Download Tracking mitotic defects via time-lapse photography

TRACKING MITOTIC DEFECTS VIA TIME-LAPSE PHOTOGRAPHY
by
Joseph Daniel Williams
A Thesis Submitted to the Faculty of
The Wilkes Honors College
in Partial Fulfillment of the Requirements for the Degree of
Bachelor of Arts in Liberal Arts and Sciences
with a Concentration in Biology
Wilkes Honors College of
Florida Atlantic University
Jupiter, Florida
May 2013
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TRACKING MITOTIC DEFECTS VIA TIME-LAPSE PHOTOGRAPHY
by
Joseph Daniel Williams
This thesis was prepared under the direction of the candidate’s thesis advisor, Dr.
Nicholas J. Quintyne, and has been approved by the members of his supervisory
committee. It was submitted to the faculty of The Honors College and was accepted in
partial fulfillment of the requirements for the degree of Bachelor of Arts in Liberal Arts
and Sciecnes
SUPERVISORY COMMITTEE:
__________________________
Dr. Nicholas J. Quintyne
__________________________
Dr. Chitra Chandrasekhar
__________________________
Dean Jeffrey Buller, Wilkes Honors College
___________
Date
ii
ABSTRACT
Author:
Joseph Daniel Williams
Title:
Tracking Mitotic Defects via Time-Lapse Photography
Institution:
Wilkes Honors College of Florida Atlantic University
Thesis Advisor:
Dr. Nicholas J. Quintyne
Degree:
Bachelor of Arts in Liberal Arts and Sciences
Concentration:
Biology
Year:
2013
As tumors generate, there is a progression in genomic instability derived from
chromosomal rearrangement and instability. Often, these manifest themselves as defects
in mitosis, frequently as lagging chromosomes, multipolar spindles, and anaphase
bridges. Lagging chromosomes are the result of inaccurate chromosomal division in
mitosis, thus jeopardizing the genome of an organism’s offspring; they derive from
several errors, such as failure of a chromosome to attach to the mitotic spindle. The goal
of this project has been to characterize the mechanisms of lagging chromosomes in the
cancer cell line UPCI:SCC103. Our laboratory’s work has shown that treatment with
certain carcinogens increase the rate of mitotic defect. To further our understanding these
defects, we are monitoring the progression of lagging chromosomes in UPCI:SCC103
cells with live cell analysis, using GFP-tagged histone H2B to track their appearance and
fate, so to distinguish between the possible causes and resolutions of this mitotic defect.
iii
DEDICATIONS
To my mother and father
To Dr. Quintyne
To my goodness, and my fortress;
my hightower and my deliverer;
my shield and whom I trust.
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TABLE OF CONTENTS
List of Figures…………………………………………………………………….vi
Introduction………………………………………………………………………..1
Methods……………………………………………………………………………6
Results……………………………………………………………………………..8
Discussion………………………………………………………………………..16
References………………………………………………………………………..20
v
LIST OF FIGURES
Figure 1……………………………………………………………………………………8
Figure 2……………………………………………………………………………………9
Figure 3……………………………………………………………………………………9
Figure 4……………………………………………………………………………….10-11
Figure 5…………………………………………………………………………………..12
Figure 6…………………………………………………………………………………..13
Figure 7…………………………………………………………………………………..14
Figure 8 …….…………………………………………………………………..………..15
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INTRODUCTION:
Cancer is a major public health problem that according to the National Cancer
Institute is diagnosed in more than 1 million Americans every year. As people age, they
are more likely to suffer from cancer and as our elderly population increases so does the
expectation that more Americans will be diagnosed as well (NCI). This fact is a direct
result of the nature of cancer to acquire mutations over extended periods of time that give
it the very harmful characteristics it possesses.
Cancer develops from normal cells that have gained certain proliferative and
immortalizing advantages through mutations. Normal healthy cells found in the body are
kept in control by homeostatic mechanisms that govern cell growth and death when
appropriate. If these mechanisms are misexpressed or damaged through mutations in the
genome of the cell, the cell will take on a pattern of gene activation that is unlike its
previous state and now poses a threat to the organism as a whole (Bertram, 2000).
Mechanisms that are involved with proliferation and cell death can sustain
damage which result in gene expression patterns that stimulate proliferation and protect
against cell death. Genes that confer these abilities due to inappropriate activation are
known as oncogenes. Activating oncogenes is a very important step of carcinogenesis
(creation of cancer) and represent either endogenous genes or exogenous genetic material
(Diamandis, 1997). In contrast to this, there are tumor suppressor genes which normally
inhibit proliferation until inactivation leads to a loss of function (Weinberg, 1991).
