Assembly and functions of cilia rely on the continuous transport of ciliary components between the cell body and the ciliary tip. This is performed by specialized molecular machines, known as Intraflagellar Transport (IFT) trains. Anterograde IFT trains are powered by kinesin-2 motors and move along the B-tubules of the microtubule doublets. Conversely, retrograde IFT trains are moved by dynein-2 motors along the A-tubules back to the cell body. The segregation of oppositely directed trains on A-tubules or B-tubules is thought to prevent traffic jams in the cilium, but the mechanism by which opposite polarity trains are sorted on either tubule is unknown. It has been reported that A-tubule and B-tubule are differentially enriched in tyrosinated and detyrosinated tubulins, but whether this difference has a role in IFT regula2on is not understood. Here, I show that CRISPR knock out of VASHL, the enzyme that detyrosinates microtubules, causes repeated collisions between oppositely directed trains and reduces the rate of ciliary growth. To test whether this is ascribable to direct interaction between IFT trains and tubulin detyrosination/tyrosination, I developed a method to reconstiute the motility of native IFT trains from Chlamydomonas reinhardtii cilia ex-vivo on synthetically polymerized microtubules enriched for either tubulin post-translational modification. I show that retrograde trains have higher affinity for tyrosinated microtubules (analogous to A-tubules), while anterograde trains for detyrosinated microtubules (analogous to B-tubules). I conclude that tubulin tyrosination/detyrosination is required for the spatial segregation of oppositely directed trains and for their smooth uninterrupted motion. My results provide a model for how IFT motility is governed by the underlying tubulin code.:Table of Contents Abstract ............................................................................................................................. 4 1. Introduc1on .................................................................................................................. 7 1.1 Cilia ............................................................................................................................................... 7 1.1.1 Cilia are ubiquitous and important cell organelle ................................................................................ 7 1.1.2 Pathologies associated with Ciliary dysfunc=on .................................................................................. 7 1.1.3 Axoneme is the structural scaffold of the cilium ................................................................................. 8 1.2 Intraflagellar transport (IFT) ....................................................................................................... 11 1.2.1 IFT complex is large macromolecular protein assembly .................................................................... 11 1.2.2 Microtubule motors drive the IFT trains ............................................................................................ 12 1.3 Bidirec<onal transport by microtubule cytoskeleton motors .................................................... 16 1.3.1 Microtubules ...................................................................................................................................... 16 1.3.2 Kinesin ............................................................................................................................................... 18 1.3.3 Dynein ................................................................................................................................................ 21 1.3.4 Mul=-motor transport ....................................................................................................................... 24 1.3.5 Cargo Logis=cs ................................................................................................................................... 27 1.4 Aims of the thesis ....................................................................................................................... 34 2. Ex-vivo Recons1tu1on of IFT train mo1lity ................................................................... 36 2.1 Capillary micropipeGe can be coupled with TIRFM for IFT train recons<tu<on ........................ 36 2.2 Ex-vivo mo<lity of IFT trains ....................................................................................................... 41 2.3 Ex-vivo trains retain their complex iden<ty ................................................................................ 43 3. Role of tubulin tyrosina1on/detyrosina1on in train sor1ng ......................................... 46 3.1 Chlamydomonas VashL encodes for axonemal tubulin detyrosina<on ac<vity ......................... 47 3.2 Anterograde IFT trains exhibit collisions in Chlamydomonas VashL mutant .............................. 48 3.3 Anterograde trains have a stronger affinity for normal ghost axonemes ................................... 51 3.4 The affinity of anterograde trains reduces for VashL ghost axonemes ...................................... 51 3.5 Retrograde trains are more likely to land on detyrosinated microtubules ................................ 53 4. Sidestepping as a IFT sor1ng mechanism ..................................................................... 57 4.1 2D tracking of IFT trains reveals off-axis stepping component ................................................... 57 4.2 IFT trains do not collide when crossing on microtubules ........................................................... 61 5. Discussion and Outlook ............................................................................................... 63 6. Materials and Methods ............................................................................................... 71 6.1 Chlamydomonas Cell Culture ..................................................................................................... 71 6.2 Crea<on of IFT-46 mNeonGreen::VashL ..................................................................................... 71 6.3 Coverslip Prepara<on ................................................................................................................. 72 6.4 Capillary pipeGe prepara<on and manipula<on ........................................................................ 72 6.5 Microtubule Polymeriza<on and polarity labeling ..................................................................... 73 6.6 Enzyme treatments of Microtubules .......................................................................................... 73 6.7 TIRF Microscopy ......................................................................................................................... 74 6.8 Analysis of TIRFM data ............................................................................................................... 74 6.9 Kymographs of Ghost Axonemes: .............................................................................................. 75 6.10 Flagella Isola<on ...................................................................................................................... 75 6.11 Western Blo_ng ...................................................................................................................... 76 6.12 Par<cle tracking in FIESTA ........................................................................................................ 76 7. Step-by-step protocol for IFT mo1lity recons1tu1on .................................................... 78 7.1 Materials .................................................................................................................................... 78 7.2 Method ...................................................................................................................................... 79 Bibliography .................................................................................................................... 86 Acknowledgements ......................................................................................................... 98 Declara1on according to §5.5 of the doctorate regula1ons ............................................ 100