Rozprawy doktorskie na temat „Arl15”

Small G-proteins of Arf-like (Arl) GTPase subfamily are shown to regulate several cellular processes including intracellular trafficking, cytoskeletal organization, organelle biogenesis, cell adhesion and migration. Around 21 genes belong to this family have been identified in human. However, the critical function of Arl14 and Arl15 in cargo transport was unclear. In this study, we have attempted to characterize the role of Arl14 and Arl15 in multiple cellular processes, including intracellular trafficking using HeLa cells. Objective I: Elucidating the role of Arl15 in modulating cell adhesion, motility and filopodia biogenesis. Our study characterized the intracellular localization of Arl15 using epitope tagged Arl15-GFP in multiple mammalian types. We have observed that Arl15-GFP localizes to Golgi, plasma membrane (PM) including filopodia, and a cohort to recycling endosomes in HeLa, A549, neuro 2a, and primary keratinocytes. Additionally, we noticed the localization of Arl15 to long extracellular tube structures (resembling tunneling nanotubes, TNTs) connecting the Neuro 2a cells. The dual localization of Arl15 to Golgi and PM is independent of the actin cytoskeleton, but it is dependent on Golgi integrity. The dissociation of Golgi using small molecular inhibitors or the expression of Arf1 dominant-negative mutant completely mislocalizes Arl15 to the cytosol. We identified a novel V80A mutation in the GTP-binding domain that turns the Arl15 into a dominant-negative form and results in a reduced number of filopodia. Depletion of Arl15 in HeLa cells causes mislocalization of cargo such as caveolin-2, STX6, and ectopically expressed GFP-GPI from Golgi and accumulation of lipid droplets. Further, Arl15 knockdown cells display reduced filopodial number, dispersion of vinculin localization (focal adhesion kinase), and enhanced soluble and receptor-mediated cargo uptake without affecting the recycling kinetics. In addition, Arl15 knockdown decreases cell migration and increases cell adhesion, and displays enhanced cell spreading. Traction force microscopy studies revealed that Arl15 depleted cells exert higher traction force and generate multiple focal adhesion points. These studies demonstrated a function to Arl15 in Golgi, which regulates cargo transport to organize membrane domains at the cell surface to control cell migration, spreading and adhesion, including filopodial biogenesis. Objective II: Studying the role of Arl14 in vesicular trafficking Studies have suggested that Arl14 regulates the movement of MHC-II vesicles along the actin cytoskeleton in dendritic cells. We have studied the localization of Arl14 using epitope tagged Arl14-GFP in HeLa cells. Arl14 localizes to REs, late endosomes, and lysosomes. Expression of Arl14S27N-GFP showed no change in its localization, indicating that Arl14S27N-GFP is not acting as a dominant negative mutant while constitutive active mutant of Arl14 (Arl14Q68L-GFP) majorly localized as punctate structures that are clustered and positive for RFP-STX13 (represents endosomal structures). Depletion of Arl14 showed an enhanced number of LAMP-1-positive lysosomes without changing the localization of lysosome biogenesis transcription factors TFEB and TFE3. Immunoblotting analysis showed no change in Rab5, STX13, Rab11, annexinA2, LAMP-1, and LAMP-2 expression in siArl14 compared to siControl. Overall, these studies showed that Arl14 localizes to endo-lysosomal organelles, and its depletion altered the number of LAMP-1 positive compartments.

