美麗海葵 Rab4 和 Rab14-L 蛋白在胞內共生所扮演的角色之研究

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(1)國立高雄大學生物科技研究所碩士論文. Involvement of ApRab4 and ApRab14-L in Aiptasia-Symbiodinium endosymbiosis. 美麗海葵 Rab4 和 Rab14-L 蛋白在胞內共生所扮演的角色之研究. 研究生：宋柏青撰指導教授：黃永森博士陳鳴泉博士中華民國 96 年 7 月.

(2) 致謝. 首先誠摯的感謝指導教授陳鳴泉博士及黃永森博士，兩位老師悉心的教導使我得以一窺海洋生物領域的深奧，不時的討論並指點我正確的方向，使我在這些年中獲益匪淺。老師對學問的嚴謹更是我輩學習的典範。兩年裡的日子，實驗室裡共同的生活點滴，學術上的討論、言不及義的閒扯、趕作業和實驗的革命情感，都感謝眾位學長姐、同學、學弟妹的共同砥礪，你們的陪伴讓我兩年的研究生活變得絢麗多彩。感謝明昌學長不厭其煩的指出我研究中的缺失，且總能在我迷惘時為我解惑，也感謝逸帆、意慧同學的幫忙，恭喜我們順利走過這兩年。實驗室的小崴、阿菜、雅繐、元鳳，當然也不能忘記妳們的幫忙及搞笑我銘感在心。女朋友在背後的默默支持更是我前進的動力，沒有她的體諒、包容，相信這兩年的生活將是很不一樣的光景。最後，謹以此文獻給我摯愛的雙親。.

(3) Involvement of ApRab4 and ApRab14-L in Aiptasia-Symbiodinium endosymbiosis Advisor(s): Dr. Yung-Sen Huanga and Dr. Ming-Chyuan Chenb a Institute of Biotechnology National University of Kaohsiung b Department of Marine Biotechnology National Kaohsiung Institute of Marine Technology Student: Po-Ching Sung Institute of Biotechnology National University of Kaohsiung ABSTRACT The establishment and maintenance of the intracellular association between marine cnidarians and their symbiotic microalgae is essential to the well being of coral reef ecosystems; however, little is known concerning its underlying molecular mechanisms. A defect in the recruitment of ApRab11 (a key regulator of endocytic recycling) to the algal symbionts- containing phagosomes is strongly correlated with their maturation arrest. To further examine the relationship between endocytic recycling and symbiosome biogenesis, I cloned and characterized two additional Rab proteins (ApRab4 and ApRab14), whose mammalian homologues are known to participate in the regulation of endocytic recycling, in the endosymbiosis system between the sea anemone, Aiptasia pulchella and its algal symbiont, Symbiodinium spp. ApRab4, and ApRab14-L are 87% and 88 % identical to human Rab4b and Rab14 protein and contains all Rab-specific signature motifs. EGFP-ApRab4 and EGFP-ApRab14-L are located in the early endocytic and phagocytic compartments and functioned in the recycling pathway in HeLa cells. When we examined our endosymbiotic system, endogenous ApRab4 and ApRab14-L were associated with the majority of resident zooxanthellae-containing phagosomes (symbiosomes). Overall, my study suggest that live algal symbionts persist inside their host cells by actively retaining ApRab4 and ApRab14-L to phagosomes for maintaining an endosymbiotic relationship with their cnidarian hosts. Keywords: Endocytic recycling; Symbiosome; Endosymbiosis; Zooxanthellae; Rab4; Rab14.

(4) 美麗海葵 Rab4 Rab4 和 Rab14Rab14-L 蛋白在胞內共生所扮演的角色之在胞內共生所扮演的角色之研究指導教授：黃永森、陳鳴泉博士國立高雄大學生物科技所國立高雄海洋科技大學海洋生物技術系學生：宋柏青國立高雄大學生物科技所. 摘要海洋腔腸動物與其共生藻之間共生關係的建立與維持，在珊瑚礁生態系統中扮演一個必須且重要的角色。但是，我們對共生關係中的分子機制，了解的卻甚少。過去文獻指出，一個調節胞飲作用再循環路徑(endocytic recycling pathway) 的蛋白 (ApRab11) ，並不參與在共生藻 (Symbiodinium)進入美麗海葵(Aiptasia pulchella)宿主達成共生的建立與維持。因此，本研究進一步的選殖了美麗海葵的同源性蛋白 Rab4 和 Rab14-L (ApRab4 and ApRab14-L)，想了解同樣是調節胞飲作用再循環路徑的蛋白，是否參與共生關係的建立和維持。ApRab4 和 ApRab14-L 在胺基酸序列上與人類 Rab4 和 Rab14 有高達 87%和 88%相同，並且包含了已知的 Rab 蛋白特有的功能性區間(functional motifs)。以綠色螢光蛋白 (EGFP) 標定 ApRab4 和 ApRab14-L 蛋白座落於早期胞飲小體(early endosomes)，並可能參與 HeLa 細胞再循環的路徑。另一方面，免疫螢光染色的分析(immunofluorescence analysis) 指出，ApRab4 和 ApRab14-L 蛋白出現在包著共生藻的特化胞噬小體 (共生小體)上。綜合以上的結果，推測 ApRab4 和 ApRab14-L 可能參與在共生藻與海葵宿主細胞間共生的建立與維持。關鍵字：共生關係；胞飲作用；再循環路徑；胞噬小體；共生小體關鍵字.

(5) Contents 1. Introduction-------------------------------------------------1 1.1. Endosymbiosis-------------------------------------------------1 1.2. Rab proteins----------------------------------------------------2 1.3. Purpose----------------------------------------------------------3. 2. Materials and Methods-------------------------------------6 2.1. Cells and the animal-------------------------------------6 2.2. Cloning of full-length ApRab4 and ApRab14 cDNA and sequence analysis---------------------------------------------6 2.3. Construction, expression, and purification of recombinant ApRab4 and ApRab14-L proteins --------------------------------7 2.4. Prepared of polyclonal antibodies---------------------------8 2.5. Western blot analysis------------------------------------------9 2.6. Transfection of mammalian cells---------------------------10 2.7. Cell image-----------------------------------------------------12 2.8. Phagocytosis assay-------------------------------------------13. 3. Results------------------------------------------------------14 3.1. Cloning of full-length ApRab4 and ApRab14 cDNA---14 3.2. Alignment of ApRab4 and ApRab14-L proteins with their relatives-------------------------------------------------------15 3.3. Characterized subcellular localization of EGFP-ApRab4 and EGFP-ApRab14-L in HeLa cells---------------------------16 3.4. Characterized of EGFP-ApRab4 labeled structures-----17.

(6) 3.5. Characterized of EGFP–ApRab14-L labeled structures18 3.6. C h a r a c t e r i z e d of antibodies by western blot analysis--------------------------------------------------------------19 3.7. Localized of endogenous ApRab4 and ApRab14-L in the host cells of zooxanthellae by immunofluorescence analysis -----------------------------------------------------------------------20 3 .8 . In v o lv e men t o f Ap Rab 4 an d Ap R ab 1 4 - L i n Phagocytosis--------------------------------------------------------21. 4. Conclusion and Discussion------------------------------23 5. References-------------------------------------------------28 6. Figures------------------------------------------------------32.

(7) 1. Introduction. 1.1. Endosymbiosis The endosymbiosis between the symbiotic dinoflagellates, and their marine cnidarian hosts is the most prominent intracellular association in the sea （W. Souter and Linden, 2000）. Symbiotic dinoflagellates, commonly known as zooxanthellae, are a taxonomically diverse group of marine microalgae, predominantly classified under the genus Symbiodinium (Birkeland, 1997). They are well known for their ability to establish intracellular symbiosis (endosymbiosis) with numerous marine cnidarians such as jellyfish, sea anemones, and corals, and forms the foundation of the highly productive and diversified coral reef ecosystem worldwide (Birkeland, 1997; Hatcher, 1990; Jackson, 1991; Connell, 1978). In such an association, individual algae grow and proliferate inside a specialized phagosome, the symbiosome, of the gastrodermal digestive cells of its cnidarian host, with the ratio of one algal cell per vacuole. However, in order to occupy this unique ecological niche, zooxanthellae first need to counteract the destructive consequences of phagocytosis, and second to find ways to take up essential nutrients across the phagosome membrane for growth and replication (Fitt and Trench, 1983).. A previous electron microscopy study of the. endosymbiosis between the jellyfish, Cassiopeia xamachana and its symbiotic alga, Symbiodinium microadriaaticum showed that, unlike the. 1.

(8) phagosomes containing food particles, dead or photosynthesis-impaired algae, the phagosomes containing live and functional algae did not show any signs of lysosomal fusion (Fitt and Trench, 1983). An earlier study on another animal-algal endosymbiosis (Hydra and Chlorella) also reported similar findings (Hohman and McNeil, 1982). These observations strongly indicate that live and functional zooxanthellae possess the ability to interfere with the normal path of phagosomal maturation to survive inside their cnidarian host cells. Unfortunately, there have been limited advances in our knowledge of the responsible molecular basis for this alga-mediated inhibition of phagosome maturation in the last two decades.. 1.2. Rab proteins The Rab small GTP-binding proteins have recently emerged as key regulators of intracellular vesicle trafficking during endocytosis and exocytosis, and several members of this family have been localized to distinct intracellular structures （Zerial and McBride, 2001；Stenmark and Olkkonen, 2001）. It is thought that each of the steps of intracellular vesicular transport is mediated by distinct sets of Rab proteins （Stenmark and Olkkonen, 2001； Martinez and Goud, 1998）. Rabs are a subfamily within the large group of small GTP-binding proteins whose most-studied member is the signalling protein Ras. The proteins are synthesized in the cytosol, but as a result of their modification with two copies of the isoprenoid gernaylgeranyl, they. 2.

