IDA3-mediated IFT Transport of the Ciliary Inner Dynein Arm I1 公开

Hunter, Emily L. (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/4f16c2806?locale=zh
Published

Abstract

Motile cilia, also termed flagella, play a vital role in development, cell signaling, and organ function.  The motility of the cilium is dependent upon the conserved axonemal dynein motors that assemble in the cytoplasm prior to transport to the distal tip of the cilium by Intraflagellar Transport (IFT).  Defective assembly of the axonemal dyneins results in numerous human diseases including Primary Cilia Dyskinesia (PCD).  We are just beginning to understand the key proteins and mechanisms required to assemble and transport ciliary dyneins, as well as other axonemal complexes.  Here, I take advantage of the Chlamydomonas mutant ida3 to study the transport and assembly of the conserved inner dynein arm I1/f dynein.  The ida3 mutant properly assembles I1 dynein in the cytoplasm but I1 dynein fails to enter the cilium for assembly in the axoneme, resulting in a slow swimming phenotype.  Cryo-ET confirms I1 dynein is the only structural defect present in ida3 axonemes.  From my dissertation work, we now know the IDA3 gene encodes a conserved ~115kD coiled-coil protein that primarily enters the cilium during ciliary growth.  Live-cell imaging and biochemical analyses reveal that during cilium growth, IDA3, along with I1 dynein, is transported by anterograde IFT to the tip of the cilium.  At the tip, IDA3 detaches from IFT and diffuses within the ciliary compartment.  IFT transport of IDA3 decreases as cilia lengthen and subsides once full-length is achieved.  Presence of the I1 dynein cargo is essential to permit processive IFT transport of IDA3 to the distal tip of the cilium.  I conclude IDA3 encodes a highly specialized and transient adapter required to mediate IFT interaction with I1 dynein for entry and transport in the assembling cilium.  IDA3 is the first example of an essential and selective IFT adapter that is regulated by ciliary length.  

Table of Contents

TABLE OF CONTENTS

            Dissertation Overview……………………………………………………………1

Chapter I: Introduction

            General Mechanisms of Ciliary Assembly…………………………………….….2

            Introduction to the Eukaryotic Cilium…………………………………………….2

            Ultrastructure of the Motile Cilium…………………………………………….…4

            Chlamydomonas reinhardtii:

A Model System for Study of Ciliary Assembly ………………………...7

            The Discovery of Intraflagellar Transport (IFT)…………………………...……11

            Intraflagellar Transport and Length Regulation of the Cilium ………………….17

            The Cargos of Intraflagellar Transport (IFT)……………………………………19

            Cargo Interaction with IFT:

 Direct Binding to IFT and the Use of IFT Adapters…………………….24

            Addressing Novel Questions:

IDA3 and I1 Dynein Assembly…………………………………………..30

Figures……………………………………………………………………32

Chapter II: Introduction to Inner Dynein Arm Assembly and ida3

            Study of Inner Dynein Arm Assembly………………………………………..…44

            The I1/f Dynein Complex………………………………………………………..44

            Introduction to ida3………………………………………………………………49

            What Role Does IDA3 Play in I1 Dynein Assembly: Potential Hypotheses…….54

            Figures……………………………………………………………………………56

 

Chapter III: Study of IDA3

            Identification and Characterization of IDA3…………………………………….70

            Inner Dynein Arm I1 is Specifically Missing in ida3 mutant axonemes………..70

            A Nonsense Mutation in IDA3 Results in Loss of I1 Dynein in the Axoneme….71

            IDA3 is Transported by Anterograde IFT within the Regenerating Cilium.….…73

            IDA3 Transport by Anterograde IFT is Dependent on Ciliary Length………….79

            IDA3 Interacts Biochemically with IC140 in the Matrix of Regenerating Cilia...80

            Stable Binding of IDA3 to IFT Requires I1 Dynein……………………………..81

            Figures……………………………………………………………………………84

Chapter IV: Implications of IDA3 and Future Directions

            Summary: Model of IDA3-mediated I1 Dynein Assembly.…………………....136

            Understanding IDA3 behavior and Interactions in the Cytoplasm……………..138

            IDA3: A model for Addressing New Questions About Ciliary Assembly……..141

                        How Are Ciliary Proteins Transported to the Basal Body?…………….141

                        How is Cargo-loading Regulated?...........................................................143

