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Opaque Polyploid Cells in Ishikawa Endometrial Cultures Are Capable of Forming Megamitochondria, Organelles Derived from the Adaptation of Fused Mitochondria Whose Capacity to Develop Gaseous Vacuoles Suggests CO2 Retention and Hypoxic Metabolism

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  • Castleton State College (retired)

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Advances in Bioscience and Biotechnology, 2021, 12, 229-255
https://www.scirp.org/journal/abb
ISSN Online: 2156-8502
ISSN Print: 2156-8456
DOI:
10.4236/abb.2021.127015 Jul. 22, 2021 229
Advances in Bioscience and Biotechnology
Opaque Polyploid Cells in Ishikawa
Endometrial Cultures Are Capable of Forming
Megamitochondria, Organelles Derived from
the Adaptation of Fused Mitochondria Whose
Capacity to Develop Gaseous Vacuoles Suggests
CO2 Retention and Hypoxic Metabolism
Honoree Fleming
Castleton State College, Castleton, VT, USA
Abstract
Opaque polyploid cells capable of forming megamitochondria are a constant
feature in colonies of Ishikawa endometrial epithelia, accounting for approx-
imately 5% -
10% of the cells. Opaque cells appear to communicate with other
opaque cells via membrane extensions and with other cells in a colony by extra-
cellular vesicles. Opaque cells form first as rectangular structures, somewhat
larger than surrounding monolayer cells. The cells eventually round up, re-
maining in the colony for 20 or more hours before detaching. The most un-
usual characteristic of Ishikawa opaque cells is their capacity to form mito-
nucleons, megamitochondria that surround aggregated chromatin. This
paper
reviews evidence that adaptations resulting in megamitochondria i
nclude a
loss of the capacity for oxidative phosphorylation leaving the adapted megami-
tochondria reliant on metabolism such as reductive carboxylation.
Keywords
Mitonucleons, Megamitochondria, Opaque Polyploid Cells, Membrane
Extensions, Extracellular Vesicles, Reductive Carboxylation, Endogenous
Biotin, Hypoxia
1. Introduction
Despite an assumption that cultured monolayer cells are mostly identical, and
How to cite this paper:
Fleming, H. (2021
)
Opaque Polyploid Cells in Ishikawa End
o-
metrial Cultures Are Capable of Forming
M
egamitochondria, Organelles Derived
from
t
he Adaptation of Fused Mitochondria
Whose
C
apacity to Develop Gaseous Vacuoles Sug-
gests CO
2 Retention and Hypoxic Metabol-
ism
.
Advances in Bioscience and Biotec
h-
nology
,
12
, 229-255.
https://doi.org/10.4236/abb.2021.127015
Received:
May 22, 2021
Accepted:
July 19, 2021
Published:
July 22, 2021
Copyright © 20
21 by author(s) and
Scientific
Research Publishing Inc.
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Open Access
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Advances in Bioscience and Biotechnology
that cells detach from a monolayer only when dying, discoveries have been made
by more closely examining the minority, exceptional cells routinely found in and
above monolayer cultures. Dr. Jinsong Liu’s laboratory [1] has been studying a
cell type called polyploid giant cells (PGCC) found in ovarian as well as in other
cancer cell lines [2] [3]. Giant cells can proliferate by amitotic means such as
asymmetric division. Polyploid cells described as opaque and found in the Ishi-
kawa endometrial cell line [4] are also capable of budding off new “normal”
cells.
Dr. Liu and colleagues characterized PGCC’s whose formation was stimulated
by CoCl2, demonstrating that the giant cells express cancer stem cell markers
together with markers characteristic of normal cells. Furthermore, the resear-
chers showed that in long term cultures, with appropriate additions to the me-
dia, PGCCs can differentiate into adipose, cartilage and bone [1], a potential for
differentiation broadened by results in a subsequent paper [5]. Spheroids derived
from single PGCCs can grow into a wide spectrum of human neoplasms, in-
cluding germ cell tumors, high-grade and low-grade carcinomas and benign tis-
sues leading these researchers to suggest that PGCC’s are cancer stem cells and
the somatic equivalents of blastomeres.
In 2011, Chaffer and colleagues [6], focusing attention on cells floating above
the monolayer as opposed to clinging to the petri dish, presented compelling
data demonstrating that this population is enriched for stem cells. They went on
to demonstrate that differentiated mammary epithelial cells can convert to a stem-
like state in a stochastic manner in culture.
This paper also reports on a small cell population with apparently outsize im-
portance observed in the Ishikawa endometrial epithelial cell line originally iso-
lated in Dr. M. Nishida’s laboratory in 1985 [7]. The cell line is capable of form-
ing hemispheres of detached cells arching over reservoirs of fluid in confluent
monolayers. Once the routine induction of that process had been harnessed [8],
it became possible to study the 20-to-24-hour differentiation for structural changes
including the role played by increasing levels of biotin as syncytia of Ishikawa
cells formed hemispheres [9] [10] [11]. Elevated levels of endogenous biotin
usually observed as a “failed” control have engendered interest in the signific-
ance of this carboxylase co-factor and/or warnings that its presence can cause
confusion in assays using biotinylated antibodies to detect specific proteins [12].
Endogenous biotin became an important factor in studies of Ishikawa differen-
tiation as the explanation for large spheroidal megamitochondria in syncytia
staining bright red following incubation with avidin peroxidase and 3-amino-
9-ethylcarbazole (AEC) as a substrate. In Ishikawa cells, mitochondria are ob-
served to fuse around aggregated chromatin forming the mitonucleon, a giant
transient organelle that stains darkly for biotin. Subsequently, endogenous biotin
was also detected in opaque cells that can be observed detaching from a colony
and forming hollow spheroids [13]. As was demonstrated early on in our studies
[14], the endogenous biotin is bound to mitochondrial carboxylases whose in-
creasing concentrations accompany mitochondrial biogenesis during dome dif-
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ferentiation.
As will be discussed, membrane extensions carrying extracellular vesicles ap-
pear to be part of the process resulting in the formation of giant opaque cells.
Ishikawa cells like many other cell lines are able to form membrane extensions
under a variety of circumstances including in cells adjusting to a change in me-
dium: monolayers transferred into serum-free medium or clustered spheroids
formed in serum-free medium transferred into serum-containing medium [15].
