Cells rely on complex, highly regulated networks of signaling proteins in order to perform the basic functions of life. Over the course of evolution, gene duplication events have led to the expansion of families of similar proteins with some shared characteristics, but whose individual members evolved to perform their own unique functions. One such family is the ARF family of small GTPases, whose members function in cellular signaling pathways spanning many cellular functions including membrane traffic, cytoskeletal regulation, cell division, and many others. Several members of the ARF family have been extensively studied in specific cellular contexts or model systems, but some ARFs remain virtually unstudied. Furthermore, it is increasingly coming to be appreciated that a single GTPase can perform multiple related or independent functions in different parts of the cell. Many of these functions have not been extensively explored. As understanding the fundamental biology underlying cell function is essential to our ability to understand the mechanisms of disease pathologies, it is imperative to understand the functions of the ARF family and the fundamental signaling networks that they regulate. In order to compare and contrast the functions of these GTPases, build signaling networks, and explore functional redundancy among ARF family members, we have begun to systematically knock out members of the ARF family using Mouse Embryonic Fibroblasts (MEFs) as our model system. Using this system, coupled with microscopy techniques, we characterized cellular functions of ARL16 and ARL10 for the first time. We discovered that ARL10 regulates cell division and mitochondrial motility, and that ARL16 regulates primary cilium formation and ciliary protein traffic. Cilia are nearly ubiquitous, specialized cellular signaling hubs which regulate many aspects of eukaryotic biology including cell division, migration, and organismal development and have been linked to several human diseases. In addition to their biological importance, cilia are an ideal structure to study signaling networks of the ARF family. In addition to ARL16, three additional ARLs have been linked to cilia (ARL3, 6 and 13B). We began to explore the connections between these ARLs in cilia and identified novel cellular functions of these proteins.
Table of Contents
Table of Contents Chapter 1: Introduction 1 Scope of the Dissertation 2 References 6 Chapter 2: ARF family GTPases with links to cilia 8 Abstract 9 Introduction 10 ARFs and ARLs 12 ARF4 17 ARL3 and ARL2 21 ARL13B 24 ARL6 28 Summary 30 Acknowledgements 33 Abbreviations 34 References 35 Figures and Tables 59 Figure 1: Model for ADP-ribosylation factor 4 (ARF4) role in rhodopsin sorting at trans-Golgi network (TGN) Figure 2: Model for ADP-ribosylation factor-like 3 (ARL3) and ARL13B function in regulating the release of lipidated cargoes in the cilium. Figure 3: Model for ADP-ribosylation factor-like 6 (ARL6 action in regulating cargo transport in the cilium Table 1: Tools for assessing mammalian cilia and ciliary function 65 Chapter 3: Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E 67 Abstract 68 Introduction 69 Results 72 Discussion 86 Materials and Methods 94 Acknowledgements 104 References 105 Figures 122 Figure 1: Phylogenetic distribution of ARL16 and the three previously known cilium-associated ARF family members (ARL3, ARL6, and ARL13) and cilia in eukaryotes Figure 2: Characterization of endogenous ARL16 localization in RPE1 cells and human retina Figure 3: Deletion of Arl16 causes defects in ciliogenesis Figure 4: Arl16 KO cells have reduced recruitment of several ciliary proteins Figure 5: Arl16 KO cells have defects in Hh signaling Figure 6: IFT140 is lost from cilia in Arl16 KO cells, but other IFTs are unchanged Figure 7: IFT140 and INPP5E accumulate at the Golgi of Arl16 KO lines Figure 8: Cells deleted for Pde6d display no defects in ciliation but loss of INPP5E from cilia with its accumulation at Golgi Figure 9: Model for ARL16 in ciliogenesis and Golgi to cilium traffic Figure S1: ARL16 protein sequences diverge