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Tubular membranes extended between monolayer cells,
from solid spheroids, and from clustered hollow
spheroids in Ishikawa endometrial cell cultures can carry
chromatin granules and mitonucleons
Author: Honoree Fleming
Affiliation: Castleton State College, retired as Dean of Education
City and State: Castleton VT 05735
Country: United States of America
Corresponding Author: Honoree Fleming
Email Address: Honoree.Fleming@Castleton.edu
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PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27895v1 | CC BY 4.0 Open Access | rec: 12 Aug 2019, publ: 12 Aug 2019
Tubular membranes extended between monolayer cells,
from solid spheroids, and from clustered hollow
spheroids in Ishikawa endometrial cell cultures can carry
chromatin granules and mitonucleons
Abstract
Membrane tubule extensions, recently recognized an important communication element in
mammalian cells are demonstrated to form in Ishikawa endometrial epithelial cells growing in
monolayers, and to extend from solid spheroids and from clustered hollow spheroids. Two
kinds of chromatin cargoes travel through these tubules. Chromatin granules can pass
through an endometrial tubule bridge extending from one monolayer fragment to another.
The passage of granules over time from one of the fragments appears to support the
self-assembly of nuclei in the other colony fragment. Similarly, in a process detected by
observing an open-ended membrane tubule extending from a solid cell spheroid a nucleus
formed over a period of 3 hours.Indications are that chromatin granules such as those
observed in the amitotic processes of epithelial dome cell formation and of hollow spheroid
cell formation are contributing to the build up of nuclei. Mitonucleons, a transient subcellular
organelle consisting of fused mitochondria intimately associated with aggregated chromatin
are also observed to pass through tubular membrane extensions. Multiple membrane
extensions can be shown to to extend from clusters of unicellular polyploid hollow spheroids
whose formation is described for the first time in this paper.
Introduction
The existence of tubular extensions between cells has emerged as a particularly exciting area of
research relatively recently in the century-plus history of cell biology. Throughout that same century,
biologists, especially those studying development, understood the importance of communication
between and among cells in multicellular organisms, particularly the role of directed morphogen
diffusion (Müller et al., 2013). The advantages of even more direct communication between cells were
obvious, but typical methods of investigation, such as those involved in fixing and sectioning tissue
were not ideal for detection of intercellular conduits, delicate structures (frequently less than a micron
wide) that stretch over distances of hundreds of microns. Even in living cultures, it’s essential to look
sharp to detect membrane tubular extensions.
Researchers studying sea urchins and cultured insect cells are credited with some of the earliest
observations of “communication” by membrane tubules, frequently called filopodia. Karp and Solursh
(1985) culturing primary mesenchyme cells from sea urchins demonstrated thin, elongated, filopodia
between cells in culture. Locke (1987) studied filopodial bridges in cultured insect cells, forming
between cells “losing” contact as they moved away from each other and suggested that such
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membrane tubules re-established or maintained cell-to-cell continuity. Tubules were also observed in the
normal course of tissue morphogenesis. Ramirez-Weber and Kornberg (1999) identified cellular
processes that they called cytonemes projecting from wing disc cells to the signaling center in
Drosophila. Relevant to the discussion in this paper, the researchers also described the formation of
similar cell-to-cell extensions in co-cultures of wing disc and signaling center cells. Much of what was
discovered for sea urchins and insects was “rediscovered” in mammalian cell cultures starting with
Ramirez and Kornberg’s observation that cultured mouse limb buds also formed cytonemes.
Despite that foreshadowing, it took some time for this new research area to gain acceptance. To get a
sense of the trauma that can accompany the appearance of an exciting but challenging “new
discovery” (or even sometimes its rediscovery [Platner 1886 and Flemming 1891]), read Monya Baker’s
news feature “How the Internet of cells has biologists buzzing” (2017). Not only does Ms. Baker clearly
and concisely describe seminal research; she evokes the conflicts and self-serving skepticism that often
accompany a challenge to orthodoxy. Scientists and their enterprising postdocs “soft-peddle” exciting
discoveries until at least some of the establishment catches up with them. Publication of a relevant
research paper from the laboratory of a prominent scientist can be an ice-breaker, but it takes time for
the word to spread. Along the way grant applications with resources on the line, sometimes bring out
the worst of “peer judgement” (is the principal investigator concerned that what appears to be a
tubule might actually be a scratch in the petri dish?). And when all else fails, there is the
hard-to-answer (and equally hard to credit) suggestion that perhaps cells have learned tricks in petri
dishes that have nothing to do with what happens in vivo
(because we know exactly what that is with
our mostly two-dimensional view of “pickled” tissue). Initial reactions to the reality of membrane
extensions, as described by Ms. Baker, illustrate the paradox of a field of study dedicated to discovery
but predisposed to reject what are perceived to be challenges to beloved orthodoxies rather than
relevant refinements. Inevitably, as has happened many times before, skepticism was mostly
overcome, hopefully without any damage to hapless graduate students or postdocs whose work on
membrane tubules appeared too early in the cycle.
Amin Rustom and his colleagues in Dr. Hans-Hermann Gerdes laboratory showed elegant pictures of
tubules stretching from cell to cell in a rat pheochromocytoma cell line (2004). The research team
demonstrated that these tubules carry endosomes, calling the structures “tunneling nanotubes” to
convey something about size and function. In that same year, Onfelt and colleagues (2004) presented
evidence that “nanotubular highways” (their preferred descriptive name for tubules) represent a novel
mechanism for intercellular communication in immunology. Additional discoveries were made in the
Gerdes laboratory where, approximately 10 years later, Gerdes and his colleagues began the important
process of comparing and contrasting the data accumulating about membrane tubules in a review that
he unfortunately was unable to see to completion (Austefijord et al. 2014).
During the decade following the introduction of tunneling nanotubes, research scientists produced
evidence that tubular extensions were not only relevant for in vivo communication
but also carried
various and important cargos from one cell to another. Lou and his team (2014) published pictures of
membrane tubules in cultured cells from human mesothelioma lung cancer and studied cultured
fragments of tumor tissue from that same disease. By maintaining the three dimensionality of tissue,
the latter technique promises a close approach to observation in vivo and tunneling nanotubes were
found to be present. In an experimental tour de force, Dr. Frank Winkler and his 39 co-researchers
published a paper in 2015 that “looked” into the brains of mice injected with cancer cells derived from
human brain tumors. Not only did they observe tubular membranes galore developing among the cells,
they pinpointed the growth of tubules (8 to be exact) that could be shown to deliver a new nucleus to
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an area in the living mouse’s brain that had suffered the targeted death of a single cell from a laser
assault, a remarkable observation (Osswald et al., 2015).