The monolayer cultures studied in this paper were not similarly stressed but ra-
ther were in logarithmic growth. The observation that the extracellular vesicles
extend up into the colony to the region where a second giant cell forms is intri-
guing circumstantial evidence that giant cells may induce formation of addition-
al giant cells by the materials that are exported. Eventually giant opaque cells
detach [13] and can migrate to other regions of the petri dish and start new co-
lonies. This may be one part of the process involved in the proliferation occur-
ring when cell aliquots achieve confluence, a routine event in successful cell cul-
ture.
To investigate the origins of opaque cells in typical Ishikawa cultures, we be-
gan following cultures hours at-a-time hoping to catch the process of formation
of an opaque cell from a typical monolayer cell. Finding a culture in which an
opaque cell was forming, photomicrographs were taken at intervals throughout
the process over a period of 20 hours (Figures 3-7). As the results demonstrate,
structural changes could be detected through most of that period. The opaque
rounded cell initially present at the start of the observation undergoes the
process of detaching from the colony during the observation. Giant opaque cells
in Ishikawa cultures are exceptional, particularly in their capacity to develop one
or more mitonucleons. Mitonucleons will be discussed in light of that exceptio-
nality and characteristics shared with other megamitochondria.
2. Results
Ishikawa endometrial monolayers are mostly made up of typical epithelial cells,
usually dominated by one or two visible nuclei and fitted together with neigh-
boring cells like tiles in a mosaic. But opaque and clearly polyploid cells are al-
ways present in cultures, sometimes in groups of 2 or 3. Opaque cells make up
approximately 5% - 10% of the monolayer population and are distinguished by
size, shape (rounded up as opposed to flat), the presence of substantial cell
membranes, and often, but not always, trailing processes such as can be seen in
Figure 1. Mitonucleons form at the center of such cells, staining brightly for
endogenous biotin as seen in the light micrographs in Figure 1 and Figure 2.
These giant cells can detach and form hollow spheroids [13] capable of migrat-
ing and reattaching elsewhere on the petri dish if fresh serum is added, or of re-
maining as hollow spheroids and becoming multicellular [16].
Figure 2 shows that enlarged cells containing mitonucleons are capable of
communicating quite specifically with each other by an extension passing through
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Figure 1. Giant polyploid cell containing mitonucleons can detach from a confluent mo-
nolayer. Prior to detachment such cells are characterised by extensions made up of extra-
cellular processes that stretch back into the colony. There appear to be three mitonucle-
ons at the center of the cell featured in this photomicrograph and multiple extracellular
vesicles trailing from two processes. Bar = 50 microns.
Figure 2. Two cells containing multiple mitonucleons appear to be exchanging material
by a cellular process extending between them. Bar = 50 micron.
the colony. The enlarged structures communicating appear to contain multiple
mitonucleons.
Figures 3-7 are photomicrographs of a typical colony of Ishikawa cells proli-
ferating as a monolayer in a culture dish. The colony was followed over a period
of 20 hours to characterize the behavior of opaque cells as compared to sur-
rounding monolayer cells. Cells have been numbered in the photomicrographs
to facilitate comparison of the colony over time. Opaque cell #3 dominates the
colony by virtue of size and shape, “glowing” with evidence of cell membranes
more substantial than the barely detectable membranes of neighboring mono-
layer cells. Two exosome-decorated processes extend from this opaque cell, the
longer process reaching deep into the colony as it passes through a cytoplasmic
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ruffle. A smudge of material seems to define the furthest reach of the process al-
though it might be expected that materials from the vesicles diffuse beyond that
point. Changes will be detected at positions 7 and 10 over the next 18 hours.
If a typical monolayer cell was the starting point for differentiation of the
second giant cell, the cell could not be detected at the beginning of our observa-
tion (Figure 3). Perhaps the process of converting a monolayer cell into an en-
larged opaque cell begins with sufficient deconstruction to render the contours
of the originating monolayer cell undetectable at some point. A discernable
structure does however emerge quickly in the region labeled “10” after only 2
hours. It is dominated by the halo of a substantial membrane forming and by
something akin to striations in the center of the differentiating structure. De
novo formation such as this, together with asymmetric division [5], may explain
how, despite their propensity to wander, enlarged opaque single cells capable of
forming mitonucleons persist as a relatively constant population in cultured
Ishikawa epithelial cells.
In Figure 3 at the start of the observation, a cell undergoing mitosis is evident
in the top left part of the colony. That there are not more mitotic figures ob-
served throughout the 20-hour observation in a culture in logarithmic growth is
worth mentioning, although after decades of examining cell cultures, I will admit
to generally being underwhelmed by the representation of mitotic figures even
Figure 3. Single opaque cell on the lower edge of the colony. Extensions made up of ve-
sicles stream from either end of giant cell #3. The longer of the two, carrying more than
10 vesicles, passes close by cells #4 and #5 through a cytoplasmic ruffle, ending close to
cell #8 to the left of a gap in the culture and cell #9 to the right of that gap. A second
much shorter extension emerges from the right side of the opaque cell ending in the cy-
toplasmic ruffle of cell #1. Cell #2 is approximately 350 microns up and to the left of cell
#3. Region 10 defined by cells #6, #8, and #9 is where the new giant cell will develop. Over
time a monolayer cell will become more clearly defined at position 7. Bar = 50 microns.
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during log growth. Some of the reason for this may lie in the evidence for other
modes of proliferation described in the past 20 or so years. Papers have been
published describing proliferation of trophoblasts and human cancer cells by
processes other than mitosis. Some of the names given to these processes in-
clude: budding [17] [18], bursting [1], depolyploidization [19] [20] and neosis
[21]. Examples of what is sometimes called asymmetric division have also been
observed in Ishikawa cells and indeed there appears to be an example of a cell
budding out of giant cell #3 (Figure 5) not long before the opaque cell detaches.
Most of what is known about amitotic proliferation is derived from experi-
ments that disrupt the mitotic cycle. Just as some cancers come back after che-
motherapy, treated cancer cells do not necessarily all succumb to chemothera-
peutic agents in vitro. Using paclitaxel, an effective chemotherapeutic agent, Liu
and his colleagues poisoned ovarian cancer cells [5]. Most of the cells died, but a
few surviving cells became polyploid giant cells by endoduplication of DNA.
