Interaction between Poly(ADP-ribose) and NuMA contributes to mitotic spindle pole assembly.
Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.
Chang
Paul
P
Koch Institute for Integrative Cancer Research, and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. pchang2@mit.edu
Coughlin
Margaret
M
Mitchison
Timothy J
TJ
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D006367:HeLa Cells; D006801:Humans; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D038501:Tankyrases / Q000235:genetics / Q000378:metabolism
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D034521:Nuclear Matrix-Associated Proteins; D011064:Poly Adenosine Diphosphate Ribose; D011993:Recombinant Fusion Proteins; D038501:Tankyrases; C050045:NUMA1 protein, human; C418109:TNKS protein, human