Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Intracellular transport is fundamental to many cellular processes- from capturing and destroying pathogens to the propagation of nerve impulses. This transport is mediated by specialized enzymes that convert the free energy of ATP hydrolysis to ‘walk’ on polymeric filaments. The microtubule filament and its associated motors- dynein and kinesin - are responsible for the long-range transport of various cellular cargoes. While these two motors move in the opposite directions, they are often simultaneously present on individual cargoes leading to bidirectional motility characterized by frequency directional reversals. How this process is regulated and what determines the direction of cargo transport remains poorly understood. Addressing these questions requires a systematic analysis where the contribution of various factors in regulating/determining the transport direction of a well-defined cargo can be elucidated. This project establishes an in vitro assay where we reconstitute bidirectional motility of large unilamellar vesicles driven by purified dynein and kinesin-3 motors. Vesicles exhibit fast runs in either direction, with a subset exhibiting directional reversals. The transport features of these vesicles are remarkably similar to that of cargoes in vivo and do not require any additional regulatory proteins/complexes. The simultaneous opposing activity of dynein and kinesin-3 leads to tugs-of-war for a finite period. Finally, we use this assay to determine how microtubule-associated proteins that have differential activity towards dynein and kinesin affect the transport direction of vesicles. MAP9 biases the direction of vesicles towards the plus-end by limiting the ability of dynein to land on microtubules. Our approach can be extended to investigate the potential biasing activities of cellular factors such as posttranslational modifications of tubulin, motor-adaptors, etc. and physical factors, such as fluidity and tension of the vesicle membrane.:1 Introduction 1.1 Microtubules, dynein, kinesin, and microtubule-associated proteins (MAPs) 1.2 Bidirectional transport: In vivo observations and models 1.3 Bidirectional transport: In vitro reconstitution 1.4 Role of membranes in modulating motor activities 1.5 Aims of the thesis 2 Purification of dynein and dynactin 2.1 Purification of dynein and dynactin from adherent mammalian cell cultures 2.2 Purification of dynein and dynactin from suspension cultures using BacMam system 2.3 Validation of dynein and dynactin function using single-molecule motility assays 3 Reconstitution and characterization of bidirectional vesicle motility 3.1 DDB-KIF16B-vesicles exhibit directional reversals in vitro 3.2 Transport direction of vesicles is dependent on the relative concentration of DDB and KIF16B 3.3 Opposing motors do not affect the velocity of the driving motor 3.4 DDB and KIF16B engage in a tug-of-war before directional reversals 3.5 Discussion 4 Characterization of bidirectional motility in the presence of MAPs 4.1 Tau does not differentially affect DDB versus KIF16B at the single-molecule level 4.2 MAP9 affects landing of DDB but not KIF16B 4.3 Motor ensembles can circumnavigate the inhibitory effects of MAPs 4.4 Tau does not bias the transport direction of DDB-KIF16B-vesicles 4.5 MAP9 biases the transport direction of DDB-KIF16B-vesicles towards the plus-end 4.6 Discussion 5 Conclusion and outlook 6 Materials and Methods 6.1 Molecular Biology 6.2 Culture of Flp-In 293 cells 6.3 Protein biochemistry 6.4 In vitro motility assays 6.5 Data processing and analysis

