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
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
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
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