Research into tubular structures continues to flourish with studies on structures known by a variety of
names including cytonemes, filopodial bridges, tunneling nanotubes, cellular bridges, nanotubular
highways, tubular bridges, and others (Sisakhtnezhad and Khosravi 2016; Vignais et al., 2017;
Yamashita et al., 2018). Yamashita and colleagues suggest that while honoring each of these names,
the entire category of structures could be called “thin membrane protrusions” a term sufficiently
inclusive to call up more than 8000 references in the NIH “Pub Med” data base. The existence of
membrane tubules actualizes the staggeringly important, and no longer underappreciated, fact that
there is significant continuity of cytoplasm and membranes in complex multicellular animal systems
(Rustom, 2016). As will be discussed, researchers have made many relevant observations about tubules
and there is clearly more to be learned. The question of names is being sorted out as additional
examples of tubules are described. Frankly the process of finding names that provide some indication
of function is ongoing even for an organelle that has been the object of study for much longer.
Mitochondria whose distinction is enlargement by fusion of smaller mitochondria have variously been
called: nebenkern, spheroidal mitochondria, and cup-shaped mitochondria. In endometrium
researchers, by serial cross sections, demonstrated that what was once thought to be multiple average
sized mitochondria were actually part of a giant mitochondrion-endoplasmic reticulum unit made
up of fused mitochondria (Armstrong et al.,1983). These structures are similar to those
observed in studies of Ishikawa endometrial epithelia where the term mitonucleon is used to
signal the intimate association of fused mitochondria with chromatin (Fleming, 2018b)
essential for differentiation of domes that elongate into tube-like structures and hollow
spheroids (Fleming, 2018a).
This paper demonstrates for the first time that mitonucleons can be carried by membrane tubules that
form in cultures of Ishikawa endometrial cells. Calibers of the tubules observed in Ishikawa epithelial
cells extend from hundreds of nanometers (almost the limit of what can be seen by light microscopy) to
approximately 1-3 micron, somewhat larger than cytonemes and tunneling nanotubes but close to what
has been observed for bronchial epithelial bridges that have been shown to carry whole cells (Zani et
al. 2010). Additionally the transport of chromatin granules between Ishikawa endometrial epithelial
cells, from spheroids and from clustered hollow spheroids formed by these cells, will be described in
this paper.
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Results
A photomicrograph of an endometrial epithelial bridge (approximately 2.5 microns wide)
extending from a monolayer Ishikawa endometrial cell to a neighboring Ishikawa colony over a
distance of around 225 microns is shown in fig. 1. The tube emerges from a nucleolus in one
cell and fuses with a cytoplasmic ruffle on the edge of a neighboring colony. Serum-free
Fig. 1 Two colonies of Ishikawa endometrial epithelial cells connected by a tubular bridge. The tubule originates from a
nucleolus in a fragment containing two nuclei has attached itself to the edge of a second colony, on the lower right, in a region
that appears to be free of nuclei in a cytoplasmic ruffle similar to structures that are called lamellopodia. Bar = 25 microns
medium had been added to this dish of cells two days previously. A low level of serum has
been recognized as one of the conditions that elicit the formation of tubular extensions (Lou
et al. 2012). The tubule itself between a cell and a thriving colony some distance away might
be the prototype for many of the tubular extensions observed in other cell cultures although
the tubules do not always originate from nucleoli.
The second not so typical membrane tubule, shown at two different times in figs. 2 and 3,
links two colony fragments in a monolayer stressed by the absence of serum from medium for
five days. Many monolayer cells detach under those conditions. The two colony fragments in
fig. 2 persisted and established a tubular connection that is a little longer (approximately 280
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microns) and a little thinner (1 to 2 microns) than the process observed in fig. 1. The
fragment on the right in fig. 2 contains a pair of unusual structures within a dramatic flourish
of membranes. What appears to be the donating colony fragment contains granules
concentrated along one of the membranes leading to the opening into the tubule.
Fig.2 Two membrane fragments in a stressed monolayer linked by a thin membrane tubular bridge. Neither fragment is typical of
what is usually observed for colonies of these cells, perhaps because the monolayer was stressed by the absence of serum for 5
days.. Bar = 50 microns
Fig. 3 shows these structures after 3 hours. An amazing transformation has occurred in the
colony fragment on the right. It has doubled in size, two apparently typical nuclei fill out
most of the membrane flourish. The concentration of granules in the colony fragment on the
left has diminished and at least three small vacuoles have appeared. Fig. 4 shows
enlargements of the structures in figs. 2 and 3, with panels a and b contrasting the anucleic
fragment seen at the start of the observation and three hours later; panels c and d contrast
the colony fragment in which nuclei form over that same time period.
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Fig. 3 The appearance of both fragments changes dramatically over the period of three hours. Most of the dark granular
material at the mouth of the tubular extension of the structure on the left has disappeared and small vacuoles have appeared.
Two nuclei are being built up in the fragment on the right. The granular outlines of the nuclei resemble images of formation of
nuclei in hollow spheroids due to chromatin streaming (Fleming, 2019) Bar = 50 microns
Recently published evidence that chromatin granules amitotically form nuclei in hollow
spheroids (Fleming, 2019) bolsters a theory that chromatin granules passing through the
membrane tubule over a period of three hours contributed to the formation of two nuclei.
Granularity is still evident in the nuclear membranes in the enlargement shown in panel 4 d.
Similar granularity was observed in nuclei formed in hollow spheroids “caught” in the process
of adhering to the surface of a petri dish during amitosis (Fleming, 2019). Dispersed chromatin
was initially shown to be involved in the differentiation of monolayer Ishikawa endometrial
epithelia into detached dome cells (Fleming 2016 a,b,c) and subsequently as the product that
appears when giant polyploid nuclei in “unicellular” hollow spheroids “deconstruct”(Fleming
2019). This paper suggests that chromatin material, specifically chromatin granules, can be
added to the list of “cargos” for thin membrane tubules that includes among other things:
prions (Gerdes, 2009; Gousset et al. 2009), retroviruses (Xu etal., 2009; Rudnicka and
Schwartz, 2009; Panasiuk etal.,) and, as will be discussed in some detail, mitochondria
(Vignais et al., 2017).
