**1. Introduction**

Mitochondria are the highly dynamic cellular organelles that form a dynamic network to regulate calcium balance, energy metabolism, and apoptotic signaling [1–8]. Mitochondria alter their morphology repeatedly through the collective actions of fission as a separation of a single organelle into multiple autonomous structures, fusion as the combination of multiple

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

structures to form single organelle, as well as movement along cytoskeletal paths [5, 9]. These combined actions occur concurrently in major cell types to regulate the cell fate.

serum (FBS; 10%) and penicillin/streptomycin (1%) at 37°C in a humidified atmosphere of 5%

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

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

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Retinal progenitor cells (HRP) were kindly donated by Dr. Harold J. Sheeldo (University of North Texas Health Science Center) and were cultured at the same condition as ARPE-19 cells. Prior to all experiments, confluent ARPE-19 cells were incubated with fresh medium for 12 h and washed with phosphate buffered saline (PBS) three times. ARPE-19 cells were incubated with an oxidant, *tert*-butyl hydroperoxide (*t*-BuOOH, 200 μM, Sigma-Aldrich, St.Louis, MO), in serum-free medium for 0.5, 1, 2, 4, 6, 8, 12, and 24 h and representative images were presented. After the treatment, medium was removed, cells were washed with PBS and harvested for future analysis. Cells were lysed for experiments, including, immunoprecipitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE), Western blot analy-

To compare with other stress environment, ARPE-19 cells were incubated under intense light

Lipids were extracted from ARPE-19 cells using cholorform/methanol (2:1, v/v) and organic solvent was evaporated under a gentle nitrogen stream and dissolved in chloroform for analy-

All experiments were repeated (*N* = 3–10 biological samples) with technical duplicate or triplicate. Statistical analysis was performed using StatView software and statistical significance

Human postmortem donor eye tissues were used following the tenets of the Declaration of Helsinki. Human AMD retinas (8 mm macular and peripheral punches), RPE (8 mm central and peripheral punches), and age-matched control eyes (*N* = 9, biological triplicate x technical triplicate) were provided by the Lions Eye Bank (Moran Eye Center, University of Utah). Phosphoproteome of macular (I), peripheral retina (II), central RPE (III), and peripheral RPE (IV) was compared to age-matched control donor eyes to determine region-specific senescence-associated molecular mechanisms during AMD progression. Phosphoproteins were enriched by charge-based spin column chromatography and resolved by 2D gel electrophoresis as previously reported [28]. In addition, trypsin digested phosphopeptides from

phosphopeptides were analyzed using mass spectrometry including MALDI-TOF-TOF and

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

was determined by variance (ANOVA) or unpaired Student's *t* test when appropriate.

/s photon flux; 7000 lx) for 1 h in serum-free media and analyzed by SDS-

immobilized metal ion chromatography. Eluted

in air as suggested by the manufacturer. Cells were used between passages 8–9.

sis, immunocytochemistry, and mass spectrometry analysis.

PAGE/Western blotting, or immunocytochemistry.

**2.2. Donor eye tissue and phosphoprotein enrichment**

whole lysates were enriched using Ga3+/TiO2

**2.3. Immunocytochemical analysis**

sis by HPLC and mass spectrometry.

CO2

(210 μmoles/m2

ESI MS/MS.

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

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 apoptosis under oxidative stress.

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 depolymerization 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 network in neurodegeneration.
