An Ana2/Ctp/Mud Complex Regulates Spindle Orientation in Drosophila Neuroblasts

Abstract

Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to orient the mitotic spindle during NB asymmetric division.

The Mechanism of Dynein Light Chain LC8-mediated Oligomerization of the Ana2 Centriole Duplication Factor

“…1, C, E, and F) (12). Previous studies demonstrated a physical interaction between the N-terminal 274 residues of Ana2 and LC8, a dynein light chain, via yeast two-hybrid screening (24). Structure function analysis indicated that Ana2 contained at least two LC8 binding sites, one within the region spanning residues 1-200 and the second within the region spanning residues 201-274.…”

“…Inset, alignments of individual Sas-4 binding domains and STAN domains between D. melanogaster and H. sapiens, D. rerio, or C. elegans reveal high percentages of invariant (first value) and similar (second value) residues. D, Ana2 and LC8 form a complex with Mud to orient the mitotic spindle during asymmetric divisions in the developing Drosophila neuroblast (24). Asymmetry is achieved, in part, via differential maturation of the centrosomes.…”

“…Asymmetry is achieved, in part, via differential maturation of the centrosomes. The daughter centrosome forms the LC8-Ana2-Mud complex that coordinates spindle alignment with cortical polarity cues to maintain a stem population (ganglion mother cell (GMC)) (53,24). E, full-length Drosophila Ana2 has an N-terminal Sas-4 binding region (13,14) and a C-terminal STAN motif (12) conserved across functional Ana2 orthologs.…”

“…It was shown in a yeast two-hybrid screen that LC8 binds two Ana2 fragments: the first fragment spanning residues 1-200 and the second spanning residues 201-274, which includes a predicted ␣-helix highly conserved across fly species (Fig. 1, E and F) (24,12). To date, there is no structural insight into the LC8-Ana2 complex.…”

“…How Ana2 and Sas-6 synergistically function remains to be determined. Although the function of Ana2 is poorly understood, recent work has demonstrated that Drosophila Ana2 interacts with the dynein light chain, LC8 (cut up (Ctp)) (24), a ubiquitous protein that binds diverse targets throughout the cell to confer or potentiate target dimerization (reviewed in Ref. 25).…”