碩士
臺灣大學
分子醫學研究所
98
ARF-like (ARL) proteins belong to ADP-ribosylation factor (ARF) GTPase family, which are involved in protein trafficking and cytoskeleton organization. Those small G proteins require guanine-nucleotide exchange factors (GEFs) to switch from GDP-bound to GTP-bound form and become active. Previous reports suggested that Syt1p is the GEF of Arf2p and is involved in vesicle trafficking. Recently, our studies showed that Syt1p also acted as a GEF for Arl1p. In this study, Syt1p was further characterized. Firstly, it has been shown that Syt1p belongs to BFA-resistant GEFs. Secondly, Syt1p can use multiple regions to interact with Arl1p. The N-terminus, Sec7 domain, and C-terminus of Syt1p can all interact with Arl1d17N form, whose N-terminal first 17 amino acids are deleted. The interactions between all of the three regions and Arl1d17N are stronger than the interaction between full-length Syt1p and Arl1pd17N. Therefore, it might hint that Syt1p is autoregulated as other GEFs of Small GTPases. Surprisingly, we next found that Syt1p has an intramolecular interaction between the C-terminal region and Sec7 domain and an intermolecular interaction between C-terminal regions, indicating that Syt1p could form dimers or oligomers and might be autoregulated. Syt1p dimerization or oligomerization is also supported by in vivo pull down results, which proved that Syt1p can interact with itself. Moreover, the N-terminus is important for the formation of dimers or oligomers. Yeast two-hybrid screen was also performed to search for putative regulators of Syt1p. However, those candidates remain to be elucidated. Besides, whether dimerization or oligomerization plays an important role in Syt1p activation or membrane tethering and whether autoinhibition truly exists in Syt1p in vivo require further investigation.

博士
國立臺灣大學
分子醫學研究所
89
ADP-ribosylation factors (ARFs) Ras-related small GTP-binding proteins (~20 kDa), were originally identified as protein cofactors for the cholera toxin-catalyzed ADP-ribosylation of GS, the stimulatory subunit for adenylate cyclase. Their cofactor activity depends on GTP-binding－ regulated by interactions with ARF-specific guanine nucleotide exchange factors (GEFs) or ARF-specific GTPase activating proteins (GAPs). Today, at least six mammalian ARF members have been identified and grouped into three classes. ARFs have been found in different subcellular localizations, and regulate several different vesicle transport steps in the exocytic and endocytic pathways－ including budding from the endoplasmic reticulum and fusion to the Golgi stacks, endosomes, and nuclear vesicles. ARF-like (ARL) proteins, which belong to another subfamily of ARFs, have been cloned recently from Drosophila, rat, human and yeast. Although ARLs and ARFs have very similar amino acid sequences, ARLs lack cofactor activity for the cholera toxin-catalyzed ADP-ribosylation. dARL1 is essential in Drosophila, while disruption of yeast ARL1 gene is not lethal. ARL1 from yeast and human were reported to localize at the Golgi complex. In this thesis, we report the biochemical characterization and subcellular localizations of two ARLs－ ARL4 and ARL5. We first cloned mouse and human ARL4 (mARL4 and hARL4) cDNA, respectively. Mice ARL4 is abundant in testis and has developmental stages-specific expression pattern. The appearance of mARL4 mRNA during embryonic development coincides with the sequential formation of somites and the establishment of brain compartmentation. Using ARL4-specific antibody in immuno-fluorescence microscopy, we found that endogenous mARL4 in cultured Sertoli and neuroblastoma cells was mainly concentrated in nuclei. When ectopically expressed in COS7 cells, ARL4-T34N mutant (predicted to be the GDP-bound form) was concentrated in nucleoli. Yeast two-hybrid screen and in vitro protein-interaction assays showed that hARL4 interacts with importin-a－required its C-terminal NLS region, and the interaction is nucleotide-independent. Like ARL2 and ARL3, recombinant hARL4 does not enhance cholera toxin-catalyzed auto-ADP-ribosylation. Its binding with GTPγS could be affected by phospholipid, and the N-terminus of hARL4, like that of ARF, is myristoylated. Our findings suggest that ARL4－ with its distinctive nuclear/nucleolar localization and developmentally regulated expression pattern－may play a unique role(s) in neurogenesis and somitogenesis during embryonic development, and in the early stages of spermatogenesis in adults, respectively. Mouse and human ARL5 (mARL5 and hARL5) were also cloned, and the hARL5 mRNA is ubiquitously expressed in many tissues and considerably abundant in liver. mARL5 mRNA is mainly present at day 7 mouse embryos, then disappears at day 11. Our findings suggest that ARL5 may have an important role(s) in the early events of embryonic development. Immunostaining of COS7 cells transfected with Myc-tagged hARL5-WT, hARL5-T35N, hARL5-Q80L, and hARL5-dC constructs provide direct evidence for its subcellular localization in the cell. Interestingly, hARL5-T35N mutant, the predicted GDP-bound form, was most concentrated in nucleoli, but sometimes present in cytosol. By yeast two-hybrid screen, hARL5 interacts with NLS-receptor, importin-a, through the putative bipartite NLS in its C-terminal region. When NLS is removed from hARL5, hARL5-dC is colocalized with mitochondria; hARL5-T35N has the same pattern when it appears in the cytosol. In addition, yeast two-hybrid screen and in vitro protein-interaction assays showed that hARL5-WT, hARL5-Q80L can interact with HP1a, and the interaction is nucleotide-dependent. The interaction was further confirmed by in vivo protein-interaction assays. hARL5 contains MIR homology sequence－ VPVLVL (residue 128~133)－ that is crucial for hARL5 binding to HP1a. As ARL2, 3, and 4, recombinant hARL5 did not enhance the cholera toxin-catalyzed auto-ADP-ribosylation. Its intrinsic GTPgS-binding activity was low. The N-terminus of hARL5 is myristoylated as are ARFs. Based on these results, we have demonstrated that hARL5, like hARL4, with its specific interaction proteins and distinctive nuclear/nucleolar localizations may have important biological functions.

