**2.3. Immunocytochemical analysis**

structures to form single organelle, as well as movement along cytoskeletal paths [5, 9]. These

To maintain the balance that regulates overall morphology and cytoskeletal stability many specialized molecules including dynamin family of proteins play critical roles [10, 11]. Growing body of evidence demonstrates that disruptions of mitochondrial network lead to multiple human pathologies, including metabolic, genetic, cardiovascular diseases, as well as neurogenerative diseases and cancers [12–15]. Several studies provided the evidence that mitochondria play a critical role in the progression of age-related diseases, including age-related macular degeneration (AMD) [16–21]. The damage of mitochondrial DNA could be the key factor involved in altered vascular endothelial growth factor (VEGF) secretion, retinal pigment

combined actions occur concurrently in major cell types to regulate the cell fate.

epithelium (RPE) dysfunction, and cell death during the progression of AMD [22, 23].

apoptosis under oxidative stress.

210 Mitochondrial Diseases

work in neurodegeneration.

**2. Materials and methods**

**2.1. Cell culture and oxidative stress treatment**

The current study aimed to examine the correlation between alterations in mitochondrial morphology and mitochondrial dysfunction. For quantitative analysis of mitochondrial morphology, we introduced the mitochondrial index that includes network size, mitochondrial content and surface area. Mitochondrial interconnectivity and elongation were determined systematically using a computational model in three dimensions, showing a mitochondrialendoplasmic reticulum (ER)-nuclear hole as open space for trafficking at the beginning of

The assessment of average circularity showed mitochondrial elongation which is sensitive to fragmented vs. normal shaped mitochondria. The average area/perimeter ratio showed normal or stressed mitochondria as a highly interconnected mass of reticular network. Previously, we observed that prohibitin translocalizes between the nucleus and mitochondria under oxidative stress conditions to influence mitochondrial dynamics [24]. We observed anterograde signaling from the nucleus to mitochondria using a prohibitin shuttle under stress in the retina, as well as the retrograde shuttling of prohibitin from mitochondria to the nucleus in the RPE. In addition, cytoskeletal reorganization, tubulin/vimentin depolymeriza-

tion and increased phosphorylations were observed in stressed mitochondria [25–27].

In this study, mitochondrial dynamics was further analyzed in mitochondrial trafficking complex using prohibitin immunoprecipitation. We found a motor-based protein complex that includes kinesin 19 (93 kDa), myosin 9 (110 kDa), and cadherin isoforms (88 kDa) to regulate the mitochondrial-nuclear communication. Finally, we have established a comprehensive mitochondrial interactome map by combining several independent sets of interaction data. Our interactome map provides an integrated information on the hidden apoptotic pathway, cytoskeletal rearrangement, nitric oxide signaling, ubiquitination, and mitochondrial net-

Retinal pigment epithelial cells (ARPE-19) were purchased from ATCC (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine To analyze mitochondrial morphology, Cells were incubated with 100 nM MitoTracker Orange (Molecular Probes). Cells were fixed using 10% formaldehyde (25 min) and the membrane was permeabilized using 0.2% Triton X-100 (20 min), followed by blocking (0.05% Tween 20, 10% FBS, 1 h) and incubation overnight at 4°C with anti-prohibitin antibody (1:500; Genemed Synthesis, San Antonio). Prohibitin was visualized using Alexa Fluor 488-secondary IgG antibody (1:700; 1 h at 25°C; Molecular Probes). The nuclei were visualized by incubation with DAPI (4, 6-diamidino-2-phenylindole) added to VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA). A fluorescence microscopy was used for image analysis (Zeiss AxioVert 200 M Apo Tome, 63×).

spectra were calibrated using a known peptide, including trypsin (842.5099, 2211.105 Da). The mass spectrum was recorded in 800–3000 Da range using Flex MALDI-TOF mass spectrometer (Bruker Daltonics, Germany, 70–75% laser intensity, 100–300 shots). Mass spectrometry data were analyzed using Flex analysis software (Bruker Dal- tonics, Germany). Peptides were identified using the Mascot software (Matrix Science) and NCBI/SwissProt database (zero mismatch cleavage, carbamidomethyl cysteine, methionine oxidation, 50–300 ppm mass tolerance). Peptide identification was evaluated based on Mascot MOWSE score, number of matched peptides, and protein sequence coverage. MOWSE score is expressed as -10logP as a

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

http://dx.doi.org/10.5772/intechopen.75994

213

The connectivity, the number of mitochondrial branch points, and the interactive 3D visualization of isosurfaces were examined to identify the contact area between mitochondria and

Mitochondria were stained using MitoTracker Orange/Red or rhodamine 123. Mitochondrial interconnectivity and elongation were analyzed systematically using computational software,

