Content uploaded by Mónica Suárez Korsnes
Author content
All content in this area was uploaded by Mónica Suárez Korsnes on Mar 25, 2017
Content may be subject to copyright.
Case study on application of computer based long term
non-intrusive single cell tracking:
Mitotic catastrophe in BC3H1 cells following yessotoxin
exposure
Monica Suarez Korsnes1, and Reinert Korsnes2,3
Norwegian University of Life Sciences (NMBU) Campus Ås, Norway
P.O. Box 5003,NO-1432 Ås, Norway
Norwegian Defence Research Establishment (FFI), Kjeller, Norway
P.O. Box 25,N-2027 Kjeller
1monica.suarez.korsnes@nmbu.no
2reinert.korsnes@.no
3reinert@korsnesbiocomputing.no
INTRODUCTION
Yessotoxin (YTX) is a marine algal toxin. It is a polycyclic ether compound produced by
dinoflagellates that accumulate in filter feeding molluscs (1-3). This toxin can cause various
cytotoxic effects depending on cell type and cell line (4). It is well known to induce multiple cell
death pathways which may overlap or cross-talk (5). The present contribution provides the first
evidence that YTX can cause genotoxicity and induce mitotic catastrophe which can lead to
different types of cell death. This work also demonstrates potential information gain from non-
intrusive computer-based tracking of many individual cells during long time (6). YTX treatment
of BC3H1 cells during their exponential growth phase causes atypical nuclear alterations and
formation of giant cells with multiple nuclei and micronuclei. These are the most prominent
morphological features of mitotic catastrophe. Activation of checkpoint kinases indicates DNA
damage response and cell cycle deregulation. Data from tracking single cells reveal that YTX
treatment suppresses a second round of cell division in BC3H1 cells. These findings suggest
that YTX can induce genomic alterations or imperfections in chromosomal segregation leading
to permanent mitotic failure. This understanding extends the list of effects from YTX and which
are of interest to control cancer and tumour progression.
AIM OF THE STUDY
An intention for this work is to lay out a case study to explore individual cell tracking as a tool
in toxicological research. Individual cell tracking is a developing technology which can
potentially provide fast diagnosis of changes in cell populations for example due to toxic
insults. It can also help to guide hypothesis formulation in pilot studies.
MATERIALS AND METHODS
BC3H1 cells were treated with 100 nM purified YTX or 0.2% methanol (controls) and analysed
by time lapse, flow-cytometry and microscopy. BC3H1 cells were plated onto 96 multiwell black
microplates (Greiner Bio-One GmbH, Germany) for time-lapse imaging. Cells were cultured in
medium (DMEM with phenol red, containing and 20\% fetal bovine serum). Cells were imaged
into Cytation 5 Cell Imaging Reader (Biotek, USA), with temperature and gas control previously
set to 37°C and 5% CO2 atmosphere. Sequential imaging of each well was taken using 10x
objective. The bright and the phase contrast imaging channel was used for image recording. A
continuous kinetic procedure was chosen where imaging was carried out with each designated
well within an interval of 5 min. for a 96 h incubation period. All cells initially inside a 580 µm X
580 µm square were subject to tracking during 30 hours using the experimental Kobio Cell
track system, https://www.korsnesbiocomputing.no. This system is based on Ada 2012 and
OpenGL.
RESULTS
This study shows results from exposing BC3H1 cells to 100 nM YTX where they due to a
spatially sparse distribution have minor contact inhibition of proliferation (Fig1). Individual cell
tracking indicates that YTX exposure then seems to lock the cells into an anti-proliferative state
after their first division (Figs 2,3) leading to the hypothesis that YTX can be genotoxic.
Additional biochemical analyses as well as morphological inspections evidence that YTX
treatment induces a permanent mitotic failure leading to mitotic catastrophe. Data from tracking
single cells allowed to visualize multipolar and assymetric cell divisions (Figure 4). The most
prominent morphological traits of mitotic catastrophe such as giants cell with multiple nuclei
and micronuclei were also observed (Figures 5,6).
Figure 6. Phase contrast image and Hoechst staining (blue fluorescent
color) of BC3H1 cells exposed to 100 nM YTX for 24, 48, 72 h. White
arrows show multiple nuclei in YTX-treated cells. The number of
multinucleated and polyploid cells tends to increase upon YTX
treatment.
References
1. Satake, M, MacKenzie, L. and Yasumoto, T. Nat. Toxins, 4 (1997)
164-167.
2. Draisci, R, Ferretti, E, Palleschi, L, Marchiafava, C , Poletti, R,
Milandri, A, Ceredi, A. and Pompei, M. Toxicon , 37 (1999) 118–1193.
3. Ciminiello, P, Dell'aversano, C, Fattorusso, E, Forino, M, Magno, S,
Guerrini, F, Pistocchi, R. and Boni, L. Toxicon, 42 (2003) 7–14.
4. Korsnes, M. S. and Espenes, A. Toxicon, 57 (2011) 947–958.
5. Korsnes, M.S, Espenes, A, Toxins, 4 (2012) 568—579
6. Korsnes, M S, Korsnes, R. Front. Bioeng. Biotechnol. (2015).3, 166.
Figure 5. Nuclear alterations visualized with Immunofluorescence labelling
of BC3H1 cells exposed to 100 nM YTX using LysoTracker Red DND-99
Staining. Upper row represents control cells. Second, third and fourth row
respectively represent cells after 24, 48, 72 h of exposure. Green arrows
show the chromosomes lined up across the equator of the spindle in
control cells. White arrows point to the micronuclei. Yellow arrows show
abnormal chromosomal segregation.
Figure 3. Cumulative distributions for time from start of exposure to first cell division for
control and exposed cells (derived from individual cell tracking). Note that exposed cells tend
to divide at least as frequent as control cells up to about eight hours from start of exposure.
This result illustrates potential methods for early detection of premature cell division based on
more extensive cell tracking as demonstrated in the present work.
Acknowledgments
This work was supported by the Norwegian University of Life
Sciences (NMBU), Department of Molecular and Cell Biology, Olav
Raagholt and Gerd Meidel Raagholts and Eckbos Legacy.
Figure1. Start images for cell tracking for 30 hours. Upper: control cells, Lower:
exposed cells. Frame size: 580 µm x 580 µm..
This work demonstrates the potential to gain information from tracking single
cells from image recordings. Following many single cells over time can
facilitate fast and robust detection of change in cell polulations due to for
example toxic exposure. Observing individual cell fate can also help to identify
novel molecular mechanisms that could be tested for therapeutic applications
of medical relevance.
CONCLUSIONS AND FUTURE PROSPECTS
Figure 2. Subset of pedigree trees for control cells (left) and exposed cells (right) as
derived from tracking all cells initially inside a 580 µm x 580 µm square during 30 h
from start of exposure. This gave 62 (51 complete) and 61 (56 complete) pedigree
trees respectively for the control and exposed cells. Twelve (12) initial (root) control
cells did not divide during the observation period and 27 exposed cells similarly did
not divide. Red, blue and green bullets represent first, second and third generation
cell division. Note that exposed cells tend only to divide one time (red bullet) if they
do not divide soon after start of exposure. The observed pedigree trees for exposed
cells contain no cell division after 20h.
Figure 4. Time-lapse video microscopy showing multipolar cell division of BC3H1 cell
exposed to 100 nM YTX. Asymmetric cell division showing generation of three
daughter cells.
This poster is an extract from Korsnes and Korsnes 2017, “Mitotic catastrophe following yessotoxin exposure”, Front.
Cell Dev. Biol. In press.