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Visualizing the Structure and Dynamics of Proteins Implicated in Long-Term Memory

  • Author(s): Palida, Sakina
  • Advisor(s): Tsien, Roger Y
  • et al.
Abstract

Synapse maturation during long-term potentiation (LTP) depends on increased protein synthesis and stabilization as well as localized extracellular matrix modification in the brain. One challenge to characterizing changes in protein dynamics is that newly synthesized proteins are generally identical to older copies, making them chemically indistinguishable. To investigate our lab’s hypothesis that long-term memory storage relies on molecules that are stable, not rapidly synthesized and degraded, the lab previously developed a genetically encoded reporter called TimeSTAMP that allows new copies of a specific protein to be labeled and tracked in a drug-dependent manner. I designed and optimized a new version of TimeSTAMP, called TS:YSOG3, that can be used for correlated light and electron microscopy (CLEM) as well as protein quantification and mass spectrometry. TS:YSOG3 consists of a Hepatitis C Virus protease fused to a split Venus and the miniSOG reporter for EM. I fused TS:YSOG3 to the N terminus of PKMζ, a truncated form of protein kinase C implicated in long-term memory, and used this construct to study PKMζ localization and dynamics at synapses. I found that both PKMζ synthesis and degradation increased after chemically-induced LTP, contrasting with turnover of the more stable structurally related kinase PKCλ. I additionally used CLEM to show that new PKMζ copies preferentially localize to the postsynaptic membrane. While most synaptic proteins turn over rapidly, on the scale of hours to days, our lab recently identified a subset of extracellular proteins in the perineuronal net (PNN) that remain highly stable over time. The PNN is an essential structure for normal brain function, and recent studies have suggested that localized erosion of the PNN at mature synapses is responsible for long-term memory maintenance. I used microscopy to examine individual PNN components and their distribution to better understand its role in memory. I found that while the PNN was previously thought to enwrap only inhibitory interneurons, a similar structure that varies in composition surrounds all neurons in the brain. I additionally identified a new strategy for genetically encoding reporters into the PNN; the link protein HAPLN1 tolerates fusion to reporters via its C terminus, and a HAPLN1-Venus fusion integrates extracellularly around neurons. I then demonstrated that this reporter could be used to track activity-dependent structural changes in the PNN in live neurons. Collectively, these results support our lab’s hypothesis for memory maintenance and provide a tool for further study of PNN dynamics in a living brain.

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