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Fig. 4 Enlargements of structures at the ends of the tubule in fig. 2 and fig. 3. Panels a and c are enlargements of the
photomicrograph taken at the start of the observation (fig. 2); panels b and d were enlarged from the photomicrograph taken
three hours later. Granules are observed concentrated at the entrance to the tubule in panel a. Some kind of structure, perhaps
scaffolding for nuclei can be detected filling about half of the membrane in panel c, along with an elaborate flourishing
membrane of about equal size. Panel d shows that most of the membrane scaffolding fills out, doubling the size of the fragment
with the formation of two nuclei.
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Fig. 5 A flourish of membranes at the end of a tubular process extending from a solid spheroid. When this tubular extension was
initially observed in a living culture, there was little structural detail in the membranes extending out from the end of the
process flowing from a solid spheroid. Dark material and a bulge of autofluorescent material can be detected in the tubule.
These materials are observed to move during the 3 hours between the observations of the structure in figs. 5 and 6. What
appears to be a nucleus is forming in the membrane flourish at the end of the tubule. Bar = 50 micron
Fig. 6 Material flowing into the membrane flourish appears to be self assembling into a nucleus. Bar = 50 micron
The tubule in fig. 5 is approximately 250 microns long, with a prominent bulge 75 microns
from the spheroid and a flourished membrane at the end of the extension. Over a period of 3
hours, the bulge moves 75 microns toward the membrane flourish (fig. 6). In that same
period, the outlines of something resembling a nucleus become visible within the flourish at
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the end of the tubular process. The material passing through the tubule is not uniform. Dark
granular material and auto-fluorescent material can be detected.
Fixing and staining tubules arising from solid spheroids reveals some of what is being
transported from solid spheroids into tubular processes. A large, perhaps polyploid, nucleus
stained with hematoxylin is apparent at the end of the process in fig. 7. A thin rim of
endogenous biotin stained with avidin-linked peroxidase and AEC surrounds the nucleus.
Hematoxylin stained granular material can be observed in the cytoplasm surrounding the
nucleus. Approximately half-way down the tubule extending from the spheroid is another
structure staining both for chromatin and for endogenous biotin. The other half of that
structure can be seen in fig. 8, showing the solid spheroid from which the tubular extension
originates with granular material “entering” the tubule and a clearer picture of the structure
traveling through the extension. Chromatin is surrounded by endogenous biotin in a structure
initially observed in syncytia formed in a monolayer of Ishikawa cells stimulated to form
domes (Fleming 2016a) and ultimately shown to result from mitochondria fusing around
aggregated chromatin (Fleming 2018a).
Fig.7 Spheroid tubule extends from a solid spheroid and ends
with a membrane flourish containing a large, perhaps
polyploid, nucleus, surrounded by thin rim of material
staining for endogenous biotin. Diffuse hematoxylin stain
surrounds the large nucleus suggesting that nucleic acid
material has been exported from the spheroid not
incorporated into the nucleus. Bar=50 microns
Figure 9 is a tubule that contains two mitonucleons that may be further along in formation. The
mitonucleon on the right appears to have begun generating and retaining gas, a process that
has been discussed in some detail for dome formation (Fleming 2018b), and that occurs in the
development of a hollow spheroid from a monolayer cell (Fleming, 2018a).
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Figure 8. Process extending from a solid spheroid. Granular material can be detected at the mouth of the tubule.
Material half way down the tubule (also visible in fig. 7) stains for the endogenous biotin of mitochondria and a
central core of chromatin characteristic of mitonucleons. Bar = 50 microns
Figure 9. Tubular membrane process extending from another solid spheroid contains two structures that stain like
mitonucleons. One of the structure appears to have already started to generate a gaseous vacuole. Bar = 50
microns
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Clusters of unicellular hollow spheroids are also capable of forming
membrane extensions
Figure 10. Clustered cells developing detaching from a
monolayer as cells show signs of becoming clustered hollow
spheroids det. Each of the developing hollow spheroids stains
for the endogenous biotin of mitonucleons. The resulting
structure is floating above the monolayer seemingly attached
by at least one membrane process. Monolayer cells are
approximately 25 microns. The largest developing hollow
spheroid is approximately 37 microns.
Bar = 50 microns
Figure 11. Attached hollow spheroids floating in medium.
Hollow spheroids result when mitonucleons, chromatin
aggregates surrounded by a fused mitochondrial structure,
retain gases forming vacuoles that compress cytoplasmic
structures between the outer membrane of the mitonucleon
and the inner membrane of the cell, a process that has been
described in detail for a single hollow spheroid (Fleming,
2018 a and b). Largest spheroid in the cluster is 75
microns.
Bar = 25 microns
Extensions similar to those emerging from monolayer fragments and from solid spheroids have
also been detected extending from clusters of hollow spheroids, shown in this paper for the first
time. Looking like a bunch of grapes and staining for endogenous biotin, a cluster of enlarging
cells is shown detaching from a monolayer colony in fig. 10. Multiple hollow spheroids appear
to form in the same way as a single hollow spheroids (Fleming 2018a), starting with monolayer
cells in which spheroidal mitochondrial structures staining for endogenous biotin appear to
completely surround aggregated chromatin material. (Fleming, 2016a). The final point of
attachment is itself a tubular membrane extending back into the monolayer. The developing
hollow spheroid cluster can float above the monolayer, as was observed for single hollow
spheroids (Fleming, 2017a), as gas vacuoles continue to expand (fig.11). As vacuoles reach
the limits of their expansion, the resulting structure looks like a “beehive” (fig 12). Each of
the hollow spheroids is outlined by endogenous biotin that has expanded with the
mitonucleon to the point where it is compressing nuclei and other cell structures into a
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spheroidal rim against the cell membrane (Fleming 2018a). Fig. 13 shows a beehive structure,
not fixed and seemingly slightly deflated but clearly showing nuclei or chromatin granules in
spheroid rims. The outermost rims surrounding vacuoles appear to be continuous at some
points.
