Molecular dissection of mitochondria-microtubule interaction and microtubule nucleation

Abstract

Mitochondria are dynamic bioenergetic organelles and their distributions are tightly coupled with energy demands. Mitochondria also interact with other cellular structures to modulate their functions and regulate their positioning. For example, in many species, mitochondria interact with the microtubule cytoskeleton, which participates in a wide variety of cellular activities including organelle trafficking, cell migration, and cell division. A large number of microtubule associated proteins have been demonstrated not only to be involved in the microtubule related cellular activities but also to play important roles in microtubule nucleation and organization. How mitochondria are distributed by microtubules and how microtubule nucleation, the first step of microtubule biogenesis, is regulated remain to be further investigated. In this thesis, I aimed to address these two questions using the fission yeast Schizosaccharomyces pombe as a model organism. The distribution of mitochondria in different organisms depends on different cytoskeletons. In neuron, mitochondria are transported by motor proteins like dynein and kinesins along microtubules while mitochondria in fission yeast have been implicated to be positioned passively by mitochondria-microtubule interactions. The detailed molecular mechanisms underlying microtubule-based mitochondria distribution remain elusive. Furthermore, whether motor proteins and actin filaments contribute to mitochondria distribution in fission yeast has not been determined. In the first part of this thesis (chapter 3), I examined the roles of microtubules, kinesins, and actin filaments in mitochondria distribution. My experimental results demonstrate that the interaction between mitochondria and microtubules is necessary and sufficient for proper mitochondria distribution and that motor proteins and actin filaments are not necessary for mitochondria distribution in fission yeast. Thus, this study establishes that the fission yeast cells use a passive microtubule dependent mechanism to distribute mitochondria. Microtubule biogenesis starts with nucleation which is initiated by the gamma tubulin ring complex (c-TuRC) at various microtubule organization centers (MTOCs). Pre-existing microtubules serve as one of such MTOCs and contribute significantly to the overall organization of microtubule arrays. The augmin complex has been reported to play a key role in noncentrosomal microtubule nucleation. However, no augmin equivalent in yeasts has been identified. The molecular mechanism underlying non-centrosomal microtubule nucleation in yeasts remains unknown. In the second part of this thesis (chapter 4), I demonstrate that the DnaJ protein rsp1p is a key contributor in non-centrosomal microtubule nucleation in fission yeast. Rsp1p localizes to pre-existing microtubules and physically interacts with the microtubule nucleation promoting factor mto1p, which is responsible for recruiting f-TuRC, to initiate non-centrosomal microtubule nucleation. Furthermore, rsp1p appears to be involved in regulating microtubule nucleation in response to heat stress. Thus, this work uncovers a new mechanism regulating non-centrosomal microtubule nucleation.