These PGCCs were capable of amitotic production of cells by “nuclear budding,
nuclear fragmentation or nuclear fission followed by cytofission” [5]. Taking the
experiment one step further, the researchers karyotyped the surviving cells and
found multiple chromosomal rearrangements including deletions and transloca-
tions as well as lower chromosome numbers than parental cells. The authors
present evidence that daughter cells acquire new karyotypes with numerous ge-
nomic alterations following a single giant cell cycle, and that the karyotypes are
not static. They change over time suggesting that an ongoing “editing” process is
addressing at least some of the variability arising from amitotic proliferation [5].
On the basis of this study, Dr. Liu and his colleagues have proposed a giant cell
cycle that includes the amitotic reproductive behavior of polyploid giant cells
and the observation that, as a result of the editing process and asymmetric divi-
sion, some progeny cells can spiral back into the typical mitotic cycle [22] [23].
After two hours, in addition to the completion of the mitosis observed in Fig-
ure 3, the photomicrograph in Figure 4 shows the outline of a large structure
forming at position 10. A couple of changes worth noticing include the demar-
cation between the developing structure and surrounding monolayer cells that
have been numbered 6, 8, and 9, and a structure like a monolayer cell emerging
at position 7. Something that might be similar to crystalline structures observed
in developing megamitochondria [25] can be observed in the interior of devel-
oping cell #10.
The extracellular vesicles are somewhat smeared (Figure 4), suggesting that
packaged materials are seeping out into the colony. Interest in extracellular ve-
sicles has taken off in the past decade, perhaps because of evidence that they
might be involved in cancer [26]-[34]. From just a sampling of the literature, the
dozen or so vesicular packets decorating the extension stretching from cell #3
into the middle of the colony may contain proteins, RNAs, DNAs, lipids or all of
the above. Work from one laboratory suggests that extracellular vesicles are in-
volved in the transfer of cancer pathogenic components, particularly micro
RNAs [35]. It is obviously an intriguing possibility, although entirely speculative,
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Figure 4. Two hours after the start of the observation, extracellular vesicles can still be
detected on the processes extending from cell #3. Some of the vesicles appear to be
spreading. The most dramatic change is taking place in the region designated #10. The
region is approximately twice as large as surrounding cells, and mostly defined after 2
hours by a border not unlike the outline around opaque cell #3, a characteristic of the
phase contrast photomicrograph. Bar = 50 microns.
that the horizontal transfer of material(s) from the vesicles exported by cell #3
might be linked to the differentiation of the second opaque spheroidal cell at po-
sition 10. As discussed in the Introduction to this paper, the very significant
quality of “stemness” seems to be communicated from one cell to another [6],
and the same may be true with regard to the capacity to form polyploid opaque
cells and mitonucleons.
Enlarged opaque cells usually undergo detachment and form floating sphero-
ids [13]. They can then reattach at some distance from the original colony, ana-
logous to the EMT process involved in cancer metastases. As a result of his expe-
riments with extracellular vesicles, [36] Gopal and colleagues believe that cells
undergoing the EMT process, as is the case for cell #3 (Figures 3-7), are repro-
grammed with regard to protein and RNA content of extracellular vesicles. The
obvious question would be whether that material can result in the induction of a
second cell (
i.e.
cell “10”) that becomes capable of forming mitonucleons and
detaching.
After an additional four hours (Figure 5), the smaller extension observed in
Figure 3 and Figure 4 has been lost, a bud has appeared in the center of the
giant cell #3, the cytoplasmic ruffle is shrinking, and cell #10 continues to fill
out.
The photomicrograph in Figure 6 shows the colony after 12 hours. Cell #10
has rounded up. It is no longer possible to observe discrete vesicles between cells
#3 and #10 and the cytoplasmic ruffle through which the extension passed has
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Figure 5. Eight hours after the start of the observations, the extracellular vesicles extend-
ing out from cell #3 toward cell #1 have mostly disappeared along with the shorter exten-
sion. The vesicles on the longer extension have continued to spread out or diffuse. The
rectangular region above cell #8 has become further defined. Concurrent with these changes
the gap to the right of the rectangular structure has disappeared. Bar = 50 microns.
Figure 6. Twelve hours after the start of the observation. Giant cell #10 is fully formed.
The cytoplasmic ruffle extending to the right from cell # 3 has disappeared. The nature of
the conduit from cell #3 to cell #7 now looks more like a “pipeline” of sorts than the
extracellular vesicle-bearing extension in Figures 3-5. Bar = 50 microns.
further contracted. Something like a stream of materials appears to be exiting
cell #3.
In the final observation at t = 20 hours (Figure 7), it is obvious that opaque
rounded structure #3 has lifted off the plate with a single visible extension
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Figure 7. Twenty hours after the start of the observations. Cell #3 has released from the
dish although it continues to be tethered to the colony by at least one extension. Evidence
for detachment includes the foreshortened appearance of the single visible extension. Cell
#3 appears to be floating above cells #4 and 5 that were clearly visible up until 20 hours, as
can be seen in Figure 6. Finally the apparent distance between cells 1 and 3 has “leng-
thened” and the apparent distance between cells 3 and 10 has “shortened.” Bar = 50 mi-
crons.
reaching back into the colony. With the focus of the microscope on the mono-
layer, it is possible to detect that opaque cell #3 is hovering above cells #4 and #5.
The extension still attaching it to the monolayer appears foreshortened and the
relationships between cell 1 and cell 3 and between cell 3 and cell 10 have changed
in ways that can only be explained by upward movement of cell #3. As already
discussed in a previous paper [13], this “behavior” is consonant with the devel-
opment of a central gaseous vacuole within mitonucleons that develop in giant
cells.
What started out as observations about rounded opaque cells in Ishikawa cul-
tures ended up providing information about vesicle-decorated membrane exten-
sions. Extracellular vesicles are on the edge of what is readily detectable by light
microscopy. While analyses of the content of extracellular vesicles have become
numerous in the past several years, extended observations of the fate of extra-
cellular vesicles in cell cultures such as this are far less common. A short and
short-lived extension, 50 microns long with three or four extracellular vesicles,
ends in a large expanse of cytoplasm on the edge of the colony surrounding cell
#1. The second process is 4 to 5 times longer, passing through a ruffle of cytop-
lasm on the edge of the colony, “carrying” approximately a dozen extracellular
vesicles. By 8 hours the smaller extension has faded completely, seemingly ab-
sorbed by the cytoplasm surrounding cell #1, without having any obvious effect.