(9) become attached to the cytosolic face of membranes with different Rabs on different membranes （Pfeffer, 2001；Chavrier and Goud, 1999）. Like Ras, they cycle between active GTP-bound and inactive GDP-bound forms （Segev, 2001）, with both transitions likely to require additional factors: GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). The GDP-bound form is also a target for a GDI (GDP dissociation inhibitor), a slightly-misnamed but remarkable protein which extracts the GDP-Rab (including its very hydrophobic isoprenoid groups) from the membrane, allowing it to return via the cytosol to its membrane of origin （Armstrong, 2000； Stein et al., 2003）.. 1.3. Purpose According to previous studies, two Aiptasia pulchella Rabs, ApRab5 and ApRab7, although like their mammalian counterparts, sequentially and transiently associated with maturing phagosomes containing inert particles, one (ApRab5) is selectively retained in, while the other (ApRab7) excluded from phagosomes containing newly acquired live zooxanthellae.. Such a. strong differential affinity of symbiosomes (phagosomes containing resident zooxanthellae) toward ApRab5 but not ApRab7 could be effectively reversed when. zooxanthellae. were. briefly. treated. with. DCMU. (3-(3,4-. dichlorophenyl)-1, 1-dimethylurea) to damage their photosynthesis systems (Chen et al., 2003-2004). Beside Rab5 and Rab7, Rab11 is another important. 3.

(10) regulator of phagosomes maturation (Cox et al., 2000). Rab11 was thought to facilitate phagosomes maturation through efficient retrieval of cell surface proteins back to the plasma membrane. A defect in the recruitment of ApRab11 (a key regulator of endocytic recycling), to the algal symbionts-containing phagosomes, was recently demonstrated and found to be strongly correlated with the maturation arrest of those phagosomes (Chen et al., 2005). Together, all these findings are consistent with the hypothesis that for both the newly formed phagosomes containing live zooxanthellae and the symbiosomes, the normal pathway of phagosome maturation has been altered by the inhabiting zooxanthellae and therefore their further development to fuse with lysosomes is inhibited. Given that symbiosomes are constantly associated with ApRab5 and therefore are likely to fuse with other ApRab5-positive vesicles (clathrin-coated vesicles, early endosomes), and that the size of symbiosomes remains relatively constant throughout the endosymbiotic relationship of zooxanthellae with their animal host, I proposed there must exist mechanisms to balance the fusion interactions (which will increase the size of the symbiosomes through the delivery of new membrane and cargos.) between symbiosomes and endocytic vesicles. Such mechanisms will require a Rab protein to function in the retrieval of membrane and cargos via vesicle budding. Since Rab11 is well known for its function in endocytic recycling, ApRab11 will serve as an ideal candidate for such a role in the regulation of. 4.

(11) vesicle budding from symbiosomes. However, since ApRab11 is not present on symbiosomes as described above, consequently, the involvement of other recycling Rabs should be seriously considered. In current study, I chose to focus on Rab4 and Rab14, two key protein factors shown to involve in the regulation of endocytic recycling (Junutual et al., 2004; McCaffrey et al., 2001; Seabra et al., 2002; Tassula et al., 2006). Complementary DNAs for Aiptasia pulchella Rab4 and Rab14 (named as ApRab4 and ApRab14) proteins were cloned and characterized. A unique finding of my study was the uncovering of two Rab14-like proteins in Aiptasia pulchella, which has never been reported on prior studies. One Rab14-like protein-encoding cDNA of Aiptasia pulchella was short in size (228 bp) and the other was 654 bp long (designated as ApRab14-S and ApRab14-L, respectively).. Complementary DNA for both Rab14-like. proteins were cloned and sequenced, but only ApRab14-L was brought to in-depth assay in current study. The results of functional assays indicated they (ApRab4 and ApRab14-L) play similar roles with their mammalian homologues in intracellular vesicular trafficking. Immunofluorescence assays showed that both ApRab4 and ApRab14-L were localized onto the symbiosomes and early phagosomes, but not detected on mature phagosomes in Aiptaisa digestive cells. Overall, the results of my study suggested that ApRab4 and ApRab14-L are involved in the establishment and maintenance of Aiptasia-Symbiodinium endosymbiosis.. 5.

(12) 2. Materials and Methods. 2.1. Cells and the animal The experimental animal, Aiptasia pulchella, was maintained in laboratory aquaria on a 14/10-h light/dark cycle at irradiance levels of 80–100 photons m-2 s-1. The animals were fed with frozen brine shrimp twice per week. Aquarium water temperature was maintained at around 26–28 ℃, and one half of aquarium water was replaced with fresh filtered seawater (1-µm Millipore) one day after each feeding. Cultured mammalian cells (HeLa cells) were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin sulfate (100 µg/ml) at 37 ℃, in a humid incubator with 5% CO2.. 2.2. Cloning of full-length ApRab4 and ApRab14 cDNA and sequence analysis To clone the full-length cDNA for A. pulchella Rab4 and Rab14 proteins, degenerate polymerase chain reaction (PCR) was first conducted to generate short cDNA fragments coding for conserved Rab protein regions（Thompson et al., 1994; Rose et al., 1998）. According to the degenerate PCR database previously established in our laboratory, we find partial Rab4 sequence and short-form Rab14 full-length sequence from Aiptasia pullchellae cDNA library. And through Rapid amplification cDNA end (RACE) technique. 6.

(13) （Frohman et al., 1988）, we acquired Aiptaisa Rab4 and Rab14 (ApRab4 and short- and long-form ApRab14) full-length cDNA sequences. The primers used for ApRab4 3’ RACE are ApRab4-F1: 5’- CCAGGAGTTACT ACAGAGGACAG -3’ and ApRab4-F2: 5’- TAAAACAGATCTTGATGCC GACAG -3’, and 5’ RACE are ApRab4-R1: 5’- GAACTTCTCTGTCGGCA TCAA -3’, ApRab4-R2: 5’- TCCAGCTGCTCCTCTGTAGTAAC -3’.. 2.3. Construction, expression, and purification of recombinant ApRab4 and ApRab14-L proteins To produce recombinant ApRab4 and ApRab14-L proteins, the coding region of ApRab4 and ApRab14 were PCR-amplified, restriction-digested, and subsequently ligated with identically restriction-digested pET28a vector (Novagen) to generate pHis-ApRab4 and pHis-ApRab14-L. The PCR primers used for the construction of histidine-tagged recombinant ApRab4 and ApRab14 proteins are: ApRab4-pET28a-f: 5’- CGGAATTCATGTCAGAGT CTTAC GACTTCCTCT -3’ (the start codon of ApRab4 is in bold letter, and EcoRI site is underlined), ApRab4-pET28a-r: 5’- CCCTCGAGGCTACTTG CTACAGTTAGCTGGGCA-3’ (the stop codon of ApRab4 is in boldface, and XhoI site is underlined) and ApRab14(L)-pET28a-f:5’-CGCGGATCCATGG CAGCGACAGGGCCGTACAA -3’ (the start codon of ApRab14-L is in bold letter, and. BamHI. site. is. underlined), ApRab14(L)-pET28a-r:. 5’-. CGGAATTCTTAACAATTACACCTGTCTTGGTTCG -3’ (the stop codon. 7.

(14) of ApRab14-L is in boldface, and EcoRI site is underlined). Templates used in the PCRs were the plasmid containing full-length ApRab4 and ApRab14-L cDNA sequence. All PCR-amplified sequences of ApRab4 and ApRab14-L were sequenced at both strands to confirm the amplification of the desired sequences and the absence of any secondary mutations. The in-frame fusion between the histidine tag and ApRab4 and ApRab14-L were also verified by nucleotide sequencing. The bacterial transformation of pHis-ApRab4 and pHis-ApRab14-L, generation of recombinant ApRab4 and ApRab14-L proteins, and their purification were conducted according to the manufacturer’s instruction (Novagen). The recombinant ApRab4 and ApRab14-L proteins were affinity-purified by nickel column as instructed by the manufacturer (Novagen).. 2.4. Prepared of polyclonal antibodies Rats were injected in the abdominal cavity with 350µl emulsion containing 100 µg recombinants ApRab4 and ApRab14-L proteins as antigens in Freund’s complete adjuvant (Sigma) mixed at 1:1. Two weeks later, injection of the animals with the same amount of antigen emulsified with Freund’s incomplete adjuvant (Sigma). After six boosts, whole blood of the animal was collected, and incubated first for 60 min at 37℃ and then at 4℃ overnight. The serum was separated from blood cells by centrifugation at 10,000 g for 10 min at 4℃. The anti-ApRab4 and -ApRab14 IgGs were. 8.

(15) purified from the serum by using AffinityPakTM Protein A Columns (PIERCE).. 2.5. Western blot analysis Cell lysates of A. pulchella were prepared by directly homogenizing the animal in 2–3 volumes of cold homogenization buffer (250 µM sucrose, 10 mM Hepes, pH 7.4, and 0.1 mM EDTA) containing a cocktail of protease inhibitors (Roche) followed by a clarification spin (2000g, 10 min, 3 times) to remove. unbroken. cells,. intact. nuclei,. and. zooxanthellae.. Protein. concentrations were determined by the Bradford method (Bio-Rad Protein Assay Dye Reagent Concentrate). Protein samples (20 µg) were resolved by SDS–PAGE in a 15% polyacrylamide gel and then blotted onto PVDF filters using a semi-dry blotting device (Bio-Rad). The membranes were first incubated with either the rat anti-ApRab4 or ApRab14-L polyclonal antiserum (1:500 dilution) followed by HRP-conjugated goat anti-rat IgG (1:10,000 dilution, Zymed Laboratories). Both antibodies were diluted in 5% milk powder, and 0.05% Tween 20 in PBS. Enhanced chemiluminescence (Western blot chemi- luminescent reagent plus, NEN) was used for the visualization of specific protein bands on X-ray film (BioMax Light, Kodak) or digitization fluorescence LAS-3000 (Fujifilm).. 9.

