|Molecular Vision 2005;
Received 15 December 2004 | Accepted 28 June 2005 | Published 14 July 2005
Confocal laser scanning microscopy imaging of dynamic TMRE movement in the mitochondria of epithelial and superficial cortical fiber cells of bovine lenses
Jacob G. Sivak
School of Optometry, University of Waterloo, Waterloo, Ontario, Canada
Correspondence to: Vladimir Bantseev, School of Optometry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1; Phone: (519) 888-4567, ext. 7091; FAX: (519) 725-0784; email: email@example.com
Purpose: Recent confocal laser scanning microscopy studies of the mitochondria of vertebrate lenses show a striking difference in the distribution and morphology of the mitochondria of lens epithelial and superficial cortical cells. This study, using confocal microscopy, was undertaken to image the movement of the mitochondria specific dye tetramethylrhodamine ethyl ester (TMRE) in the epithelium and superficial cortex of whole live bovine lens.
Methods: Cultured bovine lenses were loaded with 5 μg/ml TMRE for 15 min at room temperature. TMRE fluorescence was acquired with a Zeiss 510 (configuration META 18) confocal laser scanning microscope for 10 to 15 min using 488 nm Argon laser excitation and 505 nm long pass emission filter settings. The uncoupler of the electron transport chain potential, carbonyl cyanide m-chlorophenylhydrazone (CCCP, 32.5 μM), was used to demonstrate the fluorescent specificity of TMRE.
Results: Multidirectional dynamic movement of TMRE was observed in epithelial cells and bidirectional dynamic movement was seen in the superficial cortical fiber cells of live bovine lenses. In the epithelium, the movement of TMRE fluorescence was up to 5 μm/min whereas in the superficial cortex the observed movement was up to 18.5 μm/min. The movement of TMRE fluorescence was abolished with treatment with the uncoupler, CCCP.
Conclusions: The observed dynamics of TMRE fluorescence movement may represent actual mitochondrial movement, indicating the dynamic state of the mitochondria in both lens epithelium and superficial cortex. That this activity is found not only in the epithelium but also in the superficial cortex indicates that the superficial cortical fiber cells play a much more active role in lens metabolism than previously suspected. Alternatively, the observed movement of TMRE across a mitochondrial network could represent change in the distribution of potential across the inner membrane, presumably allowing energy transmission across the cell from regions of low to regions of high ATP demand.
The key function of mitochondria is energy production through oxidative phosphorylation and lipid oxidation . The process takes place within the mitochondrial inner membrane and includes five multi-subunit enzyme complexes. Several other metabolic functions are performed by mitochondria including urea production, heme and non-heme iron biogenesis, steroid biogenesis, intracellular Ca2+ homeostasis, and interaction with the endoplasmic reticulum. For many of these mitochondrial functions, there is only a partial understanding of the components involved, with even less information on mechanisms and regulation .
General knowledge of the structure of mitochondria has paralleled the development of techniques for the preparation of fixed biological samples for electron microscopy. Early electron microscopy studies of mitochondria of vertebrate lenses showed an absence of mitochondria in lens fiber cells and the presence of very few, short mitochondria in the epithelial cells. It became widely accepted that the lens epithelium plays the most important role in lens metabolism . It was further proposed that ions and metabolites gain access to the fiber cells, which lack organelles, including mitochondria, via gap junctions connecting the closely apposed apical membranes of lens epithelial and superficial cortical fiber cells . The hypothesis that normal fiber cell metabolism is maintained by epithelial cells has prompted numerous studies of the role of epithelial cell dysfunction and its role in lens damage.
In recent years the basic understanding of mitochondrial morphology and distribution has been enhanced by advances in confocal microscopy that permits imaging of living cells with the aid of fluorescent dye technology. Recent studies using specific fluorescent dyes and confocal microscopy of live chick , rat , and bovine lenses [7,8] show that both the epithelial cells and the superficial cortical fiber cells of the lens contain numerous metabolically active mitochondria, suggesting that the superficial cortical fiber cells play a much more active role in lens metabolism than previously suspected.
Using fluorescent and video microscopy dynamic movement of mitochondria have been shown in Acanthamoeba castellanii  and the fungus Neurospora crassa . These observations are in keeping with the view that mitochondria are related to bacteria  that form a symbiotic relationship with the cytoplasm of eukaryotic cells. However the mitochondria lack flagella, cilia, or other anatomical structures associated with bacterial movement and the mechanisms of mitochondrial movement remain obscure .
There appears to be little or no information regarding mitochondrial movement in the lens. This study, using confocal scanning laser microscopy, reports the dynamic movement of the mitochondria specific dye tetramethylrhodamine ethyl ester (TMRE) in the epithelium and superficial cortex of whole live bovine lenses.
