lab meeting 05/05/14
Jean-Ju Chung, Spatial Organization of CatSper in Sperm
- (former collaborator of Sang-He)
- Clapman lab
- results now published, to appear soon.
- ejaculated sperm not motile outside female environment. Hence ‘not fertile’.
- what’s the difference? Bicarbonate rich, Albumin. pH changes sharply from 5 vagina to 8 in cervix / 9 in mouse fallopian tube.
- consequent ‘capacitation’ of sperm cells. Go from activated motility, to hyperactivated (whip-like motion, believed to function in fusion)
- sperm require few hours to mature.
- CatSper — sperm specific Ca channel.
- Identified 4 channels in sperm.
- knockouts are male-specific infertile
- lack hyper-active motility.
- structurally more similar to K channel than other Ca / Na channels
- stay in the ER, don't become functional ion channels until mature sperm
- then localize in principle piece (main body of the tail).
- Ca specific conductance abolished in any of the knockouts. -- heterotetramer.
- progesterone activates human CatSper
How is CatSper organized?
- Conventional imaging: CatSper is localized to the principle piece. Sometimes 2 lines, sometime 3 lines or apparent helix.
- 9(doublet microtubules)+2(singlet) microtubule organization + 2 outer layers. ~300nm inner diameter
- CatSper1 arranged in 4 long parallel lines in two double rows.
- immunoEM see four doublets flanking each of the longitudinal columns.
- CatSper is quadralateral before formation of the longitudal column in immature sperm.
How does this organization affect the function of beating?
- CatSper colocalizes with Ca2+ signaling molecules like P-CaMKII, and PP2B-Ag
- CatSper still organized in Caviolin knockout.
- Caviolin not still organized in CatSper knockout.
- functionally confines tyrosine phosphorylation to the interior of the axonneme – (microtubule bundle)
- Maybe the clustering of CatSper is important to the signalling.
probing P-Tyr phosphorylation
- add heavy labels to P-Tyr from Wt and CatSpe1 nulls
- mix and mass-spec.
- 62 phosphoylation sites on 45 proteins. (previously only 7 known)
- 41 sites show 2 fold increase in P-tyr in the knockout.
- find Src family kinases target CatSper-dependent phosphorylation pathways
- Calmodulin (Ca binding) concentrated in center.
- phosphorylated Calmodulin focused in the very center after capactiation, and focused on the perifery in the CatSper1 null.
- Tyrosine phosphorylation is downstream of PKA in sperm.
- upregulation of tyrosine phsophorylation in CatSper nulls is also PKA depedent.
relation to swimming
- Capacitation generates heterogeneous sperm populations — different radial distributions of CatSper1
- Motility correlative imaging.
- all hyperactive swimming sperm have intact quadralateral structure of CatSper and focused concentration of pY.
- use fluorescently labeled sperm to trace migration through female reproductive system in transgenic mice
- CatSper null sperm don’t exit the uterus (or very few).
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