Sometimes in unison and at other times not, these two modes of misregulated gene
expression will contribute to the transformation of a healthy cell into a cancer cell.
Evidence suggests that there are six known pathways that cancer cells must disrupt.
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These mechanisms include sustained proliferative signaling, evasion of growth
suppressors, activation of metastatic ability, enabling of cell immortality, inducing
angiogenesis, and resistance to cell death (Hanahan & Weinberg, 2011). What remains
consistent through all of these is the need for an alteration in the genomic integrity of a
cell.
With all of these new abilities, the reoccurring event that renders DNA vulnerable
to damage is mitosis. After the chromosomes are replicated in S phase of the cell cycle,
mitosis occurs after further growth and preparation for division. Mitosis occurs to divide
a cell into two daughter cells and cancer cells are known to take advantage of the
mechansims that induce it as a part of their characteristic abnormal cell proliferation.
Normally, cells go through six stages in mitosis. In these stages, chromosomes are
condensed and segregated to become the genetic material for two equivalent cells. The
stages include prophase, prometaphase, metaphase, anaphase A, anaphase B, and
telophase with an additional process called cytokinesis. All of these stages are carried out
for the purpose of symmetrically separating replicated DNA into new cells. However, in
this process they can be exposed to agents and conditions that may increase the likelihood
of damage. High amounts of damage are thought to be linked to abnormalities that cancer
cells exhibit such as aneuploidy and chromosomal instability.
Chromosome missegregation is a main trait of cancerous cells that lends to their
ability to alter their gene expression and promote genetic diversity (Nicholson, 2011).
Missegregation is usually defined by chromosomal instability (CIN) and aneuploidy. The
relationship between CIN and aneuploidy is more easily understood in terms as a rate and
state; CIN is defined as a persistently high rate of loss and gain of whole chromosomes
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where aneuploidy refers to the abnormal number of chromosomes contained in a cell
(Thompson, et.al. 2010).
Throughout the mitotic cycle in cancer cells, defects arise as evidence of
inappropriate segregation of cells. More specifically, they are visually indicative of the
loss of genomic integrity of a cell and damage upon DNA through breakages or
misplacing chromosomes. Lagging chromosomes, anaphase bridges, and multipolar
spindles are examples of these defects and are characteristic of CIN.
A cell with lagging chromosomes will have a number of chromosomes or
fragments of chromosomes seemingly isolated from the main mitotic mass of
chromosomes. During mitosis, all of chromosomes in the nucleus condense and migrate
to the center of the cell and then are segregated to the peripheries of the cell when mitotic
machinery attaches them. Improper attachments of microtubules to the anchoring sites on
microtubules, called kinetochores, result in missegregation (Thompson, 2010).
Anaphase bridges are the result of two broken ends of chromosomes rejoining to
form a dicentric chromosome. During subsequent progression of mitosis, these dicentric
chromosomes may break again and leave behind fragments that are very similar to
lagging chromosomes. These mitotic defects may occur due to failure in the machinery
that performs the actual segregation of chromosomes or in failure in the DNA repair
mechanisms. Breakage fusion bridge cycles are mechanisms of DNA repair that,
although useful in maintaining genomic integrity of the cell, can lead to the inappropriate
joining of broken ends of chromosomes to one another (McClintock, 1941; Gisselsson et
al., 2000;, Saunders et al., 2000). Multipolar spindles are similar but involve the mitotic
apparatus as a whole. In this defect, an increase in spindle pole number creates a situation
3
in which a cell will divide its chromosomes amongst an increased number of progeny
cells; a cell that contains three spindle poles will divide into three new cells. Usually this
means one cell for each additional spindle pole where spindle poles are referred to as the
cellular location at which the microtubule organizing centrosomes assemble the
cytoskeletal components that drive migration of chromosomes (Brinkley, 2001).
This research of this thesis was conducted in order to characterize the mitotic
defects in UPCI:SCC103 with specific focus on lagging chromosomes. Using time-lapse
photography and fluorescence microscopy, the goal was to catalogue a series of images
that may elucidate reasonable mechanisms involved in the missegregation of
chromosomes in the hopes of finding evidence for chromosomal rescue and resolvability.
These terms are mainly used to give a name to the possible mechanism by which a cell
may restore some semblance of normal chromosomal segregation in mitosis. In tracking
these defects and possible resolvability, it was also a goal to be able to hypothesize more
on the order in which mitotic defects arise.
