DOI: 10.5061/dryad.0gb5mkm71
title: Activity-dependent mitochondrial ROS signaling regulates recruitment of glutamate receptors to synapses

description: Our understanding of mitochondrial signaling in the nervous system has
 been limited by the technical challenge of analyzing mitochondrial
 function in vivo. In the transparent genetic model Caenorhabditis elegans,
 we were able to manipulate and measure mitochondrial ROS (reactive oxygen
 species) signaling of individual mitochondria as well as neuronal activity
 of single neurons in vivo. Using this approach, we provide evidence
 supporting a novel role for mitochondrial ROS signaling in dendrites of
 excitatory glutamatergic C. elegans interneurons. Specifically, we show
 that following neuronal activity, dendritic mitochondria take up calcium
 (Ca2+) via the mitochondrial Ca2+ uniporter MCU-1 which results in an
 upregulation of mitochondrial ROS production. We also observed that
 mitochondria are positioned in close proximity to synaptic clusters of
 GLR-1, the C. elegans ortholog of the AMPA subtype of glutamate receptors
 that mediate neuronal excitation. We show that synaptic recruitment of
 GLR-1 is upregulated when MCU-1 function is pharmacologically or
 genetically impaired but is downregulated by mitoROS signaling. Thus,
 signaling from postsynaptic mitochondria may regulate excitatory synapse
 function to maintain neuronal homeostasis by preventing excitotoxicity and
 energy depletion.
description: # Activity-dependent mitochondrial ROS signaling regulates recruitment of
 glutamate receptors to synapses
 [https://doi.org/10.5061/dryad.0gb5mkm71](https://doi.org/10.5061/dryad.0gb5mkm71) The enclosed data files include raw images and image streams used as representative data in the main and supplemental figures. These images are from a larger subset of images from which the fluorescence and transport quantification originated. These quantifications and their corresponding data statistics and summary are included for each figure as an Excel (.xlsx) file. These data files are grouped by their corresponding figures or videos (e.g., raw images and quantification files corresponding to Figure 1 are in the folder named “Figure 1 – source data 1”). Thirteen files of raw data are included. ## Description of the data and file structure ### Figure 1 – source data 1: Included in the zipped archive: 1\. Raw image streams (*RAW.tif) 2\. Annotated image streams (*ROI.tif) 3\. Raw fluorescence quantification and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Raw image streams show changes in mitoGCaMP fluorescence. Raw fluorescence values were analyzed using a custom Excel module (available at GitHub- see link in Related Works section); see Materials and Methods – Code/Software). In the annotated image stream (*ROI.tif), the white box indicates the region from which individual frames were used as representative images in Figure 1. Raw quantifications, statistical tests, and data summaries for Figure 1 (Figure1_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (F, arbitrary units) quantified from microscopy image streams using the image analysis software MetaMorph (see Code/Software). The calculated metrics represented in Figure 1’s data tables were calculated using a custom excel module that is available on GitHub (link in the Related Works section). These metrics include mitoGCaMP fluorescence include normalized fluorescence (F/Fmin), the normalized amplitude of a mitoGCaMP event (maximum fluorescence value normalized to the minimum fluorescence value [Maximum F/Fmin]), and total mitoGCaMP activity throughout the imaging session (summation of fluorescence normalized to baseline fluorescence [Total Activity Norm. to Baseline]).  ### Figure 2 – source data 1: Included in zipped archive: 1\. Raw, single channel maximum projections of GLR-1 located to the plasma membrane (*SEP.tif) and mitochondria (*tdTomato.tif) used to make the composite image shown in Figure 2A 2\. Raw maximum projections capturing mito-roGFP fluorescence resulting from 488 and 405 nm excitation per sample (*RAW.tif) 3\. An annotated version of each maximum projections (*ROI.tif) 4\. Raw GCaMP image streams (*ImageStream.tif) 5\. Raw fluorescence quantification and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Raw maximum projections are 2D images created via the projection of pixels with the highest fluorescence values for each plane of a 3D image stack (z-stack) into a single image. In the annotated image (*ROI.tif), the white box indicates the region that is shown as representative data in Figure 2. Raw GCaMP image streams show changes in GCaMP fluorescence. Raw fluorescence values were analyzed using a custom Excel module (available on GitHub; see Materials and Methods – Code/Software). Raw quantifications, statistical tests, and data summary for Figure 2 (Figure2_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (arbitrary units) quantified from microscopy images using the image analysis software MetaMorph (see Code/Software). The metric calculated for the data table related to Figure 2C include the average fluorescence within a region of interest (ROI) for various time points after photobleaching (e.g., 0, 2, 4 minutes post-photobleaching) from which the 0 min. post-photobleaching is subtracted from all timepoints (‘Fluorescence Recovery After Photobleaching’). Additionally, a similar metric was calculated the data table related to Figure 2E by normalizing ‘Fluorescence Recovery After Photobleaching’ to the fluorescence of the ROI prior to photobleaching to calculate the ‘Percentage of Fluorescence Recovery After Photobleaching’. Raw quantifications related to Figure 2G represent the number of transport events per minute manually quantified using kymographs (see Code/Software). The calculated metrics represented in data tables for panels 2I-2J were calculated using a custom excel module that is available on GitHub. These metrics the normalized amplitude of a GCaMP event (maximum change in fluorescence value normalized to the minimum fluorescence value [Maximum deltaF/Fmin]) and total GCaMP activity throughout the imaging session (summation of fluorescence normalized to baseline fluorescence [Total Activity Norm. to Baseline]).  ### Figure 2 Supplement 1 – source data 1: Included in zipped archive: 1\. Puncta or normalized fluorescence, transport velocity quantifications, and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Raw quantifications, statistical tests, and data summaries for Figure 2 Supplement 1 (Figure2_Supplement1_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (F, arbitrary units) quantified from microscopy image streams using the image analysis software MetaMorph (see Code/Software). Puncta fluorescence was quantified as previously described [PMID: 24360545]. Transport velocities for panel C were calculated using kymographs and the ImageJ plug-in KymoAnalyzer as described in the methods, and the average transport velocity for each worm was compared between conditions. ### Figure 3 – source data 1: Included in zipped archive: 1\. Raw maximum projections capturing mito-roGFP fluorescence resulting from 488 and 405 nm excitation per sample at given time points or in different treatment conditions(*RAW.tif). 2\. An annotated version of each maximum projections (*ROI.tif) 3\. Raw fluorescence quantification and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Raw maximum projections are 2D images created via the projection of pixels with the highest fluorescence values for each plane of a 3D image stack (z-stack) into a single image. In the annotated image (*ROI.tif), the white box indicates the region that is shown as representative data in Figure 3. Raw quantifications, statistical tests, and data summaries for Figure 3 (Figure3_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (F, arbitrary units) due to excitation at 488 nm (FEx488) or 405 nm (FEx405) quantified from microscopy image streams using the image analysis software MetaMorph (see Code/Software). The mito-roGFP fluorescence ratio (mito-roGFP Fratio) was calculated by dividing FEx488 by FEx405.  ### Figure 3 Supplement 1– source data 1: Included in zipped archive: 1\. Raw fluorescence quantification and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Raw quantifications, statistical tests, and data summaries for Figure 3 Supplement 1 (Figure3_Supplement1_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (F, arbitrary units) due to excitation at 488 nm (FEx488) or 405 nm (FEx405) quantified from microscopy image streams using the image analysis software MetaMorph (see Code/Software). The mito-roGFP fluorescence ratio (mito-roGFP Fratio) was calculated by dividing FEx488 by FEx405.  ### Figure 4 – source data 1: Included in zipped archive: 1\. Raw maximum projections capturing mito-roGFP fluorescence resulting from 488 and 405 nm excitation per sample with different treatments and at different time points (*RAW.tif) 2\. An annotated version of each maximum projections (*ROI.tif) 3\. Raw fluorescence quantification and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Raw maximum projections are 2D images created via the projection of pixels with the highest fluorescence values for each plane of a 3D image stack (z-stack) into a single image. In the annotated image (*ROI.tif), the white box indicates the region that is shown as representative data in Figure 4. Raw quantifications, statistical tests, and data summaries for Figure 4 (Figure4_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (F, arbitrary units) due to excitation at 488 nm (FEx488) or 405 nm (FEx405) quantified from microscopy image streams using the image analysis software MetaMorph (see Code/Software). The mito-roGFP fluorescence ratio (mito-roGFP Fratio) was calculated by dividing FEx488 by FEx405.  ### Figure 5 – source data 1: Included in zipped archive: 1\. Raw maximum projections capturing SEP or GFP fluorescence at various time point following SEP/GFP photobleaching per sample (*RAW.tif) 2\. An annotated version of each maximum projections (*ROI.tif) 3\. Raw kymographs depicting GFP location in a photobleached neurite (*Kymo_RAW.tif) 4\. Annotated kymographs (*Kymo_ROI.tif) 5\. Raw fluorescence quantification and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Raw maximum projections are 2D images created via the projection of pixels with the highest fluorescence values for each plane of a 3D image stack (z-stack) into a single image. In the annotated image (*ROI.tif), the white box indicates the region that is shown as representative data in Figure 5. Raw quantifications, statistical tests, and data summary for Figure 5 (Figure5_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (arbitrary units) quantified from microscopy images using the image analysis software MetaMorph (see Code/Software). The metric calculated for the data tables related to Figure 5C and 5E include the average fluorescence within