeled following the protocol of the pHrodo particle labeling kit (Invitrogen, USA) with some modifications. Briefly, DCV virions were purified and resuspended in 3 mL 100 mM sodium bicarbonate buffer (component F). After centrifugation at 110 000× g for 2 h, the pellet was resuspended in the pHrodo dye solution (component D), followed by incubation for 45 min at room temperature (no light). Subsequently, pHrodo-labeled DCV virions were rinsed with PBS and centrifugation at 110 000× g for 2 h. The pellet was resuspended in PBS. S2 phagocytosis of pHrodo-labeled DCV virions was performed. At different time after phagocytosis (10 min, 25 min, 45 min, 60 min, 90 min, 2 h, 4 and 10 h), S2 cells were collected . After washes for three times with PBS, cells were incubated with 1% paraformaldehyde and the phagocytosis percentage was analyzed by flow cytometry using the CELLQuest program (Becton Dickinson, USA) as described above. Statistical Analysis

Interaction between the Rab6 and Actin Proteins in Drosophila melanogaster S2

The numerical data from three independent experiments were analyzed by one-way ANOVA to calculate the mean and standard deviation of triplicate assays.

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The Drosophila melanogaster S2 cell line, which exhibits phagocytic activity, and DCV were used in this study to investigate the roles of Rab6 in the antiviral phagocytosis in invertebrates. Phylogenetic analysis showed that the Rab6 protein was highly conserved in animals (Figure 2a), suggesting that the function of Rab6 might be preserved during animal evolution. The results indicated that shrimp Rab6 shared 88 and 91% identities with human and fruit fly Rab6, respectively. The results showed that the Rab6 and actin proteins were detected in S2 by Western blot, indicating that the two genes were expressed in fruit fly (Figure 2b). To investigate the interaction between the fruit fly Rab6 and actin proteins, the plasmids encoding these genes were cotransfected into S2 cells. It was observed that Rab6 was bound with the actin protein while the control GFP could not bind to actin (Figure 2c), indicating the existence of the interaction between Rab6 and actin in fruit fly. Shrimp Rab6 regulated antiviral phagocytosis of shrimp hemocytes through its interaction with actin. Therefore, the existence of the interaction between Rab6 and actin in fruit fly suggested that Rab6 was involved in the antiviral phagocytosis of S2.

RESULTS

Roles of the Interaction between Rab6 and Actin in the Antiviral Phagocytosis of Shrimp Hemocyte

It was reported that Rab6 regulated the hemocytic phagocytosis through interaction with β-actin in shrimp.10 To elucidate the molecular events mediated by Rab6 during phagocytosis, the interaction between Rab6 and actin was characterized. The fulllength Rab6 gene was truncated to construct the deletion mutants (Figure 1a). After the cotransfection of the full-length actin gene and Rab6 mutants into insect Hi 5 cells, all the Rab6 deletion mutants were expressed at comparable levels (Figure 1b). The results showed that the actin and Rab6 proteins were simultaneously detected in the coimmunoprecipitated (Co-IP) complex when the full-length actin and full-length Rab6 were cotransfected into cells (Figure 1c). However, the Rab6 mutants were not detected in the Co-IP complex when the Rab6 mutants were cotransfected with the full-length actin, respectively (Figure 1c), indicating that the truncated Rab6 could not bind to actin. As a control, the GFP was not bound to actin (Figure 1c). On the basis of these data, it could be concluded that there might be an interaction between actin and Rab6 proteins. To investigate the effects of Rab6 on actin conformation and antiviral phagocytosis, the expression of Rab6 was silenced by Rab6-specific-siRNA (Rab6-siRNA). Northern and Western

The Antiviral Phagocytosis Mediated by Rab6 in Fruit Fly S2 Cells

The function of Rab6 protein in antiviral phagocytosis of S2 cells was investigated. S2 phagocytosis against DCV was evaluated following the silencing of Rab6 gene expression with Rab6-specific-siRNA, the overexpression of Rab6 and the rescued expression of Rab6 gene in Rab6-knocked-down S2 4840

