Canoe binds RanGTP to promote Pins(TPR)/Mud-mediated spindle orientation.
Regulated spindle orientation maintains epithelial tissue integrity and stem cell asymmetric cell division. In Drosophila melanogaster neural stem cells (neuroblasts), the scaffolding protein Canoe (Afadin/Af-6 in mammals) regulates spindle orientation, but its protein interaction partners and mechanism of action are unknown. In this paper, we use our recently developed induced cell polarity system to dissect the molecular mechanism of Canoe-mediated spindle orientation. We show that a previously uncharacterized portion of Canoe directly binds the Partner of Inscuteable (Pins) tetratricopeptide repeat (TPR) domain. The Canoe-Pins(TPR) interaction recruits Canoe to the cell cortex and is required for activation of the Pins(TPR)-Mud (nuclear mitotic apparatus in mammals) spindle orientation pathway. We show that the Canoe Ras-association (RA) domains directly bind RanGTP and that both the Canoe(RA) domains and RanGTP are required to recruit Mud to the cortex and activate the Pins/Mud/dynein spindle orientation pathway.
Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.
Brett
B
Wee
Christopher A
CA
Johnston
Kenneth E
KE
Prehoda
Chris Q
CQ
Doe
D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism* / Q000648:ultrastructure; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000502:physiology*; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D008565:Membrane Proteins; C411670:Mud protein, Drosophila; D009419:Nerve Tissue Proteins; C405164:Pins protein, Drosophila; C092747:cno protein, Drosophila; D020931:ran GTP-Binding Protein