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

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Presentation on theme: "Identification of novel cell division proteins: functional proteomics of the midbody Ahna Skop."— Presentation transcript:

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

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 Several conserved components known midbody


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

9 577 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

10 159 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 Midbody LC/LC Tandem Mass Spec Data 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 cytokinesis earlylate sterile 25% 12% 38% 11% 24% 14% 8% 16% 12% M-phase mitmei alignunc WT seggonad RNAi Phenotypes Observed 10 20 30 40

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

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

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

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

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

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 chromosomes/kinetochores nuclear matrix mitotic spindles

21 Acknowledgments University of California, Berkeley Jen Banks Priya Prakash Budde Renée Deehan Sharat Gadde Petr Kalab Tom Maresca Aaron Van Hooser Sadie Wignall Scripps John Yates Hongbin Liu Ahna Skop Barbara Meyer

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