            IDA3: A New Way to Think About IFT Adapters…………………………..…147

            ODA16 v. IDA3………………………………………………………………...148

            Identification of Additional IFT Adapters……………………………………...151

                        ODA8 as a Candidate ODA Adapter…………………………………...152

                        PF27 as a Potential Radial Spoke IFT Adapter………………………...152

            Concluding Remarks……………………………………………………………154

            Figures…………………………………………………………………………..155

Appendix I: Materials and Methods………………………………………………....170

Appendix II: IDA3 Construct Information …………………………………………188

References……………………………………………………………………………...191

List of Figures and Tables

Chapter I

Figure 1. Ultrastructure of the motile cilium…………………………………………..32

Figure 2. Dikaryon rescue in Chlamydomonas reinhardtii…………………………….34

Figure 3. TIRF microscopy using Chlamydomonas……………………………………36

Figure 4. Composition of the IFT complex…………………………………………….38

Figure 5. Intraflagellar Transport in Chlamydomonas reinhardtii……………………..40

Figure 6. Model of known and potential IFT cargo binding sites……………………...42

Chapter II

Figure 7. I1/f dynein subunit composition………………………………………………56

Figure 8. Dikaryon rescue of I1 dynein assembly occurs from the distal tip……….......58

Figure 9. Dikaryon rescue of axonemal I1 assembly requires protein synthesis………..60

Figure 10. Transport of I1 to the tip of the cilium requires Kinesin-2…………………..62

Figure 11. IC140::GFP is transported by anterograde IFT……………………………...­­­64

Figure 12. The ida3 mutant is defective in I1 dynein entry into the ciliary compartment……………………………………………………………………………..66

Figure 13. Potential hypotheses for the role of IDA3 in I1 dynein assembly…………..68

Chapter III

Figure 14. I1 dynein is specifically missing in ida3 axonemes………………………...84

Figure 15. I1 dynein is absent in ida3 axonemes when examined by electron microscopy……………………………………………………………………………….86

Table I. Primers used to map the ida3 mutant to Creo03.g205000……………………..88

Figure 16. The ida3 mutant contains a single base transversion that results in a premature stop codon………………………………………………………………………………..90

Table II. IDA3 constructs used for ida3 transformation………………………………...92

Figure 17. Transformation of ida3 with IDA3-tagged constructs rescues I1 dynein assembly in the axoneme………………………………………………………………..94

Figure 18. I1 dynein assembly is rescued in the axoneme of intragenic revertants of ida3; oda2 cells……………………………………...…………………………………………96

Table III. Sequencing analysis of ida3; oda2 intragenic revertants…………………….98

Figure 19. Extragenic reversion of ida3; oda2 results in the assembly of truncated IC140 in the axoneme………………………………………………………………………….100

Figure 20. Analysis of IDA3 protein structure and conservation……………………...102

Figure 21. IDA3 is present in the cytoplasm but does not assemble in the axoneme….104

Figure 22. IDA3 localizes to the basal bodies…………………………………………106

Figure 23. IDA3 localizes to distinct puncta in the cytoplasm………………………...108

Figure 24. Analysis of cilia length during regeneration………………………………..110

Figure 25. IDA3 selectively enters the cilium during regeneration……………………112

Figure 26. Immunofluorescence analysis detects IDA3::HA in the matrix compartment of the regenerating cilium………………………………………………………………114

Figure 27. IDA3 is transported by anterograde IFT in the regenerating cilium……….116

Figure 28. Characterization of IDA3 behavior in the regenerating cilium…………….118

Figure 29. Single particles of IDA3 are transported by IFT to the tip of the cilium…..120

Figure 30. Kymogram gallery of IDA3 behavior in the cilium………………………..122

Figure 31. IDA3 transport by anterograde IFT is regulated by ciliary length…………124

Figure 32. IDA3 transport is regulated by changes in cilium length and not the need to assemble I1 dynein in the axoneme……………………………………………….……126

Figure 33. IDA3 interacts with IC140 of the I1 dynein complex in the matrix of the regenerating cilium………………………………………………………………….….128

Figure 34. IDA3 interacts with IC140 in the cytoplasm………………………….……130

Figure 35. Modeling IDA3-mediated I1 dynein interaction with IFT…………………132

Figure 36. Efficient IFT transport of IDA3 requires I1 dynein………………………..134

Chapter IV

Figure 37. IDA3 mediates IFT entry and transport of the I1 dynein complex in a cilium-autonomous manner…………………………………………………………………….155

Figure 38. IDA3 is transported along the rootlet microtubules………………………..157

Figure 39. The potential role of IDA3 diffusion in IFT-cargo loading and cilium length regulation………………………………………………………………………….……159

Figure 40. IDA3 is phosphorylated in the regenerating matrix………………………..161

Figure 41. IDA3 attachment to IFT may be regulated by phosphorylation……………163

Figure 42. Potential outcomes for study of IDA3 phosphorylation mutants…………..165

Figure 43. The proximal radial spokes in the pf27 axoneme assemble asynchronously from the rest of the axoneme during ciliary regeneration………………………………167

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