Calcium/Calmodulin-Dependent Protein Kinase II Serves as a Biochemical Integrator of Calcium Signals for the Induction of Synaptic Plasticity by Jui-Yun Chang Department of Biochemistry Duke University Date:_______________________ Approved: ___________________________ Ryohei Yasuda, Co-Supervisor ___________________________ G. Vann Bennett, Co-Supervisor ___________________________ Richard G. Brennan ___________________________ Kenichi Yokoyama Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University 2016 ABSTRACT Calcium/Calmodulin-Dependent Protein Kinase II Serves as a Biochemical Integrator of Calcium Signals for the Induction of Synaptic Plasticity by Jui-Yun Chang Department of Biochemistry Duke University Date:_______________________ Approved: ___________________________ Ryohei Yasuda, Co-Supervisor ___________________________ G. Vann Bennett, Co-Supervisor ___________________________ Richard G. Brennan ___________________________ Kenichi Yokoyama An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University 2016 Copyright by Jui-Yun Chang 2016 Abstract Repetitive Ca2+ transients in dendritic spines induce various forms of synaptic plasticity by transmitting information encoded in their frequency and amplitude. CaMKII plays a critical role in decoding these Ca2+ signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ decoding in spines remain elusive. Here, I measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging with millisecond temporal resolution. Following each repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. This signal integration, at the time scale of seconds, critically depended on Thr286 phosphorylation. In the absence of Thr286 phosphorylation, only by increasing the frequency of repetitive Ca2+ elevations could high peak CaMKII activity or plasticity be induced. In addition, I measured the association between CaMKII and Ca2+/CaM during spine plasticity induction. Unlike CaMKII activity, association of Ca2+/CaM to CaMKII plateaued at the first Ca2+ elevation event. This result indicated that integration of Ca2+ signals was initiated by the binding of Ca2+/CaM and amplified by the subsequent increases in Thr286-phosphorylated form of CaMKII. Together, these findings demonstrate that CaMKII functions as a leaky integrator of repetitive Ca2+ signals during the induction of synaptic plasticity, and that Thr286 phosphorylation is critical for defining the frequencies of such integration. iv Dedication This dissertation is dedicated to my Mom, my Dad, and Chien-Pin, for whose continued support I am extremely grateful. v Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................ xii List of Figures ............................................................................................................................ xiii List of Reaction Schemes ...........................................................................................................xvi List of Abbreviations ............................................................................................................... xvii Acknowledgements ................................................................................................................... xxi Chapter 1. Introduction ......................................................................................................... 1 1.1 Long-term potentiation .................................................................................................... 1 1.1.1 History .......................................................................................................................... 1 1.1.2 LTP and memory ......................................................................................................... 1 1.1.3 Basic properties of LTP ............................................................................................... 2 1.1.4 LTP induction mechanism ......................................................................................... 3 1.2 Ca2+/calmodulin-dependent protein kinase II (CaMKII) ............................................ 4 1.2.1 CaMKII in LTP, learning and memory ..................................................................... 4 1.2.1.1 Pharmacology inhibition of CaMKII activity ................................................... 4 1.2.1.2 Genetics manipulation of CaMKII activity ....................................................... 6 1.2.2 Temporal and spatial expression of CaMKIIα in mice brain ................................ 7 1.2.3 Molecular architecture of CaMKII ............................................................................ 8 1.2.4 CaMKII activation mechanism ................................................................................ 12 1.2.5 Phosphorylation at Thr286 induces calmodulin trapping ................................... 13 vi 1.2.6 CaMKII: a frequency decoder of Ca2+ oscillation in vitro ..................................... 14 1.2.7 CaMKII translocation and binding to NMDA-receptors ..................................... 15 1.2.7.1 CaMKII translocation to PSD ........................................................................... 15 1.2.7.2 Discovery of CaMKII/NR2B complex ............................................................. 16 1.2.7.3 CaMKII/NR2B complex in LTP ........................................................................ 17 1.2.8 Other CaMKII binding proteins in PSD ................................................................. 19 1.2.9 Potentiation of AMPA-receptors by CaMKII ........................................................ 19 1.2.9.1 AMPA-receptors phosphorylation by CaMKII ............................................. 20 1.2.9.2 Insertion of AMPA-receptors to synapse ....................................................... 20 1.2.10 Gating of CaMKII activity by protein phosphatase activity ............................. 22 1.3 Synaptic structural plasticity ........................................................................................ 23 1.3.1 Structural basis of LTP .............................................................................................. 23 1.3.2 Biophysical basis of the synapse-specific signaling .............................................. 26 1.3.3 Ca2+ signaling during synaptic plasticity ............................................................... 26 1.3.4 Signaling molecules underlying LTP ..................................................................... 27 1.4 Experimental rationale and specific aims ................................................................... 30 Chapter 2. Materials and methods ..................................................................................... 34 2.1Experimental animals ..................................................................................................... 34 2.2 Preparation ...................................................................................................................... 34 2.3 Plasmids ........................................................................................................................... 35 2.3 Two-photon fluorescence lifetime imaging ................................................................ 35 vii 2.3.1 CaMKII activity imaging .......................................................................................... 36 2.3.1.1 Characterize CaMKII activation in HeLa cells ............................................... 37 2.3.2 Binding of Ca2+/CaM to CaMKII .............................................................................. 38 2.4 2pFLIM data analysis ..................................................................................................... 39 2.4.1 Camuiα ....................................................................................................................... 39 2.4.2 CaM/CaMKII FRET biosensor ................................................................................. 40 2.5 Two-photon glutamate uncaging ................................................................................. 41 2.6 Measurements of structural plasticity ......................................................................... 42 2.7 Functionality of Camuiα sensor ................................................................................... 43 2.8 Calcium imaging ............................................................................................................ 43 2.9 Electrophysiology ........................................................................................................... 44 2.10 Simulation of CaMKII kinetics scheme ..................................................................... 45 2.11 Statistical analysis ......................................................................................................... 49 Chapter 3. Kinetics of CaMKII activation during spine plasticity measured with millisecond temporal resolution ..............................................................................................