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Figure 3. Antiviral phagocytosis mediated by Rab6 in fruit fly S2 cells. (a) Real-time PCR detection of Rab6 mRNA using the total RNA extracted from S2 cells at different times after transfection. Lane headings showed the time after transfection. The treatments used for transfection were indicated at the top. S2 cells only were used as a control. Columns represented the mean of triplicate assays. Statistically significant differences between treatments were represented by asterisks (*P < 0.05 and **P < 0.01). (b) Detection of Rab6 protein at different times after transfection by Western blot using the Rab6-specific antibody. β-actin was used as a control. Headings showed the treatments used for transfection. (c) Percentage of S2 phagocytosis against inactivated DCV. The Rab6 gene in S2 cells was silenced, overexpressed or rescued, followed by evaluation of S2 phagocytic activity against virus at different times (shown at the top). DCV was inactivated and labeled with FITC and phagocytosis was examined by flow cytometry. The treatments used for transfection were shown at the top. Lane headings indicated the time after transfection. S2 cells only were used as a control. Columns represented the mean of triplicate assays. Statistically significant differences between treatments were indicated by asterisks (*P < 0.05 and **P < 0.01). (d) Effects of Rab6-regulated phagocytosis on virus infection. The Rab6-silenced, overexpressed or rescued S2 cells were infected with DCV. At different times postinfection, the MOI of DCV in S2 cells was evaluated. Numbers at the top indicated the time postinfection. Lane headings indicated the treatments used for transfection. Noninfected S2 cells were used as a control. Columns represented the mean of triplicate assays within ±1% standard deviation. (e) Effects of Rab6 on actin conformation in S2 cells. The Rab6 gene was silenced by Rab6specific siRNA and the expression of Rab6 in Rab6-silenced S2 cells was rescued by transfection of the plasmid containing the mutated Rab6. S2 cells only were used as a control. At 36 h after transfection, S2 cells were stained with rhodamine-phalloidin (red) and DAPI (blue) and examined by confocal microscopy (up; scale bar, 20 μm). Assays were conducted on three occasions. Enlarged images were shown at the top. At the same time, the S2 cells treated with Rab6-specific siRNA and the control S2 cells were examined with an optical microscope (down; scale bar, 50 μm). (f) Timecourse evaluation of Rab6 in phagocytosis against DCV. S2 cells were transfected with the Rab6-specfic siRNA or the Rab6-specific siRNA+the plasmid containing the mutated Rab6, respectively. S2 cells with mutation-siRNA were used as a control. At 36 h after transfection, inactivated FITClabeled DCV virions (green) were added to the cells. At different times after phagocytosis, the cells were incubated with rhodamine-phalloidin (red) and DAPI (blue) to label actin and the nucleus, respectively. Images were obtained with confocal microscopy. All assays were repeated on three occasions. Numbers indicated the time for phagocytosis against DCV. Lane headings indicated the labeling targets. Scale bar, 10 μm.

cells. Quantitative real-time PCR results showed that Rab6 mRNA was reduced dramatically at 24 and 36 h after transfection of Rab6 gene-specific siRNA (Rab6-specific-

siRNA) and was normally transcribed when treated with a single-nucleotide variation in the Rab6-specific siRNA (mutation-siRNA) (Figure 3a), indicating that the Rab6-specifc 4842

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Figure 4. Mechanism of Rab6 activity in S2 phagocytosis against DCV. (a) RNAi-induced knock down of Rab5. Rab5 mRNA abundance was quantified by real-time RT-PCR in S2 cells pretreated with Rab5-siRNAs or Rab5-mutation-siRNA (left). Rab5 protein was detected by Western blotting using a Rab5-specific antibody (right). Numbers indicated the time in hours after treatment with siRNA. Control, S2 cells without treatment. (b) Silencing of LAMP1 gene by siRNA. Transcription of the LAMP1 gene was evaluated by real-time quantitative PCR (left). Western blot was used to detect the LAMP1 protein using a LAMP1-specific antibody (right). Numbers indicated the time in hours after treatment with siRNA. Control, S2 cells without treatment. (c) The effects of Rab5, Rab6 and LAMP1 on phagocytosis against FITC-labeled DCV (top) or pHrodo-labeled DCV (bottom). At 24 h after treatment of Rab5-siRNA, Rab6-siRNA or LAMP1-siRNA, S2 cells were collected and incubated for different times with FITC-labeled DCV (top) or pHrodo-labeled DCV (bottom). The percentage of phagocytosis was evaluated by flow cytometry. Rab6 overexpressing and rescued S2 cells were included in the assays. Three biological replicates were performed for all assays. Columns represented the mean of triplicate assays. Statistically significant differences between treatments were indicated by asterisks (*P < 0.05 and **P < 0.01). Lane headings indicated the time for phagocytosis.