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The second extension is also fading. The cytosol ruffle it is passing through has
contracted substantially, leaving what looks more like a “pipeline” similar to the
structure connecting two giant cells with mitonucleons in Figure 2. The optics
have changed from discrete vesicular packets into something that looks like a
flow of material out of giant cell # 3 to the region of the colony near cells #7 and
# 10. By 20 hours, it is clear that cell #3 has detached and a new opaque cell has
formed at position 10. As was described [13], the potential of a detached cell is to
form a hollow spheroid that can float to a new location to reattach. If reattach-
ment does not occur, chromatin streaming from giant nuclei in the cytoplasmic
rim of the hollow spheroid results in a multicellular spheroid [16]. The clearest
evidence of detachment can be observed by a comparison of Figure 6 and Fig-
ure 7. The apparent distance from cell #3 to cell #10 has decreased from 260 mi-
crons to 192 microns, while the apparent distance from cell #1 to cell #3 has in-
creased from 138 microns to 195 microns.
3. Discussion
3.1. Mitonucleons
Mitonucleons were first observed in Ishikawa cells at regular intervals through-
out an epithelial monolayer within hours of imposing conditions that stimulate
the formation of domes (fluid-filled hemispheres) [8]. Hybrid and transient,
mitonucleons are formed by the fusion of multiple mitochondria around aggre-
gated nuclei in syncytia [9]. Three or four mitonucleons form within a syncy-
tium, fill with gas, and elevate separately so that, for a short period of time, they
create multiple protuberances in an “expanding” syncytial apical membrane that
most likely includes endoplasmic reticulum membranes associated with the
giant mitochondria, an association observed in tissues such as endometrium
[37], in the adrenal cortex [38] and in human hepatocytes [25].
Small gas bubbles forms within the chromatin of aggregated nuclei inside the
mitonucleon. At least one of the gases in that bubble could be nitric oxide, a ga-
seous neurotransmitter, that has been shown to form a lethal bubble in nuclei of
cells that are extremely stressed at low temperatures [39]. Within the mito-
nucleon, the bubble does not cause cell death but creates an image that has been
called an “optically clear” nucleus, a structure observed in cancerous tissue or in
endometrium during pregnancy [9] [40]. Gaseous neurotransmitters can acti-
vate enzymes that might be responsible for later fragmentation of DNA. The
chromatin aggregate is gradually compressed in a separate compartment up against
the unfurling apical membrane of the syncytium as a larger vacuole forms within
structures created by fused mitochondria [9]. Each mitonucleon vacuole, to-
gether with the bolus of chromatin elevating against the apical membrane, forms
a structure that looks like a “signet ring,” another structure frequently associated
with sectioned biopsied cancer tissue. Our results show that these cells represent
two different stages brought about by the activity of mitonucleons.
Some combination of pressure and perhaps of activated enzymes and/or a
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change in pH explosively fragments the chromatin and the DNA filaments slip
out of membrane protuberances back into the common structure forming an
array throughout the syncytium as the breached mitonucleons fade [10]. Such an
array opens fragmented DNA to the kinds of epigenetic alterations believed to be
involved in differentiation [41]. For a period of time the fundamental distinction
between cytosol and nucleus is lost. Chromatin fragments reassociate relatively
rapidly forming an irregular mass out of which the nuclei of dome cells emerge,
arranging as a layer in what is now the envelope of apical and basal syncytial
membranes elevating over accumulating fluid.
Such a process of nuclear proliferation is even more controversial than the
amitotic processes already discussed in the Results section. In dome formation,
nuclei are “produced” out of a mass of reassociated chromatin [11]. Similarly
deconstructed chromatin can stream through hollow spheroids [17] and mem-
brane extensions [15]. Streams of chromatin fragments passing through the
envelope of a hollow spheroid look like the seams of a basketball. It is difficult to
predict how such streams might look in fixed sectioned tissue. Certainly gas
filled spheroids would be collapsed. Since the earliest observations of “exploded”
chromatin we have speculated that proteins and DNA associating and dispersing
may have something in common with “nuclear envelope limited chromatin”
mysterious structures observed in all classes of tissue in mammals [42] [43] for
almost 50 years.
Chromatin streaming has all of the problems associated with the amitotic
processes of budding and bursting as described for giant cells together with the
“complexity” introduced by the necessity of stitching fragmented chromosomes
back together again, once the material associates into nuclei. What exactly guar-
antees that progeny cells contain complements of genetic material and chromo-
somal structure identical to the parental genome? The answer may be that they
do not, but that there is an “editing” process that can correct enough of the
problems that arise so that cells can function. Research has been accumulating
for some time demonstrating that cells from tumors and cancer cell lines fre-
quently contain more or fewer chromosomes than a typical non-cancer cell, a
condition that has been called aneuploidy [44]. The additional problem when
chromosomes are fragmented, as has been shown in cell lines treated with che-
motherapeutic agents, is that karyotypes of surviving cells display what Heng
and associates have called “genome chaos” [45]. Not predictably for cell biolo-
gists schooled in the belief that mitotically inherited chromosome complements
are an absolute “must” for progeny success, these researchers show that some
cells survive even with a chaotic genome. When the genomes are karyotyped
every day for more than a week, as was done in this paper by Liu and colleagues
[45], significant changes are observed. The peak of fragmentation is measured
on day 1. Genome chaos peaks between days 2 and 3. “Normal” genomes repre-
sent 50% of what is measured on day 5, peaking on day 8. Approximately 0.1%
of the cells in a typical experiment survive and, as described by the authors,
some stable clonal populations will be produced from dynamic reorganized ge-
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nomes after a few more weeks. All of this suggests that a persistent editing
process exists that is capable of bringing enough order to a chaotic genome so
that it once again becomes capable of mitosis, but probably is still not identical
to the genome of the progenitor cell (evolutionary biologists may find all of this
rather exciting, cell biologists less so). Ongoing and effective genome editing and
the ultimate survival of a small population of cells makes amitosis by chromatin
streaming more acceptable in certain circumstances and there are advantages. It
is a process that can produce dozens of “new” cells in a very short period of time
so that chromatin streaming might represent an energy and time-efficient mode
for proliferation of multiple terminally differentiated cells [15].
