Microtubule organization is critical for cell function. Nearly all dividing animal cells use the centrosome as a microtubule organizing center (MTOC), where microtubules tether chromosomes to the spindle poles to facilitate the correct segregation of DNA between daughter cells. By contrast, differentiated cells organize their microtubules in a wide variety of patterns, and establish specific noncentrosomal locations as MTOCs to achieve these microtubule arrangements. While much is known about how centrosomes organize microtubules, little is known about the composition of noncentrosomal MTOCs (ncMTOCs), how these sites are designated, or how they organize microtubules. A simple hypothesis is that an ncMTOC is essentially the MTOC features of a centrosome targeted to a different cellular location. Our studies suggest that the ncMTOC that forms at the apical surface of the polarized C. elegans intestine is in fact different in its composition and protein requirements for microtubule organization than the centrosome. We have examined the localization of several CRISPR-tagged microtubule- and MTOC-associated proteins in the intestinal ncMTOC, and observe three classes of proteins: (1) only at the centrosome (SPD-2/Cep192, SPD-5), (2) only at the ncMTOC (PTRN-1/CAMSAP, NOCA-1/Ninein), and (3) at whichever location is the active MTOC (?-TuRC proteins, AIR-1/Aurora A). To test the requirement of several factors in intestinal ncMTOCs, including AIR-1 and the ?-TuRC components GIP-1 and Mozart, we have optimized the ZIF-1/ZF protein degradation system to allow for the removal of early essential proteins from tissues of interest. We find evidence that the microtubule nucleation complex ?-TuRC must be intact for the recruitment of ?-TuRC components to the ncMTOC. Surprisingly, microtubules are still made and correctly localized to the apical ncMTOC even when ?-TuRC and AIR-1 are compromised, and we are currently analyzing the dynamics of these microtubules. These results suggest that differentiated cells use novel mechanisms to create a functional ncMTOC that organizes their microtubules.

Affiliation:
- Dept. of Biology, Stanford University, Stanford, CA