Influenza A virus (IAV) is an important human pathogen that causes epidemic and pandemic events of flu. The study of the viral life cycle and its interactions with the infected host is crucial for the development of novel therapeutic strategies. IAV has an eight-part segmented RNA genome organized in viral ribonucleoprotein complexes (vRNP), which are replicated in the nucleus of the cell. De novo synthesized vRNPs need to leave the nucleus to reach the cytosol for viral assembly, budding and release. Several pathways have been implicated in nuclear export of vRNPs, including CRM1, apoptosis activation and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling cascade. Mitochondria are crucial organelles for the maintenance of cellular homeostasis, since they are responsible for the regulation of metabolism, apoptosis, calcium homeostasis and innate immunity. Their functions are tightly regulated by dynamic changes in mitochondrial morphology. Given their importance, many viruses modulate mitochondria to promote cellular environments favoring their proliferation. IAV has been shown to fragment mitochondria to decrease the antiviral immune response. Our lab identified a candidate modulator of mitochondrial morphology and IAV infection: the host GTPase Arl17. Our work demonstrated that depletion of Arl17 leads to a reduction in viral titers and promotes mitochondrial fragmentation, regardless of infection. Interestingly, this phenotype was not accompanied by alterations in IFNβ1 expression and mitochondrial unfolded protein response activation (UPRmt). However, ATP levels were significantly reduced in the absence of Arl17. Additionally, we showed that Arl17 is required for regular vRNP nuclear export. In its absence, we observed a delay in vRNPs nuclear export that was CRM1- and ERK1/2-independent. Therefore, influence of Arl17 on the induction of apoptosis should be further investigated as its inhibition could explain vRNPs nuclear export delay and it could be the element that links mitochondria and vRNPs nuclear export.