Figure 12. The endogenous biotin previously shown to be
bound to mitochondrial carboxylases highlights the
membranes of a cluster of hollow spheroids. The focus of the
microscope is on the surface of the multiple spheroid
structure. The structure may be in the process of becoming
anchored to the petri dish by a structure at the top of the
beehive. The entire structure is larger than 250 by 200
microns
Bar = 25 microns
Figure 13. A cluster of hollow spheroids whose irregular
shapes suggest deflation. In a single hollow spheroid, nuclear
and cytosolic material are pressured against the cell wall by
developing internal vacuoles. This photomicrograph
demonstrates that clustered hollow spheroids are similarly
structured with the possibility that the rims of individual
hollow spheroids can run together within a cluster.
Bar = 25 microns
A structure that looks like a nucleus can be observed in the interstitial space between two of
the beehive “chambers” in fig. 13. It may be relevant to emphasize that the cluster is made
up not of multicellular hollow spheroids but rather of polyploid hollow spheroids that are in
effect “single cells,” albeit extraordinary cells whose monolayer attachments to each other
survive through the period of time between the release of a cluster of what were monolayer
cells to the appearance of what is a cluster of hollow spheroids.
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Figures 10 through 13 illustrate the variability in sizes of hollow spheroids, an obvious
function of the increasing size of the gas vacuoles. Furthermore, one especially large vacuole
in the structure in fig. 13 suggests that vacuoles themselves may fuse.
Figure 14. Photomicrograph focused on the upper surface of a cluster of hollow spheroids. As fig. 8 shows, the expectation is
that nuclear material will be on the surface of the hollow spheroids as is apparently the case in this example. And, in fact, the
longest of the tubular extensions is emerging from a structure resembling a nucleolus. Bar = 50microns
A cluster of hollow spheroids can, under appropriate conditions such as the addition of fresh
medium with added glucosamine but without serum, “bristle” with multiple membrane
extensions from the surface of a structure that is, as already described, mostly vacuoles
surrounded by rims of cytoplasm containing nuclei (figs. 14 and 15).
Figure 14 is a photomicrograph of the upper most surface of this three-dimensional cluster of
hollow spheroids. Two prominent extensions arise from nucleoli (much like the extension
shown in fig. 1). Focusing down from the surface of the cluster, fig. 15 brings the ends of
these extensions into focus. One of the extensions is capped by a flourish of membranes much
like what was observed in fig 5. The other process ends with two “rectangular” cargoes. The
presence of an electron lucent structure in the midst of the membrane flourish, and in one of
the rectangular packages, suggesting that the rectangular package may be an as yet unfurled
membrane flourish.
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Figure 15. Honeycomb of hollow spheroids bristling with tubular membrane extensions. Fillapodia extend out from
the edges of some of the hollow spheroids and these also have tubular membrane extensions. The hollow
spheroids are approximately 40 microns. An enlargement of the tubule extending from the right side of the
honeycomb is shown in fig. 16.
Filipodia extending out from the structure are sprouting additional shorter tubules. Tubular
extensions range from barely visible by light microscopy (hundreds of nanometers) to micron
size. No details of structure can be detected in the hollow spheroids that cap off the ends of two
extensions. Experiences with single hollow spheroids (Fleming, 2018a) suggest that one
possible explanation is that nuclei compressed against the rim of the structure are on the other
side of the spheroid. Interestingly, there is, what appears to be debris associated with each of
the spheroids.
The intriguing tubule extending from the right side of the cluster, enlarged in fig. 16, contains a
bolus of material, as well as a membranous flare and is capped by a hollow spheroid. Granular
material comes into focus in the membrane flare. The sum total of these various extensions
suggest that this intimidatingly complex beehive structure is prepared to “put down roots” using
some of the structures that have been the subject of this paper. The clusters of hollow
spheroids, in figs.11 through 16 outlines one way in which a cluster of cells may successfully
move from attached monolayer to mobile structure to re-attaching mass, an important “trick” in
the playbook of cancer metastases.
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Figure 16. Scanning the 35mm photomicrograph of a membrane flourish in fig. 15 at a higher resolution reveals that the
contents of the flourish are granular.
Discussion
Research over the past 20 years has put to rest any doubts about the existence of membrane
tubules and their importance in cellular physiology. The field is indebted to the authors of
comprehensive reviews (Sisakhtnezhad and Khosravi 2016; Vignais et al. 2017; Yamashita et al.
2018) who have documented the appearance of tubular membrane extensions in more than 20
different cell lines as well as in tissue cultures and in a living brain. The evidence in this
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paper demonstrates, for the first time, that such structures are also formed by endometrial
epithelial cells (Ishikawa cell line from the laboratory of Nishida et al., 1985).
What tubules carry
The initial function of membrane tubules was understood to be the passage of morphogens,
and perhaps ions such as calcium, from one cell to another. But intriguing bulges led
scientists early on to investigate whether larger cargoes could pass through tubules or
bridges. Not only was this discovered to be the case: researchers demonstrated physiological
changes accompanying at least some of the cargoes, highlighting the importance of
membrane tubules to cell communality. Among multiple examples, it was shown that
endothelial progenitor cells took on a cardiomyogenic phenotype as a result of the transport
of organelles, including mitochondria, through membrane tubules that form between the two
cell types in co-culture (Koyanagi et al. 2005). Spees and colleagues (2006) demonstrated
that mitochondrial transfer through membrane tubules from adult stem cells can rescue
aerobic respiration in mammalian cells with nonfunctional mitochondria. In a co-culture of
bone marrow derived mesenchymal and endothelial cells, it was shown that the transfer of
mitochondria from endothelial to cancer cells through tunneling nanotubes modulates
chemoresistance of the cancer cells (Pasquier et al., 2013). One more observation is that the
transfer of mitochondria via tunneling nanotubes can rescue apoptotic PC12 cells. (Wang &
Gerdes, 2015)
Another important fact about membrane tubules, learned early on, is that their numbers
increase when cells are stressed. Donghui et al. (2005) demonstrated that hydrogen peroxide
increased intercellular connections in astrocytes. Low serum together with high glucose
levels resulted in increased numbers of tubules in mesothelioma cells (Lou et al., 2014) and
tubule formation was stimulated in ovarian (Desir et al., 2016), as well as in colon cancer
cells (Lou et al., 2018), by hypoxia. In vivo
Osswald and colleagues (2015) showed that a
microtube (another name for the larger membrane extensions containing microtubules) could
be observed growing to the region of a laser damaged brain cell within 12 hours of the event;
with a “replacement nucleus” traveling through the tubule over the next 24 hours.