Transport of different organelles along the Microtubule cytoskeleton is carried out mainly by motor proteins Dynein and Kinesin. The tubulin monomers in Microtubules are organized in such a way that the generate polarity (a minus and a plus end) that is recognized by Motor proteins. Dynein usually acts with a binding partner, Dynactin, and is in charge of moving cargoes to the minus end of microtubules (mainly towards the center of the cell). There are different kinesins, the most studied is Kinesin-1, which moves cargoes towards the plus end of microtubules. In order to fulfil their function Motors usually bind to their cargoes indirectly through adaptor proteins. Chapter 1 explains the general concepts related to a group of Adaptors that recognize the small GTP-ases, Rabs, in cargoes that need to be transported under certain physiological circumstances and help recruiting the Dynein/Dynactin complexes to them so they can move in the minus end direction. This family of Adaptors is called Rab-Dynein-Dynactin (RDD) adaptors and in this project I focused on two of them: BicD2 and RILP. In chapter 2, I will focus on BicD2 and its role in Golgi morphology. BicD2 is an RDD adaptor that mediates binding of Dynein/Dynactin to Rab6-positive vesicles. Some mutations in BicD2 have been associated to Golgi apparatus morphology disruption, but the mechanism is unclear. It has been suggested that mutated BicD2 abnormally binds Dynein/Dynactin, sequestering this motor complex, producing Golgi disruption indirectly since this organelle depends heavily on minus-directed transport to maintain its localization and structure. I test this hypothesis and conclude that even when most pathological mutations disrupt the Golgi, a Dynein/Dynactin-mediated mechanisms is probably true only to some of them, proposing alternatives mechanisms such as Rab6 abnormal accumulation and non-Golgi related mechanisms of pathogenesis. In chapter 3, I will focus on RILP and its role in autophagosome movement. RILP is an RDD adaptor that mediates binding of Dynein/Dynactin to Rab7-positive vesicles such as Lysosomes. During autophagy, autophagosomes (which are LC3-positive) are formed mainly in the ER and mature to finally fuse with the Late Endosomes or Lysosomes (both acidic) in the center of the cell. It has been described by our lab that RILP can transport LC3-vesicles in axons. Nevertheless, these vesicles are acidic, which suggest these LC3-vesicles are already fused with either Lysosomes or Late endosomes. I will work under the Hypothesis that RILP can move autophagosomes in early stages (before fusion with Lysosomes or Late endosomes) in non-neuronal cells. I show that RILP can move autophagosomes to the center and FYCO1 (a Kinesin-1 adaptor) can move them to the periphery. RILP-mediated movement of autophagosomes depends on Rab7 activation status and seems to be controlled by PKA. I proposed a phosphorylation in Rab7 as a control mechanism. Finally, the discovery of 3 LC3 interacting regions (LIRs) in the RILP molecule is discussed and their contribution to autophagosome movement is analyzed. My results highlight the relevance of RDD proteins in physiological and pathological context.

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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

The complex natural product paclitaxel (Taxol®), first isolated from Taxus brevifolia, is a member of a large family of taxane diterpenoids. Paclitaxel is extensively used for the treatment of solid tumors, particularly those of the breasts and ovaries. In order to obtain additional information about the mechanism of action of paclitaxel and the environment of the paclitaxel-binding site, several fluorescent analogs of paclitaxel were synthesized, and their biological activities have been evaluated. For the investigation of possible synergistic effects, concurrent modifications on selected positions have been performed and their biological evaluation were studied. / Ph. D.

Prostate cancer is the most common non-skin cancer for men in America. The androgen receptor exerts transcriptional activity and plays an important role for the proliferation of prostate cancer cells. Androgen receptor ligands bind the androgen receptor and inhibit its transcriptional activity effectively. However, prostate cancer can progress to hormone refractory prostate cancer (HRPC) to avoid this effect. Chemotherapies are currently the primary treatments for HRPC. Unfortunately, none of the available chemotherapies are curative. Among them, paclitaxel and docetaxel are two of the most effective drugs for HRPC. More importantly, docetaxel is the only form of chemotherapy known to prolong survival in the HRPC patients. We hypothesized that the conjugation of paclitaxel or docetaxel with an androgen receptor ligand will overcome the resistance mechanism of HRPC. Eleven conjugates were designed, synthesized and biologically evaluated. Some of them were active against androgen-independent prostate cancer, but they were all less active than paclitaxel and docetaxel. Discodermolide is a microtubule interactive agent, and has a similar mechanism of action to paclitaxel. Interestingly, discodermolide is active against paclitaxel-resistant cancer cells and can synergize with paclitaxel, which make it an attractive anticancer drug candidate. Understanding the bioactive conformation of discodermolide is important for drug development, but this task is difficult due to the linear and flexible structure of discodermolide. Indirect evidence for the orientation of discodermolide in the tubulin binding pocket can be obtained from fluorescence spectroscopy of the discodermolide tubulin complex. For this purpose, we designed and synthesized a simplified fluorescently labeled discodermolide analog, and it was active in the tubulin assembly bioassay. In addition, a conformationally constrained discodermolide was designed to mimic the bioactive conformation according to computational modeling. The synthetic effort was made, but failed during one of the final steps. / Ph. D.