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First published online 14 November 2006
doi: 10.1242/jcs.03254

Journal of Cell Science 119, 4926-4934 (2006)
Published by The Company of Biologists 2006
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ER vesicles and mitochondria move and communicate at synapses

Sergej L. Mironov* and Natalya Symonchuk

DFG-Center `Molecular Physiology of the Brain', Department of Neuro- and Sensory Physiology, Georg-August-University, Göttingen, Humboldtallee 23, 37073, Germany


Figure 1
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  		Fig. 1. Identification and continuity of ER in the respiratory neurons. (A) EYFP-calreticulin-transfected and Mag-Fura-2-stained neuron. Fluorescence images of Mag-Fura-2 and EYFP-calreticulin were coded by red and green, respectively, and were merged with yellow pixels indicating an overlap (42% in this image, mean 44±5% in seven cells). (B) Independent decreases in lumenal Ca2+ in the soma and in the dendrite after the local application of thapsigargin (Tg, 1 µM). Shown are ratioed images of Mag-Fura-2 fluorescence (350 or 380 nm excitation) measured before (top two panels) and 2 minutes after thapsigargin (lower two panels). Changes at application sites are 2x-enlarged in the right upper-corner of each panel. The arrows indicate the positioning of the application pipette. Similar responses were measured in four other neurons. Bar, 10 µm.

 

Figure 2
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  		Fig. 2. Motility of ER vesicles. (A) Red image shows the dwell areas of ER vesicles that were obtained as maximal values in a stack of 300 images in the Mag-Fura-2-stained neurons. A green-coded image shows the first frame and indicates initial positions of the vesicles (in the overlay all green pixels are yellow). (B) Fluorescence scans in the dendrite taken along the white line in A. The red and green traces show the dwell areas and positions of the vesicles, respectively. (C) Mean profiles across the vesicles and their dwell areas obtained by averaging linescans for 17 vesicles in dendrites. (D) Trajectories of ER vesicles in one representative dendrite. The traces were repositioned for presentation to begin in one point. Note the episodes of `wiggling' (small irregular displacements by <0.2 µm) and transportation events. (E) Kymographs of ER vesicles in the `x'-t plane where `x' corresponds to the one-dimensional curvilinear path in the dendrite. The colours of traces are the same as used in presenting the trajectories in D. Horizontal episodes correspond to the `wiggling' of the vesicles and inclined displacements represent the episodes of directed transport where velocity is given by the slope of kymographs as exemplified in the last trace. (F) Instantaneous velocities of ER vesicles and their approximation by the sum of three Gaussian curves.

 

Figure 3
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  		Fig. 3. ER, mitochondria and synaptic vesicles. (A) Colocalisation of ER and synaptic vesicles. The uppermost panel shows the merged images of Mag-Fura-2 (red) and FM 1-43 (green). The overlap between images was 25% (mean 27±6% in six cells). The middle panel presents the fluorescence profiles in the dendrite which indicate positions of ER vesicles (red) and synapses (green). The lowermost panel shows the mean profiles obtained by averaging the data in 15 ROIs that contained both ER and synaptic vesicles. (B) Mitochondria and synaptic vesicles. The uppermost panel shows the images of TMRE (red) and FM 1-43 fluorescence (green). In this image, the coincidence of pixels that corresponded to the positions of mitochondria and synapses was 24% (mean 27±5% in seven cells). The lower graphs show the linescans in dendrites and corresponding mean profiles obtained from 18 ROIs where the positions of synaptic vesicles overlapped with those of mitochondria. (C) Colocalisation of mitochondria and ER vesicles presented as an overlay of Mag-Fura-2 (green) and TMRE images (red). The overlap between positions of ER vesicles and mitochondria in this neuron was 34% (mean 37±6% in seven cells). The middle and the lowermost graphs show respectively the linescans along dendrites and the mean profiles that were obtained by averaging the data for 16 pairs of particles. Bar in all upper panels, 10 µm.

 

Figure 4
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  		Fig. 4. Synaptic activity, lumenal [Ca2+] and mitochondrial potential. Neurons were stained with FM 1-43 and either Mag-Fura-2 (A-C) or TMRE (D-F). The representative traces (>=5 pairs collected in >=4 neurons) were obtained from the signals measured in the overlapping spots of FM 1-43 and either Mag-Fura-2 or TMRE fluorescence. Changes in [Ca2+]ER are presented as ratios of Mag-Fura-2 signals at 350 or 380 nm. Decreases in TMRE fluorescence indicate mitochondrial depolarisations. Note that all applied stimuli, 45 mM KCl (A,D), hypoxia (B,E), 100 µM t-ACPD (C), and 1 µM kainate (F) decreased the fluorescence of FM 1-43, indicating exocytosis.