The movement of nuclear material has been reported in other systems. Zani et al. (2010)
reported migration of whole cells through what the authors called type 2 epithelial bridges
whose width was in the range of one or more microns and whose composition included
microtubules along with actin. Antanavičiūtė and colleagues (2015) described the movement
of DAPI stained vesicles containing nucleic acids through tubules. The results in this paper
indicate that chromatin granules and mitonucleons can also pass through tubular membranes.
Origins of tubules in endometrial cells
Although tubular membrane extensions are being reported for the first time in cultured
human endometrial cells, it appears that the origins of some tubular membranes were initially
observed more than 50 years ago. Researching structures in human endometrium during the
reproductive cycle, electron microscopists discovered an actin-based structure in nucleoli
that they showed to be a compact whorled arrangement of tubules around an electron-lucent
core (Clyman, 1963; Terzakis, 1965). Researchers called this a nucleolar channel system and
showed that its appearance was related to the human reproductive cycle with the number of
such structures peaking on day 19 of the cycle (More et al. 1974). More recently the
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formation of these systems has been shown to be dependent on a threshold level of
progesterone (Nejat etal., 2014).
Researchers assumed that the nucleolar channel system acted within the cell of origin, having
detected on occasion that a tubule extended from the nucleolus out into the cytoplasm (Wang
Tzuneng & Schneider, 1992). Tubules were not reported to extend any further, perhaps
because their fragility, together with their small diameter, made survival during tissue
processing unlikely. Some of the tubules shown in this paper clearly emerge from a nucleolus.
Nucleolar channel membrane systems may or may not be unique to endometrial cells and to
oocytes where they have also been detected (Funaki etal., 1995). But it is the case, even in
Ishikawa cell culture, that not all membrane tubules can be traced back to nucleoli.
The membrane tubule in fig. 2 illustrates that fact. What appears to be the originating
colony fragment does not contain anything remotely resembling a nucleus. Dark granules,
mostly concentrated at the “mouth” of the extension, appear to be the origin of the tubule
that is attached to another monolayer fragment containing some kind of internal structure
amidst a flourish of membranes. Over a period of three hours, granular material at the mouth
of the tubule diminishes, while the structure to which the extension is attached doubles in
size with the formation of two “typical” nuclei filling most of the membrane flourish. On the
basis of this observation, the best guess is that one fragment of a colony is “donating” nuclear
material to a second, perhaps “healthier,” fragment to complete assembly of nuclei. Actin
filaments associated with cell membranes are suspected of involvement in membrane tubular
extensions by a complex process that includes actin polymerization (Drab et al., 2018).
The movement of chromatin granules through a tubular membranes is also the most
reasonable explanation for the changes that occur in the membrane flourish in figs. 5 and 6.
The flourish appears mostly featureless in fig. 5 but, after 3 hours (fig. 6), is observed to
contain the “granular outlines” of a nucleus similar to what was found for nuclei in
reattaching hollow spheroids seemingly stopped in the midst of amitosis (Fleming, 2019).
Chromatin streaming in hollow spheroids (Fleming, 2019) produces colonies of nuclei that
eventually position themselves equidistantly in dome or spheroid and then form membranes.
Despite skepticism about whether amitosis can achieve precise distribution of the parental
genome into daughter cells, reports of different forms of amitosis in cultured mammalian
cells (reviewed Fleming, 2016c) continue to appear. At the same time, it has become
obvious, particularly for cancer cells, that such precise distribution does not actually occur as
evidenced by a large population of aneuploid cells that are found (reviewed, Fleming, 2019).
So perhaps that rationale for rejecting a proliferative process widely “used” by successful
lower forms of life can be put to rest.
Credible reports of what might be called niche amitosis responsible for production of normal
progeny cells from giant cells have been observed by many researchers (reviewed in Fleming,
2016c and in Fleming, 2019). Liu and colleagues made an important advance in such studies
by isolating polyploid giant cancer cells induced by hypoxia in human ovarian cancer cell lines
and demonstrating that the polyploid nuclei give rise to regular sized cancer cells by amitotic
processes described as “budding, splitting, and bursting” (Zhang et al., 2014). In a recent
paper, Liu and his colleagues proposed the existence of a cell cycle relevant to giant cells
that “integrates” amitotic processes such as these with the more familiar process of cell
proliferation by mitosis (Niu et al.,2016).
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Origins and function of chromatin granules
Amitosis by chromatin streaming discussed in this paper appears to be outside the cell cycle,
although it mostly appears to involve polyploid nuclei. The process resulting from the
fragmentation of nuclei into chromatin granules has been observed during dome
differentiation (Fleming 2016 a,b,c) and during conversion of a single cell hollow spheroid
into a multicellular hollow spheroid (Fleming, 2019). Fundamental changes within an
aggregate of nuclei, including, most probably, a change in pH, results in an “explosive”
deconstruction of chromatin into granules, a process strikingly like the phenomenon that
Székvölgyi and colleagues (2008) were able to demonstrate for isolated nuclei. In predomes
and in the shell of hollow spheroids, chromatin granules become DNA filaments whose
epigenetic alteration is probably essential for differentiation. The dispersed chromatin then
coalesces into a mass from which arises a colony of nuclei that is the basis for dome or
spheroid cells. Formed as a cluster, nuclei eventually move apart and generate or perhaps
sequester cytosol from what resembles a giant multinucleated cell, ultimately forming cell
membranes around each nucleus. Not only is nuclear assembly from chromatin granules a kind
of amitotic proliferation, it also constitutes a significant exception to cell theory (Fleming
2016 a,b,c; Fleming 2019).
Results to date suggested that chromatin streaming might be another kind of niche amitosis,
responsible for “assembly-line” proliferation of multiple nuclei in adult organisms (Fleming,
2019). The process is obviously more efficient than mitosis, producing dozens of cells over
approximately the period of time required for completion of a round of mitosis (Fleming,
2016c). The fact that domes can eventually form gland-like structures highlight the possibility
(Fleming, 2016c) that streaming chromatin granules may be responsible for production of cells
such as those that make up endometrial glands, structures that will flourish for a relatively
short period before being replaced. Furthermore such a process might be involved when the
demands for proliferation are at their greatest and trillions of cells are being produced during
the 40 week gestation period of a fertilized human zygote (Fleming, 2019).