The mitochondria specific dye tetramethylrhodamine ethyl ester (TMRE), penicillin, streptomycin, and dialyzed fetal bovine serum were obtained from Invitrogen (Burlington, ON, Canada). All other chemicals were obtained from Sigma (St. Louis, MO).
Eye dissection and treatment
Bovine eyes, obtained from a local abattoir, were dissected, and the lenses were excised within 1-5 h post-mortem under sterile conditions, as described previously . Briefly, the lenses were placed in a three part chamber made from glass, silicon rubber, and a metal base and immersed in 21 ml of culture medium consisting of Medium 199 with Earle's salts, 100 mg/l L-glutamine, 3% dyalized fetal bovine serum, 2.2 g/l sodium bicarbonate, 5.96 g/l HEPES, and 1% antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin). The cultured lenses were incubated at 37 °C and 4% CO2 for 48 h prior to experimental use and lenses that were damaged during dissection were excluded from the study.
To demonstrate the fluorescence specificity of TMRE, the uncoupler of the electron transport chain potential, carbonyl cyanide m-chlorophenylhydrazone (CCCP), was used. Lenses were transferred for treatment into glass vials containing 32.5 μM CCCP in 10 ml serum free M199 and incubated for 30 min. The lenses were then rinsed three times with physiological saline and culture medium.
Confocal microscopy and 3D reconstruction
To investigate mitochondrial movement, the mitochondria specific fluorescent dye tetramethylrhodamine ethyl ester (TMRE) was used. TMRE is a lipophilic, cell permeable, cationic, nontoxic, fluorescent dye that specifically stains live mitochondria. TMRE is accumulated specifically by the mitochondria in proportion to membrane potential . Whole lenses were loaded with TMRE by bathing the lens for 15 min at room temperature in 10 ml serum free M199 containing 5 μg/ml TMRE. Lenses were then mounted in 1% agarose on glass bottom plates as described previously . Time series and z-stacks of lens epithelial and superficial cortical fiber cells were acquired with a Zeiss 510 (configuration Meta 18) confocal laser scanning microscope (Carl Zeiss Inc., Toronto Canada) equipped with an inverted Axiovert 200M microscope and 40x water immersion C-Apochromat objective (NA 1.2). The combination of an 488 nm Argon laser and 505 long pass emission filters were used to visualize TMRE fluorescence. Subsequent image restoration and analysis (such as the length of mitochondria and rate of movement) was done with commercial LSM510 VisArt and Physiology software packages (version 3.2; Carl Zeiss Inc., Jena, Germany). The representative length of mitochondria was measured from one end of the most visible structure to the other end using the measure function of software. The rate of movement (expressed in μm/sec) was determined by noticing the initial position (position zero) of moving mitochondria in the first X, Y gallery of time series, to the final movement observed during the time series by measuring the length in microns (μm).
The morphology and distribution of the mitochondria between lens epithelial and superficial cortical fiber cells is very different. In epithelial cells, up to 10 μm dense numerous perinuclear mitochondria, resembling an interconnected network were seen (Figure 1A). These dense mitochondria appear to fill the whole volume of the lens epithelial cell (Figure 2). In the superficial cortex the mitochondria were not as dense (Figure 1B), were more elongated (up to 65 μm), distinctly separated, and often branched (Figure 3).
Dynamic multidirectional movement of TMRE was observed in epithelial cells and bidirectional movement was seen in the superficial cortical fiber cells. In the epithelium, the movement of TMRE fluorescence was up to 5 μm/min (Figure 4) whereas in the superficial cortex the observed movement was up to 18.5 μm/min (Figure 5). The movement of TMRE fluorescence was abolished in both the lens epithelium (Figure 6) and the superficial cortex (Figure 7) following treatment with the uncoupler of the electron transport chain potential, CCCP.
The vertebrate lens is a cellular structure that consists of two types of cells organized in distinct spatial patterns; the epithelial monolayer that covers the anterior surface and fiber cells that comprise the bulk of the lens. Cell division in the lens is restricted to the epithelial cells. This monolayer can be divided into three compartments, related to lens anatomy and rate of cell division. Central epithelial cells overlying the anterior suture regions of the lens normally undergo little or no mitosis . Intermediately located epithelial cells undergo limited mitosis whereas epithelial cells at the lens equator show the fastest mitotic rate. These equatorial epithelial cells give rise to terminally differentiated fiber cells in a process that continues throughout life, resulting in a steady increase in tissue volume.