In a second example of mitonucleon-dependent differentiation, one or more
mitonucleons form at the center of a single multinucleated monolayer epithelial
cell. The cell containing mitonucleons detaches from neighboring cells in the
colony and then from the substrate, floating up into the medium above the mo-
nolayer where it forms a hollow spheroid [13]. The giant gas vacuole forms
within the inner mitonucleon membrane, pushing cell contents including po-
lyploid nuclei into a rim of cytoplasm between the outer mitochondrial mem-
brane and the cell membrane [13]. The hollow spheroid is mobile and can reat-
tach elsewhere on the petri dish, releasing gas bubbles and becoming a locus of
monolayer growth, a process that evokes the cyclical nature of the epithelial to
mesenchymal transition (EMT) involved in cancer metastases. In the dish, it
may be one of the mechanisms whereby cells proliferate to form a confluent
monolayer. If the spheroid does not reattach, a function of adding serum to the
culture medium, the polyploid unicellular spheroid can become multicellular
[16] and it does so by chromatin streaming through the cytoplasmic shell. In its
entirety, this process results in cell “mobility,” a capacity with important func-
tions such as wound healing but one that can be catastrophic as in highly metas-
tatic cancers.
This paper is designed to shed light on the earliest stages of differentiation of
opaque cells capable of forming mitonucleons and to advance a theory on the
nature of metabolism in mitonucleons that can form gas vacuoles of various siz-
es [9]; some that quickly fall apart as in dome formation and some that have the
capacity for an independent existence as is true for the beehive of multiple
spheroids recently described [16].
3.2. Megamitochondria
Mitonucleons appear to be a subset of organelles called megamitochondria by
Tadeo Wakabayashi in an extensive review [24]. He summarized work from his
and other laboratories, referencing more than 50 years of experimental literature
on super-sized mitochondria and cataloging more than 100 observations of me-
gamitochondria in diseased or normal tissue, experimentally induced by exercise
and hormones or brought on by drug and even toxin administration. The sheer
volume of observations, including examples of megamitochondria in healthy as
opposed to dying cells, indicates that megamitochondria are not simply dam-
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aged organelles, as has sometimes been assumed. Electron micrographs such as
those published by Wakabayashi [24] demonstrate that the internal structure of
megamitochondria is substantially different from the “typical” mitochondria
engaged in oxidative phosphorylation in aerobic organisms. While the functions
of megamitochondria appear to be diverse, Wakabayashi stresses a role for these
adapted organelles in reestablishing homeostasis when there is a break-down in
the mitochondrial electron transport system, suggesting that the organelles are
adapting to neutralize reactive oxygen species and/or free electrons resulting
from the break-down. Such a mechanism may go a long way toward explaining
the presence of megamitochondria in so many different kinds of diseased tissues:
they may develop as a result of the diseased state as opposed to being a cause of
it.
Knowledge of giant mitochondria stretches at least back to 1883, as referenced
by Charles Bowen [46] in a paper on what were then called nebenkern (giant
spherical mitochondria). Furthermore, early researchers following nebenkern
formation in living spermatids recognized that the megamitochondria were
transient organelles involved in tail elongation. The giant mitochondria in
spermatids, together with microtubules, organize the cytoskeletal dynamics re-
sponsible for the elongation of spermatid tails from 7 microns to almost 2 cen-
timeters [47] [48]. It is probable that endoplasmic reticulum is involved in this
process given that the volume of mitochondria is preserved while its surface area
is enlarged.
Electron microscopy provides a window into megamitochondrial structure,
although, because of their size, it is often essential to employ serial sectioning
and model building to obtain the complete picture [49]. Of particular interest for
researchers of endometrial epithelial cells, megamitochondria make an appear-
ance in the epithelial lining of the uterus as a part of the reproductive cycle. Fol-
lowing up on a report by Gompel [50] describing giant mitochondria, Armstrong
and colleagues [37] investigated giant mitochondria in uterine tissue as a func-
tion of that cycle, employing serially sectioned tissue. By this method, megami-
tochondria were found in 12 of 14 endometrial specimens collected around the
time of ovulation. The report demonstrated that the giant mitochondria were
invested with endoplasmic reticulum studded with ribosomes. Armstrong rec-
orded that the appearance of giant mitochondria occurs in the middle of the
cycle, declining rapidly after day 18. On the basis of mitonucleon involvement in
gland-like differentiation in vitro [9] [10] [11], it seems reasonable to suggest
that the megamitochondria observed in tissue are also involved in gland forma-
tion for the secretory phase.
Addressing both the size and the 3-dimensional complexity of giant mito-
chondria, space-filling models were derived from three-dimensional transmis-
sion electron microscopy (TEM) [51]. An informative sequence of changes in
the formation of megamitochondria was established by Sun and colleagues [52],
who treated HeLa cells with etoposide, a disruptor of the electron transport sys-
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tem, and recorded changes over a period of 16 hours. They established the lea-
kage of cytochrome C from mitochondria into cytoplasm as an early measurable
change in cells treated with this toxin, a phenomenon also observed as an early
signal of impending apoptosis. As already described, structures such as optically
clear nuclei and signet ring cells, as well as chromatin fragmentation, accompany
mitonucleon formation, as they are reported to accompany apoptosis, but that
they result not in cell death but in cell differentiation [9] [10] [11]. In Suns se-
quence, the leakage of cytochrome C out of mitochondria into the cytoplasm is
followed by the uncoupling of oxidative phosphorylation as measured by the loss
of mitochondrial membrane potential, essential for oxidative phosphorylation.
Fifteen hours after administration of the toxin, researchers observed changes in
the internal structures of mitochondria, specifically the transformation of inter-
nal membranes from the “typical” christae structure into separate vesicular ma-
trix compartments that can fill with gases and merge. A “snapshot” of the cells
after the administration of etoposide revealed four categories of giant mito-
chondria, different from normal and presumably forming sequentially over a pe-
riod of 16 hours: in one of those variations, normal christae membranes coexist
with vesicular membranes; some mitochondria are observed to be fully vesicular;
some of these vesicular mitochondria are also swollen; and finally some vesicular
mitochondria are so swollen that almost all internal membranes have been pushed
to the edges of the expanding organelle [52].
