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PNAS 110 (4): 1482-1487

Copyright © 2013 by the National Academy of Sciences.

Increased neuronal activity fragments the Golgi complex

Desiree A. Thayer, Yuh Nung Jan, and Lily Yeh Jan1

Department of Physiology, and Howard Hughes Medical Institute, University of California, San Francisco, CA 94158


Figure 01
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  		Fig. 1. The Golgi complex fragments under hyperexcitable conditions. (A) Neurons were cultured under normal and hyperexcitable conditions (elevated potassium concentration, high K). Immunostaining with anti-GM130 (green) and anti-MAP2 (blue) with 3D reconstruction of anti-GM130 signal. The color of the distinct Golgi fragments corresponds to the relative size of the fragment. (Scale bar: 10 μm.) (B) Quantification of number, surface area (μm2), and volume (μm3) of distinct Golgi fragments from reconstructed anti-GM130 fluorescent signal. Data shown are median and IR (controls: 7 DIV, n = 10; 10 DIV, n = 9; 14 DIV, n = 10; 17 DIV, n = 21; high K: 7 DIV, n = 10; 10 DIV, n = 8; 14 DIV, n = 9; 17 DIV, n = 17).

 

Figure 02
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  		Fig. 2. Prolonged treatment with bicuculline (20 μM) or removal of APV (200 μM DL-APV) fragments the Golgi complex. (A) Immunostaining of cultured hippocampal neurons (≥21 DIV) with cis-Golgi marker anti-GM130 (green) and anti-MAP2 (blue) with 3D reconstruction of Golgi staining. (Scale bar: 10 μm.) (B) Quantification of number, surface area (μm2), and volume (μm3) of distinct Golgi fragments from reconstructed anti-GM130 fluorescent signal. Application of TTX (1 μM) before bicuculline to block synaptic transmission. Data shown are median and IR (control, n = 10; Bic, 1 d, n = 7; Bic, 3 d, n = 6; APV, 1 d, n = 9; APV, 3d, n = 7; TTX+Bic, 1 d, n = 5). For Bic, 1 d, P < 0.1.

 

Figure 03
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  		Fig. 3. Golgi fragmentation is visualized with the trans-Golgi marker TGN38 as well as the cis-Golgi marker GM130. (A) Immunostaining of hippocampal neurons (≥21 DIV) with anti-TGN38 (green) and anti-MAP2 (blue) with 3D reconstruction of anti-TGN38 signal. (Scale bar: 10 μm.) (B) Quantification of number, surface area (μm2), and volume (μm3) of distinct Golgi fragments from reconstructed anti-TGN38 fluorescent signal. Data shown are median and IR (control, n = 10; Bic, 1 d, n = 9; APV, 1 d, n = 8; APV, 3d, n = 7).

 

Figure 04
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  		Fig. 4. Golgi fragmentation occurs during bicuculline treatment and reverses after return to normal medium. Cultured hippocampal neurons were transfected with Mgat2–EGFP and myristoylated Td-Tomato. Individual neurons were imaged, then treated with bicuculline for 1 d. Bicuculline was removed and neurons were imaged again after 2 d in normal medium. (A) Examples of two neurons with fragmentation of the Golgi complex after 1 d with bicuculline, then reversal of fragmentation 2 d after bicuculline removal. (Scale bar: 15 μm.) (B) Example control neuron showing change in Mgat2–EGFP signal but lack of fragmentation. (C) Summary of data from bicuculline-treated (green, n = 12) and control (black, n = 4) neurons.

 

Figure 05
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  		Fig. 5. Pretreatment with CaMK II/IV inhibitor KN-93 blocks bicuculline-induced Golgi fragmentation. (A) Immunostaining of hippocampal neurons (≥21 DIV) with anti-GM130 (green) and anti-MAP2 (blue) with 3D reconstruction of anti-GM130 signal. The color of the distinct Golgi fragments corresponds to the relative size of the fragment. (Scale bar: 10 μm.) (B) Quantification of number of distinct Golgi fragments from reconstructed anti-GM130 fluorescent signal. Data shown are median and IR (control, n = 10; Bic, 1 d, n = 7; KN-93+Bic, 1 d, n = 5; Bic, 3 d, n = 6). (C and D) Quantification of surface area (μm2) and volume (μm3) of Golgi fragments from reconstructed images.

 

Figure 06
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  		Fig. 6. Okadaic acid causes Golgi fragmentation in cultured hippocampal neurons. Cultured hippocampal neurons (17 DIV) were treated with okadaic acid (10 nM) or FK506 (100 nM) for 1 d, then returned to normal culture medium for an additional day. (A) Immunostaining with anti-GM130 (green) and anti-MAP2 (blue) with 3D reconstruction of anti-GM130 signal. The color of the distinct Golgi fragments corresponds to the relative size of the fragment. (Scale bar: 10 μm.) (B) Quantification of the number of distinct Golgi fragments from reconstructed anti-GM130 fluorescent signal. Data shown are median and IR (control, n = 11; OA, 1 d, n = 10; OA, 2 d, n = 9). (C) Proposed Golgi fragmentation pathway during neuronal hyperactivity.

 

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