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Ahna Skop Identification of novel cell division proteins: functional proteomics of the midbody The phases of cytokinesis or cleavage The Midbody -formed from microtubules that span the spindle midzone in late anaphase that are bundled by the cleavage furrow - midbody function is poorly characterized Length(early) ~3-5um Length (late) ~1um Analogous division remnants midbody Several conserved components known Rationale Midbodies easy to purify Enriched source of proteins relevant to cell division Function-based secondary screen possible Midbody purification -sychronize CHO cells in cytokinesis -Stabilize MTs and actin -Isolate midbodies in a hypotonic lysis buffer (PIPES, Triton) by vortexing vigorously -Pellet The Strategy Tandem Liquid 4x ChromatographyTandem Mass Spec (LC/LC-MS/MS) (i.e no gel cutting) Isolate Midbodies Synchronize CHO cells BIG LIST of proteins RNAi homologs to test for function Batch BLAST to find C. elegans homologs Midbody LC/LC Tandem Mass Spec Data 577 Mammalian Proteins: REMOVED 418 from list (!!!) Nuclear contaminants (1 nucleus/60-70 midbodies) histones/chromatin proteins (15%) splicing factors (6%) Ribosomal proteins (12%) mitochondrial proteins (25%) heat shock proteins (6%) transcription/translation factors Midbody LC/LC Tandem Mass Spec Data 159 Mammalian Proteins: 52 known cell division proteins in mammal, yeast, amoebae, plant, Drosophila or C.elegans. 107 proteins previously uncharacterized with respect to cell division 90% had C. elegans homologues Ahna RNAied OVER 171 homologous or similar genes 88% had meiotic or mitotic defects! Functional Classes of Mammalian Midbody Proteins Secretory Kinases Actin Binding Other MT binding Structural 38% 40 30 RNAi Phenotypes Observed 25% 24% 20 16% 14% 12% 16% 12% 11% 8% 10 early late cytokinesis gonad sterile mit mei seg M-phase align unc WT RNAi examples K04D7.1-RACK1 -anchor for PKC and other signaling enzymes, dynamin dsRNA is injected into a histone & tubulin::GFP strain The C. elegans gonad is a syncytium Top Focal Plane oocytes germline nuclei Mid Focal Plane rachis common cytoplasm oocytes The C.elegans gonad is partially cellularized like the Drosophila embryo an incomplete form of cytokinesis syncytial germline membrane An example of a gonad cytokinesis defect F09C3.1-IQGAP WT gonad -midfocal plane F09C3.1 RNAi -midfocal plane T05E11.3-Endoplasmin -most abundant ER protein metaphase congression failure lagging chromosomes Conclusions - good news • Functional proteomic analysis of the mammalian midbody revealed conserved cell division proteins • Most midbody components identified function in cytokinesis and related processes • Common membrane-cytoskeletal dynamic mechanisms appear to underlie cell division, vesicle trafficking and neuronal function Caveats • Purification procedures are never perfect purity/sensitivity trade-off effects of drugs, detergents • Data analysis is necessarily somewhat subjective what to choose for functional studies • Multiple functions of identified proteins complicated functional analysis Other potential cellular proteomes • mitotic spindles • chromosomes/kinetochores • nuclear matrix Acknowledgments University of California, Berkeley Jen Banks Priya Prakash Budde Renée Deehan Sharat Gadde Petr Kalab Tom Maresca Aaron Van Hooser Sadie Wignall Ahna Skop Barbara Meyer Scripps John Yates Hongbin Liu