Highly variable morphology and distribution of mitochondria is observed in different types of cells. In growing cells mitochondria are frequently found as extremely dynamic structures with tubular sections dividing in half, branching, and fusing to form a complex network . In differentiated cells, such as cardiac muscle or kidney tubules, mitochondria are often localized in specific cytoplasmic regions rather than randomly distributed . In this study, three dimensional reconstruction of the lens epithelium indicates that mitochondria may form a complex network (Figure 2) similar to that of other mammalian cells . In contrast to the lens epithelium the mitochondria of the superficial cortical fiber cells were not as dense, were distinctly separated and often branched (Figure 3 and Figure 5). Despite such variability between the two cell types, no obvious difference in morphology or distribution is observed between lenses of different species, including fish and mammals [7,18]. The random distribution of the mitochondria throughout the terminally differentiated superficial cortical fiber cells (Figure 1B, Figure 3, and Figure 5) also contrasts with the more localized distribution seen in other differentiated cells such as those of cardiac muscle or kidney tubules.
Using confocal laser scanning microscopy of excised bovine lenses stained with the mitochondria specific dye tetramethylrhodamine ethyl ester (TMRE), this study is the first to show the dynamic movement of TMRE in both lens epithelial cells and in the superficial cortex.
General observations of mitochondria go back as far as the middle of the nineteenth century. The observed structures were often called granules and in 1898 a German microbiologist Carl Benda coined the name "mitochondrion" from Greek mitos "thread" and khondrion "little granule". In living cells the mitochondria are dynamic structures continually moving and changing their size and shape. One of the earlier observations of the mitochondrial dynamic reported in the literature goes back to 1915 when Lewis and Lewis using cultured embryonic chick cells and light microscopy reported movement of the mitochondria in living cells . These observations supported suggestions that mitochondria were related to bacteria, foreshadowing widespread acceptance of the endosymbiotic theory of mitochondrial descent from prokaryotic cells that were symbiotically established in the cytoplasm of eukaryotic cells . Early clues to the mechanisms of mitochondrial distribution and movement emerged from studies of the cytoskeleton. Microscopic analysis revealed colocalization of mitochondria with certain cytoskeletal components. In particular, many studies documented colocalization of mitochondria with microtubules in diverse cell types including mammalian neurons , cultured fibroblasts , and the protozoan Acanthamoeba castellanii . Involvement of microtubules was further supported by the observation that mitochondria redistribute in cultured mammalian cells treated with agents that disassemble microtubule networks [21,22]. Furthermore, disruption of microtubules by certain conditional mutations in genes encoding tubulins (the building blocks of microtubules) caused aberrant mitochondrial distribution in S. pombe, providing genetic evidence suggesting that microtubules position mitochondria in this organism . A pivotal advance in identifying the molecular basis of organelle movement on microtubules was the discovery of the microtubule based motor proteins, kinesin and cytoplasmic dynein . These proteins appear to bind microtubules and transduce chemical energy (ATP) into mechanical work to power polarized movement of the mitochondria along microtubules . Both proteins can bind and transport "cargo" in the form of vesicles, organelles, or other proteins, and mitochondria appear to be among the favored cargoes. In particular, several different members of the kinesin superfamily have been localized preferentially to mitochondria in animal cells. A recent study of rat lens showed the existence of a microtubule based motor system containing both kinesin and dynein in the elongating fiber cells . Presence of this system was attributed to the transportation of important membrane proteins and organelles to the target regions during increased cell growth that accompanies elongation of the secondary fiber cells. A large number of microtubules were regularly arranged into bundles parallel to the long axis of fiber cells, a morphological observation similar to that of the distribution of the mitochondria seen in the superficial cortical fiber cells (Figure 1B) and may represent the machinery responsible for the observed rapid organelle movement.
The observed dynamics of TMRE fluorescence movement may represent actual mitochondrial movement, indicating the dynamic state of the mitochondria in both lens epithelium and superficial cortex. That this activity is found not only in the epithelium but also in the superficial cortex indicates that the superficial cortical fiber cells play a much more active role in lens metabolism than previously suspected. Alternatively, the observed movement of TMRE across a mitochondrial network could represent change in the distribution of potential across the inner membrane, presumably allowing energy transmission across the cell from regions of low to regions of high ATP demand.
In summary, this report describes a new observation regarding the dynamic properties of the mitochondria of the lens. Further research will probe the relation between mitochondrial dynamics and the microtubule based motor system. Furthermore, the effects of CCCP on mitochondrial dynamics suggest that this approach may be useful in evaluating lens integrity.
We would like to thank Kelley Moran for her assistance in this project related to eye dissection. This work was supported by the an operating grant to JGS from the Natural Sciences and Engineering Research Council of Canada (NSERC) and a grant from the Canadian Foundation for Innovation to VB.
