For asymmetric divisions to proceed properly, stem cells must accomplish the following: A) establish proper polarity, B) build a functional mitotic spindle using centrosomes that steer and anchor it, and C) properly align the spindle along a defined polarity axis using forces generated by astral MTs-cortical interactions. If executed properly, stem cells divide asymmetrically to produce a differentiating daughter cell. However, defects in this process lead to missegregation of cell fate determinants. If both cells retain stem cell identity, this can, under certain circumstances, lead to tumorogenesis. Mutations in polarity genes (Lgl, Dlg, Scrib) cell fate determinants (Pros, Numb, Mira, Brat), and centrioles/centrosomes (sas-4, asl, aurora A, polo) can all lead to cell overgrowth and malignant transformation. Our lab is interested understanding a unique feature of stem cell asymmetric division the asymmetric activity of the two centrosomes. To investigate how this asymmetry is achieved we are determining the protein composition of the two centrioles/centrosomes. Preliminary data suggest that the composition does differ in several waysthe presence of Polo on the active centrosome and the presence of Centrobin on the inactive centrosome in interphase. Further investigation is underway to analyze all the centriole proteins, and some centrosome components. For this project it was necessary to establish protocols for proper live cell imaging of two SC populations, the neural SCs and the germline SCs. We are now able to follow our SCs for 48hrs with little damage to the tissue and cells. We are currently analyzing both SC populations in wild-type and mutant centrosome backgrounds to determine how specific mutations alter SC division and function.
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