(16) 2.6. Transfection of mammalian cells The. expression. construct. encoding. either. EGFP-ApRab4. or. EGFP-ApRab14-L fusion protein (EGFP tagged at the N terminus of ApRab4 or ApRab14-L) were constructed in two steps. First, the coding sequence of ApRab4 and ApRan14-L were PCR-amplified from pApRab4 and pApRab14-L (ApRab4 and ApRab14-L cDNA in YEA yT&A vector) using the sense primer, ApRab4-pEGFPc1-f: 5’-CGGAATTCCATGTCAGAGTCT TACGACTTCCTCT-3’,ApRab14(L)-pEGFPc1-f: 5’-GCGGAATTCTATGG CAGCGACAGGGCCGTACAA-3’ (EcoRI site is underlined, the start codons of ApRab4 and ApRab14 are bolded) and the antisense primer, ApRab4-pEGFPc1-r: 5’-CGGGATCCCTACTTGCTACAGTTAGCTGGGC A-3’, ApRab14(L)-pEGFPc1-r:5’-GCGGGATCCTTAACAATTACACCTGT CTTGGTTCG-3’ (BamHI site is underlined, the stop codons of ApRab4 and ApRab14-L are bolded). Then, after restriction digestion with EcoRI and BamHI, the PCR product was ligated with identically pre-digested pEGFPc1 vector (Clontech) to create pEGFP-ApRab4 and pEGFP-ApRab14-L plasmid. To construct the plasmid encoding EGFP-tagged mutant ApRab4 and ApRab14-L. proteins. where. S22. or. S26. were. mutated. to. N. (pEGFP-ApRab4-S22N, pEGFP-ApRab14-L-S26N) and Q67 or Q71 were mutated to L (pEGFP-ApRab4-Q67L, pEGFP-ApRab14-L-Q71L), eight partially overlapping mutagenic primers, ApRab4-S22N-f: 5’-CACGGGAAA AAACTGCATCCTGCATCAGTTTAT-3’, ApRab4-S22N-r: 5’-GCAGGAT. 10.

(17) GCAGTTTTTTCCCGTGCCAGCACTTC-3’, Rab4-Q67L-f: 5’-CACTGCT GGCCTAGAACGTTTCAGGT CAGTAAC-3’, ApRab4-Q67L-r: 5’-TGAA ACGTTCTAGGCCAGCAGTGTCCCATATCT-3’ and ApRab14(L)-S26N-f: 5’-TGTTGGTAAGAACTGTCTCCTTCACCAATTCAC-3’, -S26N-r:. ApRab14(L). 5’-GAAGGAGACAGTTCTTACCAACACCCATATCAC-3’,. ApRab14(L)-Q71L-f:5’-TACGGCAGGACTAGAACGATTCAGGGCTGT AAC-3’,. ApRab14(L)-Q71L-r:. 5’-TGAATCGTTCTAGTCCTGCCG. TATCCCATATCT-3’ (mutagenic bases were boxed, overlapping regions were underlined), were synthesized and paired with pEGFPc1-f and pEGFPc1-r. in. two. separate. PCRs. using. pEGFP-ApRab4. and. pEGFP-ApRab14-L as template to generate two overlapping cDNA fragments. Then a second round of PCR using the purified cDNA fragments generated above as the templates and pEGFPc1-f and pEGFPc1-r as the primers was conducted to generate complete ApRab4 and ApRan14-L coding sequence containing the desired single amino acid mutation. This cDNA was ligated with pEGFPc1 vector (Clontech) pre-digested with EcoRI and BamHI to. create. pEGFP-ApRab4-S22N,. pEGFP-ApRab14-L-S26N. and. pEGFP-ApRab4-Q67L,. pEGFP-ApRab14-L-Q71L. plasmid.. Sequencing was performed to verify the intended amplification and the absence of any secondary mutations in the coding sequence of ApRab4 and ApRan14-L. For cell transfection, both newly confluent HeLa cells were seeded at 2 x 105 cells to each well of a six-well culture plate one day before. 11.

(18) transfection was conducted. Two µg of either pEGFP or pEGFP-ApRab4 or ApRan14-L was introduced into these cells using FuGENE 6 transfection reagent (Roche). Fluorescence images of living cells were immediately taken without prior fixation using a Nikon ECLIPSE E400 microscope equipped with appropriate filter sets and a digital imaging device (Evolution VF COOLED COLOR, Media Cybernatics).. 2.7. Cell image To determine the identity of pEGFP-ApRab4 and pEGFP-ApRab14-L positive structures, sub-confluent HeLa cells were first transfected using FuGENE 6 transfection reagent (Roche) with the plasmid encoding the fusion protein. At 16-h post-transfection, cells were incubated either with 1 µM transferrin-Texas Red (Sheff, 2002; Widera et al., 2003) for 30 min at 37 ℃ to label all recycling endosomes, or with 5 mg/ml dextran-TRICT (70 kDa) for 10 min and 30 min at 37 ℃ to label early and late endocytic compartments, respectively, or with 75 nM LysoTracker-red for 15 min to label acidic compartments, or TR-C5-ceramide (Invitrogen) for 30 min to label sphingolipids (the Golgi apparatus). Fluorescence images of living cells were immediately taken without prior fixation using a Nikon ECLIPSE E400 microscope equipped with appropriate filter sets and a digital imaging device (Evolution VF COOLED COLOR, Media Cybernetics). To investigate the subcellular localization of endogenous ApRab4 and ApRab14-L, endodermic. 12.

(19) tissues of symbiotic A. pulchella were fixed by acidic solution (acetic acid : glycerol : filtered seawater = 1:1:13 ) 10 min and dissociated into single cells by pipetting as described (David, 1973). The generated cell suspension was transferred onto poly-lysine coated glass slides and allowed to adhere to the slides for 30 min at room temperature. DCMU (3-(3, 4-dichlorophenyl) -1, 1-dimethylurea)-treated animals were processed in the same way. These cells were brought through standard immunostaining procedures with the rat anti-ApRab4 and ApRab14-L polyclonal IgGs used at 1:30 (ApRab4) or 1:60 (ApRab14-L) dilution and the Cy3- conjugated goat anti-rat IgG (Zymed Laboratories) at 1:1000 dilution. Both antibodies were diluted in 1x PBS containing 0.1% Triton X-100. The cells were imaged with a Nikon ECLIPSE E400 microscope equipped with a TRICT filter set and a digital imaging device (Evolution VF COOLED COLOR, Media Cybernatics).. 2.8. Phagocytosis assay Animals were starved at least for 3 days before being challenged with either 2 μm latex beads (Sigma), live zooxanthellae freshly isolated from symbiotic A. pulchella, or zooxanthellae immediately heat-killed after isolation. All particles diluted in frozen Brine shrimp solution were injected into the gut of the animal in a volume of 20 μl through a standard white micropipette tip. All injections and incubations were conducted at room temperature under ambient illumination. For each phagocytosis assay,. 13.

(20) approximately 1x105 particles were injected. After fixed time intervals, three animals. from. each. challenging. experiment. were. processed. for. immunofluorescence.. 3. Results. 3.1. Cloning of full-length ApRab4 and ApRab14 cDNA Two partial cDNA clones with high sequence homology to human Rab4 and Rab14 were initially identified in a degenerate RT-PCR cloning project conducted previously to identify most Aiptasia Rab proteins. Nested primers were then designed and used in combination with vector primers to PCR-amplify the missing 5’ and 3’ sequences by the RACE approach as described under “Materials and methods”. Contigs of 854 bp (ApRab4), 746 bp (ApRab14-S) and 1230 bp (ApRab14-L) in length were thus assembled, and that continuity were confirmed by the amplification of the sequence from the Aiptasia cDNA library using primers corresponding to the extreme 5’ and 3’ ends of the assembled sequence. As shown in Fig. 1, the ApRab4 cDNA contains a 145-bp 5’ untranslated region (UTR), a 654-bp open reading frame (nucleotide 145–799), a 55-bp 3’ UTR, a putative polyadenylation signal (ATTAAA, nucleotide 805–811), and a 28-bp poly (A) tail. On the other hand, the ApRab14-L cDNA contains a 97-bp 5’ untranslated region (UTR), a 654-bp open reading frame (nucleotide 97–751), a 479-bp 3’ UTR, a putative. 14.

(21) polyadenylation signal (AAAATT, nucleotide 1191–1196), and a 22-bp poly (A) tail (Fig. 3). Finally, the ApRab14-S cDNA contains a 97-bp 5’ untranslated region (UTR), a 228-bp open reading frame (nucleotide 97–325), a 379-bp 3’ UTR, a putative polyadenylation signal (ATATTG, nucleotide 358–363) as shown in Fig. 2. The deduced ApRab4 and ApRab14-L proteins are both 218 amino acids (24 kDa) in length, while ApRab14-S is 75 amino acids (8.3 kDa) long.. 3.2. Alignment of ApRab4 and ApRab14-L proteins with their relatives Blastp search of the GenBank database showed that the deduced amino acid sequences of these cDNAs were highly homologous to those of known Rab4 and Rab14 proteins, with the highest overall sequence identity (87% and 88%) to human Rab4 and Rab14 proteins, respectively. Multi- alignment analysis further revealed the extensive homology ApRab4 and ApRab14-L with Rab4 and Rab14 proteins from several model organisms, indicating that these proteins share similar three-dimensional conformation (Fig. 4, 5). In additional, sequence features shared by all GTP-binding proteins, in particular, the members of the Rab family, are all present on the deduced protein, including the four functional motifs involved in GTP-binding (G1–G4), the five Rab- specific motifs (RabF1–5), and a double cysteine (CPANC)directed of ApRab4 and (CNC)-directed prenylation of ApRab14-L signal at. 15.