Another focus of this research was to try and induce mitotic defects by creating
abnormal gene expression. Using shRNA as a method to mediate knockdown in genes
specific to intracellular transport, it was our goal to try to observe possible genetic bases
by which mitotic defects could be agitated. Genes that were used were targeted were
Arp1, p150Glued, p27, and KIF5A.
Arp1 is an integral subunit of the dynactin complex which is involved in the
mechanism by which dynactin binds to a variety of subcellular structures. Dynactin
works alongside dynein which is a motor protein associated with intracellular transport.
Along with Arp1, p150Glued and p27 are also subunits of dynactin where p150Glued is
4
thought to mediate interactions with microtubule-based motors and p27 which is thought
to act as an adaptor protein that engages dynactin to other subcellular structures
(reviewed in Schroer, 2004). KIF5A is member of kinesin family proteins that are
microtubule-dependent molecular motors important in neuronal function (Nakajima,
2012). All of these genes were chosen because of their association with intracellular
transportation in the cell which hypothetically may contribute to possible mechanisms of
chromosomal resolvability.
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METHODS:
Cell Culture.
The oral squamous cell carcinoma line UPCI:SCC103 was acquired from the
University of Pittsburgh Cancer Institute. The cells were grown in 10 mL of M10
medium: Dulbeccos’s Modified Eagle’s medium (Sigma Chemical Company; St. Louis,
MO) with added 10% FBS (Hyclone; Logan, UT), L-Glutamine (MP Biomedicals, Solon,
OH), Gentamycin (MP Biomedicals), and non-essential amino acids (Chemicon,
Temecula, CA). Cells were seeded from progenitor plates following a 2-3 mL PBS wash,
followed by incubation with 0.05% trypsin-EDTA solution (MP Biomedicals) for 5
minutes at 37oC. These cells could then be divided to seed new plates, to make
coverslips, or seed onto glass bottom petri dishes.
Fixed Cell Analysis
Cells were seeded onto 22 mm2 coverslips at an initial density of 4.5 x 105 cells
per coverslip and allowed to incubate 24 hours. They were then treated with 2 mL of 20oC MeOH for 5 minutes, and then treated with 4,6-diamidino-2-pheylindole (DAPI;
Sigma) for 30 seconds to stain the chromatin. The coverslips were then mounted onto
glass slides with phenylene diamine (Sigma) dissolved in glycerol and sealed with nail
polish with color preference given to fire hydrant red, or bubblegum pink, when
available. Cells were observed through an Olympus IX-81 Inverted Fluorescence
Microscope (100x oil-immersion objective; N.A.=1.65, Olympus America, Inc.; Center
Valley, PA). Images were captured using Hammamatsu C4742-95 High resolution
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Cooled-CCD Camera (Hammamatsu; Bridgewater, NJ) and Slidebook version 5.0
software (Intelligent Imaging Innovation Inc., Denver, CO).
Gene Knockdown Transfection
100 μL of OPTI-MEM (Life Technologies, Grand Island, NY) per cover slip was
added to a snap cap tube. FuGENE (Roche, Indianapolis, IN) transfection reagent was
added for Arp1, p150Glued, p27, and KIF5A at 3 μL per slip. Liquid was mixed by tapping
the tube and allowed to sit for 5 minutes. The shRNA concentration was then added to
the tube in the following quantities: for Arp1 0.46 μL at 2.45 g/ μL, for p150Glued 10.36
μL at 0.11 g/ μL, for p27 0.5 μL at 2.3 g/μL, and for KIF5A 0.83 μL at 1.38 g/ μL.
Components were mixed by tapping tubes again and allowed to sit for 15 minutes.
Respective volumes were added to each cover slip and incubated for 24 hours before
DAPI stain was added as above.
Live Cell Analysis – GFP Expression
Cells were seeded on glass bottom 35 mm2 petri dishes at a density of 4.5 x 105
cells/coverslip and 2 μL of CellLight Histone H2B (BacMAM 2.0, Life Technologies)
was immediately added. The dishes were allowed to incubate for 72 hours. Cells were
observed via microscopy as above. Time-lapse images were captured at rates of 1-5
image/minute for varying amounts of time that were dependent upon decreasing GFP
expression.