siRNA was highly specific. When the Rab6 was overexpressed, the Rab6 mRNA was significantly increased (Figure 3a). To exclude the effect of internal S2 Rab6, the expression of Rab6 gene was inhibited by Rab6-specific siRNA and simultaneously rescued with the transfection of the plasmid containing Rab6. The results revealed that the expression of Rab6 in Rab6knocked-down S2 cells was recovered compared with the control (Figure 3a). Western blot and real-time PCR analyses yielded essentially similar results (Figure 3b). Under conditions in which the expression of Rab6 gene was silenced, overexpressed or rescued, evaluation of S2 phag-

ocytosis of FITC-labeled DCV indicated that the phagocytic percentage of S2 cells was significantly decreased at 24 h after transfection with the Rab6-specific-siRNA, whereas there was no difference in the phagocytic percentage in the presence of the mutation-siRNA at all the time points examined (Figure 3c). These data indicated that Rab6 silencing led to significantly decreased phagocytic activity. Rab6 gene overexpression resulted in significantly increased phagocytic percentage compared with the control (Figure 3c), suggesting that the overexpression of Rab6 enhanced phagocytosis. Interestingly, when expression of Rab6 was rescued in Rab6-silenced S2 cells, 4843

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the phagocytic percentage returned to normal levels (Figure 3c), indicating that the recombinant Rab6 protein functioned in S2 phagocytosis. Therefore, these data demonstrated that Rab6 played a crucial role in the regulation of phagocytosis. To evaluate the effects of phagocytosis on virus infection, the Rab6-silenced, Rab6-overexpressing and Rab6-rescued S2 cells were infected with DCV. The results showed that the MOI of DCV in S2 cells was significantly decreased in the Rab6overexpressing S2 cells and in the Rab6-rescued S2 cells, while the MOI was increased in the Rab6-silenced S2 cells at 48 h postinfection (Figure 3d). The results clearly indicated that the regulation of phagocytosis by Rab6 protein significantly affected virus infection. To elucidate the mechanism of antiviral phagocytosis mediated by Rab6 protein, the Rab6 gene was silenced or rescued in the Rab6-silenced S2 cells and then examined by fluorescence microscopy. S2 cells were used as a control. The results showed that the number of the Rab6-knocked-down S2 cells by Rab6-specific-siRNA was identical to that of the control S2 cells at a density of 1 × 105 cells/mL (Figure 3e, down), indicating that there was no toxicity effect with the Rab6 knockdown on S2 cells. It was revealed that Rab6 depletion led to an absence of criss-crossed actin filaments through the cell compared with the control (Figure 3e, up). When Rab6 expression was knocked down, S2 filamentous actin was distributed around the outside of cells. However the appearance of actin filaments was identical to that of the control when the silenced Rab6 was rescued in S2 cells (Figure 3e, up). These data indicated that Rab6 was required in the actin filament organization of Drosophila, similar to the requirement of shrimp actin. Time-course analysis of phagocytosis against DCV showed that the antiviral phagocytic activity of the S2 cells treated with Rab6-specific-siRNA was decreased by comparison with that of the control (Figure 3f). From the above results, it was concluded that the Rab6 protein could enhance the phagocytotic activity against virus infection through manipulation of actin conformation in fruitfly.

(Figure 4c, top), demonstrating the involvement of Rab6 in phagocytosis. It is well-known that phagocytosis is completed in a low pH environment. Therefore, the pHrodo dye-based phagocytosis assay was conducted to detect late stage phagocytosis. The results showed that the percentage of phagocytic S2 cells against pHrodo-labeled DCV was not affected by silencing of Rab5 or LAMP1 gene during the entire phagocytosis process (Figure 4c, bottom), which was consistent with phagocytosis assays using FITC-labeled DCV (Figure 4c, top). When the Rab6 gene expression was inhibited, S2 phagocytic activity was significantly decreased during phagocytosis (Figure 4c, bottom). Furthermore, overexpression or rescue of Rab6 gene expression led to increased or recovered phagocytic activity (Figure 4c, bottom). These data demonstrated that Rab6 protein played essential roles throughout the entire phagocytic process.

