Figure S1. Schematic rationale of FRAP experiments.
(A) FRAP under basal conditions. Without activity, GluR1 is located in an extrasynaptic mobile pool on a spine. After photobleaching a spine, the photobleached GluR1-SEP is replaced by dendritic GluR1-SEP and recovers to ~100% after 30 min. GluR2 is in a synaptic immobile pool as well as in an extrasynaptic mobile pool on a spine. Photobleaching a spine leads to a recovery of only an extrasynaptic mobile pool. (B) FRAP after LTP induction. After LTP induction, GluR1 moves to a synaptic immobile pool on a spine, and photobleaching a spine does not lead to a complete recovery. GluR2 does not move into a synapse and its recovery after photobleaching a spine is similar to the basal condition.
Figure S2. Glutamate-uncaging-evoked LTP and GluR1-SEP fluorescence recovery on spines.
Images shown are high magnification of . Note the substantial increase in GluR1-SEP fluorescence in LTP spine, which does not show a complete recovery after photobleaching, while GluR1-SEP recovers completely at the nearby spine.
Figure S3. FRAP of spine SEP-tagged AMPARs following glutamate uncaging-evoked LTP relative to pre-LTP values.
(A) GluR1-SEP fluorescence recovery on spines. Immobile fraction corresponds to 44% and 1% of pre-LTP GluR1-SEP on spines in potentiated (n = 14, 10 cells) and non-potentiated spines (n = 11, 7 cells), respectively. (B) GluR2-SEP fluorescence recovery on spines. Immobile fraction corresponds to 25% and 28% of pre-LTP GluR2-SEP on spines in potentiated (n = 8, 3 cells) and non-potentiated spines (n = 8, 4 cells), respectively.
Figure S4. GluR1-SEP in intracellular acidic compartments is protected from photobleaching.
(A) Experimental design and an example of the change in GluR1-SEP fluorescence signal after bath application of NH4Cl. Top and bottom panels show dual-color images and pseudo-color-coded SEP intensity images, respectively. (B) Experimental design and an example of the changes in GluR1-SEP fluorescence signal after photobleaching and subsequent bath application of NH4Cl. Note the heterogeneous distribution of GluR1-SEP fluorescence, indicating that GluR1 is located in subcellular compartments. (C) Mean changes in GluR1-SEP on dendrites after bath application of NH4Cl (7 cells). (D) Mean changes in GluR1-SEP on dendrites after photobleaching and subsequent NH4Cl bath application (4 cells). (E) Comparison of mean changes in GluR1-SEP on dendrites and spines caused by NH4Cl bath application (dendrite: 7 cells for no photobleaching and 4 cells for photobleaching, p = 0.79; spine: n = 17, 5 cells for no photobleaching and n = 15, 3 cells for photobleaching, p = 0.43). Changes in GluR1-SEP fluorescence was calculated as a difference between NH4Cl – baseline ( no photobleaching ) or NH4Cl – post-bleaching ( photobleaching ).
Figure S5. Four possible routes for AMPAR trafficking to synapses during LTP and consequences of spine photobleaching in each case.
AMPARs are delivered to synapses during LTP either by (A) lateral diffusion from a spine surface extrasynaptic pool; (B) lateral diffusion from a dendrite surface extrasynaptic pool; (C) directly from intracellular stores through exocytosis; (D) lateral diffusion from a newly exocytosed extrasynaptic pool. Note that only (A) permits photobleached AMPARs to move to a synaptic pool.
02: Movie. Spatiotemporal dynamics of GluR1-SEP exocytosis before, during and after two-photon glutamate uncaging-evoked LTP.
White filled circle indicates the location of a laser pulse for glutamate uncaging. Note that GluR1-SEP exocytosis is highly compartmentalized to a stretch of dendrite and occurs after LTP induction (images acquired at ~2 sec/frame).