Recently tomography enabled a complete survey of megamitochondria and
normal mitochondria in the livers of deceased patients with non-alcoholic fatty
liver disease [25]. Using 4 livers in the study, researchers examined every mito-
chondrion and concluded that the internal configuration of what they called
giant mitochondria “is both multifold and dramatically distinct from their nor-
mal-sized counterparts.” They observed elongated giant mitochondria, intrami-
tochondrial crystalline structures, and spheroidal giant mitochondrial structures.
An important consideration is that in cells that form megamitochondria, typical
mitochondria are also found. In one cell, researchers counted almost 2400 mi-
tochondria. Of that number, 55% were giant, the rest were normal, suggesting
that both adapted and normal mitochondria present in a single cell may also be
active in that cell. In the research into dome formation in Ishikawa cells, while it
was certainly the case that all of the syncytial nuclei were subsumed into mito-
nucleons, it was also the case that additional mitochondria staining for carbox-
ylases did not fuse, remaining small particulate organelles arrayed throughout
the syncytium [9]. Variations in metabolism between these structures may be
complementary.
A flurry of research during the golden age of the electron microscope in the
middle of the last century established that giant mitochondria were also found in
organs specializing in anabolic activity such as the adrenal gland that synthesizes
most of the steroid hormones [53]. In their studies of the rat adrenal gland, Ca-
nick and Purvis also found a mix of giant and typical mitochondria [54]. Synthe-
sis and release of steroid hormones such as cortisol is stimulated by trophic
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hormones from the pituitary gland. Time-dependent changes in relative amounts
of the two kinds of mitochondria could be elicited by removing the pituitary
gland. Under those circumstances, researchers detected an increase in size of li-
pid droplets and in the volume of giant mitochondria, possibly a build-up of raw
materials. Megamitochondria comprise 8% of the total mitochondrial volume in
control animals, a value that increased to 65% with the swelling that accompa-
nies hypophysectomy. It has been found that administration of ACTH causes the
gradual reversal of these effects of hypophysectomy over a period of 9 days as it
restores synthesis of steroid hormones. As described for endometrium, there
have been numerous observations in the adrenal gland of the importance of en-
doplasmic reticulum membranes extending from giant mitochondria.
The contents of vacuoles in megamitochondria, some of which are so large as
to be called cavitations [38], has largely remained an open question. Our results
with mitonucleons leads to the suggestion that these vacuoles are formed by the
retention of CO2 and perhaps other gases within megamitochondria. Such a pos-
sibility was suggested in the systems described in Ishikawa cells by observing the
force of an enlarging vacuole that unfurls the apical membrane of a syncytium
undergoing differentiation [9] or the “hollowing out” of mitonucleons at the
center of a detached cell [13]. Additional clues that megamitochondria with at-
tached endoplasmic reticulum must be retaining gas include the rapidity with
which membranes unfurl [9]; the observation that vacuoles rapidly formed can
be readily dispelled as gaseous bubbles when hollow spheroids adhere to a petri
dish [13], and the observation that vacuoles can become enormous to the point
of “bursting” when synthesis of steroid hormones is stopped by the administra-
tion of an enzymatic inhibitor [38]. Additionally, it is probable that vacuolar
pressure against the chromatin mass in mitonucleons results in pyknotic chro-
matin. Nothing seems to explain these various observations as well as the forma-
tion and expansion of a gaseous vacuole or its dissolution.
The kind of structural change brought about by nebenkern, or even mito-
nucleons during dome formation, does not appear to explain the presence of
megamitochondria in the adrenal gland. Perhaps ready access to CO2 makes
“stored” CO2 in megamitochondria valuable in anabolic tissue such as the
adrenal gland. The gland uses cholesterol as the starting point for synthesis and
secretion of various steroid hormones. All 27 carbon atoms of cholesterol are
derived from acetyl and there are indications that cells of the adrenal glands re-
sponsible for synthesizing steroids are enriched for the same CO2-fixing enzymes
detected in dome formation as demonstrated by the work of Paul and Laufer
[55], who correlated the presence of high levels of endogenous biotin with the
ability to synthesize steroids in specific cells in the adrenal gland.
3.3. Composition of Gas Vacuoles
The theory then to explain the formation of megamitochondria such as mito-
nucleons is that the fusion of multiple typical mitochondria into a giant mito-
chondrion and the association with endoplasmic reticulum may in some in-
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stances fundamentally alter the structure so that CO2 diffuses out from the
adapted structure slowly, if at all, and gas fills up vesicular matrix compart-
ments that begin to coalesce into larger vacuoles within the organelle itself.
Vacuoles can enlarge and create force and vacuoles can store CO2. Is the for-
mation of megamitochondria about achieving one or both of these desired
ends?
The chief metabolic gas produced by any cell is CO2, however the widespread
assumption that CO2 diffuses passively through biological membranes, as it does
through lipid bilayers in the laboratory, has consistently worked against any sus-
picion that it can be retained. That assumption was successfully challenged when
Boron and colleagues [56] demonstrated differences between apical and basal
membrane permeabilities for CO2 using isolated gastric glands. The research
methods of Endeward and Gros [57] demonstrated just how much variability
(two orders of magnitude) there can be in biological membranes with regard to
CO2 permeability. These researchers and their colleagues proved that some
membranes offer substantially more resistance to CO2 diffusion than others [58]
[59]. Furthermore the laboratory has demonstrated an amazing reciprocal rela-
tionship between increasing pressure required for CO2 to pass through a mem-
brane and declining levels of O2 consumption [60].