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DISCUSSION Phagocytosis is a complex actin-dependent process that plays a critical role in the immune response to pathogen infection.34,35 It has been reported that phagocytosis functions as part of the host-defense mechanisms through the uptake and degradation of infectious pathogens and contributes to inflammation and the immune response. Currently, effective phagocytosis is known to consists of distinct steps that include membrane invagination, coated vesicle formation, directed vesicle trafficking, formation of the phagocytic cup and engulfment of particles36−38 Among these steps, particle internalization and phagosomal maturation are two essential steps.39 Phagosome maturation involves programmed changes in membrane fission and fusion events with endosomes, lysosomes and possibly other endomembrane organelles. The process of phagosome maturation depends on regulators of vesicular traffic, such as the Rab GTPases, SNARE proteins and fission complexes. As documented, early phagosomes are characterized by the presence of Rab5, which integrates the targeting, tethering and fusion of early endosomes.40 Early phagosomes eventually divest themselves of early markers, as they acquire the hallmarks of late endosomes. It is evident that the small GTPase Rab7A is a characteristic marker of the late endosome and is known to mediate the traffic between phagosomes and late endosomes or lysosomes.41 In addition to Rab5 and Rab7, late phagosomes/endosomes acquire a variety of transmembrane proteins and lipids typical of late endosomes/lysosomes, such as lysosome associated membrane protein (LAMP1).42 In this present investigation, it was revealed that Rab6, belonging to the Rab GTPase family, was required for normal rates of phagocytic uptake during phagocytosis against virus infection in invertebrates. Regardless of different organisms or specific molecular mechanisms, all phagocytic processes ultimately lead to the spatial and conformational changes of the actin cytoskeleton. In theory, the actin cytoskeleton may be involved in some or all of the steps. Therefore the host actin is a critical effector in phagocytosis against pathogen invasion. It has been documented that the formation and organization of actin into functional higher-order networks is regulated by a wide variety of actin-binding proteins,43−47 which can be classified into three main categories: proteins regulating actin assembly and disassembly, proteins altering the structure of the actin network and proteins using actin as a scaffold, physical support or track.43−46 In this study, the results showed that, after the

Mechanism of Rab6 in Phagocytosis

To elucidate the mechanism of Rab6 in phagocytosis, Rab5 (a marker of the early phagosome) and LAMP1 (a marker of the late phagosome), as well as Rab6, were characterized in S2 cells. The Rab6 expression was knocked down as described above. To silence the expression of Rab5 and LAMP1 genes, RNAi experiments were performed using sequence-specific siRNAs. Real-time quantitative PCR results showed that the expressions of Rab5 and LAMP1 were knocked down compared with the controls at 24 h after the siRNA treatment (Figure 4a and b, left). Western blot analysis demonstrated significant decreases in Rab5 and LAMP1 proteins in response to the sequencespecific-siRNA treatments (Figure 4a and b, right). Following silencing of the expression of Rab5 or LAMP1 genes by siRNA, the phagocytic percentages of S2 cells against FITC-labeled DCV did not change during the entire phagocytic process (Figure 4c, top), indicating that the two marker proteins were not critical factors in phagocytosis. Following silencing of Rab6 gene expression, evaluation of S2 phagocytosis using FITC-labeled DCV indicated that the percentage of phagocytic S2 cells was significantly decreased during phagocytosis compared with the control (Figure 4c, top), while the overexpression or rescue of Rab6 gene expression led to increased or recovered phagocytic activity 4844