from ARF1 in key motif residues Figure S2: ARL16 localizes to cytosol and mitochondria Figure S3. Knock out of Arl16 in MEFs and screening of markers Figure S4. A number of ciliary features are unchanged between WT and Arl16 KO lines Figure S5: Pde6d CRISPR design Chapter 4: The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and Cilia to Regulate Ciliogenesis and Ciliary Protein Traffic 143 Abstract 144 Introduction 145 Results 149 Discussion 160 Materials and Methods 169 Acknowledgements 178 References 180 Figures and Tables 197 Figure 1: ELMOD1 and ELMOD3 show overlapping, but also distinctive, localization patterns in mouse retinal photoreceptor cells Figure 2: Elmod1 and/or Elmod3 KO in MEFs cause decreased ciliation Figure 3: Elmod1 or Elmod3 KO causes loss of ARL13B, ARL3, and INPP5E from cilia Figure 4: INPP5E and IFT140 accumulate at the Golgi in Elmod1 or Elmod3 KO lines Figure 5: The ciliation defect in Elmod1 or Elmod3 KO cells can be reversed upon transient expression of activated ARL3 or ARL16 Figure 6: Model for ELMOD1 and ELMOD3 function as regulators of ciliogenesis and traffic of key ciliary cargoes Figure S1: Generation of Elmod1 KO, Elmod3 KO, and DKO MEFs Figure S2: Cellular markers unchanged in Elmod1, Elmod3, or DKO cells Figure S3: Elmod1 KO, Elmod3 KO, or DKO MEFs exhibit no evident defects in cell cycle, rootlet morphology, or centrosome separation Figure S4: ELMOD1-myc is expressed to much higher levels than is ELMOD1- myc in MEFs Figure S5: Neither Elmod1 nor Elmod3 KO alters the extent of CEP164 recruitment and CP110 release from the basal body, despite the decrease in ciliation Figure S6: Gli3 recruitment to cilia is unaffected by deletion of Elmod1 or Elmod3 Figure S7: IFT140 colocalizes with Rootletin in both WT and KO cells Figure S8: IFT88 and IFT140 localization to cilia in ELMOD1/3 KO lines is unchanged Figure S9: Number and sizes of focal adhesions are unchanged in ELMOD1 and ELMOD3 KO lines Figure S10: Expression of myc-tagged ELMOD1 and ELMOD3 in WT MEFs Figure S11: ARL13B expression is reduced in Elmod1 KO, Elmod3 KO, and DKO lines Table 1: Summary of phenotypes found in MEFs deleted for Elmod1 KO, Elmod2 KO, Elmod3 KO, and Elmod1/Elmod3 DKO MEFs using CRISPR-Cas9 Chapter 5: ARL3 223 Background 2224 Results 226 Discussion 229 Materials and Methods 231 Figures 235 Figure 1: Knock out of ARL3 in MEFs Figure 2: Microtubules, Golgi, and Mitochondria are not obviously affected by the loss of ARL3 Figure 3: Arl3 KO cells are multinucleated and have increased midbodies Figure 4: Arl3 KO have increased focal adhesion and stress fiber staining Figure 5: Arl3 KOs have traffic related phenotypes: enlarged early endosomes and Golgi enrichment of INPP5E Figure 6: INPP5E is specifically lost from cilia while SMO is enriched in Arl3 KO cells References 241 Chapter 6: Phenotyping of ARL6 and ARL10 Knockouts 246 ARF Family Introduction 247 ARL10 Results 247 ARL10 Summary 251 ARL6 Results 252 ARL6 Summary 254 Materials and Methods 254 Figures 258 Figure 1: ARL10 KO, guides, alleles, and predicted protein sequences Figure 2: Mitochondria, Golgi, Actin, and microtubules are unchanged by the loss of ARL10 Figure 3: ARL10 KOs ciliate normally and have normal recruitment of ARL13B Figure 4: ARL10 KOs have supernumerary centrosomes and multinucleation Figure 5: ARL10 localizes to mitochondria and overexpression causes mitochondrial clustering Figure 6: ARL6 KO guides and predicted protein sequences Figure 7: ARL6 KO cells ciliate normally Figure 8: Actin, Golgi, and mitochondria are unchanged by the loss of ARL6 Figure 9: ARL6KO cells have supernumerary centrosomes and multinucleation References 259 Chapter 7: Discussion 269 Rationale and Challenges 270 Summary and Future Directions 272 Concluding Remarks 286 Figures and Tables 288 Figure 1: Cellular Locations of ARF Family Proteins Figure 2: ARL16, ELMOD1, ELMOD3, and ARL3 function in a common pathway to regulate ciliogenesis and ciliary protein traffic Table 1: Comparison of ciliary phenotypes of ARFs in cilia References 291
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