(22) the C-terminal end （Stenmark and Olkkonen, 2001；Pereira-Leal and Seabra, 2000- 2001）. When the same set of Rab4 and Rab14 proteins sequences were subjected to phylogenetic analysis, ApRab4 and ApRab14-L were found to be more closely related to Xenopus tropicalis (frog) and Caenorhabditis elegans (worm), respectively (Fig. 6).. 3.3. Characterized subcellular localization of EGFP-ApRab4 and EGFP-ApRab14-L in HeLa cells To determine whether the homology of ApRab4 and ApRab14-L to others Rab4 and Rab14 proteins would extend to the functional level, I investigated the intracellular localization of ApRab4 and ApRab14-L. To this end, I constructed a series of recombinant plasmids encoding either EGFP-tagged. ApRab4,. ApRab14-L. protein. (pEGFP-ApRab4,. pEGFP-ApRab14-L), or their mutants (including pEGFP- ApRab4-S22N, pEGFP-ApRab4-Q67L,. pEGFP-ApRab14-L-. S26N,. and. pEGFP-ApRab14-L-Q71L) and expressed them in HeLa cells. The G1 (Ser/Thr to Asn) mutants of ApRab4 and ApRab14-L (ApRab4-S22N and pEGFP-ApRab14-L-S26N) are likely to retain an inactive, GDP-bound conformation, while their G2 (Gln to Leu) mutants (pEGFP-ApRab4-Q67L and pEGFP-ApRab14-L-Q71L) are supposed to be in constitutively active, GTP-bound conformation.. As shown in Fig. 8, EGFP fluorescence of. GTP-bound ApRab4 was mainly associated with vesicular structures of. 16.

(23) irregular sizes dispersed throughout the cytoplasm. On the contrary, GDP-bound ApRab4 displayed a diffusive distribution pattern in the cytoplasm. With regard to ApRab14-L, according to EGFP fluorescence, GTP-bound ApRab14-L not only localized on the irregular vesicular structures throughout the cytoplasm, but also accumulated at one side of the nucleus. Finally, GDP-bound ApRab14-L showed strong tendency to cluster around the nucleus.. 3.4. Characterized of EGFP-ApRab4 labeled structures To further identify the localization of ApRab4 on endocytic pathway, HeLa cells were transfected with the plasmid encoding EGFP-ApRab4. Different species of endocytic compartments were labeled with Texas red-conjugated transferrin（Ghosh and Maxfield, 1995）, TRITC-conjugated dextran, or LysoTracker, by the procedure described under “Materials and methods”. Co-localization of ApRab4 with dextran was apparent as indicated by strong overlapping signals after labeling for 10 minutes but significantly reduced when the labeling was prolonged for 30 minutes (Fig. 10). This result suggested that ApRab4 localizes preferentially to early endosomes. When the degree of co-localization between transferrin, and EGFP-ApRab4 signals were examined, high levels of co-localization were noted when HeLa cells were labeled with transferrin for 30 minutes to saturate all recycling endosomes (Fig. 9). This result suggested that ApRab4 also localizes to. 17.

(24) recycling endosomes. Finally, as shown in Fig. 12, there was no overlapping between the green fluorescence signals of EGFP-ApRab4 and the red fluorescence signals of LysoTracker, which indicated that ApRab4-positive vesicles are not acidic compartments. Overall, the results of my study suggested ApRab4 is most likely present on both early endosomes and transferrin-positive recycling endosomes.. 3.5. Cha ra cterize d of EGFP–ApRab14-L labeled structures To determine the identities of EGFP-ApRab14-L -positive structures and the role of ApRab14-L in the trafficking between trans-Golgi network and early endosomes, HeLa cells were first trasfected with the plasmid encoding EGFP-tagged ApRab14-L, and then incubated with TRITC- dextran, transferrin-Texas Red, LysoTracker, or TR-C5- ceramide to specifically label early, late endosomes, recycling endosomes, lysosomes and the Golgi apparatus. As shown in Fig. 9 and 11, EGFP signals of GTP-bound ApRab14-L and ApRab14-L were highly co-localized with the structures labeled by 10-min incubation of either dextran or transferrin. Such a high degree of co-localization gradually disappeared when the incubation period was prolonged to 30 minutes (Fig. 9.11). When ApRab14-expressing HeLa cells were stained with TR-C5-ceramide to label the Golgi apparatus, it was found that GDP-bound ApRab14 displayed a very extensive co-distribution with the Golgi apparatus, whereas the wild type ApRab14 only partially. 18.

(25) overlapped with the red TR-C5- ceramide signals (Fig. 13). Finally, as observed for ApRab4, there was no overlap between the red signals of LysoTracker and the green signals of EGFP-ApRab14 (Fig. 12). Overall, the results of my study suggested that EGFP-ApRab14 protein was present on both early endosomes and the Golgi apparatus.. 3.6. Cha ra cterize d of antibodies by western blot analysis To investigate the potential involvement of ApRab4 and ApRab14-L in the regulation of phagosome maturation in Aiptasia phagocytic cells, two specific rat polyclonal antisera, one specific for ApRab4, the other for ApRab14, were prepared as described in the ”Materials and Methods” using the corresponding affinity-purified histidine-tagged ApRab proteins as the immunogens, and used as specific probes for the detection of endogenous ApRab4 and ApRab14-L proteins, respectively. To determine whether these antibodies can specifically recognize ApRab4 and ApRab14-L in highly complex protein mixtures, I prepared and analyzed total Aiptasia tissue homogenates by Western blot using the rat anti-ApRab4 and -ApRab14-L antisera as the primary antibodies. As revealed in Fig. 7, the rat anti-ApRab4 antiserum recognized a single protein band of approximately 24 kDa in size, which is in close agreement with the expected size of ApRab4. On the other hand, the rat anti-ApRab14-L antiserum strongly reacted with two protein bands of approximately 24 kDa and 8 kDa in size, both of which are. 19.

(26) consistent with the calculated sizes of ApRab14-L and ApRab14-S, respectively. Together, the results of my Western blotting analyses strongly suggested that the two antisera I have prepared are capable of specifically detect their corresponding antigens, and therefore are suitable for immunocytochemical studies, which I described below.. 3.7. Localized of endogenous ApRab4 and ApRab14-L in the host cells of zooxanthellae by immunofluorescence analysis To examine whether ApRab4 and ApRab14-L are involved in the maintenance of endosymbiotic association of zooxanthellae with their host sea anemones, symbiotic A. pulchella was processed for ApRab4 and ApRab14-L immunofluorescence analysis (Fig. 14) with or without prior treatment of the photosynthesis inhibitor, DCMU, and the percentages of symbiosomes (phagosomes containing resident zooxanthellae) associating with either ApRab4 or ApRab14-L were then determined. In control animals (0-h DCMU treatment), a major proportion of symbiosomes were found to possess detectable ApRab4 (84%) and ApRab14-L (76%) staining signals, and a sharp decrease (more than four folds) in the positive counts of either ApRab4- or ApRab14-L- associated symbiosomes were obtained after only 1-h DCMU treatment (Fig. 16). This finding of my study demonstrated clearly that the association of ApRab4 and ApRab14-L with the symbiosomes is brought about by a yet unknown effect exerted by the healthy. 20.

(27) zooxanthellae dwelling inside.. 3.8. Involvement of ApRab4 and ApRab14-L in Phagocytosis To investigate the potential involvement of ApRab4 and ApRab14-L in the establishment and/or maintenance of the endosymbiotic association of zooxanthellae with their native animal host A. pulchella, immunofluorescence analysis was employed to examine the association of ApRab4 and ApRab14-L with the phagosomes containing newly internalized latex bead. As shown in Fig. 15, ApRab4 and ApRab14-L staining signals were apparently associated with latex bead-containing phagosomes. Noticeably, these signals were distributed as discrete patches around the phagosomes. As short as 15 minutes after phagocytosis, approximately 74% and 80% of phagosomes containing latex beads were already associated with ApRab4 and ApRab14-L,. respectively.. The. percentage. of. ApRab4-. or. ApRab14-L-positive phagosomes gradually decreased to respective 16% and 12% of the phagosome population at the end of the phagocytosis assays (45 minutes) (Fig. 17). These results strongly suggested that both ApRab4 and ApRab14 preferentially associate with early phagosomes, and therefore are likely participating in the early phases of phagosomes maturation through regulating vesicle budding from the maturing phagosomes. On another phagocytosis assay by feeding live or heat-killed zooxanthellae, 50% and 60% of 15 minutes old phagosomes containing heat-killed zooxanthellae were. 21.

(28) positive for ApRab4 and ApRab14-L signals. They were decreased to 20% and 50% in 30-minutes old phagosomes and further down to 15% and 10% in 45-minutes old phagosomes as shown in Fig. 18. For feeding with live zooxanthellae, there are 45% and 55% of 15-minutes-old phagosome population which were detected ApRab4- and ApRab14-L-signals around phagosomes. But not decreased whether on 30-minutes-old phagosomes or 45-minutes-old phagosomes, they were still highly retained on them, as shown in Fig. 19.. 22.

(29) 4. Conclusion and Discussion. In this work, cDNAs for ApRab4 and two isoforms of ApRab14 have been successfully cloned and partially characterized. (Fig. 1-3). These full-length cDNA sequences contain 5’ and 3’ UTR, start and stop codon, poly (A) tails, Rab family specific conserved domains and double cysteine at 3’ end of coding region (except ApRab14-S had no double cysteine). Multi-alignment analysis of ApRab4 and ApRab14-L indicated that they are highly homologues with human Rab4 and Rab14. Therefore, ApRab4 and ApRab14-L were belonged to family of Rab protein (Fig. 4-6). In my attempt to clone the A. pulchella homologue to ApRab14, two ApRab14-like sequences were detected. The longer one, ApRab14-L, is 654 bp in length, while the shorter one, ApRab14-S, 228 bp. The amino acid sequence of ApRab14-S was essentially identical to that of ApRab14-L N-terminus with two exceptions. The first one occurred at the 51th amino acid (Thr/Pro) and the second being the last 5 amino acids of ApRab14-S. Because of the 51th amino acid alteration, the folding of ApRab4-S is probably changed to a significant level. In addition, since ApRab14-S had no double cysteine at its C-terminus, subsequently prenylation is not likely. I proposed that ApRab14-S may function by interacting with ApRab14-L. According to the EGFP report analysis described above, ApRab4 was present on the transferrin-positive structures (Fig. 9) and early endosomes. 23.