a region of interest (ROI) for various time points after photobleaching (e.g., 0, 2, 4 minutes post-photobleaching) from which the 0 min. post-photobleaching is subtracted from all timepoints then normalized to the fluorescence of the ROI prior to photobleaching to calculate the ‘Percentage of Fluorescence Recovery After Photobleaching’. Raw quantifications related to Figure 5H represent the number of transport events per minute manually quantified using kymographs (see Code/Software). ### Figure 5 Supplement 1 – source data 1: Included in zipped archive: 1\. Raw fluorescence and transport velocity quantifications, and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Transport velocities for panel A and B were calculated using kymographs and the ImageJ plug-in KymoAnalyzer as described in the methods, and the average transport velocity for each worm was compared between conditions. Raw quantifications for panels C and D (Figure5_Supplement1_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (arbitrary units) quantified from microscopy images using the image analysis software MetaMorph (see Code/Software). The metric calculated for the data tables related to panels C and D include the average fluorescence within a region of interest (ROI) for various time points after photobleaching (e.g., 0, 2, 4 minutes post-photobleaching) from which the 0 min. post-photobleaching is subtracted from all timepoints then normalized to the fluorescence of the ROI prior to photobleaching to calculate the ‘Percentage of Fluorescence Recovery After Photobleaching’. ### Figure 6 – source data 1: Included in zipped archive: 1\. Raw maximum projections capturing SEP or GFP fluorescence at various time point following SEP/GFP photobleaching per sample (*RAW.tif) 2\. An annotated version of each maximum projections (*ROI.tif) 3\. Raw kymographs depicting GFP location in a photobleached neurite (*Kymo_RAW.tif) 4\. Annotated kymographs (*Kymo_ROI.tif) 5\. Raw fluorescence quantification and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Raw maximum projections are 2D images created via the projection of pixels with the highest fluorescence values for each plane of a 3D image stack (z-stack) into a single image. In the annotated image (*ROI.tif), the white box indicates the region that is shown as representative data in Figure 6. Raw quantifications, statistical tests, and data summary for Figure 6 (Figure6_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (arbitrary units) quantified from microscopy images using the image analysis software MetaMorph (see Code/Software). The metric calculated for the data table related to Figure 6C include the average fluorescence within a region of interest (ROI) for various time points after photobleaching (e.g., 0, 2, 4 minutes post-photobleaching) from which the 0 min. post-photobleaching is subtracted from all timepoints then normalized to the fluorescence of the ROI prior to photobleaching to calculate the ‘Percentage of Fluorescence Recovery After Photobleaching’. Raw quantifications related to Figure 6F represent the number of transport events per minute manually quantified using kymographs (see Code/Software). ### Figure 6 Supplement 1 – source data 1:  Included in zipped archive: 1\. Raw fluorescence quantification and corresponding statistics and summary (*Quantification_DataSummary.xlsx) Raw quantifications, statistical tests, and data summary for Figure S5 (Figure6_Supplement1_SourceData1_Quantification_DataSummary.xlsx) relate to measures of fluorescence (arbitrary units) quantified from microscopy images using the image analysis software MetaMorph (see Code/Software). The metric calculated for the data table related to panel A include the average fluorescence within a region of interest (ROI) for various time points after photobleaching (e.g., 0, 2, 4 minutes post-photobleaching) from which the 0 min. Post-photobleaching is subtracted from all timepoints then normalized to the fluorescence of the ROI prior to photobleaching to calculate the ‘Percentage of Fluorescence Recovery After Photobleaching’. Raw quantifications related to panel B represent the number of transport events per minute manually quantified using kymographs (see Code/Software). ### Video 1 – source data 1:  Included in zipped archive: 1\. A video created from a raw mitoGCaMP image stream (*RAW.avi) ### Video 2 – source data 1: Included in zipped archive: 1\. A video created from a raw GLR-1::GFP image stream (*RAW.tif) 1\. Annotated video showing the region used to create the corresponding kymograph (*RAW.tif) ## Sharing/Access information All data has been generated by the authors for the purpose of this publication and is not available elsewhere. ## Code/Software Maximum projections of z-stacks and kymographs representing single, 20-pixel-wide user-specified line scans of image streams were created using the microscopy and image analysis software MetaMorph (Molecular Devices). Raw fluorescence values were extracted from user-specified regions on all fluorescent raw images or maximum projections using MetaMorph’s ‘Region Measurement’ tool (see Materials and Methods for more details) Kymographs rendered in MetaMorph were imported into ImageJ and ~10 transport events per kymograph were manually traced then analyzed with the ImageJ plug-in KymoAnalyzer (see Materials and Methods).  Raw fluorescence values were analyzed using a custom Excel module (available on GitHub; see Materials and Methods – Code/Software).