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Ahna Skop Identification of novel cell division proteins:

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1 Ahna Skop Identification of novel cell division proteins:
functional proteomics of the midbody

2 The phases of cytokinesis or cleavage

3 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

4 Analogous division remnants
midbody Several conserved components known


6 Rationale Midbodies easy to purify
Enriched source of proteins relevant to cell division Function-based secondary screen possible

7 Midbody purification -sychronize CHO cells in cytokinesis
-Stabilize MTs and actin -Isolate midbodies in a hypotonic lysis buffer (PIPES, Triton) by vortexing vigorously -Pellet

8 The Strategy Tandem Liquid Chromatography- Tandem Mass Spec 4x
(LC/LC-MS/MS) (i.e no gel cutting) 4x Isolate Midbodies Synchronize CHO cells BIG LIST of proteins RNAi homologs to test for function Batch BLAST to find C. elegans homologs

9 577 Mammalian Proteins: Midbody LC/LC Tandem Mass Spec Data
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

10 159 Mammalian Proteins: Midbody LC/LC Tandem Mass Spec Data
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!

11 Functional Classes of Mammalian Midbody Proteins
Actin Binding Secretory MT binding Kinases Other Structural

12 RNAi Phenotypes Observed
40 38% RNAi Phenotypes Observed 30 25% 24% 20 16% 16% 14% 12% 12% 11% 8% 10 early late gonad mit mei seg align unc cytokinesis sterile M-phase WT

13 RNAi examples K04D7.1-RACK1 -anchor for PKC and other signaling enzymes, dynamin dsRNA is injected into a histone & tubulin::GFP strain

14 rachis common cytoplasm
The C. elegans gonad is a syncytium Top Focal Plane oocytes germline nuclei rachis common cytoplasm Mid Focal Plane oocytes

15 The C.elegans gonad is partially cellularized
like the Drosophila embryo  an incomplete form of cytokinesis syncytial germline membrane

16 An example of a gonad cytokinesis defect
F09C3.1-IQGAP WT gonad -midfocal plane F09C3.1 RNAi -midfocal plane

17 -most abundant ER protein
T05E11.3-Endoplasmin -most abundant ER protein metaphase congression failure lagging chromosomes

18 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

19 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

20 Other potential cellular proteomes
mitotic spindles chromosomes/kinetochores nuclear matrix

21 University of California, Berkeley
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

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