Identification of an Aurora-A/PinsLINKER/Dlg spindle orientation pathway using induced cell polarity in S2 cells.
Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. Here, we develop a method to construct cortical polarity in a normally unpolarized cell line and use this method to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals) -dependent spindle orientation. We identify a previously unrecognized evolutionarily conserved Pins domain (Pins(LINKER)) that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized Pins(TPR) domain has no function alone, but placing the Pins(TPR) in cis to the Pins(LINKER) gives dynein-dependent precise spindle orientation. This "induced cortical polarity" assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity.
Johnston
Christopher A
CA
Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.
Hirono
Keiko
K
Prehoda
Kenneth E
KE
Doe
Chris Q
CQ
D000818:Animals; D064127:Aurora Kinases; D018797:Cell Cycle Proteins; D002460:Cell Line; D016764:Cell Polarity; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology* / Q000196:embryology; D004398:Dyneins / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010766:Phosphorylation; D011418:Prophase; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; C513192:Lis-1 protein, Drosophila; D008869:Microtubule-Associated Proteins; C405164:Pins protein, Drosophila; D025521:Tumor Suppressor Proteins; C070350:dlg1 protein, Drosophila; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D004398:Dyneins