Figure 1. TIRFM imaging of activity-sensitive PSD-95 palmitoylation. (A) Compared with epifluorescent microscopy (Epi; green), TIRFM selectively reveals punctae from GFP-tagged PSD-95 (WT) (top; red) but not palmitoylation-deficient PSD-95 (CS) (bottom; red) in cultured hippocampal neurons. To define dendritic morphology, we coexpressed mCherry (Epi; blue). (B) TIRFM preferentially visualizes PSD-95 (WT)–GFP punctae as compared with PSD-95 (CS)–GFP or GFP alone. n = 10 neurons; ***, P < 0.001. Comparable expression levels of PSD-95 (WT)– and PSD-95 (CS)–GFP in transfected neuron culture were confirmed. (C) TIRFM tracks synaptic PSD-95. PSD-95 punctae (green) visualized by TIRFM apposed presynaptic synaptophysin and VGLUT1 and overlapped postsynaptic NR1. (D) PSD-95–GFP dynamics were analyzed by time-lapse TIRFM imaging. Inhibition of glutamate receptor activity with 10 mM Kyn increased PSD-95 (WT)–GFP intensity within 2 h. In contrast, the palmitoylation-deficient mutant PSD-95 (CS) did not change. Kymographs represent the changes in the intensity of PSD-95–GFP over 2 h. White lines indicate the regions used for the kymographs. (E) Synaptic accumulation of PSD-95 depends on newly occurring palmitoylation. Fluorescent intensities of PSD-95–GFP (WT and CS), GFP containing a C-terminal prenylation CaaL motif of Rac1 (GFP-CLLL), and synaptophysin-GFP (Syn-GFP) at 2 h after the indicated treatments were quantified. The intensity of PSD-95 (WT)–GFP but not other membrane-targeting proteins significantly increased upon 10 mM Kyn or 2 µM TTX treatment. Coapplication of 100 µM 2-BP with Kyn completely inhibited Kyn-induced increase of PSD-95–GFP intensity. n = 3–8 experiments; ***, P < 0.001 compared with control. (B and E) Error bars indicate SD. Bars: (A) 10 µm; (C and D) 5 µm.
Figure 1.

TIRFM imaging of activity-sensitive PSD-95 palmitoylation. (A) Compared with epifluorescent microscopy (Epi; green), TIRFM selectively reveals punctae from GFP-tagged PSD-95 (WT) (top; red) but not palmitoylation-deficient PSD-95 (CS) (bottom; red) in cultured hippocampal neurons. To define dendritic morphology, we coexpressed mCherry (Epi; blue). (B) TIRFM preferentially visualizes PSD-95 (WT)–GFP punctae as compared with PSD-95 (CS)–GFP or GFP alone. n = 10 neurons; ***, P < 0.001. Comparable expression levels of PSD-95 (WT)– and PSD-95 (CS)–GFP in transfected neuron culture were confirmed. (C) TIRFM tracks synaptic PSD-95. PSD-95 punctae (green) visualized by TIRFM apposed presynaptic synaptophysin and VGLUT1 and overlapped postsynaptic NR1. (D) PSD-95–GFP dynamics were analyzed by time-lapse TIRFM imaging. Inhibition of glutamate receptor activity with 10 mM Kyn increased PSD-95 (WT)–GFP intensity within 2 h. In contrast, the palmitoylation-deficient mutant PSD-95 (CS) did not change. Kymographs represent the changes in the intensity of PSD-95–GFP over 2 h. White lines indicate the regions used for the kymographs. (E) Synaptic accumulation of PSD-95 depends on newly occurring palmitoylation. Fluorescent intensities of PSD-95–GFP (WT and CS), GFP containing a C-terminal prenylation CaaL motif of Rac1 (GFP-CLLL), and synaptophysin-GFP (Syn-GFP) at 2 h after the indicated treatments were quantified. The intensity of PSD-95 (WT)–GFP but not other membrane-targeting proteins significantly increased upon 10 mM Kyn or 2 µM TTX treatment. Coapplication of 100 µM 2-BP with Kyn completely inhibited Kyn-induced increase of PSD-95–GFP intensity. n = 3–8 experiments; ***, P < 0.001 compared with control. (B and E) Error bars indicate SD. Bars: (A) 10 µm; (C and D) 5 µm.

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