The microtubule cytoskeleton is a critical regulator of cell differentiation, and must be spatially organized in order to fulfill its cellular functions. Although the concept that microtubules are organized by specific sites called microtubule organizing centers (MTOCs) has been appreciated for more than 50 years, the vast majority of research on MTOCs has focused on the centrosome. While all animal cells use centrosomes as MTOCs during mitosis, MTOC function is reassigned to non-centrosomal sites during cell differentiation. For example, non-centrosomal MTOCs (ncMTOCs) form at the apical membrane of epithelial cells, down the length of axons and dendrites in neurons, and at the nuclear envelope in myotubes and microtubules are critical for neurogenesis, muscle function, and in morphogenesis and polarization of most tissues including the heart, brain, and intestine. Despite their ubiquity and importance in differentiated cells in vivo, little is known about mechanisms of ncMTOC establishment or the identity of ncMTOC components in an organism, in part due to the lack of an appropriate genetic model. This proposal tests the central hypothesis that ncMTOCs are composed of site-specific adapters and microtubule minus end proteins, with adapters linking microtubules through their minus ends to polarity complexes that mark cellular locations.
Our aims will address the composition and mechanisms of establishment of ncMTOCs, the two significant knowledge gaps in this field using cutting edge genetic and proteomic tools in the model organism C. elegans. We identified interactors with the exclusive ncMTOC component PTRN-1/Patronin and will uncover the role of these conserved interactors in ncMTOC establishment (Aim 1). We will identify novel ncMTOC components using our recently adapted proximity labeling technique TurboID, applied for the first time in C. elegans, and a high throughput tissue- and pathway-specific forward genetic screening strategy (Aim 2). Finally, we will test specific models for ncMTOC establishment using a tissue specific degradation strategy that we have optimized (Aim 3). Proper microtubule organization is essential for normal development and cell function and hyperactive MTOC function at the centrosome is a hallmark of some cancers. Thus, the molecules uncovered in these studies could provide potential therapeutic targets as well as shed light on an important, but understudied topic in cell and developmental biology.
All animal cells use microtubules during division to create the mitotic spindle and during differentiation for cell polarity, shape, and transport. The proposed research will define the mechanisms that control how microtubules become spatially organized to carry out these fundamentally different processes. An understanding of the mechanisms that control microtubule organization should provide therapeutic insight into the growing number of specific birth defects and human cancers that have been linked to defects in the microtubule cytoskeleton.