(30) (Fig. 10). These results indicate that EGFP-ApRab4-decorated structures are indeed recycling endosomes, and consequently, normal endocytic recycling will likely be inhibited by GDP-bound ApRab4.. Examination in. EGFP-ApRab14-L expressing cells showed that ApRab14-L was present on dextran-, transferrin-, or TR-C5-ceramide-positive structures (Fig. 9, 11, 13). And, when GDP-bound EGFP-ApRab14-L was expressed in HeLa cells, the green fluorescence mostly overlapped with TR-C5- ceramide staining signals. This result suggested that ApRab14 in its GDP-bound state will locate on the Golgi apparatus (Fig. 13). All results suggested that ApRab14-L participate in the recycling pathway and membrane trafficking between the Golgi apparatus and early endosomes. The intracellular localization patterns of ApRab4 and ApRab14-L that I have observed are consistent with previous mammalian studies, and therefore, their function may be similar to their mammalian homologues. To determine whether or not the rat anti-ApRab4 and the rat anti-ApRab14-L antiserum that I have prepared can specifically recognize ApRab4 and ApRab14-L in total Aiptasia tissue homogenates, Western blot analysis was executed, as shown in Fig. 7. A major band approximately 24 kDa in size was detected by the rat anti-ApRab4 antiserum, which was close to the calculated molecular weight of ApRab4 protein. Western blot by using the rat anti- ApRab14-L antiserum revealed two major bands. One was approximately 24 kDa and the other was 8 kDa in size, which were in close. 24.

(31) agreement with the calculated molecular weight of ApRab14-L and ApRab14-S proteins, respectively. Therefore, it was concluded that the two antisera were able to specifically recognize the three intended proteins in Aiptasia total protein preparation. Although the rat anti-ApRab14-L polyclonal antibody reacted both with ApRab14-S and ApRab14-L, but sequence analysis of ApRab14-S protein was shown there are no double cysteine in its C-terminus. All previous studies report that all Rabs require a double C-terminus cysteine to anchor to their target membrane (Casey and Seabra, 1996). So I proposed ApRab14-S protein can not anchor to membrane by itself. The signals detected on organelle membrane by the rat anti-ApRab14-L polyclonal antibody could come from two potential sources, one from ApRab14-L, and the other from the complex between ApRab14-S and ApRab14-L. But no research has reported that Rabs can interact with each other and form a complex. Therefore, there is high probability that the signals detected on organelle membrane are from ApRab14-L protein. Accordingly, it should be appropriate to use the rat anti-ApRab14-L antibody be in immunofluorescence assays to detect ApRab14-L protein. In my phagocytosis assays (Fig.17, 18, 19), phagosomes containing newly acquired dead algae seemed to follow a maturation path similar to that taken by phagosomes containing latex beads—quick acquisition of and then gradual removal of ApRab4 and ApRab14-L from phagosome membrane, except that the former appeared to develop at a bit faster pace. A different. 25.

(32) picture emerged when ApRab4 or ApRab14-L association with phagosomes containing newly internalized live zooxanthellae was examined. Although a general decreasing trend was followed, significantly lower percentages of these phagosomes were positive for ApRab4 and ApRab14-L. In all cases, the positive counts (in percentage) of ApRab4- and ApRab14-L-associated phagosomes containing newly internalized live zooxanthellae ranged from a mere quarter to one-half of those for the heat-killed zooxanthellae-containing counterparts. These results were shown that ApRab4 and ApRab14-L were maintained in early stage of phagosome maturation and indicated normal phagosome maturation pathway was interfered by live zooxanthellae. That‘s a reason which ApRab4 and ApRab14-L protein is discovered on the symbiosomes (modified phagosomes by live zooxanthellae), but not on the phagosomes containing dead zooxanthellae. Results of phagocytosis assays and immunofluorescence assay showed that ApRab4 and ApRab14-L were likely involved in early stage of phagosome maturation and were present on the zooxanthellae-housing symbiosomes in Aiptasia digestive cells. The combined results described above. suggested. that. ApRab4. and. ApRab14-L. function. on. Aiptasia-Symbiodinium endosymbiosis and are involved in the regulation of endocytic recycling and vesicle transport between symbiosomes and the Golgi apparatus of Aiptasia host cells. Overall, the results of above experiments strongly suggest that both ApRab4 and ApRab14-L play critical. 26.

(33) roles in the endocytic recycling pathway and the biogenesis of symbiosomes in the Aiptasia-Symbiodinium endosymbiosis.. 27.

(34) 5. References Armstrong, J. (2000). How do Rab proteins function in membrane traffic (Review)? Int. J. Biochem. Cell Biol. 32: 303- 307. Birkeland, C. (1997). Life and Death of Coral Reefs, Chapman and Hall, New York. Casey, P.C. and Seabra M.C. (1996). Protein prenyltransferases (minireview). J. Biol. Chem. 271: 5289-5292. Chavrier, P. and B. Goud (1999). The role of ARF and Rab GTPases in membrane transport (Review). Curr. Opin. Cell Biol. 11: 466- 475. Chen, M.C. et al (2003). Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae. Biochem. Biophys. Res. Commun. 308: 586- 595. Chen, M.C. et al (2004). Molecular identification of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae. Biochem. Biophys. Res. Commun. 324: 1024- 1033. Chen, M.C. et al (2005). ApRab11, a cnidarian homologue of the recycling regulatory protein Rab11, is involved in the establishment and maintenance of Aiptasia-Symbiodinum endosymbiosis . Biochem. Biophys. Res. Commun. 338: 1607- 1616. Connell, J.H. (1978). Diversity of tropical rainforests and coral reefs, Science. 199: 1302–1310. Cox, D. et al (2000). A Rab11-containing rapidly recycling compartment in. 28.

(35) macrophages that promotes phagocytosis. Proc. Natl acad. Sci. U.S.A. 97: 680-685. David, C. N. (1973). A quantitative method for maceration of hydra tissue, Roux’s Arch. Dev. Biol. 171: 259-268. Desjardins, M. and G. Griffiths (2003). Phagocytosis: latex leads the way (Review). Curr. Opin. Cell Biol. 15: 498- 503. Fitt, W.K. and R.K. Trench (1983). Endocytosis of the symbiotic dinoflagellate Symbiodinium microadariaticum freudenthal by endodermal cells of the scyphistomae of Cassiopeia xamachama and resistance of the algae to host digestion. J. Cell Sci. 64: 195- 212. Frohman, M.A. et al (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. U. S. A. 85: 8998- 9002. Ghosh, R.N. and F.R. Maxfield (1995). Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HEp2 cells. J. Cell Biol. 128: 549- 561. Hatcher, B.G. (1990). Coral reef primary productivity: a hierarchy of pattern and process. Trends Ecol. Evol. 5: 149–155. Hohman, T.C. et al (1982). Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis. J. Cell Biol. 94: 56- 63. Jackson, J.B.C. (1991). Adaptation and diversity of reef corals. BioScience 41: 475–482.. 29.

(36) Junutula, J.R. et al (2004). Rab14 is involved in membrane trafficking between the Golgi complex and endosomes. Mol. Biol. Cell. 15: 22182229. Martinez, O. and B. Goud (1998). Rab proteins (Review). Biochim. Biophys. Acta. 1404: 101- 112. McCaffrey, M.W. et al (2001). Rab4 affects both recycling and degradative endosomal trafficking. FEBS letters. 495: 21-30. Nybakken, J.W. (2001). Marine Biology/5th. Benjamin Cummings. San Francisco. Pereira-Leal, J.B. and M.C. Seabra (2000). The mammalian Rab family of small GTPase: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. J. Mol. Biol. 301: 1077- 1087. Pereira-Leal, J.B. and M.C. Seabra (2001). Evolution of the Rab family of small GTP-binding proteins. J. Mol. Biol. 313: 889- 901. Pfeffer, S.R. (2001). Rab GTPases: specifying and deciphering organelle identity and function (Review). Trends. Cell Biol. 11: 487- 491. Rose, T.M. et al (1998). Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 26: 1628- 1635. Seabra, M.C. et al (2002). Rab GTPases, intracellular traffic and disease. (Review) Trends. Mol. Med. 8: 23- 30.. 30.

(37) Segev, N. (2001). Ypt and Rab GTPase: insight into functions through novel interactions (Review). Curr. Opin. Cell Biol. 13: 500- 511. Sheff, D. et al (2002). Transferrin receptor recycling in the absence of perinuclear recycling endosomes. J. Cell Biol. 156: 797- 804. Stein, M.P. et al (2003). Rab proteins and endocytic trafficking: potential targets for therapeutic intervention. Adv. Drug. Deliv. Rev. 55: 14211437. Stenmark, H. and V.M. Olkkonen (2001). The Rab GTPase family (Review). Genome Biol. 2: 3007.1- 3007.7. Tassula, P.C. et al (2006). Rab14 is part of the early endosomal clathrin-coated TGN microdomain. FEBS letters. 580: 5241-5246. Thompson, J.D. et al (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673- 4680. W. Souter, D. and O. Linden (2000). The health and future of coral reef systems. Ocean Coast. Manage. 43: 657- 688. Widera, A. et al (2003). Mechanisms of TfR-mediated transcytosis and sorting in epithelial cells and applications toward drug delivery. Adv. Drug. Deliv. Rev. 55: 1439- 1466. Zerial, M. and H. McBride (2001). Rab proteins as membrane organizers (Review). Nat. Rev. Mol. Cell Biol. 2: 107- 117.. 31.

