Abstract
Homeostatic mechanisms maintaining high levels of adhesion molecules in synapses over prolonged periods of time remain incompletely understood. We used fluorescence recovery after photobleaching experiments to analyze the steady state turnover of the immobile pool of green fluorescent protein-labeled NCAM180, the largest postsynaptically accumulating isoform of the neural cell adhesion molecule (NCAM). We show that there is a continuous flux of NCAM180 to the postsynaptic membrane from nonsynaptic regions of dendrites by diffusion. In the postsynaptic membrane, the newly delivered NCAM180 slowly intermixes with the immobilized pool of NCAM180. Preferential immobilization and accumulation of NCAM180 in the postsynaptic membrane is reduced after disruption of the association of NCAM180 with the spectrin cytoskeleton and in the absence of the homophilic interactions of NCAM180 in synapses. Our observations indicate that the homophilic interactions and binding to the cytoskeleton promote immobilization of NCAM180 and its accumulation in the postsynaptic membrane. Flux of NCAM180 from extrasynaptic regions and its slow intermixture with the immobile pool of NCAM180 in the postsynaptic membrane may be important for the continuous homeostatic replenishment of NCAM180 protein at synaptic contacts without compromising the long term synaptic contact stability.
Highlights
Neurons are assembled into circuits via specialized contacts, called synapses, that are essential for information processing, learning, and storage in the brain
The largest isoform of neural cell adhesion molecule (NCAM), NCAM180, accumulates in the postsynaptic membrane and recruits the spectrin scaffold, NMDA-type glutamate receptors, and Ca2ϩ/calmodulin-dependent protein kinase ␣ (CaMKII␣) [25,26,27], whereas polysialic acid attached to NCAM restrains the signaling through GluN2B-containing NMDA receptors, a process that is required for synaptic plasticity and learning [28]
NCAM180GFP Diffuses to Sites of Interneuronal Contacts— To visualize the behavior of NCAM180 in live neurons, cultured hippocampal neurons were transfected with NCAM180 tagged with the GFP inserted between its transmembrane and second fibronectin type III domains to ensure that the tag does not interfere with the interactions mediated by the extracellular and intracellular domains of NCAM [12]
Summary
Cultures of Hippocampal Neurons—Cultures of hippocampal neurons were prepared from 1–3-day-old C57BL/6J or NCAM-deficient (NCAMϪ/Ϫ) mice from homozygous breeding pairs and maintained on glass coverslips as described [29]. Confocal Laser Scanning Microscopy and Photobleaching— Transfected hippocampal neurons were imaged on the LSM510 laser scanning microscope (Zeiss). Data Analysis of FRAP Experiments—For analysis, the average pixel intensity of the photobleached region of the neurite for each time point before and after photobleaching was measured from the original stored images using LSM510 software. Mathematical Model of the Diffusion of NCAM180GFP and the Kinetics of Binding to Scaffold Proteins—To predict the behavior of NCAM180GFP molecules in the membrane, we defined F, B, and G to be, respectively, the number of free (bleached), bound (bleached), and green fluorescent molecules normalized by the total number. To test whether the inclusion of binding and dissociation improved the model fit, we used the lack-of-fit test with degrees of freedom (2, n Ϫ 3) for each data set from each individual time lapse experiment, where n is the sample size. For 26 of 27 data sets, the resulting p value was Ͻ10Ϫ6, indicating that the model with binding and diffusion was statistically superior to the model with only diffusion
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