Results in this paper suggest, for the first time, that chromatin granules may also be involved
in building up individual nuclei on an “as needed” basis. The monolayer fragments in fig. 2,
challenged by the addition of medium without serum, are also with nuclei. Over time,
granular material traveling from one fragment to the other appears to have contributed to
the assembly of two nuclei in one of the fragments. Similarly in figs 5 and 6, a tubular
process appears to be “delivering” materials into a membrane flourish resulting in a structure
that looks like a nucleus. As many will surely ask, is there an in vivo
analogue to this process?
The amazing work of Winkler and colleagues already described in this paper documents a
fascinating example of nature “swapping out” a nucleus via a membrane tubule seemingly
formed precisely for that process (Osswald et al. 2015). It is possible to wonder whether the
nucleus being delivered was formed early on in the tubular process from chromatin granules.
Mitonucleons are also observed in Ishikawa epithelial tubules
In addition to streams of chromatin granules that appear to“build” nuclei (figs.2,3; 5,6),
fused mitochondria as detected by staining for endogenous biotin also pass through
endometrial epithelial tubules. In the unfixed, unstained process in figs. 5 and 6, a
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significant concentration of mitochondria was suggested by an auto-fluorescent bulge moving
through the tubule apparently behind, but probably admixed with, dark granular material.
When processes were stained for endogenous biotin as well as for chromatin, a familiar
maroon structure was observed created by the salmon stained endogenous biotin of
mitochondria enveloping hematoxylin stained chromatin. This structure called a mitonucleon
was initially observed in dome formation (Fleming, 2016a) where it was shown to be a
transient organelle able that results in retention of gases that compress aggregated chromatin
into pyknotic-like structures (Fleming, 2016 a,b,c). As has been highlighted in previous
papers this nuclear compression resembles the earliest stages of apoptosis except that cell
differentiation rather than cell death is the outcome.
By following the formation of mitonucleons in monolayer cells, the process of formation of
detached hollow spheroids could be described (Fleming 2018a). This is a structure that has
intrigued cancer researchers for more than a couple of decades, without much being
understood concerning its origins. As the gas vacuole within the mitonucleon in a single
monolayer cell expands, it pressures polyploid nuclei against the cytoplasmic membrane and
the developing spheroid detaches from the monolayer, having been converted from an
anchored into a mobile “cell”. Researchers studying spheroid formation in pancreatic cancer
cell lines (Feng et al. 2017), elicited structures capable of growth as various kinds of
spheroids by an alternative method called high-throughput single-cell derived sphere
formation. The method is like cloning but uses a sophisticated device to guarantee single cell
distribution in conditions favoring non-attachment (Chen et al., 2015). Cells that can
proliferate under such conditions interact with each other to form spheroids. In Feng’s
laboratory, cultures of “single cell-derived” tumor spheroids, appear to contain unicellular
(characterized by a rim of cytoplasm) hollow spheroids and multicellular (golf-ball-like)
hollow spheroids, as well as solid spheroids. Such results are consistent with the observation
in Ishikawa endometrial cells that a unicellular hollow spheroid is the starting point for
differentiation of a multicellular hollow spheroid (Fleming, 2019). Multicellular hollow
spheroids may well proliferate inward to form solid spheroids, a process observed for
pancreatic spheroids (Feng et al., 2017) but not yet observed in Ishikawa spheroids. What has
been observed is that hollow spheroids can proliferate outward by forming additional
attached hollow spheroids (Fleming, 2018a).
Spheroids for other epithelial cell lines sometimes named according to tissue origins such as
neurospheres and mammospheres frequently arise in cancer cell lines from specific tissues. A
common observation with regard to spheroids is that cells from such structures form more
tumors in compromised animal models than the same number of the parent cells grown as a
monolayer. This observation has led to the suggestion that spheroids are enriched for
tumorigenic cells sometimes called tumor stem cells (Dontu et al.2003). Studies linking
spheroids to tumorigenicity include research done with ovarian cancer cell lines. As few as
2000 cells from a dissociated spheroid of epithelial ovarian cancer cells, injected into immune
deficient mice, elicited the formation of a tumor. Even a bolus of 10,000 parent cells from
monolayers (even monolayers resulting from the outgrowth of solid spheroids) was not found
to be as tumorigenic (Liao et al., 2014). In research that supports the tumorigenic capacity of
spheroid cells, Espina et al. (2014) showed that mammospheres arising spontaneously from
human ductal cancer tissue fragments consistently generated mammary xenograft tumors in
an immune-compromised mouse.
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In Ishikawa endometrial cells, the formation of mitonucleons and the ensuing generation of
gas vacuoles converts a monolayer cell into a floating spheroid with giant polyploid nuclei
pressured within a rim of cytoplasm between the outer membrane of the mitonucleon and the
cell membrane, fairly exotic as cells go. If fetal calf serum is added to medium containing
such structures, the spheroid can reattach, releasing bubbles of gas as it does so and forming
an attached cell with one or more giant nuclei (Fleming, 2018a). Without that intervention,
activity within the cytoplasmic rim of unicellular hollow spheroids, apparently initiated by the
generation of small vacuoles in the region of polyploid giant nuclei, converts typical giant
nuclei into chromatin granules that can be observed streaming through the cytoplasmic rim
(Fleming, 2019). The chromatin granules become an array of filaments, open no doubt to
essential epigenetic changes, and then re associates to form colonies of nuclei that separate,
become bounded by membranes and ultimately fill the cytoplasmic rim of a hollow spheroid
in a structure some have called “golf ball” spheroids. (Fleming, 2019).
Mitonucleon involvement in metastases
The observation that the mitonucleon is responsible for hollow spheroid formation in Ishikawa
endometrial epithelia (Fleming, 2018a), together with the fact that spheroid cells have been
frequently shown to be more tumorigenic than monolayer cells, suggests circumstantially that
mitochondria or some form of fused mitochondria, characteristic of mitonucleons, are
involved in tumorigenicity. More direct evidence for mitochondrial involvement in cancer
metastases has been provided by some very clever experiments. Porporata and colleagues
(2014) pursued the existence of a metabolic phenotype associated with tumor metastases in
their ovarian carcinoma cell line. Their selection process resulted in the creation of a cell
line that was significantly more invasive than the parental cells and that contained giant
mitochondria not observed in the parent cell. In another approach, Farnie and associates
(2015), studying breast cancer cell lines, used a fluorescent tag to separate out cell
populations with higher than average levels of mitochondrial material (mito high) and those
with lower than average mitochondrial material (mito low). Their mito high breast cells
formed mammospheres more efficiently and showed a 2.4 fold enrichment in tumor-initiating
cell activity over the mito-low cells. And, in an approach dependent on membrane tubules, Lu
and colleagues (2017) demonstrated that the transfer of mitochondria from a highly invasive
kidney cancer cell line to a less invasive kidney cell line via tubules increased the invasiveness
of the latter.