碩士
國立臺灣大學
分子醫學研究所
93
ADP-ribosylation factor like (ARL) proteins are one subfamily of ADP-ribosylation factor (ARF) small GTP binding protein family. Little is known about a human ARL protein, ARL5. Here we characterized the biologic properties and functions of hARL5 and described several novel observations. ARL5 expressed ubiquitously in many mammalian cell lines. In COS-7 cells, endogenous ARL5 and overexpressed GFP-ARL5 localized to centrosomes through out the cell cycle with exception of midbody localization during cytokinesis. By yeast two-hybrid screening, a microtubule plus-end binding protein EB1 was identified as an interacting partner of ARL5. ARL5 also interacted with EB1 in vivo. To investigate the biologic functions of ARL5, RNAi knockdown was performed. In ARL5 knockdown COS-7 cells, the microtubule regrowth from and anchoring at centrosomes were delayed and disordered similar to the defects caused by EB1 knockdown. Furthermore, overexpressed wild type and constitutively active form (ARL5Q80L) of ARL5 were diffused and partially localized to endosomes in COS-7 cells. The heterogeneous distributions of ARL5 constitutively inactive form (ARL5T35N) at endosomes, mitochondria, and aggresome-like compartments provided a distinct feature of ARL5. In addition, ARL5T35N diminished mitochondria membrane potential through its C-terminal region when it localized to mitochondria. Taken together, we infer that ARL5 might regulate different cellular processes, including centrosome-mediated microtubule nucleation and anchoring, vesicle trafficking, mitochondrial function, and protein degradation.

碩士
國立臺灣大學
分子醫學研究所
88
ADP-ribosylation factors（ARFs）are highly conserved ~20-kDa guanine nucleotide-binding proteins, which are believed to participate in vesicular trafficking in both endocytic and exocytic pathways. Several ARF-like proteins（ARLs）have been cloned, but their biological functions are still unknown. To characterize their molecular properties, we prepared ARL4- and ARL5-specific antibodies. In addition, human ARL4 mutants G2A, T34N, Q79L and deletion of C-terminus were respectively constructed. By library screening, we also cloned mouse ARL5 cDNA and genomic DNA. Our results indicated that mouse ARL5 mRNA was notably abundant in liver and heart. The appearance of mouse ARL5 mRNA during early embryonic development suggested that ARL5 might have played a fundamental role in embryogenesis. Using ARL4-specific antibody, we demonstrated that mouse ARL4 protein mainly presented in heart and colon. Furthermore, yeast two-hybrid screening showed that human ARL5 Q80L mutant, which was predicted to be in the GTP-bound state, interacted with α-catenin, heterochromatin protein 1α, N-methylpurine-DNA glycosylase and α-1-antichymotrypsin. Our findings proposed that ARL5 was possibly involved in nuclear functions. We provide some previous observation about mammalian ARL4 and ARL5 in this thesis.

碩士
國立臺灣大學
分子醫學研究所
106
Arf-like proteins (Arls) are important regulators involved in a diversity of biological events, especially vesicle trafficking. In Saccharomyces cerevisiae, Arl1 acts to recruit a golgin protein Imh1 to the trans-Golgi network (TGN). However, besides the role of Imh1 as a high-copy suppressor of Ypt6, an important Rab protein mediating endosome-to-Golgi trafficking, less is known about it physiological significance. In previous studies (Hsu et al., 2016), they demonstrated that the Unfolded-Protein Response (UPR) augmented the activity of Arl1 and the subsequent Imh1 recruitment, but it remained to be elucidated on its specific functions under ER stress. Here, we showed that the UPR-activated Arl1 and Imh1 act to specifically maintain the endosome-to-Golgi trafficking when cell encounters ER stress. Moreover, this regulation does not simply depend on GARP complex recruitment, an important tethering complex, to affect retrograde trafficking. We found that the first five amino acids at Imh1 N-terminus is required for its function, as it contributes to the recruitment of Sft2, a tetra-spanning membrane protein involved in vesicle fusion. The N-terminus of Sft2 is responsible for facilitating the transport of Snc1 and Tlg1 and promoting retrograde trafficking. Together, our study is one of the first to demonstrate the physiological function of golgin Imh1 and elucidate the importance of the trafficking machinery in response to ER stress.

