Control of Transmembrane Protein Diffusion within the Postsynaptic Density Assessed by Simultaneous Single-Molecule Tracking and Localization Microscopy

Abstract

Postsynaptic transmembrane proteins are critical elements of synapses, mediating trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules, and flux of Ca and other ions. Positioning and mobility of each member of this large class of proteins is critical to their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD) strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out of the synapse is critical for activity-dependent plasticity. However, factors that control receptor mobility within the boundaries of the synapse are not well understood. Notably, PSD scaffold molecules accumulate in domains much smaller than the synapse. Within these nanodomains, the density of proteins is considerably higher than that of the synapse as a whole, so high that steric hindrance is expected to reduce receptor mobility substantially. However, while numerical modeling has demonstrated several features of how the varying protein density across the face of a single PSD may modulate receptor motion, there is little experimental information about the extent of this influence. To address this critical aspect of synaptic organizational dynamics, we performed single-molecule tracking of transmembrane proteins using universal point accumulation-for-imaging-in-nanoscale-topography (uPAINT) over PSDs whose internal structure was simultaneously resolved using photoactivated localization microscopy (PALM). The results provide important experimental confirmation that PSD scaffold protein density strongly influences the mobility of transmembrane proteins. A protein with a cytosolic domain that does not bind PSD-95 was still slowed in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize receptors even in the absence of binding. Because numerous proteins thought to be involved in establishing PSD structure are linked to disorders including autism and depression, this motivates further exploration of how PSD nanostructure is created. The combined application PALM and uPAINT should be invaluable for distinguishing the interactions of mobile proteins with their nano-environment both in synapses and other cellular compartments.

The Axon Initial Segment: An Updated Viewpoint

“…However, fluorescence and electron microscopy do not show a dense accumulation of actin inside the AIS: actin forms rings connected by spectrin, as in the rest of the axon (Xu et al, 2013;D'Este et al, 2017), as well as patches made of short intertwined filaments (Fig. 3 A, B) (Watanabe et al, 2012;Jones et al, 2014;Leterrier et al, 2017b). Diffusion of the microtubuleassociated protein tau is impaired at the AIS in an isoformdependent manner (Li et al, 2011;Zempel et al, 2017).…”

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

“…Tracking and estimation of the instantaneous diffusion was performed as described for the PALM imaging. Synapses were identified based on widefield Homer1c-mCherry signal as described [85]. Synaptic tracks were defined as tracks in which 80% of the localizations were located within the border of the synapse.…”

Transcellular Nanoalignment of Synaptic Function

“…The PSD is extremely dense with protein, and modeling suggests that the even higher density within nanodomains likely provides obstacles to mobility of bulky receptors even not bound to scaffolds (Santamaria et al, 2010). Indeed, synaptic diffusion rates of both lipids (Renner et al, 2009) and transmembrane proteins (Li and Blanpied, 2016) are size-dependent, and even proteins without domains that bind PSD-95 diffuse more slowly within portions of the PSD where the scaffold is denser (Li and Blanpied, 2016). This effect, termed macromolecular crowding, may be particularly important in controlling receptor exit rate during episodes of plasticity (Li et al, 2016), and in concert with binding may contribute to the pattern of receptors.…”