(38) 6. Figures ApRab4. full length cDNA. AGCAGTGGTA T CAAC GC AG AGT GACC AT TACGG C C GGGG AG AG AAT T GT GGGTAA A C AT C AG A C G T G A A G G T T TAT T T T G TA A A G T G T TA A A A C T T T G G A T T G A A AT TA A G T C G AT C TA A A G G C T T G A A A A C AC A A G C AT T T GT C A T G T C AG A G T C T T AC G AC T T C C T C T T C A AG T T T C T C G T G AT T G G A M S E S Y D F L F K F L V I G AGT G CT GGC A CG GG AAA AT C AT GC A T CCT GC AT C AGT T T ATT GAA S A G T G K S C I L H Q F I E GG AAA AT T T AAAC AAG AT T C C AGT C AT AC A AT T G GT GT T GAAT T T G K F K Q D S S H T I G V E F GGTT CC AAA A TT AT T AAT GT T GGAG GC AAAT CT GT G AAGT T GC AG G S K I I N V G G K S V K L Q AT AT GGGACACT GCT GGCCAAG AAC GTTTCAGGT CAGT AACC AGG I W D T A G Q E R F R S V T R AGT T ACT AC AG AGG AG C AG CT GG AG CT CT ACT T GTTT AT G AC AT A S Y Y R G A A G A L L V Y D I T C C AGT C GT GA AA C C T T C AAT T C CT TG AC AA AC T G GT T G AC AG AT S S R E T F N S L T N W L T D G C AAG A AC T C T AG C AAG T C C C AAC A T T GT G AT AAT T CT T GTT G GT A R T L A S P N I V I I L V G AAT AAAAAAG AT CTT GAT GCCGAC AGAG AAGT T ACCTT CTTAG AA N K K D L D A D R E V T F L E GCC AG C AG AT TT GCAC AGG AAAAT G ACT T GAT TTT CTT GGAG AC A A S R F A Q E N D L I F L E T AGT GC AT T ATCT GGAG AAAAT GT GGAG G AAG GT TTTTT AAAAT GC S A L S G E N V E E G F L K C T CAAG AAC AATT CT AAAC AAAAT T GAAT CAGGT GAACTT GACCC A S R T I L N K I E S G E L D P GAC AG AAT G G GTT CAGGT AT T C AAT AT GGT G AC C C AT C AT T ACG A D R M G S G I Q Y G D P S L R CGAACCTTACGACCTGGAAGAGGAAGAGGAGAATCAAGGAGCAAG R T L R P G R G R G E S R S K T GCCCAGCT AACT GT AGCAAGTA GTGAAC C ATT AAAT GATAT AAA C P A N C S K * CAGTCGAAAAAAAAAAAAAAAAAAAAAAAAAAAA. 10 55 100 145 190 15 235 30 2 80 45 325 60 370 75 415 90 460 105 505 120 55 0 135 595 150 640 165 6 85 180 730 195 775 210 82 0 217 854. Fig. 1. Primary sequences of A. pulchella Rab4 (ApRab4). The start codon and a proceeding in-frame stop codon are in bold letters. The stop codon is indicated by an asterisk. The putative polyadenylation signal is underlined.. 32.

(39) ApRab14-L full length cDNA GGCCATT 7 A C G G C C G G G A AT C A C G A G AT C C G C C AT C T T T C C TA A A G T C T T C T T 52 C C G G AT T T C TA ATATA AT T TA ATAT T C C C T TA C C A G G T C G C A A A G 97 A T G G C A G C G A C A G G G C C G T A C A A T T A T T C C T A T AT A T T C AA G T A T 142 M A A T G P Y N Y S Y I F K Y 15 ATT ATT AT AGGT GAT AT GGGTGTTGGT AAGT CAT GTCT CCTTCAC 187 I I I G D M G V G K S C L L H 30 C A AT T C A C A G A A A A G A A AT T C AT G G C A G A C T G C C C T C A C A C A A T T 232 Q F T E K K F M A D C P H T I 45 G GT GT AG AGT T T G G C AC A AG A AT AA T AG AG G T AG CT G G AC A A A AG 277 G V E F G T R I I E V A G Q K 60 AT C A AG C T T CAG AT AT G G G AT AC G G C AG G AC A AG A A C G AT T C AG G 322 I K L Q I W D T A G Q E R F R 75 G C T G T A A C T A G A A G C T A C T A C A G A G G G G C A G C C G G C G C T C T T AT G 367 A V T R S Y Y R G A A G A L M 90 G T AT AT G A C A T T A C A A G G A G A A G T A C AT A C A A C C AT C T G A G T AG C 412 V Y D I T R R S T Y N H L S S 105 T G G C T G A C A G AT G C T AG A A AT C T A A C C A A C C C T A AT A C A G T A AT A 457 W L T D A R N L T N P N T V I 120 TTTCTAATTGGTAACAAATCAGATTTGGACGCACAGAGAGATGTT 502 F L I G N K S D L D A Q R D V 135 A C T T AT G A G G A A G C A A A A C A G T T T G C T G A A G A A A A T G G T C T G T T A 547 T Y E E A K Q F A E E N G L L 150 T T T T G T G A A G C T A G T G C A A A A A C A G G T G A A A AT G T T G A A G AT G C T 592 F C E A S A K T G E N V E D A 165 T T C T T AG A A A C T G C A A A A A A G AT T T A C C A A A AT AT T C A A G AT G G G 637 F L E T A K K I Y Q N I Q D G 180 AG T T T G G A C C T C A AT G C A G C C G A G T C C G G AG T G C A A C A C A A A C C C 682 S L D L N A A E S G V Q H K P 195 AT G A C A G G C A G AC C T G C C A AT G C C C T G A A C A C G G AT C AG C C AT C G 727 M T G R P A N A L N T D Q P S 210 A A C C A A G A C A G G T G T A AT T G T T A A T G A AT T G A G T AT G C T G T A C A G 772 N Q D R C N C * 217 T C T T T TA C A AT G TA AT TAT C A A G G A C A A A C AT C C C C T G C A A G T C A 817 C G TA A A C A A AT C AT G A C AT G T T T C T C TA A G G C T TA C T G T T G TA A C 862 AT C T C T T C T C AT TAT TATA A ATA C A AT C TA G A A C A A A G G A A A A A C 907 A A A A A G T T T G A A C TATA G TAT C A G C A A A A C TAT T T T T T T G A G G T G 952 A C A C A A A C T T G A C A ATA C T C ATA A ATA G C T T T G TATA G TATAT TA 997 G G TA ATAT G T C T G TA A A G C C T C C AT T T C G AT TAT TA G C A A C T G G A 1 0 4 2 G A G A A G A AT T TA AT T C AT G A ATA A ATA C T G G TA G A A AT T C TA A G T 1 0 8 7 C AT C T G G A C T G C C C G T C TAT T T T G C T C A A C TA G G G C C G G C C A G C C 1 1 3 2 C T C C C C A AT C C C C T TA G G G C C G C TA C G C A G G C TA C TAT T TAT C T C 1 1 7 7 C T TA A G C T G A AT C A A A AT T TA C A A G T TA A G G A A A A A A A A A A A A A A 1 2 2 2 1230 AAAAAAAA. Fig. 2. Primary sequences of A. pulchella Rab14-L (ApRab14-L). The start codon and a proceeding in-frame stop codon are in bold letters. The stop codon is indicated by an asterisk. The putative polyadenylation signal is underlined.. 33.

(40) ApRab14-S full length cDNA GGCCATT. 7. ACGGCCGGGAATCACGAGATCCGCCATCTTTCCTAAAGTCTTCTT. 52. CCGGATTTC TAATATAATTTAATATTCCCTTACCAGGTCGCAAAG. 97. ATGGCAGCGACAGGGCCGTACAATTATTCCTATATATTCAAGTAT. 142. M. A. A. T. G. P. Y. N. Y. S. Y. I. F. K. Y. ATTATTATAGGTGATATGGGTGTTGGTAAGTCATGTCTCCTTCAC I. I. I. G. D. M. G. V. G. K. S. C. L. L. H. CAATTCACAGAAAAGAAATTCATGGCAGACTGCCCTCACACAATT Q. F. T. E. K. K. F. M. A. D. C. P. H. T. I. GGTGTAGAGTTTGGCCCAAGAATAATAGAGGTAGCTGGACAAAAG G. V. E. F. G. P. R. I. I. E. V. A. G. Q. K. ATCAAGCTTCAGATATGGGATACGGCAGGCTACTATTTATCTCCT I. K. L. Q. I. W. D. T. A. G. Y. Y. L. S. P. TAAGCTGAATCAAAATTTACAAGTTAATGAAAAACATATTGAATT. 15 187 30 232 45 277 60 322 75 367. * CTCATTTGATTATAAACTCCTTGCAGTTAGTCTCTTGTGTCTTCA. 4 12. CATTGTAGCTAGATTAATGTGATGACACCGGAAATAGCTGCAAGG. 457. AGCCTAACTGAACGAAGCTTAACTGTTGTTACAGTAAAATATATT. 502. T GCT GTGGTTAAAAT TAC ATT GTATT CAGGATTTGAATTTTTTTT. 5 47. CAATAAGATAAGACAGCTCTTCATTTGTTAAAATGGCTGATAAAC. 592. TACACTAATTGACAGTGAATGTAATTTGAATATTGTCATAGCAAC. 637. CCAGAATTCTACATTTCCATGACAACCACTGNTGTCTATAACATT. 682. GTCATGGTAACTTAGAAGAAGT. 704. Fig. 3. Primary sequences of A. pulchella Rab14-S (ApRab14-S). The start codon and a proceeding in-frame stop codon are in bold letters. The stop codon is indicated by an asterisk. The putative polyadenylation signal is underlined.. 34.

