Identification of novel cell division proteins:functional proteomics of the midbody

Ahna Skop

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 characterizedLength(early) ~3-5umLength (late) ~1um

Analogous division remnants

Several conserved components known

midbody

Rationale

Midbodies easy to purifyEnriched source of proteins relevant to cell divisionFunction-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

Tandem Liquid Chromatography-

Tandem Mass Spec(LC/LC-MS/MS)

(i.e no gel cutting)

The Strategy

BIG LISTof proteins

Batch BLAST to find C. elegans homologs

RNAi homologsto test for function

Isolate Midbodies

Synchronize CHO cells

4x

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 division90% 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!

Functional Classes of Mammalian Midbody Proteins

Actin Binding

Secretory

MT binding

Kinases

Other

Structural

cytokinesis

early late

sterile

25%

12%

38%

11%

24%

14%

8%

16% 16%

12%

M-phase

mit mei align unc

WT

seggonad

RNAi Phenotypes Observed

10

20

30

40

K04D7.1-RACK1-anchor for PKC and other signaling enzymes, dynamin

RNAi examples

dsRNA is injected into a histone & tubulin::GFP strain

TopFocal Plane

MidFocalPlane

The C. elegans gonad is a syncytium

rachiscommon

cytoplasm

germline nuclei

oocytes

oocytes

The C.elegans gonad is partially cellularized like the Drosophila embryo

an incomplete form of cytokinesis

syncytial germline membrane

F09C3.1-IQGAP

WT gonad-midfocal plane

F09C3.1 RNAi-midfocal plane

An example of a gonad cytokinesis defect

laggingchromosomes

metaphasecongression

failure

T05E11.3-Endoplasmin-most abundant ER protein

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

• chromosomes/kinetochores

• nuclear matrix

• mitotic spindles

Acknowledgments

University of California, Berkeley

Jen Banks Priya Prakash BuddeRenée DeehanSharat GaddePetr Kalab Tom MarescaAaron Van Hooser Sadie Wignall

Scripps

John YatesHongbin Liu

Ahna Skop

Barbara Meyer