Mechanisms controlling microtubule organization during cell differentiation

PROJECT SUMMARY/ABSTRACT The microtubule cytoskeleton is a critical regulator of cell differentiation and must be spatially organized by subcellular sites called microtubule organizing centers (MTOCs) to fulfill cellular functions. Since the discovery of MTOCs over 50 years ago, the majority of MTOC research has focused on the centrosome, an organelle that organizes microtubules during animal cell mitosis. However, MTOC function is reassigned to non- centrosomal sites during cell differentiation: non-centrosomal MTOCs (ncMTOCs) form at the apical membrane of epithelial cells, the axons and dendrites of neurons, and the nuclear envelope in skeletal muscle cells, and non-centrosomal microtubules are critical for the development and function of the tissues that these cell types comprise. The generation of non-centrosomal microtubule networks is associated with the simultaneous inactivation of MTOC function at the centrosome and defects in centrosome inactivation are prevalent in epithelial cancer and linked to invasive cell behavior. Thus, a dramatic reorganization of the microtubule cytoskeleton is a fundamental aspect of cell differentiation, and my lab has used support from NIGMS over the last 5 years to uncover mechanisms of ncMTOC establishment and centrosome inactivation. We established C. elegans as an in vivo model of non-centrosomal microtubule organization, identifying and characterizing ncMTOCs at the apical surface of intestinal epithelial cells and at the tips of the dendrites in sensory neurons. By developing tools for tissue-specific degradation and proximity labeling, we found that essential MTOC proteins can vary across subcellular locations and cell types and identified novel ncMTOC components, including the spectraplakin protein VAB-10B and primary microcephaly protein WDR-62. We uncovered the modes and mechanisms by which MTOC function is removed from the centrosome and identified an essential SPD-5-based module of proteins required for centrosomal MTOC function across cell types. With these technical and conceptual advances in hand, our goal over the next 5 years is to answer fundamental questions related to microtubule organization: 1. How do differentiated cell types build specialized microtubule networks? We will use VAB-10B and WDR-62 as genetic handles to define comprehensive proximity maps of ncMTOCs across cell types with the goal of understanding how ncMTOCs first arise during differentiation; 2. How and why is MTOC function inactivated at the centrosome? We will uncover the molecular mechanism by which SPD-5 transforms the centrosome into an MTOC and test the physiological relevance of centrosome inactivation; 3. How do compound microtubules form? Using novel genetic tools, we will determine how microtubules build onto one another to form doublet microtubules, conserved features of organelles called cilia. These questions are fundamental to our understanding of cell differentiation and leverage unique properties of C. elegans to address this important, but understudied topic in cell and developmental biology.