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RESULTS:
Tracking Defects in UPCI:SCC103:
One focus of this thesis was to examine cancerous cells during mitosis for
evidence of resolvability and rescue of chromosomal defects with emphasis on lagging
chromosomes. Many time-lapse images were capture and the most promising slides
followed and movements of individual chromosomes tracked. First, however, it was
important to gain an understanding of what the different mitotic defects were and how
they appeared under the microscope. From DAPI staining of UPCI:SCC103 oral
carcinoma cells, very defining images were found. Figures 1, 2, and 3 represent the
characteristic images that would have been counted when scoring mitotic defects.
Figure 1 – Lagging Chromosome in Mitotic UPCI:SCC103
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Figure 2 – Anaphase Bridge in Mitotic UPCI:SCC103
Figure 3 – Multipolar Spindle in Mitotic UPCI:SCC103
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After becoming familiar with the different mitotic defects, the next phase of research was
to collect visual evidence of mitotic cells that may be exhibiting chromosomal
resolvability and rescue. Figure groups 4 through 6 represent the best series of images
collected. In them, green and blue dots indicate chromosomes of interest.
Figure 4a – Lagging Chromosome Rescue
Figure 4b – Lagging Chromosome Rescue
Figure 4c – Lagging Chromosome Rescue
Figure 4d – Lagging Chromosome Rescue
Figure 4e – Lagging Chromosome Rescue
Figure 4f – Lagging Chromosome Rescue
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Figure 4g – Lagging Chromosome Rescue
Figure 4h – Lagging Chromosome Rescue
Figure 4i – Lagging Chromosome Rescue
Figure 4j – Lagging Chromosome Rescue
The images from Figure 4 show image captures in roughly 20 minute intervals of
a cell in metaphase. They appear to reveal two lagging chromosomes moving towards the
main mitotic mass. By the seventh frame, we see the chromosome on the top-right
continues its trajectory while its counterpart in the top-left takes a position further away
from the metaphase plate.
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The next images of a cell in mitotic progression in Figure 5 group (below) show a
chromosome that initially seems to be in the main mitotic mass. Upon further viewing,
the same chromosome retreats from its position to one that is more laterally left and away
from the metaphase plate.
Figure 5a – Lagging Chromosome
Figure 5b – Lagging Chromosome
Figure 5c – Lagging Chromosome
Figure 5d – Lagging Chromosome
Figure 5e – Lagging Chromosome
Figure 5f – Lagging Chromosome
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Figure 6 group of images (below) shows a cell in metaphase that appears to show
rescue of a lagging chromosome. Located in the bottom right, the chromosome clearly
moves to a lateral and vertical center in reference to the mitotic mass.
Figure 6a – Lagging Chromosome Rescue
Figure 6b – Lagging Chromosome Rescue
Figure 6c – Lagging Chromosome Rescue
Figure 6d – Lagging Chromosome Rescue
Figure 6e – Lagging Chromosome Rescue
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Figure 6f – Lagging Chromosome Rescue
Agitating Defects in UPCI:SCC103:
The second main focus of this thesis was to build a foundation by which the mode
and mechanism of lagging chromosome missegregation could be agitated through
knockdown experiments. It was a goal of particular interest to better characterize the
possibility of resolvability and rescue. In order to first do this, a detailed account of the
mitotic defects that occur in UPCI:SCC103 needed to be made. In separate counts in
which sample size of 475 mitotic cells were observed, the frequency of lagging
chromosomes, anaphase bridges, and multipolar spindles are reported (Figure 7).
Rates of Mitotic Defects in
UPCI:SCC103
40%
30%
20%
10%
0%
10.46%
8.63%
4.9%
Lagging
Chromosomes
Anaphase Bridges
Control
Multipolar
Spindles
Figure 7 – Differing Rates of Mitotic Defects in UPCI:SCC103
The next step in agitating mitotic defects was to induce knockdown expression of
genes involved in intracellular transport. The genes chosen for this experiment were
Arp1, p150Glued, p27, and KIF5A. Mitotic defects were then scored for each of the
knockdown populations (Figure 8).
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Figure 8 – Effects of Genetic Knockdown on Mitotic Defects in UPCI:SCC103
The graph reflects the observed differences in mitotic defects after each individual
knockdown. Arp1 and p150Glued were the only observable knockdowns that had an effect
on mitotic defects; 50 mitotic cells were sampled in each trial. However, in p27 and
KIF5A, less than 10 mitotic cells were observed in each trial with no noticeable defects
observed.
