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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 8, 2000 as doi:10.1096/fj.00-0028fje. |
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Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA
2Correspondence: Department of Cell and Molecular Biology, Northwestern University Medical School, 305 E. Chicago Ave., Chicago, Illinois 60611, USA. E-mail: g-buehler{at}nwu.edu
SPECIFIC AIMS
My finding that mammalian cells may use their centrioles to detect and locate microscopic sources that emit 800 nm near-infrared light pulses pointed to mitochondria as the best candidates as the natural emitters of such light pulses. The present study tested the hypothesis that the autofluorescence of mitochondria expresses some of the fundamental properties of other known generators of pulse trains, including the existence of two levels of light emission, a threshold level of excitation that causes them to leap from a low level of emission to a high level and a refractory period during which they return to the ground state.
PRINCIPAL FINDINGS
1. The excitation-induced enhancement of fluorescence
Using a self-designed microspectrograph (Fig. 1
), the crucial finding of this
article is the observation that one or several peaks of the
autofluorescence spectrum of the mitochondria of live mammalian cells
flared up threefold or more if the cells were irradiated for 2–3 min
with ultraviolet light (
=365 nm) (Fig. 2
) Subsequently the peaks remained stable at this new level of intensity
for 30 min or longer. Within the limits of resolution of ±10 nm of the
microspectrograph, the location of the emission peaks did not change.
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There are numerous wavelengths capable of stimulating the autofluorescence of mitochondria. These wavelengths appear to be related to the absorption spectra of flavins and NADH. This article and earlier results of Brock et al. emphasize the wavelengths of 365 nm and 532 nm because they appear to excite an additional mechanism that enhances the mitochondrial fluorescence with increasing time.
2. Reversibility of level transition
The effect was reversible: 1) the induced increase
of fluorescence could be reversed by turning off the excitation light
for a certain period of time, and 2) the effect could be
restimulated using the same cells. Therefore, the effect was not due to
UV damage of the cells. As shown in detail in the full article, it
appears that the enhancement of autofluorescence is due to an increase
of the quantum efficiency of the fluorescence mechanism. Based on these
results, one may characterize the effect as ‘reversible, excitation
light-induced enhancement of fluorescence’. To simplify the text, we
shall use the acronym ‘RELIEF’.
3. Other systems expressing RELIEF
In addition to 3T3 and CV1 cells, we found that cells from other
placental mammals such as HeLa cells and BHK cells expressed RELIEF, as
well. Not only cultured cells, but also cells isolated directly from
tissue expressed RELIEF. For example, rat liver homogenates expressed
RELIEF very strongly.
When we tested cells from organisms other than placental mammals, we did not find expression of RELIEF. The tested cell types included avian cells (chick embryo fibroblasts SL-29), amphibian cells (homogenates of Xenopus laevis liver), insect cells (Drosophila melanogaster cell line Kc167), nematode cells (intact worms and homogenates of Caenorhabditis elegans), yeast cells (Saccharomyces cerevisiae), and slime mold cells (Dictyostelium discoideum).
4. Evidence for mitochondrial origin of RELIEF
RELIEF was found to be insensitive to drugs that inhibit protein
synthesis or cytoskeletal elements. In contrast, it was inhibited by
drugs that inhibited mitochondrial function. The addition of 15 mM KCN
(30 min preincubation), 40 mM Na-azide (30 min preincubation), or 2.7
mM 2,4-dinitro-phenol (10 min preincubation) caused the fluorescence
spectra to fade rather than to increase their intensity. The same
result was found for rhodamine 123, a highly specific vital fluorescent
dye for mitochondria that causes stained mitochondria to burst under
the prolonged UV irradiation conditions of the RELIEF assay. The above
drugs not only inhibited transition to the active level of fluorescence
that is characteristic for RELIEF, they altered significantly the
ground level of fluorescence of the cells, too.
Since the above 15–30 min exposures of cells to the mitochondrial inhibitors were not sufficient to deprive the cells of ATP, the results suggest that RELIEF may be an expression of intact mitochondria and not merely an energy-dependent phenomenon.
Consistent with this interpretation that RELIEF originated in the mitochondria, cells or cell fractions without mitochondria such as red blood cells (the author’s own) or isolated nuclei of CV1 cells did not express RELIEF. On the other hand, destructive cell treatments that left mitochondria, or at least their inner membrane, intact continued to support RELIEF. For example, cell homogenates generated by intense douncing of rat liver expressed RELIEF very intensely. Likewise, incubation of CV1 cells with 1% of the non-ionic detergent NP40 did not inhibit RELIEF, although the time required to reach the active level was increased by fourfold for up to 24 min.
Preliminary studies showed that preparations of mitochondria from mouse liver did not express RELIEF. Instead, the fluorescence faded. However, after suspending the mitochondria in normal culture medium to which 10 mM Na-pyruvate was added, the fluorescence recovered within 4 min and, subsequently, expressed RELIEF.
CONCLUSIONS
RELIEF is an intriguing phenomenon for various reasons. 1) The high photochemical activity of the ultraviolet light should either cause the fluorescence intensity to fade as it destroys the fluorochromes or alter the fluorescence spectra as it creates new compounds such as eximers. In contrast, RELIEF appears to increase the intensity of certain emission peaks without changing their wavelengths. 2) One may suspect that RELIEF is accompanied by or even the result of irreversible cell damage caused by the ultraviolet irradiation. In contrast, it is reversible with a refractory period of several minutes. 3) If the mitochondria give rise to RELIEF, one may expect that all eukaryotic cells express it. In contrast, testing a variety of mammalian and nonmammalian cells suggested that only placental mammalian cells were capable of expressing RELIEF under the described experimental conditions.
The 365 nm photons that triggered the unknown function underlying RELIEF are hardly its natural trigger. It seems more likely that they reflect the height of an energy barrier that was overcame ‘by force’ in the above experiments, whereas the cells under normal conditions can be expected to bypass it by a specific enzymatic reaction whenever they trigger the unknown function.
To aid future search for the unknown function underlying RELIEF, one may try to design its profile as follows. It appears to 1) mediate the transition between a resting and an active state (bistability), 2) be protected by a threshold, and 3) recover from the transition with a certain refractory period (refractory behavior).
Nerve excitation and other pulsating phenomena shares the above three
properties with the unknown function, albeit on a very different time
scale. Therefore, the results may point to the existence of an
excitation mechanism for (mammalian?) mitochondria, which in turn may
be involved in the generation of pulsating (light?) signals of these
mitochondria and, thus, of the corresponding cell types. This
hypothesis is illustrated in Fig. 3
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0028fje 
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