SUMMARYIntracellular calcium ion (Ca 2+ ) elevation on the left side of the mouse embryonic node or zebrafish Kupffer's vesicle (KV) is the earliest asymmetric molecular event that is functionally linked to lateral organ placement in these species. In this study, Ca 2+ /CaMdependent protein kinase (CaMK-II) is identified as a necessary target of this Ca 2+ elevation in zebrafish embryos. CaMK-II is transiently activated in approximately four interconnected cells along the anterior left wall of the KV between the six-and 12-somite stages, which is coincident with known left-sided Ca 2+ elevations. Within these cells, activated CaMK-II is observed at the surface and in clusters, which appear at the base of some KV cilia. Although seven genes encode catalytically active CaMK-II in early zebrafish embryos, one of these genes also encodes a truncated inactive variant (aKAP) that can hetero-oligomerize with and target active enzyme to membranes. aKAP, b2 CaMK-II and g1 CaMK-II antisense morpholino oligonucleotides, as well as KVtargeted dominant negative CaMK-II, randomize organ laterality and southpaw (spaw) expression in lateral plate mesoderm (LPM). Left-sided CaMK-II activation was most dependent on an intact KV, the PKD2 Ca 2+ channel and g1 CaMK-II; however, aKAP, b2 CaMK-II and the RyR3 ryanodine receptor were also necessary for full CaMK-II activation. This is the first report to identify a direct Ca 2+ -sensitive target in left-right asymmetry and supports a model in which membrane targeted CaMK-II hetero-oligomers in nodal cells transduce the left-sided PKD2-dependent Ca 2+ signals to the LPM.

In order to evaluate links between Ca 2+ /calmodulin (CaM)-dependent protein kinase type II (CaMK-II) and cell cycle progression, CaMK-II binding partners were sought in proliferating cells by epitope-tag tandem mass spectrometry. One protein identified was the gelsolin family member, flightless-I (Fli-I). Fli-I is not a CaMK-II substrate, but binds directly and preferentially to constitutively active (T 287 D) CaMK-II over inactive CaMK-II. Fli-I gradually enters the nucleus upon CaMK-II inhibition and is retained in the cytosol by T 287 D CaMK-II. CaMK-II inhibition and Fli-I overexpression suppress transcription of b-catenin dependent transcriptional reporters, whereas Fli-I suppression enhances their transcription. These findings support a novel mechanism whereby cytosolic CaMK-II influences b-catenin dependent gene expression through Fli-I.

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