The diffusion of CO2 through the membranes of typical mitochondria is more
rapid than diffusion through most other membranes, an observation from Gros’s
laboratory that makes sense for the organelle that will produce CO2 rapidly by
oxidative phosphorylation of glucose. The question becomes what happens when
normal mitochondria together with endoplasmic reticulum fuse into megami-
tochondria. Researchers such as Tandler and Hoppel [61] and Wakabayashi [24]
were early champions of the notion that megamitochondria form by the fusion
of normal mitochondria, a process evident in our studies of dome formation
[62]. Wakabayashi did extensive studies on the identity of megamitochondrial
lipids to try to understand the underlying biochemical changes that would result
in fusible organelles. Wakabayashi [24] compared lipids of megamitochondria
with normal mitochondria and found increases in phosphatidylethanolamine
and acidic phospholipids as well as in the ratio of unsaturated to saturated fatty
acids, changes that might increase fusibility as shown by recent work on phos-
phatidylethanolamine, phosphatidic acid, and cardiolipin, a negatively charged
lipid unique to mitochondria [63] [64]. But in light of results demonstrating that
cholesterol is an important variable [58] in determining diffusibility of CO2
through a membrane, the increases in cholesterol that Wakabayashi found in his
studies [16] might be most relevant to the possibility of CO2 retention by mega-
mitochondria. Such lipid changes together with the close investment of enlarg-
ing mitochondrion by a cistern of endoplasmic reticulum [37] could affect pas-
sage of gases, particularly since ER itself is believed to be the site of cholesterol
synthesis. Although absolute lipid content has not been measured for Ishikawa
cells forming mitonucleons, other data suggest that lipids are involved in mito-
nucleon formation. Fatty acids such as butyrate stimulate the formation of domes
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and therefore of mitonucleons more than three-fold [65]. Furthermore the in-
crease in endogenous biotin accompanying mitonucleon formation was ulti-
mately shown to be linked to increases in mitochondrial CoA carboxylases [14],
biotin-containing enzymes involved in fatty acid synthesis such as proprionyl
CoA carboxylase.
For it to be true that megamitochondria are adapted so that CO2 retention is
the source of vacuoles, it is essential that the formation of megamitochondrial
membranes significantly slows the exit of CO2 and also that the CO2 be produced
by a metabolic pathway other than oxidative phosphorylation, a process reliant
on O2 and the typical christae structure of the internal membranes of normal
mitochondria. Together with the loss of mitochondrial membrane potential, the
nature of the changes in the inner membrane structure of typical mitochondria
that fuse into megamitochondria suggest a point beyond which the chemiosmo-
sis essential to oxidative phosphorylation [66] would no longer be possible.
Beyond that point, increasing gas levels suggest an alternative kind of metabol-
ism. Otto Warburg maintained that cancer cells must use alternative metabolic
pathways because of an observation of decreased uptake of O2 by cancer cells.
DeBerardinis and his associates [67] extended Warburg’s observation by de-
monstrating that although cancer cells utilize glucose, most of the molecule ends
up being excreted as lactate rather than being metabolized by oxidative phos-
phorylation. In that paper, the researchers presented evidence for the potential
of glutamine metabolism as an alternate pathway.
Much has been learned about glutamine metabolism since then. An important
piece to the puzzle is experiments demonstrating that glutamine can be processed
by reductive carboxylation, bypassing oxidative phosphorylation. Experiments
with a melanoma cell line able to proliferate under extreme hypoxia established
that reductive carboxylation of alpha ketoglutarate generated from metabolism
of glutamine can become much more important than oxidative metabolism as a
pathway for production of citrate [68]. The reaction uses an NADP+/NADPH-
dependent isocitrate dehydrogenase (IDH2) located in mitochondria. Mullen
and colleagues [69] showed that tumor cells with defective mitochondria are able
to use glutamine-dependent reductive carboxylation as the major pathway of ci-
trate metabolism. Reductive glutamine metabolism also occurs in cytosol and
depends on a second isocitrate dehydrogenase (IDH1) [70].
In fully formed megamitochondria, internal membranes are converted from
christae into separate vesicular matrix compartments that can fill with gases
[62]. The compartments merge, creating a vacuole that is so large it pushes all
membranes to the edge of the megamitochondria [62], as has been shown in elec-
tron micrographs. At some point, and it is probably early in the process, mega-
mitochondrial membranes will be unable to carry on oxidative phosphorylation,
suggesting that adapted mitochondria may only be capable of reductive carbox-
ylation. Shim and colleagues [71] [72] established a significant connection be-
tween glutamine and megamitochondria with the discovery that glutamine, the
substrate for reductive carboxylation, is essential for the induction of megami-
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Advances in Bioscience and Biotechnology
tochondria. In fact, megamitochondria may be the organelle best suited for re-
ductive carboxylation, especially if its adaptations make it more or less imper-
vious to oxygen. Depending on the extent of reductive carboxylation, such a
possibility suggests that “pools” of CO2 might increase or decrease depending on
whether catabolism or anabolism is the dominant form of metabolism. And it
seems reasonable to suggest that the pools of retained CO2 would be supportive
of reductive carboxylation.
It would be hard to overstate the advantages of such a possibility, especially
for mitochondria whose principal function is anabolism since, as Zimorski
et al.
[73] recently wrote, “the synthesis of biomass costs thirteen times more energy
per cell in the presence of oxygen than in anoxic conditions.” Because of the
ready diffusion of CO2 into and out of typical mitochondria, there has not been
too much emphasis on supply, but demands for CO2 would appear to be highly
variable. The fact that the adrenal gland needs to excrete steroid hormones on
demand and then cease excretion makes the idea of an adapted mitochondria
that can store CO2 seem even more reasonable. Some kind of release of CO2
from vacuoles in a dissolved state must be imagined since dissolved CO2 has
been shown to be the primary substrate for IDH [74]. The same paper concluded
that experiments on the reductive carboxylation of pyruvate catalysed by what
the authors call the malicenzyme also uses dissolved CO2 as the primary sub-
strate.
There probably is much more to be learned about reductive carboxylation. Du
and colleagues [75] have demonstrated it to be the major metabolic pathway in
retinal pigment epithelium Additionally reductive carboxylation is enhanced in
detached epithelial cells [76]. And it is noteworthy that, as reported in a study by
Labuschagne
et al.
[77], the increase in reductive carboxylation is accompanied
by morphological changes in mitochondria. As already mentioned, glutamine is
essential for the formation of megamitochondria in Drosophila [71] [72]. One
very recent paper even describes an effect of glutamine on the origin and func-
tion of cancer cell extracellular vesicles [78].
