Investigating dynein-based transport of intracellular pathogens
Cytoplasmic dynein is a major microtubule-based motor which powers the long-distance transport of organelles, vesicles and other cytoplasmic contents from the cell periphery towards the centre. While dynein typically moves cellular cargoes, it can also be hijacked by pathogens that rely on intracellular transport for parts of their replicative cycle. This phenomenon is well exemplified by the obligate intracellular bacterium Orientia tsutsugamushi, which exploits dynein and microtubules to reach its subcellular niche at the perinucleus. Similarly, the neurotrophic virus Herpes Simplex Virus type 1 (HSV-1) undergoes dynein-dependent movement from the axonal tip of sensory neurons to the nucleus. Although the ability of these pathogens to co-opt dynein motors is well documented, the molecular mechanisms underlying this process have remained elusive. In particular, it is not known how dynein motors are recruited to incoming bacteria or viral particles and how transport is regulated. In Chapter 3 of this dissertation, I investigate the mechanisms underpinning the dynein-based transport of Orientia tsutsugamushi. Using a combination of biochemistry and cell biology, I discovered an interaction between the bacterial surface protein ScaC and the activating adaptor BICD2, which has a known role in connecting dynein to its cargoes. I further demonstrate that ScaC binds the C-terminus of BICD2 and relieves its autoinhibition to promote the formation of motile dynein-dynactin complexes. In Chapter 4, I present the results of a collaborative project in which I leveraged the cell-based assays I developed during my PhD to study the function of the dynein regulator LIS1. Finally, in Chapter 5, I investigate the mechanisms of dynein hijacking by HSV-1. Previous studies have indicated that the viral tegument protein UL36 is responsible for the recruitment of both dynein and kinesin motors. I demonstrate that ectopically expressed UL36 exhibits different transport behaviours in different cell types and discuss potential strategies to investigate how motor preference is specified. Additionally, I present my efforts to develop and optimize a pipeline to study HSV-1 transport in iNeurons cultured in microfluidic devices.
molecular biology, cytoskeleton, host-pathogen interactions
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