Mitochondrial size and morphology were analyzed using the software connected to Image J software (Ruben Dagda: http://imagejdocu.tudor.lu/doku.php?id=macro:mitpophagy\_ mitochondrial\_morphology\_content\_lc3\_colocalization\_macro). We chose the region of interest using the polygon selection tool to analyze mitochondrial morphology. Individual red, green, and blue channels were obtained from the RGB images, and then the red and blue channels were closed. The grayscale was extracted from the red channel and the pixels were inverted to photographic channel. The Threshold function determined maximal and minimal pixel values. To understand mitochondrial structure and function, 12 mitochondrial indexes, including (1) mitochondrial area, (2) cellular area, (3) mitochondrial content, (4) perimeter, (5) circularity, (6) average perimeter, (7) average mitochondrial area, (8) average circularity, (9) area/ perimeter, (10) area/perimeter normalized to minor axis, (11) minor axis, (12) area/perimeter normalized to circularity, were evaluated in ARPE-19 cells under oxidative stress conditions. Mitochondrial shape, including fused, fragmented, tubular, swollen, branched, uniform, and perinuclear clustering, was examined quantitatively. Mitochondrial filaments in three dimensions and mitochondrial parameters in ARPE-19 cells were calculated mathematically using Image J and Imaris (v8.2.1) software. The connectivity, the number of mitochondrial branch points, and the interactive 3D visualization of isosurfaces were examined to identify the contact

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

including Mito-Morphology macro, Mitograph 2.0, Imaris 8.2.1., and SOAX 3.6.1.

probability value to compute the composite probability P.

**2.6. Quantitative analysis of mitochondrial morphology**

between mitochondria and other organelles.

**2.7. Mitochondrial mapping in AMD**

other organelles.

#### **2.4. Immunoprecipitation**

ARPE-19 cells were rinsed (Modified Dulbecco's PBS) and lysed using immunoprecipitation (IP) lysis buffer (pH 7.4) containing Tris (25 mM), NaCl (150 mM), EDTA (1 mM), NP-40 (1%), glycerol glycerol (5%), and protease inhibitor cocktail by incubating on ice for 5 min with periodic sonication (3 × 5 min), followed by centrifugation (13,000 × *g*, 10 min). Proteins (1 mg/mL, 200–400 μL) were loaded for immunoprecipitation and nonspecific bindings were avoided using control agarose resin cross-linked by 4% bead agarose. Amino-linked protein-A beads were used to immobilize anti-prohibitin antibody with a coupling buffer (1 mM sodium phosphate, 150 mM NaCl, pH 7.2), followed by incubation (room temperature, 2 h) with sodium cyanoborohydride (3 μL, 5 M). Columns were washed using a washing buffer (1 M NaCl), and protein lysate was incubated in the protein A-antibody column with gentle rocking overnight at 4°C. The unbound proteins were spun down as flow-through, and the column was washed three times using the washing buffer (1 M NaCl) to remove nonspecific binding proteins. The prohibitin-interacting proteins were eluted by incubating with elution buffer for 5 min at room temperature. The eluted proteins were equilibrated with Laemmli sample buffer (5X, 5% β-mercaptoethanol). Eluted proteins were separated using SDS–PAGE and visualized using Coomassie blue (Pierce, IL) or silver staining kit (Bio-Rad, Hercules, CA). Prohibitin and p53 were visualized using Western blot analysis. Proteins were identified by mass spectrometry analysis.

#### **2.5. Mass spectrometry analysis**

Protein bands were excised into 1 × 1 × 1 mm cubes. The Coomassie-stained or silver-stained gel pieces were incubated using a Coomassie destaining buffer (200 μL of 50% MeCN in 25 mM NH4 HCO3 , pH 8.0, room temperature, 20 min) or silver destaining buffer (50% of 30 mM potassium ferricyanide, 50% of 100 mM sodium thiosulfate, 5–10 min). The gel pieces were dehydrated (200 μL, MeCN) and vacuum-dried (Speed Vac, Savant, Holbrook, NY). Proteins were reduced (10 mM DTT, 100 mM NH4 HCO3 , 30 min, 56°C) and were alkylated (55 mM iodoacetamide, 100 mM NH4 HCO3 , 20 min, room temperature in the dark). Proteins were digested using trypsin (13 ng/μL sequencing-grade from Promega, 37°C, overnight) in NH4 HCO3 (10 mM) containing MeCN (10%). The peptides were enriched using a buffer (50 μL, 50% MeCN in NH4 HCO3 , 5% formic acid, 20 min, 37°C). Dried peptides were dissolved in the mass spectrometry sample buffer (5–10 μL, 75% MeCN in NH<sup>4</sup> HCO3 ,1% trifluoroacetic acid). Alpha-cyano-4-hydroxycinnamic acid (5 mg/mL, Sigma-Aldrich, St. Louis, MO) was freshly dissolved in a matrix buffer (50% MeCN, 50% NH<sup>4</sup> HCO3 , 1% triflouroacetic acid) and centrifuged (13,000× *g*, 5 min). The peptide-matrix mixtures (0.5 μL) were spotted onto the MALDI target plate (Ground steel, Bruker Daltonics, Germany). Mass spectrometer and all spectra were calibrated using a known peptide, including trypsin (842.5099, 2211.105 Da). The mass spectrum was recorded in 800–3000 Da range using Flex MALDI-TOF mass spectrometer (Bruker Daltonics, Germany, 70–75% laser intensity, 100–300 shots). Mass spectrometry data were analyzed using Flex analysis software (Bruker Dal- tonics, Germany). Peptides were identified using the Mascot software (Matrix Science) and NCBI/SwissProt database (zero mismatch cleavage, carbamidomethyl cysteine, methionine oxidation, 50–300 ppm mass tolerance). Peptide identification was evaluated based on Mascot MOWSE score, number of matched peptides, and protein sequence coverage. MOWSE score is expressed as -10logP as a probability value to compute the composite probability P.