In an important and possibly underappreciated observation, Tamura and colleagues (2011)
studied hollow spheroids just beyond the leading edge of invasive colon cancer. The
researchers looked for correlations between the presence of hollow spheroids and the
clinicopathological characteristics of 314 patients with colorectal cancer from whom the
cancers were removed. By serial cross-sectioning they identified 96 patients having complete
hollow spheroids beyond the invasive front. Furthermore, the research team demonstrated
that the presence of hollow spheroids was an independent risk factor for metastases and
predictive of shorter survival time for patients. The invasion appears to involve lymph nodes
Tamura and colleagues suggest that solid spheroids invaded normal tissue and were hollowed
out by apoptosis. Results with Ishikawa cells suggest an alternative, possibly more efficient
process. An invading mitonucleon could be responsible for generating a hollow spheroid if
delivered by a membrane tubular process from a structure that has invaded lymph fluid.
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Results in this paper show that chromatin and mitochondria in endometrial tubules can form
mitonucleons, and that tubular cellular bridges can accommodate the relatively compact
mitonucleon whose width is not much larger than that of a nucleus. Once it has been
introduced, the mitonucleon can expand into a single cell hollow spheroid and then into a
multicellular hollow spheroid (Fleming 2016 a,b,c and Fleming 2019) making it a little like a
“cellular trojan horse.” Mitonucleons that generate hollow spheroids would represent a
previously unknown invasive structure, something of a hybrid between the well documented
forms of single tumor cell and collective tumor cell invasion (Khalil et al.,2017)
Are invading hollow spheroids precursors to micropapillary cancer?
It may be relevant to this discussion that in colon, as well as in other cancers, nests of
epithelial cells are found whose growth pattern suggests origins different from surrounding
cancer cells, a phenomenon that has been called micropapillary cancer. Interest in
micropapillary cancer has been growing because in addition to colon, it has been observed in
lung, breast, urinary bladder, stomach and other cancers (Vyas et al.,2019). As reported by
Haupt and colleagues (2007), micropapillary carcinoma is an aggressive variant of cancer
associated with frequent lymphovascular invasion and poor clinical outcome. Having
reviewed approximately 375 cases of colorectal cancer, Verdu and colleagues (2011) detected
a micropapillary component in cancers for 60 patients. Noting a greater depth of invasion and
more positive lymph nodes in cancers with a micropapillary component the authors concluded
that colorectal micropapillary carcinoma is more aggressive than conventional colorectal
adenocarcinoma.
In addition to Tamura’s observations of hollow spheroids invading normal tissue, other
sightings have suggested that hollow spheroids are involved in human cancers. In 1987, Allen
and colleagues reported on multicellular aggregates found in the peritoneal fluid of patients
with ovarian cancer, describing clusters that included “spheroids with a central lumen
surrounded by a cell monolayer.” Such hollow spheroids were subsequently demonstrated
capable of giving rise to monolayers (Burleson et al., 2006). Reviewing research on spheroids
found in malignant ascites, Shield et al.(2009) characterized them as capable of tumorgenesis
in vivo
and as exhibiting a reduced response to chemotherapeutic drugs when compared to
monolayers in vitro
.
Hollow spheroids have been harder to find in the blood of cancer patients despite extensive
studies demonstrating that tumor cells and tumor cell clusters are present in a significant
proportion of patients with cancer, both before and after the removal of a primary
tumor.(Allard et al., 2004). Denes and colleagues (2017) observing that most available
methods to detect cancer tumor cells include steps that destabilize or eliminate spheroids,
developed a new light scatter flow cytometry blood test that allowed them to search for and
find hollow spheroids in 46.3% of metastatic patients but not in the blood of normal subjects,
nor in patients with non-metastatic cancer. Finding evidence of regions of hypoxia in these
structures, a condition favoring metastases, the research team suggested that hollow
spheroids might afford a premetastatic niche for tumor stem cells. Along these same lines,
Hou and colleagues have suggested that tumor stem cells in circulating clusters might have a
survival advantage (Hou et al. 2017).
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Are clusters of hollow spheroids involved in metastases?
The characteristics of a unique structure of clustered hollow spheroids is described for the
first time in this paper. An obvious question is whether anything like it has already been seen
in vivo
, an inquiry that turns up a surprisingly rich history of research on tumor cell clusters
mostly isolated from blood, and stretching back more than 50 years (Hong et al.2016; Umer et
al.,2018). Almost consistently, starting with Watanabe in 1954, researchers have concluded
that tumor cell clusters are more likely to contribute to metastases than single tumor cells.
Liotta et al. (1973) showed that the injection of single tumor cells and tumor cell clumps (or
clusters as they have been called recently) resulted in many metastatic foci, but clumped
tumor cells produced a significantly greater number of metastatic foci than did the same
number of cells in single-cell form and larger-sized clumps produced still more metastatic foci
than smaller-sized clumps. Aceto and colleagues (2014) presented evidence that tumor cell
clusters are up to 50- fold more likely to form metastases. Furthermore they demonstrated
that the clusters detach as such from tumors, rather than forming from single tumor cells in
the bloodstream, suggesting that their cohesion may identify a novel and potentially
targetable step in the blood-borne dissemination of cancer. The relevance of tumor cell
clusters in metastases was recently reviewed by Cheung & Ewald (2016) with a model of
metastatic dissemination that highlights the activities of clusters of tumor cells that retain
epithelial properties. Overall, there appears to be recognition that at least some tumor cell
clusters may be more than the sum of their parts relating perhaps to discussions about the
distinct phenotypic and molecular characteristics of cells in tumor clusters or microemboli in
comparison with single cancer tumor cells (Umer et al.,2018).
Single hollow spheroids were difficult to detect in vivo
because of their relative “fragility,”
and it is probable that spheroid clusters are similarly fragile. In fact, the cluster in fig. 14
appears to be deflating, a fact that provided us with evidence of its intriguing internal
structure but might ultimately result in a structure that is no longer recognizable.