(41) ApRab4.PRO fish.PRO fly.PRO frog.PRO human.PRO mouse.PRO rat.PRO. : : : : : : :. * 20 * 40 * MSESYDFLFKFLVIGSAGTGKSCILHQFIEGKFKQDSSHTIGVEFGSKIINVGGK MSETYDFLFKFLVIGSAGTGKSCLLHQFIENKFKQDSNHTIGVEFGSRVVNVGGK MSETYDYLFKFLIIGSAGSGKSCLLHHFIESKFKDDSSHTIGVEFGSRIVNVGGK MSETYDFLFKFLVIGSAGTGKSCLLHQFIESKFKQDSNHTIGVEFGSRIVNVGGK MAEDRHFLFKFLVIGSAGTGKSCLLHQFIENKFKQDSNHTIGVEFGSRVVNVGGK MAETYDFLFKFLVIGSAGTGKSCLLHQFIENKFKQDSNHTIGVEFGSRVVNVGGK MAETYDFLFKFLVIGSAGTGKSCLLHQFIENKFKQDSNHTIGVEFGSRVVNVGGK M E yd5LFKFL6IGSAG3GKSC6LHqFIE KFKqDS HTIGVEFGS466NVGGK. : : : : : : :. 60 * 80 * 100 * SVKLQIWDTAGQERFRSVTRSYYRGAAGALLVYDISSRETFNSLTNWLTDARTLA TVKLQIWDTAGQERFRSVTRSYYRGAAGALLVYDITSRETYNALTNWLTDARTLA SVKLQIWDTAGQERFRSVTRSYYRGAAGALLVYDATSRDSFNALTNWLNDARTLA SVKLQIWDTAGQERFRSVTRSYYRGAAGALLVYDIASRETYNALTNWLTDARTLA TVKLQIWDTAGQERFRSVTRSYYRGAAGALLVYDITSRETYNSLAAWLTDARTLA TVKLQIWDTAGQERFRSVTRSYYRGAAGALLVYDITSRETYNSLAAWLTDARTLA TVKLQIWDTAGQERFRSVTRSYYRGAAGALLVYDITSRETYNSLAAWLTDARTLA 3VKLQIWDTAGQERFRSVTRSYYRGAAGALLVYDi SRe35N L WLtDARTLA RabF2. ApRab4.PRO fish.PRO fly.PRO frog.PRO human.PRO mouse.PRO rat.PRO. G2. RabF3. RabF4. : : : : : : :. 110 110 110 110 110 110 110. : : : : : : :. 165 165 165 165 165 165 165. RabF5. : : : : : : :. 120 * 140 * 160 SPNIVIILVGNKKDLDADREVTFLEASRFAQENDLIFLETSALSGENVEEGFLKC SPNIVIILCGNKKDLDADREVTFLEASRFAQENELMFLETSALTGENVEEAFLKC SPNIVILLVGNKKDLEEARDVTFLEASTFAQENELIFLETSAKTGENVEEAFLKC SPNIIIILCGNKKDLDADREVTFLEASRFAQENELMFLETSALTGENVEEAFLKC SPNIVVILCGNKKDLDPEREVTFLEASRFAQENELMFLETSALTGENVEEAFLKC SPNIVVILCGNKKDLDPEREVTFLEASRFAQENELMFLETSALTGENVEEAFLKC SPNIVVILCGNKKDLDPEREVTFLEASRFAQENELMFLETSALTGENVEEAFLKC SPNI666L GNKKDLd ReVTFLEASrFAQENeL6FLETSAl3GENVEEaFLKC. : : : : : : :. * 180 * 200 * SRTILNKIESGELDPDRMGSGIQYGDPSLRRTLRPGRGRGESRSKCPANCS-KARSIPNKIESGELDPERMGSGIQYGDASLRQ-IRQPRG---SAAQTKQQCN--C SKTILAKIETGELDPERIGSGIQYGGAALRNLQTRQRS------INKPDCTCRV ARTILSKIESGELDPERMWSGIQYGDASPRH-AKHSHG---TQQQSRQQCN--C ARTILNKIDSGELDPERMGSGIQYGDASLRQ-LRQPRS---AQAVAPQPCG--C ARTILNKIGSGELDPERMGSGIQYGDISLRQ-LRHARS---AQAVAPQPCG--C ARTILNKIDSGELDPERMGSGIQYGDISLRQ-LRQPRS---AQAVAPQPCG--C 43Il KI 3GELDPeR6gSGIQYGd slR r C. G3. ApRab4.PRO fish.PRO fly.PRO frog.PRO human.PRO mouse.PRO rat.PRO. 55 55 55 55 55 55 55. RabF1. G1. ApRab4.PRO fish.PRO fly.PRO frog.PRO human.PRO mouse.PRO rat.PRO. : : : : : : :. G4. : : : : : : :. P. Fig. 4. Multi-alignment of ApRab4 protein with selected Rab4 relatives. Amino acid residues identical in all Rab4 proteins are shown on a black background; residues conserved in most members are shown on a gray background. Sequence motifs that are highly conserved among Rab proteins are indicated by solid lines and labeled. Functional motifs involved in. 35. 217 213 213 213 213 213 213.

(42) GTP-binding are marked with solid lines and numbered (G1-G4). Sequence motifs that are highly conserved in Rab family proteins are indicated by solid lines and labeled (RabF1–RabF5). The putative C-terminal double cysteine prenylation signal is underlined and labeled with the letter P. The GenBank accession numbers of the selected Rab4 proteins are: AAP97171 (human); AAL11725 (mouse); AAH44974 (frog); NP 523777 (fly); NP 001004002 (fish); and NP 059051 (rat).. 36.

(43) ApRab14-L. fish.PRO fly.PRO frog.PRO human.PRO mouse.PRO worm.PRO. : : : : : : :. ApRab14-L. fish.PRO fly.PRO frog.PRO human.PRO mouse.PRO worm.PRO. : : : : : : :. ApRab14-L. fish.PRO fly.PRO frog.PRO human.PRO mouse.PRO worm.PRO. : : : : : : :. ApRab14-L. fish.PRO fly.PRO frog.PRO human.PRO mouse.PRO worm.PRO. : : : : : : :. * 20 * 40 * MAATGPYNYSYIFKYIIIGDMGVGKSCLLHQFTEKKFMADCPHTIGVEFGTRIIE MT-TAPYNYSYIFKYIIIGDMGVGKSCLLHQFTEKKFMADCPHTIGVEFGTRIIE MTA-APYNYNYIFKYIIIGDMGVGKSCLLHQFTEKKFMANCPHTIGVEFGTRIIE MA-TAPYNYSYIFKYIIIGDMGVGKSCLLHQFTEKKFMADCPHTIGVEFGTRIIE MA-TAPYNYSYIFKYIIIGDMGVGKSCLLHQFTEKKFMADCPHTIGVEFGTRIIE MA-TAPYNYSYIFKYIIIGDMGVGKSCLLHQFTEKKFMADCPHTIGVEFGTRIIE MTA-APYNYSYIFKYIIIGDMGVGKSCLLHQFTEKKFMADCPHTIGVEFGTRIIE M aPYNYsYIFKYIIIGDMGVGKSCLLHQFTEKKFMA1CPHTIGVEFGTRIIE G1 RabF1 60 * 80 * 100 * VAGQKIKLQIWDTAGQERFRAVTRSYYRGAAGALMVYDITRRSTYNHLSSWLTDA VSGQKVKLQIWDTAGQERFRAVTRSYYRGAAGALMVYDITRRSTYNHLSSWLTDA VDDKKIKLQIWDTAGQERFRAVTRSYYRGAAGALMVYDITRRSTYNHLSSWLTDT VSGQKIKLQIWDTAGQERFRAVTRSYYRGAAGALMVYDITRRSTYNHLSSWLTDA VSGQKIKLQIWDTAGQERFRAVTRSYYRGAAGALMVYDITRRSTYNHLSSWLTDA VSGQKIKLQIWDTAGQERFRAVTRSYYRGAAGALMVYDITRRSTYNHLSSWLTDA VSGQKIKLQIWDTAGQERFRAVTRSYYRGAAGALMVYDITRRSTYNHLSSWLADA V gqK6KLQIWDTAGQERFRAVTRSYYRGAAGALMVYDITRRSTYNHLSSWLtDa G2 RabF4 RabF5 RabF2 RabF3 120 * 140 * 160 RNLTNPNTVIFLIGNKSDLDAQRDVTYEEAKQFAEENGLLFCEASAKTGENVEDA RNLTNPNTVIILIGNKADLEAQRDVTYEEAKQFAEENGLLFLEASAKTGENVEDA RNLTNPSTVIFLIGNKSDLESTREVTYEEAKEFADENGLMFLEASAMTGQNVEEA RNLTNPNTVIILIGNKADLEAQRDVTYEEAKQFAEENGLLFLEASAKTGENVEDA RNLTNPNTVIILIGNKADLEAQRDVTYEEAKQFAEENGLLFLEASAKTGENVEDA RNLTNPNTVIILIGNKADLEAQRDVTYEEAKQFAEENGLLFLEASAKTGENVEDA KSLTNPNTAIFLIGNKADLEDQRDVPYEEAKAFAEENGLTFLECSAKTGSNVEDA 4nLTNPnTvI LIGNK DLe qRdVtYEEAK FAeENGL FlEaSAkTG NVEdA G4 G3 * 180 * 200 * FLETAKKIYQNIQDGSLDLNAAESGVQHKPMTGRPANALNTDQPSNQDRCNC- : FLEAAKKIYQNIQDGSLDLNAAESGVQHKP-TAPQGGRLSSDAQPQKEGCS-C : FLETARKIYQNIQEGRLDLNASESGVQHRPSQPSRTS-LSSEATGAKDQCSC- : FLEAAKKIYQNIQDGSLDLNAAESGVQHKP-SAPQGGRLTSEPQPQREGCG-C : FLEAAKKIYQNIQDGSLDLNAAESGVQHKP-SAPQGGRLTSEPQPQREGCG-C : FLEAAKKIYQNIQDGSLDLNAAESGVQHKP-SAPQGGRLTSEPQPQREGCG-C : FLETAKQIYQNIQDGSLDLNAADTGVQPKQNLPRAAE------NNGKKDCNC- : FLE A4kIYQNIQdGsLDLNAae3GVQh4p l C P. : : : : : : :. 55 54 54 54 54 54 54. : : : : : : :. 110 109 109 109 109 109 109. : : : : : : :. 165 164 164 164 164 164 164. 217 215 215 215 215 215 210. Fig. 5. Multi-alignment of ApRab14-L protein with selected Rab14 relatives. Amino acid residues identical in all Rab14 proteins are shown on a black background; residues conserved in most members are shown on a gray background. Sequence motifs that are highly conserved among Rab proteins are indicated by solid lines and labeled. Functional motifs involved in. 37.