In the history of the planet, the current levels of oxygen have only been around
for the past 500 million years [73]. Elevated oxygen levels resulting in the evolu-
tion of oxidative phosphorylation threatened processes that had evolved in the
absence of O2 such as the conversion of gaseous nitrogen into ammonia. I began
my professional life studying the enabling adaptations that allow some species of
cyanobacteria to convert N2 to NH3 even in the presence of O2. The process in-
volves a 20 to 24 hour differentiation [79] of approximately 10% of vegetative
cells into heterocysts, a cell type that maintains a “relatively anoxic microenvi-
ronment in a filament that is predominantly oxic.” [80] And this is not the only
example of cellular adaptation for enzymes whose activity is lost in the presence
of O2. Researchers have shown that anaerobic localities called microoxic niches
exist in chloroplasts of Chlamydomonas reinhardtii to protect the activity of
[FeFe] hydrogenase from O2 inactivation, a gas that the organelle itself generates
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[81]. One more example of structural changes resulting in an anoxic environ-
ment is actually to be found in megamitochondria in a single-celled green prot-
ist.
A mutant of Euglena gracilis without chloroplasts, seemingly wholly depen-
dent on oxidative phosphorylation, is nevertheless able to function if the elec-
tron transport system is poisoned or if the organism is placed under anaerobic
conditions. In these adverse conditions, growth immediately ceases, but without
any further changes in medium, Euglena adapt and begin proliferating again af-
ter approximately 20 hours. Sharpless and Butow [82] established the induction
of a unique NADP+ dependent pyruvate dehydrogenase, insensitive to antimy-
cin or cyanide poisoning and markedly stimulated by AMP. However, the in-
duced enzyme is inactivated by O2, as researchers found when they tried to pu-
rify the enzyme. Its activity was lost as soon as adapted mitochondria were bro-
ken open [83].
3.4. Conclusions
The inescapable conclusion is that the adapted structure of Euglena megamito-
chondria protects the newly induced enzyme from inactivation by O2. Can we
assume similar functions for megamitochondria in higher organisms? Do me-
gamitochondria within opaque giant cells achieve localized hypoxia by virtue of
an adapted membrane structure fortified with cholesterol? The observation that
megamitochondria retain gases even as evidence of their capacity for oxidative
phosphorylation dramatically decreases [52], together with the observations of a
reciprocal relationship between membrane permeability to CO2 and O2 uptake
[61], suggests that adapted mitochondria may be mostly hypoxic.
Finally, this paper demonstrates how opaque multinuclear cells arise in an
Ishikawa culture in a process that appears to be an exception to what Virchow
posited about all cells arising from other cells. It cannot be proven that a mono-
layer cell did not exist in region 10 before the start of the observation, but for-
mation of the opaque cell does not appear to be dependent on any prior struc-
ture that can be detected. What is observed is a single opaque cell capable of
forming a mitonucleon being built up over a period of hours with circumstantial
evidence that it develops under the influence of extracellular vesicles arising
from another opaque multinuclear cell. Less conclusively but worth mentioning,
it appears that a monolayer cell forms at the same time.
If the mitonucleons that form in these extraordinary cells are structures that
protect O2-sensitive enzymes, it may even be possible that the biochemical en-
zymes essential for H2 formation may be inducible and active under some cir-
cumstances in megamitochondria. It would be hard to overestimate how useful
it would be to adapt some of a cell’s population of mitochondria so that they are
hypoxic or even anoxic, capable of more efficient anabolism, capable of storing
CO2 and perhaps even capable of generating H2 with the capacity to restore
NADP+ to a reduced state.
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Advances in Bioscience and Biotechnology
4. Materials and Methods
Ishikawa endometrial epithelial cells were grown in phenol-red free MEM sup-
plemented with 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin,
and 0.25 mg amphotericin B (GIBCO, Grand Island, NY). The cell line estab-
lished by Nishida and colleagues [7] from an endometrial adenocarcinoma was
obtained from Dr. Erlio Gurpide’s laboratory at Mt. Sinai Hospital in New York.
Cells seeded at an approximate density of 5 × 105 cells/cm2, were grown for 1 - 2
weeks in MEM containing 5% calf serum (CS). The experiments were performed
while cells were in logarithmic growth phase. Dishes were marked so that we
could return to the colony of interest at the desired intervals. Structures were
viewed using an Olympus inverted stage microscope at powers of 200× and
400×.
The presence of stainable amounts of biotin was used to detect the presence of
mitonucleons as in Figure 1 and Figure 2, cultures were fixed by adding 4% pa-
raformaldehyde in phosphate buffered saline (PBS) to the culture dish. After 10
min, the cells were washed gently four times with 5 - 10 ml PBS. A solution of
1% Triton X-100 was added to the cells to permeabilize the membrane. Again
after 5 min, the culture was washed with successive changes of PBS. After wash-
ing, cells were exposed to a 1:200 dilution of Extravidin conjugated horsera-
dish peroxidase (HRP) (Signa) for 30 min. After further washing, a solution of
3-amino-9-ethylcarbazole (AEC), prepared by dissolving 20 mg of AEC in 2.5 ml
of dimethylformamide and diluting with 47.5 ml of 50 mM potassium acetate
adjusted to pH 5.0, was added to the cells together with 0.25% H2O2. This solu-
tion was incubated at 37˚C for 45 min to allow color to develop. The AEC solu-
tion was removed, and the cultures were examined and then stored in the pres-
ence of PBS at 4˚C. If avidin linked to peroxidase is not added to the cultures,
there is no reaction. If avidin without peroxidase is added first to the cultures,
followed by avidin linked to peroxidase, staining is not observed. Staining does
not occur if avidin HRP is not added to the cultures prior to AEC indicating that
an endogenous peroxidase is not responsible for the staining. To ensure that
avidin was reacting with biotin, we stained domes using streptavidin linked to
horseradish peroxidase as well as primary antibody to biotin and secondary an-
tibody linked to horseradish peroxidase. Staining occurred under all circums-
tances, indicating that avidin does indeed react with biotin that is endogenously
present in the cell in significant amounts.
Conflicts of Interest
The author declares no conflicts of interest regarding the publication of this paper.
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... Mitonucleons, physical evidence of mitochondrial interaction with nuclei, are temporary organelles with consequential effects. I recently presented evidence [52] that their structure must cause a change in mitochondrial metabolism from aerobic to anaerobic with the possibility that the resulting anoxic organelle may generate energy and metabolites using the alternative pathway for generation of citrate that includes CO 2 fixation. In that paper, I also suggested that CO 2 does not as readily pass out of mitonucleons as it does out of mitochondria that have not fused. ...
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