 

Figure 5
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  		Fig. 5. Ca2+ exchange between mitochondria and ER. ER vesicles and mitochondria were labelled with Mag-Fura-2 and Rhod-2, respectively. KCl (45 mM) (A), hypoxia (B), 100 µM t-ACPD (C), 1 µM thapsigargin (D), 1 µM CCCP (E) and 3 µM Taxol (F) were applied as indicated. The traces were obtained from fluorescence changes in the areas that contained both ER vesicles and mitochondria. The data are representative of at least five trials for each protocol performed in neurons from three different cell batches.

 

Figure 6
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  		Fig. 6. Spatiotemporal interactions between mitochondria and ER. (A) Mean Mag-Fura-2 signals in ER vesicles measured during Ca2+ release from mitochondria because of 1 µM CCCP versus separation of ER vesicles and mitochondria. Peak (open bars) and resting ratios (grey bars) of fluorescence at 350 nm/380 nm were cumulated into the 0.5 µm-wide bins (n>7 for each), averaged and plotted as histograms where the vertical bars show standard deviations. The asterisks indicate the significance (**P<0.01; *P<0.05). Exocytotic sites were locally depolarised with 45 mM KCl and representative measurements are demonstrated in the inset where the four small panels show ROIs with blue-, green- and red-coded images representing corresponding images of FM 1-43, Mag-Fura-2 and Rhod-2, and their overlay (Bar, 1.5 µm). Red and green curves show the relative changes in Mag-Fura-2 and Rhod-2 fluorescence which was measured in single organelles as indicated near the frames. (B) The dependence of relative peak changes in Rhod-2 fluorescence because of 1 µM thapsigargin on the distance between ER vesicles and mitochondria. The coding of frames in B is similar to that in A. (C) Interference between the movements of mitochondria and ER vesicles. The left panel shows two typical trajectories of ER vesicles and mitochondria in dendrites that are also presented as kymographs in the right panel. Note a bilateral suppression of movements of mitochondria and ER vesicles upon their approach as shown by the couple of traces in the upper panel that are representative for 33 stops of mitochondria by ER vesicles and 11 stops of ER vesicles by mitochondria analysed in 27 cells. The lowermost part of the figure shows the absence of correlation between the movements of organelles that did not come close (the occasional pauses in movements here were caused by interruptions of the directed transport of organelles).

 

Figure 7
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  		Fig. 7. Modulation of exocytosis by mitochondria and ER vesicles. Synaptic vesicles, ER and mitochondria stained with FM 1-43, Mag-Fura-2 or Rhod-2. The ROIs are shown in the insets by blue-, green- and red-coded images, respectively (Bars, 1 µm). Synaptic activity was induced by locally applying high-K+ solutions as indicated by the horizontal bars. The application pipette was positioned 2 µm from the spots of FM 1-43 fluorescence. The time-course of exocytosis was measured as a derivative of the relative FM 1-43 fluorescence, -d({Delta}F/Fo)/dt. Shown are representative experiments (>=5 trials for each protocol) performed in neural processes at sites that contained ER vesicles and mitochondria (A,B,E,F), only ER vesicles (C) and only mitochondria (D). Note two additional peaks in B that corresponded to spontaneous Ca2+ releases from ER, each accompanied by exocytosis. In the experiment shown in E, 1 µM CCCP was first applied to the bath for 2 minutes and then exocytosis was locally evoked. In F, 1 µM thapsigargin was first applied to the bath for 2 minutes and then a local membrane depolarisation was applied. Note also a weaker fluorescence of Mag-Fura-2 (excited at 350 nm) and Rhod-2 after the inhibition of Ca2+ uptake into the corresponding organelle.

 

Figure 8
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  		Fig. 8. Changes in exocytosis because of communication between mitochondria and ER vesicles. Synaptic vesicles, ER and mitochondria stained with FM 1-43, Mag-Fura-2 or Rhod-2. The ROIs are shown in the insets by blue-, green- and red-coded images, respectively (Bar, 1 µm). Exocytosis was induced by local applications of high-K+ solutions as indicated by the horizontal bars and its time-course was measured as the derivative of relative FM 1-43 fluorescence, -d({Delta}F/Fo)/dt. Shown are representative experiments performed at synaptic sites that contained both ER vesicles and mitochondria in the control (A,B) and 5 minutes after addition of 5 µM nocodazole (C) and 0.1 µM taxol (D) to the bath. Note separation of the initially contacting ER vesicles and mitochondria after nocodazole, the shortening of exocytosis in the presence of nocodazole and its prolongation by taxol.

 

Figure 9
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  		Fig. 9. Modulation of the respiratory motor output in vivo after modification of contacts between ER and mitochondria. Shown are the effects of 5 µM nocodazole (A) and 0.1 µM taxol (B). The two traces in each panel present the respiratory motor output ({int}XII) and the membrane current (Im) recorded at the holding potential of -40 mV. The episodes marked by asterisks are expanded in the lower part of each panel. Note differential effects of nocodazole and taxol on the inhibitory and excitatory synaptic currents (the upward and downward deflections in current traces, respectively) and synaptic drives, which represent a correlate of respiratory motor activity.

 

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© The Company of Biologists Ltd 2006