(44) GTP-binding are marked with solid lines and numbered (G1-G4). Sequence motifs that are highly conserved in Rab family proteins are indicated by solid lines and labeled (RabF1–RabF5). The putative C-terminal double cysteine prenylation signal is underlined and labeled with the letter P. The GenBank accession numbers of the selected Rab4 proteins are: NP 057406 (human); NP 080973 (mouse); NP 001007505 (frog); NP 477171 (fly); NP 958903 (fish); and NP 446041 (rat).. 38.

(45) Rab4 human rat mouse fish frog ApRab4 fly. 13.3 12. 10. 8. 6. 4. 2. 0. Rab14 human mouse frog fish. ApRab14-L worm. fly. 11.8 10. 8. 6. 4. 2. 0. Fig. 6. Phylogenetic tree of selected Rab4 and Rab14 proteins. The GenBank accession numbers of the selected Rab4 proteins are: AAP97171 (human); AAL11725 (mouse); AAH44974 (frog); NP 523777 (fly); NP 001004002 (fish); and NP 059051 (rat) and the selected Rab14 are NP 057406 (human); NP 080973 (mouse); NP 001007505 (frog); NP 477171 (fly); NP 958903 (fish); and NP 446041 (rat).. 39.

(46) rApRab4. Ap. rApRab14-L. Ap. Fig. 7. Western blot analysis of ApRab4 and ApRab14-L in Aiptasia protein sample. The rat anti-ApRab4 and anti- ApRab14-L polyclonal antibodies (1:500 dilution) were used in combination with a HRP-conjugated goat anti-mouse IgG secondary antibody (1:10,000 dilution) to detect ApRab4 and ApRab14-L proteins. Ap, 14µg of total Aiptasia protein; rApRab4 and rApRab14-L, affinity-purified recombinant histine-tagged ApRab4 and ApRab14-L proteins as positive control. The recombinat protein contains additional 34 amino acid residues than its parental, WT-type protein. Protein fractions were prepared as described in “Material and Methods”, and equal. 40.

(47) amount of protein extracts were fractionated in 15% SDS-PAGE gel. Positive protein bands were visualized by chemiluminescence using the Renaissance Western blot reagent from NEN.. 41.

(48) ApRab4-WT. ApRab14-L-WT. ApRab4-S22N. ApRab14-L-S26N. ApRab4-Q67L. ApRab14-L-Q71L. Fig. 8. EGFP reporter analyses of ApRab4 and ApRab14-L (wild-type and mutant forms) in HeLa cells. Plasmid encoding either the EGFP-ApRab4, EGFP-ApRab14-L or their mutant forms were introduced into HeLa cells using FuGENE 6. Bars: 10µm. 42.

(49) ApRab4. Transferrin. ApRab14-L. Merge. Transferrin. Merge. Fig. 9. Co-localization of EGFP-ApRab4 and -ApRab14-L with exogenous transferrin.. Plasmids. encoding. either. the. EGFP-ApRab4. or. EGFP-ApRab14-L fusion proteins were introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteen hours after transfection, cells were incubated with 1µM transferrin-Texas Red for 30 min at 37℃ to label transferring-containing recycling compartments. Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Arrows indicate the overlapping signals. Bars: 10µm. 43.

(50) ApRab4. dextran-TRICT (10 min). Merge. ApRab4. dextran-TRICT (30 min). Merge. Fig. 10. Co-localization of EGFP-ApRab4 with dextran- TRICT (70 kDa). Plasmid encoding the EGFP-ApRab4 fusion protein was introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteen hours after transfection, cells were incubated with dextran-TRICT (70 kDa) for 10 min and 30 min at 37℃ to label early and late stages of endosomes. Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Arrows indicate the overlapping fluorescence signals. Bars: 10µm. 44.

(51) ApRab14-L. dextran-TRICT (10 min). Merge. ApRab14-L. dextran-TRICT (30 min). Merge. Fig. 11. Co-localization of EGFP-ApRab14-L with dextran- TRICT (70 kDa). Plasmid encoding the EGFP-ApRab14-L fusion protein was introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteen hours after transfection, cells were incubated with dextran-TRICT (70 kDa) for 10 min and 30 min at 37℃ to label early and late stages of endosomes. Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Arrows indicate the overlapped signals. Bars: 10µm. 45.

(52) ApRab4. LysoTracker-red. Merge. ApRab14-L. LysoTracker-red. Merge. Fig. 12. Co-localization of EGFP-ApRab4 and -ApRab14-L with LyoTracker. Plasmid encoding the EGFP-ApRab4 and EGFP-ApRab14-L fusion proteins were introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteen hours after transfection, cells were incubated with 75 nM LysoTracker- red for 15 min at 37℃ to label acidic compartments. Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Arrows indicate the non-overlapping signals. Bars: 10µm. 46.

(53) ApRab14-L. ApRab14-L-S26N. TR-C5-ceramide. Merge. TR-C5-ceramide. Merge. TR-C5-ceramide. Merge. Fig. 11.. ApRab14-L-Q71L. Fig. 13. Co-localization of wild-type EGFP-ApRab14 and its mutants with TR-C5-ceramide.. Plasmids. encoding. either. the. EGFP-ApRab14. or. EGFP-ApRab14-L-S26N and ApRab14 -L-Q71L fusion proteins were introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteen. 47.

(54) hours after transfection, cells were incubated with TR-C5-ceramide for 30 min at 4℃ to label sphingolipids. Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Bars: 10µm. 48.

(55) ApRab4. N. ApRab14-L. N. Fig. 14. Immunofluorescence analysis of endogenous ApRab4 and ApRab14-L in Aiptasia host cells housing zooxanthellal symbionts. The polyclonal rat anti-ApRab4 and ApRab14-L were used as 1st antibody to label and the Cy3-conjugated goat anti-mouse IgGs was used as 2nd antibody to visualize endogenous ApRab4 and ApRab14-L in zooxanthellae- containing Aiptasia cells. ApRab4 and ApRab14-L staining signals were detected on phagosomes containing lived zooxanthellae. Nucleus was labeled by H33258 staining. Arrows and the letter N indicate the nuclei and the phagosome membrane, respectively. Bars: 10µm. 49.

(56) ApRab4. N. ApRab14-L. N. Fig. 15. Intracellular distribution of endogenous ApRab4 and ApRab14-L in Aiptasia phagocytes. Identification of Aiptasia phagocytes was aided by the presence of latex beads (blue fluorescence). Fixed cells were stained for either ApRab4 or ApRab14-L with rat anti-ApRab4 or rat anti-ApRab14-L polyclonal as the primary antibody, a Cy3-conjugated goat anti-mouse IgGs as the secondary antibody. Nucleus was labeled by H33258 staining. Arrows and the letter N in these patterns indicate the phagosomes and the nucleus, respectively. Bars: 10µm. 50.

(57) 100 90 ) % (s tn u o ce v iit so p. 80 70 60 50 40 30 20 10 0. 0 min. 30 min. 60 min. length of incubation (min). Fig. 16. Immunofluorescence analysis of endogenous ApRab4 and ApRab14 association. with. resident. zooxanthellae-. containing. phagosomes. (symbiosomes) in DCMU-treated or untreated Aiptasia cells. The rat anti-ApRab4 and rat anti- ApRab14-L IgGs were affinity-purified and used to label, and the Texas Red-conjugated goat anti-rat IgGs was used to visualize endogenous ApRab4 and ApRab14-L in zooxanthellae-containing Aiptasia cells. ApRab4 and ApRab14-L associated with the symbiosomes as a function of the duration of DCMU treatment. ApRab4(□), ApRab5(▲), ApRab7(◇), and ApRab14-L(○). 51.

(58) 90 80 70 ) (% 60 st nu oc 50 ev it 40 is op 30 20 10 0. 15 min. 30 min. 45 min. length of incubation (min). Fig. 17. Phagocytosis of latex bead showed the tendency of association of ApRab4 and ApRab14-L with the phagosomes. ApRab4 and ApRab14-L were detected on punctate structures and on latex-bead containing phagosomes in Aiptasia digestive cells. Fixed cells were stained for ApRab4 and ApRab14-L with either the rat anti-ApRab4 or the rat anti-ApRab14-L IgGs as the primary antibody (1:50 dilution), a Texas Red-conjugated goat anti-rat IgGs was used as the secondary antibody (1:1000 dilution). ApRab4 (□), ApRab5 (▲), ApRab7 (◇), and ApRab14 (○). 52.

(59) 70 60 ) % ( 50 tsn u 40 o c ev it is 30 o p. 20 10 0 15 min. 30 min. 45 min. length of incubation (min). Fig. 18. Phagocytosis of heat-killed zooxanthellae showed the tendency of association of ApRab4 and ApRab14-L with the phagosomes in Aiptasia digestive cells. Fixed cells were stained for ApRab4 and ApRab14-L with either the rat anti-ApRab4 or the rat anti-ApRab14-L IgGs as the primary antibody (1:50 dilution), a Texas Red-conjugated goat anti-rat IgGs was used as the secondary antibody (1:1000 dilution). ApRab4 (□), ApRab5 (▲), ApRab7 (◇), and ApRab14 (○). 53.

(60) 70 60 ) % ( 50 tsn u 40 o c ev it is 30 o p 20. 10 0 15 min. 30 min. 45 min. length of incubation (min). Fig. 19. Phagocytosis of live zooxanthellae showed the tendency of association of ApRab4 and ApRab14-L with the phagosomes. Fixed cells were stained for ApRab4 and ApRab14-L with either the rat anti-ApRab4 or the rat anti-ApRab14-L IgGs as the primary antibody (1:50 dilution), a Texas Red-conjugated goat anti-rat IgGs was used as the secondary antibody (1:1000 dilution). ApRab4 (□), ApRab5 (▲), ApRab7 (◇), and ApRab14 (○). 54.

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