1. Frey TG, Mannella CA. The internal structure of mitochondria. Trends Biochem Sci 2000; 25:319-24.
2. Wallace DC, Brown MD, Melov S, Graham B, Lott M. Mitochondrial biology, degenerative diseases and aging. Biofactors 1998; 7:187-90.
3. Kinsey VE, Reddy DV. Studdies on the crystalline lens. XI. The relative role of the epithelium and capsule in transport. Invest Ophthalmol 1965; 34:104-16.
4. Goodenough DA, Dick JS 2nd, Lyons JE. Lens metabolic cooperation: a study of mouse lens transport and permeability visualized with freeze-substitution autoradiography and electron microscopy. J Cell Biol 1980; 86:576-89.
5. Bassnett S, Beebe DC. Coincident loss of mitochondria and nuclei during lens fiber cell differentiation. Dev Dyn 1992; 194:85-93.
6. Bantseev VL, Herbert KL, Trevithick JR, Sivak JG. Mitochondria of rat lenses: distribution near and at the sutures. Curr Eye Res 1999; 19:506-16.
7. Bantseev V, Cullen AP, Trevithick JR, Sivak, JG. Optical function and mitohondrial metabolic properties in damage and recovery of bovine lens after in vitro carbonyl cyanide m-chlorophenylhydrazone treatment. Mitochondrion 2003; 3:1-11.
8. Bantseev V, McCanna D, Banh A, Wong WW, Moran KL, Dixon DG, Trevithick JR, Sivak JG. Mechanisms of ocular toxicity using the in vitro bovine lens and sodium dodecyl sulfate as a chemical model. Toxicol Sci 2003; 73:98-107.
9. Baumann O, Murphy DB. Microtubule-associated movement of mitochondria and small particles in Acanthamoeba castellanii. Cell Motil Cytoskeleton 1995; 32:305-17.
10. Steinberg G, Schliwa M. Organelle movements in the wild type and wall-less fz;sg;os-1 mutants of Neurospora crassa are mediated by cytoplasmic microtubules. J Cell Sci 1993; 106:555-64.
11. Margulis L. Archaeal-eubacterial mergers in the origin of Eukarya: phylogenetic classification of life. Proc Natl Acad Sci U S A 1996; 93:1071-6.
12. Yaffe MP. The machinery of mitochondrial inheritance and behavior. Science 1999; 283:1493-7.
13. Sivak JG, Stuart DD, Herbert KL, Van Oostrom JA, Segal L. Optical properties of the cultured bovine ocular lens as an in vitro alternative to the Draize eye toxicity test: preliminary validation for alcohols. Toxicology Methods 1992; 2:280-94.
14. Scaduto RC Jr, Grotyohann LW. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 1999; 76:469-77.
15. McAvoy JW. Cell division, cell elongation and the co-ordination of crystallin gene expression during lens morphogenesis in the rat. J Embryol Exp Morphol 1978; 45:271-81.
16. Bereiter-Hahn J. Behavior of mitochondria in the living cell. Int Rev Cytol 1990; 122:1-63.
17. Porter KR, Bonneville MA. Fine structure of cells and tissues, With the collaboration of Susan A. Badenhausen in transmission electron microscopy and Peter Andrews in scanning electron microscopy. 4th ed. Philadelphia, Lea & Febiger; 1973.
18. Bantseev V, Moran KL, Dixon DG, Trevithick JR, and Sivak, JG. Optical properties, mitochondria and sutures of lenses of fishes: a comparative study of nine species. Can J Zool 2004; 82:86-93.
19. Lewis MR, Lewis WH. Mitochondria (and other cytoplasmic strutures) in tissue culture. Am J Anat 1915; 17:339-401.
20. Yaffe MP, Harata D, Verde F, Eddison M, Toda T, Nurse P. Microtubules mediate mitochondrial distribution in fission yeast. Proc Natl Acad Sci U S A 1996; 93:11664-8.
21. Heggeness MH, Simon M, Singer SJ. Association of mitochondria with microtubules in cultured cells. Proc Natl Acad Sci U S A 1978; 75:3863-6.
22. Ball EH, Singer SJ. Mitochondria are associated with microtubules and not with intermediate filaments in cultured fibroblasts. Proc Natl Acad Sci U S A 1982; 79:123-6.
23. Vale RD. Intracellular transport using microtubule-based motors. Annu Rev Cell Biol 1987; 3:347-78.
24. Vale RD, Fletterick RJ. The design plan of kinesin motors. Annu Rev Cell Dev Biol 1997; 13:745-77.
25. Lo WK, Wen XJ, Zhou CJ. Microtubule configuration and membranous vesicle transport in elongating fiber cells of the rat lens. Exp Eye Res 2003; 77:615-26.