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A CaMKIIβ signaling pathway at the centrosome regulates dendrite patterning in the brain

Abstract

The protein kinase calcium/calmodulin-dependent kinase II (CaMKII) predominantly consists of the α and β isoforms in the brain. Although CaMKIIα functions have been elucidated, the isoform-specific catalytic functions of CaMKIIβ have remained unknown. Using knockdown analyses in primary rat neurons and in the rat cerebellar cortex in vivo, we report that CaMKIIβ operates at the centrosome in a CaMKIIα-independent manner to drive dendrite retraction and pruning. We also find that the targeting protein PCM1 (pericentriolar material 1) localizes CaMKIIβ to the centrosome. Finally, we uncover the E3 ubiquitin ligase Cdc20-APC (cell division cycle 20–anaphase promoting complex) as a centrosomal substrate of CaMKIIβ. CaMKIIβ phosphorylates Cdc20 at Ser51, which induces Cdc20 dispersion from the centrosome, thereby inhibiting centrosomal Cdc20-APC activity and triggering the transition from growth to retraction of dendrites. Our findings define a new, isoform-specific function for CaMKIIβ that regulates ubiquitin signaling at the centrosome and thereby orchestrates dendrite patterning, with important implications for neuronal connectivity in the brain.

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Figure 1: CaMKIIβ restricts the elaboration of dendrites.
Figure 2: CaMKIIβ regulates dendrite patterning in vivo.
Figure 3: CaMKIIβ operates specifically at the centrosome to control dendrite patterning.
Figure 4: The centrosomal targeting protein PCM1 localizes CaMKIIβ to the centrosome.
Figure 5: CaMKIIβ phosphorylates Cdc20 at Ser51 and thereby inhibits centrosomal Cdc20-APC activity in neurons.
Figure 6: CaMKIIβ-induced phosphorylation of Cdc20 promotes Cdc20 dispersion from the centrosome, leading to the restriction of dendrite elaboration.

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Acknowledgements

We thank M. Ericsson for assistance with electron microscopy experiments and J. Blenis, G. Corfas and members of the Bonni laboratory for discussions and critical reading of the manuscript. This work was supported by US National Institutes of Health grant NS051255 (A.B.), a Ruth L. Kirschstein National Research Service Award (National Cancer Institute), a Brain Science Foundation grant (A.H.K.) and a Human Frontier Science Program Long-term Fellowship (Y.I.).

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Authors

Contributions

A.B. directed and coordinated the project. S.V.P., A.H.K. and Y.I. designed and performed in vivo experiments, biochemical assays and morphological analyses. J.T.W.-G. prepared mass spectrometry samples and completed analyses of Cdc20 phosphorylation in S.P.G.'s laboratory. A.M. contributed molecular reagents. The manuscript was written by S.V.P. and A.B. and critically reviewed and commented on by all authors.

Corresponding author

Correspondence to Azad Bonni.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 (PDF 6487 kb)

Supplementary Video 1

Live Imaging Analyses of Control Vector and T287D CaMKIIβ-transfected Neurons. Granule neurons were transfected on DIV1 with the T287D CaMKIIβ expression plasmid or control vector together with the GFP expression plasmid. Starting at DIV2, granule neuron dendrites were assessed by live spinning disk confocal imaging every eight hours over a 48 hr interval in an environment-controlled chamber. At each time point, neurons were imaged every ten minutes for a one hour period. Representative neurons at DIV 2 + 24 hrs are shown. Expression of T287D CaMKIIβ reduced dendrite extension events and promoted dendrite retraction events. (MOV 801 kb)

Supplementary Video 2

Live Imaging Analyses of Control U6 and CaMKIIβ RNAi-transfected Neurons. Granule neurons were transfected on DIV0 with the CaMKIIβ RNAi or control U6 plasmid together with the GFP expression plasmid. Starting at DIV2, granule neuron dendrites were assessed by live spinning disk confocal imaging every eight hours over a 48 hr interval in an environment-controlled chamber. At each time point, neurons were imaged every ten minutes for a one hour period. Representative neurons at DIV 2 + 24 hrs are shown. CaMKIIβ knockdown promoted dendrite extension events and reduced dendrite retraction events. (MOV 1199 kb)

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Puram, S., Kim, A., Ikeuchi, Y. et al. A CaMKIIβ signaling pathway at the centrosome regulates dendrite patterning in the brain. Nat Neurosci 14, 973–983 (2011). https://doi.org/10.1038/nn.2857

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