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(3) Pfeffer, S.; Aivazian, D. Targeting Rab GTPases to distinct membrane compartments. Nat. Rev. Mol. Cell Biol. 2004, 5 (11), 886− 896. (4) Zhang, J.; Fonovic, M.; Suyama, K.; Bogyo, M.; Scott, M. P. Rab35 controls actin bundling by recruiting fascin as an effector protein. Science 2009, 325 (5945), 1250. (5) Rink, J.; Ghigo, E.; Kalaidzidis, Y.; Zerial, M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 2005, 122 (5), 735−749. (6) Segev, N. Ypt and Rab GTPases: insight into functions through novel interactions. Curr. Opin. Cell Biol. 2001, 13 (4), 500−511. (7) Zerial, M.; McBride, H. Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2001, 2 (2), 107−117. (8) Kitano, M.; Nakaya, M.; Nakamura, T.; Nagata, S.; Matsuda, M. Imaging of Rab5 activity identifies essential regulators for phagosome maturation. Nature 2008, 453 (7192), 241−245. (9) Niedergang, F.; Chavrier, P. Signaling and membrane dynamics during phagocytosis: many roads lead to the phagos (R) ome. Curr. Opin. Cell Biol. 2004, 16 (4), 422−428. (10) Wu, W.; Zong, R.; Xu, J.; Zhang, X. Antiviral phagocytosis is regulated by a novel Rab-dependent complex in shrimp Penaeus japonicus. J. Proteome Res. 2008, 7 (01), 424−431. (11) Grosshans, B. L.; Ortiz, D.; Novick, P. Rabs and their effectors: achieving specificity in membrane traffic. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (32), 11821−11827. (12) Markgraf, D. F.; Peplowska, K.; Ungermann, C. Rab cascades and tethering factors in the endomembrane system. FEBS Lett. 2007, 581 (11), 2125−2130. (13) Pan, X.; Eathiraj, S.; Munson, M.; Lambright, D. G. TBCdomain GAPs for Rab GTPases accelerate GTP hydrolysis by a dualfinger mechanism. Nature 2006, 442 (7100), 303−306. (14) Caron, E.; Hall, A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 1998, 282 (5394), 1717−1721. (15) Harris, E.; Cardelli, J. RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion. J. Cell Sci. 2002, 115 (18), 3703−3713. (16) Cuttell, L.; Vaughan, A.; Silva, E.; Escaron, C. J.; Lavine, M.; Van Goethem, E.; Eid, J. P.; Quirin, M.; Franc, N. C. Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis. Cell 2008, 135 (3), 524−534. (17) Smith, A. E.; Helenius, A. How viruses enter animal cells. Science 2004, 304 (5668), 237−242. (18) Goley, E. D.; Ohkawa, T.; Mancuso, J.; Woodruff, J. B.; D’Alessio, J. A.; Cande, W. Z.; Volkman, L. E.; Welch, M. D. Dynamic nuclear actin assembly by Arp2/3 complex and a baculovirus WASPlike protein. Science 2006, 314 (5798), 464−467. (19) Muller, M. P.; Peters, H.; Blumer, J.; Blankenfeldt, W.; Goody, R. S.; Itzen, A. The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b. Science 2010, 329 (5994), 946. (20) Ingmundson, A.; Delprato, A.; Lambright, D. G.; Roy, C. R. Legionella pneumophila proteins that regulate Rab1 membrane cycling. Nature 2007, 450 (7168), 365−369. (21) Vieira, O. V.; Bucci, C.; Harrison, R. E.; Trimble, W. S.; Lanzetti, L.; Gruenberg, J.; Schreiber, A. D.; Stahl, P. D.; Grinstein, S. Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase. Mol. Cell. Biol. 2003, 23 (7), 2501− 2514. (22) Rojas, R.; Van Vlijmen, T.; Mardones, G. A.; Prabhu, Y.; Rojas, A. L.; Mohammed, S.; Heck, A. J. R.; Raposo, G.; Van Der Sluijs, P.; Bonifacino, J. S. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7. J. Cell Biol. 2008, 183 (3), 513− 526. (23) Duclos, S.; Diez, R.; Garin, J.; Papadopoulou, B.; Descoteaux, A.; Stenmark, H.; Desjardins, M. Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages. J. Cell Sci. 2000, 113 (19), 3531−3541.

cotransfection of the plasmid containing Rab6 gene and the plasmid containing actin gene into cultured cells to overexpress the two genes, the Rab6 and actin proteins were simultaneously detected in the immunoprecipitated protein complex, suggesting that there might be an interaction between Rab6 and acin proteins. It has been reported that Rab6 can be directly interacted with myosin II in mammalian.48,49 Therefore the interaction between Rab6 and actin in invertebrates, as revealed in this study, might be indirect. The Rab6, actin and myosin proteins might be linked in a multiprotein complex through indirect interactions. In this context, the Rab6 protein and cytoskeleton proteins, for example actin and myosin, can form a protein complex and execute certain physiological functions. In our study, it was presented that the Rab6 protein played an important role in the formation of actin stress fibers during phagocytosis. The results showed that the depletion of Rab6 by sequence-specific- siRNA led to a significant decrease in phagocytic activity. However, RNAi assays indicated that silencing of Rab5 or LAMP gene expression had little effect on the phagocytosis. The data in our study demonstrated that the Rab6 protein, which was essential for the correct conformation of the actin cytoskeleton, played very important roles in the regulation of phagocytosis in invertebrates. Therefore our study contributed a novel and important insight into the mechanism of regulation of phagocytosis by small G protein in an actin-dependent manner, in which the direct binding of Rab6 with the cytoskeleton protein actin led to the actin filament remodeling. This rearrangement of actin conformation mediated by Rab6 might result from the polymerization and/or depolymerization of actin. This issue merited to be further investigated. In this study, the sequence analysis of Rab6 revealed that the Rab6 protein was highly conserved in animals. It could be speculated that the molecular events of phagocytosis mediated by the Rab6 protein in invertebrates might be preserved in higher mammals during evolution. In this context, the Rab6mediated mechanism of regulation of phagocytosis merited further investigation in higher vertebrates in order to expand the currently limited information available describing innate immune responses against pathogen infection in vertebrates.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-571-88981129. Fax: 86-571-88981129. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (30830084), the Hi-Tech Research and Development Program of China (863 program of China) (2010AA09Z403) and Project of Ministry of Agriculture, China (201103034).

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