Additionally, It has long been understood that blood-borne cancer cells and cancer cell
clusters can get hung up in capillaries as they pass through the blood system. On the basis of
size alone, clusters of hollow spheroids released in vivo
would be expected to quickly become
sequestered. In one recent study, comparing numbers of cancer tumor cells in portal and
arterial blood during a pancreatic tumor resection, a significant uptake (approximately 40%)
of putative cancer cells was demonstrated by researchers, who suggested that tumor cell
structures might have become caught up in liver or lung compartments (Vilhav et al.,2018).
As was true in the detection of single hollow spheroids, the best place to begin to look for
delicate structures such as these may well be in carefully handled malignant ascites fluid. It
is relevant that Kashima and associates (2010) examining ascites fluid in a woman with cancer
of the large intestine described small papillary clusters with a smooth surface showing
peripherally located cytoplasm surrounding a rare central lumen. The researchers dubbed
these "inside-out" cell clusters and concluded that the patient had micropapillary cancer.
Given a clear, concise description that is so evocative of hollow spheroids, it is reasonable to
wonder whether clusters of hollow-spheroids might also be found.
In the long history of looking for tumor cell clusters in the blood of cancer patients (Hou et
al., 2017; Umer etal., 2018),it is possible that research groups have found structures whose
silhouettes resemble that of a hollow spheroid cluster. Larger clusters are sometimes called
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CTM an acronym for circulating tumor microemboli. As new methods of isolation, reviewed
by Umer and colleagues (2018) continue to be tested, the potential destruction of cancer
cells and clusters is an ongoing concern (Yu et al., 2011). Glaves and associates (1988) studied
dissemination of cells from human renal adenocarcinomas using density gradients, a gentler
method than some separation strategies currently used. The pictures published as a result of
this study contain at least one example of a structure that could correspond to the hollow
spheroid clusters shown in this paper. Staining for endogenous biotin such as was done for
the cluster of hollow spheroids in fig.10 might be of some help in making an identification.
Molnar and colleagues (2001) similarly found clustered tumor cells in the peripheral blood of
colon cancer patients. Furthermore, they identified what they characterized as dendritic-like
processes emanating from such clusters, leaving us with a tantalizing “maybe” as to whether
what they identified bears any similarity to the structures shown in this paper.
Hollow spheroid clusters have distinct phenotypic characteristics that might support a role for
them as metastatic agents. Foremost, upon release from the monolayer they can be
transported in fluids. Yet, as is shown in figs.15 and 16, they possess abundant resources to
reattach and send out filopodia, believed by some to “drive cancer cell invasion” (Jacquemet
etal., 2015). An additional relevant question would be whether structures like this could
become dormant, perhaps because of adaptive metabolism, surviving in metastatic niches for
years before “springing” to life? With all the work underway to study tumor cell clusters,
researchers might want to keep in mind the potential importance of hollow spheroids and
clustered hollow spheroids. If such “inside” out cell structures comprise at least one kind of
metastatic structures, their vulnerability can be probed in vitro
. It might be possible to
identify a toxin that attacks the particular physiology of the “inside” out cell structures,
inhibiting their ability to spread cancer.
Materials and Methods
The subject of this paper is the formation of tubular membrane protrusions by Ishikawa endometrial epithelial cells. Tubules
have been observed to form in monolayers as well as to extend from solid spheroids. Examples are shown in figs. 1 through 7.
The tubules carry granules that contribute to “building” nuclei and they carry mitonucleons, transient organelles essential to the
formation of predomes (Fleming, 2016a,b,c) and unicellular hollow spheroids(Fleming, 2018a). In both of those examples,
vacuole formation within the mitonucleon compresses chromatin aggregates and initiates the amitotic process of chromatin
streaming. The revelation in this paper is that mitonucleons can transit through tubules. Whereas figs. 1 through 5 are
photomicrographs of living culture, the mitonucleon was detected by fixing and staining cultures containing membrane tubules.
Fixation was achieved by adding 4% paraformaldehyde 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 washing, cells were exposed to
a1:200 dilution of Extravidin conjugated horse-radish peroxidase (HRP) (Sigma) 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 .25% H2O2. This solution was incubated at 37oC
for 45 min to allow color to develop. The AEC solution was removed, and the cultures were examined and then stored in the
presence of PBS at 4oC. Controls were carried out to ensure that the stain was reacting specifically with endogenous biotin. 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 antibody linked to horseradish peroxidase. Staining occurred under all circumstances, indicating that avidin does
indeed react with biotin that is endogenously present in the cell in significant amounts. The endogenous biotin had previously
been shown to be associated with mitochondrial carboxylases. (Fleming etal., 1998)
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Cells for all of these experiments were grown in phenol-red free Minimal Essential Medium with 10% calf serum. Additionally 2
mM glutamine was added. Culture conditions eliciting formation of tubules were similar to those used by Lou and colleagues
(2014) except that phenol-red-free Minimum Essential Medium contained no serum, but did contain 2 mM glutamine in addition to
fresh glucose. Serum-free medium was added to confluent monolayers, a condition that stresses the cells and result in the
formation of tubular membrane extensions. Solid spheroids were formed when trypsinized Ishikawa monolayers were
resuspended in serum-free medium in petri dishes . Spheroids formed in that manner were carefully transferred to dishes to
which additional fresh serum-free medium was added Spheroids were monitored for the formation of membrane tubular
extensions. Solid and hollow spheroids were transferred into petri dishes using techniques least likely to disrupt their delicate
three dimensional structures. In point of fact, medium bathing the monolayer was simply decanted, carefully in a laminar flow
hood. Some of the hollow spheroids maintained without serum spontaneously initiated amitotic cell proliferation as previously
described (Fleming, 2019). Cells were viewed using an Olympus inverted stage microscope at powers of 100X, 200X and 400X.
Tubule formation was observed in cultures starved for serum. The most dramatic tubule forming activity was observed for
clustered Ishikawa hollow spheroids, a structure whose formation is documented in the second half of the paper. The formation
of a cluster of hollow spheroids follows almost the identical process for the formation of a single hollow spheroid (Fleming,
2018a). As was true for the single hollow spheroid, its formation proceeds from the formation of mitonucleons except that
multiple cells containing mitonucleons detach together, remain together as a cluster, and eventually form a structure with the
appearance of a beehive, gaseous vacuoles surrounded by rims containing nuclear material within cytosol, including mitochondria
as detected by the presence of endogenous biotin.
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