碩士
國立臺灣大學
分子醫學研究所
94
ADP-ribosylation factors (ARFs) and ARF-like proteins (ARLs) are members of the ARF family, which play essential roles in intracellular membrane trafficking. These proteins are regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) to cycle between active GTP-bound form and inactive GDP-bound form. ARLs are ~40–60 % identical to each other and to ARFs. Despite their high similarity to ARFs, only ARL1 has been shown to be involved in vesicular transport. ARL1 is localized in the Golgi complex and may participate in trans-Golgi network (TGN) to plasma membrane or endosome to TGN trafficking pathway. In the first part, we investigated the function of ARL1 by using constitutively active (Q71L) or inactive (T31N) ARL1. Discrepant with previous studies, we found that overexpressed ARL1Q71L specifically disperses Golgin-245 from Golgi apparatus. Over-expressed ARL1T31N disrupted trans-Golgi proteins but not cis/medial Golgi and endosome proteins. Moreover, VSVG transport was blocked in ARL1Q71L overexpressed but not in ARL1 knockdown cells which implied that ARL1 itself may not directly participate in the VSVG transport. In the second part, we attempted to find possible GAP for ARL1. Among our three candidates, ARFGAP1 did not interact with ARL1 in yeast two-hybrid assay but endogenous ARL1 were dissociated from the Golgi in cells with overexpressed ARFGAP1, which we considered an indirect consequence of general effect on Golgi proteins. Centaurin α1 neither interacted with ARL1 nor affected ARL1 localization. ARAP1 interacted with ARL1Q71LdN, but we could not detect the change of ARL1 localization when ARAP1 overexpressed. Whether ARAP1 could be GAP for ARL1 needs to be investigated further. In the third part, we identified an ARL1Q71L interacting protein, CIB1. We showed that ARL1 interacted with CIB1 in a Ca2+-dependent manner. Furthermore, CIB1 was ubiquitously expressed in cells especially colocalized with ARL1 in the Golgi apparatus in particular conditions. In addition, Golgi localization of CIB1 was not dependent on ARL1, and vice versa. Unfortunately, even though the interaction and colocalization of ARL1 and CIB1 were observed, we could not demonstrate the functional relevance between these two proteins.

博士
長庚大學
生物醫學研究所
103
The small GTPase Arf-like protein 1 (Arl1) is well known for its role in intracellular vesicular transport at the trans-Golgi network (TGN). In here, we used differential affinity chromatography combined with mass spectrometry to identify Arf-interacting protein 1 (including arfaptin-1a and arfaptin-1b) as an Arl1-interacting protein. Yeast two-hybrid demonstrated that arfaptin-1 directly interacted with Arl1 through their arfaptin homolog (AH) domain. Arl1 knockdown caused arfaptin-1 dispersed from the Golgi apparatus. Expression of the wild-type Arl1, but not Arl1TN or wild-type Arf1 in Arl1- knockdown cells restored the Golgi localization of arfaptin-1. The Golgi dissociations of arfaptin-1 and Arl1 were temporally coupled in response to brefeldin-A treatment. These results demonstrated that Arl1 medicates the Golgi targeting of arfaptin-1. We also characterized a novel function for arfaptin-1 in Arl1-mediated retrograde transport. Using a Shiga-toxin subunit B (STxB) transportation assay, we demonstrated that knockdown of arfaptin-1 accelerated the retrograde transport of STxB from the endosome to the Golgi apparatus, whereas Arl1 knockdown inhibited STxB transport compared with control cells. Arfaptin-1 overexpression, but not an Arl1 binding-defective mutant (arfaptin-1b-F317A), consistently inhibited STxB transport. Exogenous arfaptin-1 expression did not interfere with the localization of the Arl1-interacting proteins golgin-97 and golgin-245 to the TGN and vice versa. Moreover, we found that the N-terminal region of arfaptin-1 was involved in the regulation of retrograde transport. Our results show that arfaptin-1 is an Arl1 effector and acts as a negative regulator in Arl1-mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.