**2.7. Mitochondrial mapping in AMD**

Mitochondrial signaling in a network-based interactome map between genome, proteome, and metabolome of AMD was established using proteome data and bioinformatics software. The protein-protein interaction was established using the Münich Information Center for Protein Sequence (MIPS), Biomolecular Interaction Network Database (BIND), the Database of Interacting Proteins (DIP), the Molecular Interaction Database (MINT), the Protein Interaction Database (IntAct), and STRING.

Interaction mapping of prohibitin was determined using immunoprecipitation, followed by mass spectrometry analysis. Prohibitin binding proteins in the RPE were connected using STRING 10.0 software (http://string-db.org/).

Prohibitin interactions were confirmed using eight sources that include neighborhood, gene fusion, co-occurence, high-throughput interaction experiments, databases, homology, conserved co-expression, and published knowledge, including ExPASy (http://www.expasy. org/proteomics /protein–protein\_interaction), MIPS (http://mips.helmholtz-muenchen.de/ proj/ppi/), and Pubmed database (http://www.ncbi.nlm.nih.gov/pubmed).

AMD and oxidative biomarkers interactome were established using protein–protein interaction map software and databases, including STRING 10.0 (http://string-db.org/), MIPS and iHOP (http://www.ihop-net.org/UniPub/iHOP/) (**Figure 8**). Proteins found in AMD or oxidative stress conditions were added to establish the AMD interactome. Protein interactions were presented using eight categories, including neighborhood (green), gene fusion (red), cooccurrence (dark blue), co-expression (black), binding experiments (purple), databases (blue), text mining (lime), and homology (cyan). Protein interactions were determined and confirmed by genomic context, high-throughput experiments, co-expression, and previous publications in Pubmed. Protein database analysis showed the region-specific phosphorylation of specific proteins in AMD eyes. The interactome between AMD proteome was compared to the retina/ RPE proteome under stress conditions.

The genome regulatory network was connected to the proteome network using Uniprobe and JASPAR. Protein phosphorylations were examined by phosphoprotein/peptide enrichment, followed by mass spectrometry analysis. Phosphorylations were compared to Phospho. ELM, and PhosphoSite. The metabolome mapping was established using KEGG and BIGG databases.

Our results showed that the connectivity, the number of mitochondrial branch points, and the interactive isosurfaces were altered at the contact sites between mitochondria and other organelles. Under extended oxidative stress (1, 8, 24 h) and intense light (7000 lx, 1 h), we observed a decrease in mitochondrial size, presence of fragmented filaments (red arrows), and holes on the organelle contact sites. Under intense light condition, mitochondria in ARPE-19 cells were

**Figure 1.** Quantitative analysis of mitochondrial morphology: representative images of MitoTracker Orange-labeled mitochondria from ARPE-19 cells exposed to *t*-BuOOH for 0.5–24 h or light for 1 h are shown here. A. ARPE-19 cells under oxidative stress were analyzed by immunocytochemistry using MitoTracker. B. Mitochondrial content was represented by 2D graph (radius/intensity) showing decreased size and fragmentation pattern under stress conditions.

Mitochondrial Trafficking by Prohibitin-Kinesin-Myosin-Cadherin Complex in the Eye

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Next, mitochondrial perimeter vs. circularity was examined to determine the correlation between mitochondrial morphology and oxidative stress (**Figure 2**). We hypothesized that some mitochondrial indexes that include circularity and perimeter ratio may represent mitochondrial dynamics. We calculated mitochondrial area, perimeter, minor axis, and circularity

The average mitochondrial area/perimeter ratio normalized to the minor axis suggests that specific conditions may induce mitochondrial swelling (**Figure 3**). Time-dependent decrease of minor axis and mitochondrial area/perimeter normalized to the circularity was noticed under stress condition. Our previous proteomic study demonstrated that tubulin/vimentin

To understand mitochondrial dynamics in detail, mitochondrial trafficking complex was examined. Subcellular fractionation, immunoprecipitation using primary prohibitin antibody, native gel, and mass spectrometry analysis suggest that motor protein complex may determine mitochondrial dynamics and retrograde signaling under stress conditions. Molecular motor

to conclude that specific mitochondrial ratio correlated positively with stress kinetics.

depolymerization and phosphorylations increased in stressed mitochondria [25].

decreased and fragmented as shown in oxidative stress (1–8 h).

C. Mitochondria in ARPE-19 cells were presented in 3D structure using Image J software.
