656A Poster - 10. Cell biology: Cytoskeleton, organelles and trafficking
Thursday April 07, 2:00 PM - 4:00 PM

Authors:
Ryan O'Neill ¹; Afeez Sodeinde ²; Frances Welsh ³; Carey Fagerstrom ¹; Brian Galletta ¹; Nasser Rusan ¹

Affiliations:
1) National Heart, Lung, and Blood Institute, NIH; 2) Yale, New Haven, CT; 3) University of Washington, Seattle, WA

Keywords:
a. cytoskeleton; a. genome evolution

Centrosomes are the major microtubule organizing center (MTOC) of the cell, ensuring proper spindle formation and chromosome segregation during cell division. Hundreds of proteins make up the centrosome, including many proteins that form the pericentriolar material (PCM) which nucleates microtubules. The specific requirements for MTOC activity varies across cell types, a concept we are just starting to investigate and understand. In this study, we took an evolutionary cell biological approach to gain insight into cell type-specific regulation of centrosome proteins, reasoning that gene duplication could lead to the evolution of centrosome gene duplicates with cell type-specific functions. We first used BLAST to screen 35 sequenced Drosophila genomes for duplications of centrosome genes, finding five that were duplicated at least once. Here, we focus on the duplication of Spd-2 in D. willistoni: we refer to the parental gene as Spd-2A and the new copy as Spd-2B. In D. melanogaster, Spd-2 is known to function by organizing PCM in neuroblasts and spermatocytes. We found that Spd-2B is rapidly evolving and lacks the C-terminal 116 amino acid tail of Spd-2A. To explore expression and function we made GFP-tagged Spd-2A and Spd-2B transgenes, including their D. willistoni native regulatory elements, in D. melanogaster. Spd-2A is ubiquitously expressed, whereas Spd-2B is only expressed in spermatogenesis. Consistent with their expression, Spd-2A, but not Spd-2B, rescues spd-2^- mutant neuroblast PCM. In contrast, Spd-2B rescues spd-2^- mutant spermatocyte PCM, whereas even when ectopically expressed Spd-2A fails to do so. Thus, after gene duplication Spd-2A lost the ability to organize PCM in spermatocytes, leading to complementary functions for Spd-2A and Spd-2B. We further mapped the changes responsible for this difference in function, finding that removing the C-terminal tail from Spd-2A allowed it to properly organize spermatocyte PCM, whereas adding a C-terminal tail to Spd-2B prevented it from organizing spermatocyte PCM. We infer a model where the C-terminal tail of Spd-2 mediates a priming step that precedes PCM recruitment. Together, these results show that somatic and germline cells have different requirements for PCM, and suggest that Spd-2 is differentially regulated across cell types to satisfy these distinct requirements.