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DISCUSSION:
Tracking Defects in UPCI:SCC103:
In tracking mitotic defects using an oral cell carcinoma, a focus of this thesis was
to examine cancerous cells during mitosis in the attempt to find evidence of resolvability
and rescue of chromosomal defects with emphasis on lagging chromosomes. With this
evidence, continued research into the mechanism of lagging chromosome rescue may
provide the hope of therapies that may target these defects and slow the progression of
tumor formation that is present due to a compromised genome. What was observed in this
project may help in that process.
In Figure 4, there is plausible evidence for a mechanism by which a cell may
rescue lagging chromosomes which are present during mitosis. It is clear that two
chromosomes that are distal from the mitotic mass are migrating towards the center of the
metaphase plate (Figure 4a-g) but whether there is a mechanism that exists is unclear.
However, the chromosomes begin to deviate in their trajectories (Figure 4h-j) which
might suggest that for whatever reason both were migrating, one of the chromosomes
failed to properly continue on its course.
In Figure 5, a chromosome appears to lose its position and migrates away from
the center of the mitotic mass. It seems to take a mirrored trajectory compared to the
chromosome in Figure 4h-j and could possibly be a reenactment of the mechanism (or
failure of) that moved the chromosomes. Either way, it may lead to evidence of a specific
mode of migration or rescue.
Figure 6 shows an observation of a lagging chromosome that is migrating from a
more lateral position to a more centered one. What these images seem to portray is rescue
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of lagging chromosomes but they appear to move from a position that is similar to the
chromosomes in the previous figures. This may suggest that if rescue is occurring, the
positions of the chromosomes from the previous images are in fact in a place outside
from normal chromosomal migration. If this is indeed the case, then this might be
evidence that chromosomes lag, that they do this via faulty segregation mechanisms or
from the failure of a rescue mechanism, and that if in an abnormal position, chromosomes
can migrate to a normal one.
Agitating Defects in UPCI:SCC103:
As seen by the data collected from the preliminary-study knockdown trials of the
genes Arp1, p150Glued, p27, and KIF5A, there were mixed results. The knockdowns of
p27 and KIF5A using shRNA exhibited a severe decrease in mitotic cells; on average,
less than 10 were observed in total where 475 mitotic cells were viewed under control
conditions. However, results were obtained from Arp1 and p150Glued knockdowns and 50
mitotic cells were sampled at each observation. The absence of results in the p27 and
KIF5A may be inconclusive, however, the data concerning Arp1 and p150Glued may help
to clarify possible mechanisms of chromosomal rescue.
Arp1 and p150Glued are both subunits of the dynactin binding complex which
interacts with the dynein motor for intracellular, minus-ended directed transport that takes
its cargo to the center of the cell (Wang, 1995). As can been seen in mitotic cells,
chromosomes migrate toward the center of the cell during metaphase before mitosis
completes. With this in mind, there was an almost 300% increase in the occurrence of
lagging chromosomes in mitotic cells with the knockdown expression in contrast to the
17
characteristic occurrence in the control cells. It is possible that the rescue of migrating
lagging chromosomes is directed by the function of dynein motors. Arp1 and p150Glued
are integral pieces of dynactin binding functionality between dynein and cargo and the
data collected would support this if dynein motors are involved in a mechanism of
chromosome rescue.
The frequency of anaphase bridges stayed consistent between the knockdowns
and the control group. The frequency of multipolar spindles showed response to the gene
expression with more than 300% reduced occurrences in the Arp1 and p150Glued groups
compared to the control. It may be difficult to determine the cause of these results as
multipolar spindles and anaphase bridges were not a main focus of this thesis but perhaps
it gives information to the order of progression in mitotic defect formation. To speculate,
it may follow that lagging chromosomes are the first defect to occur or that as a result of
the appearance of a progenitor defect, lagging chromosomes build in frequency.
Solidifying what has been gathered from this thesis may still need further
experimentation but it seems probable that evidence for chromosomal rescue can be
hypothesized from the experiments performed. What would need to occur to add more
weight to the assumptions made are observations of full mitotic cycles, over expression
of the dynactin complex, and further trials of the previous experimentation.
The data collected was limited due to the conditions by which the cells were
observed which were not at an optimal, incubation-like setting. There is equipment
available that allows for cells to have an adequate environment for proliferation and
growth, such as heated or enclosed microscopes, and such equipment would be ideal for
18
further research. It would allow further observation to fully understand the fate of lagging
chromosomes and whether or not they resolved.
In an effort to understand the exact mechanism and cellular machinery that may
lend to the rescue of lagging chromosomes, an introduction of full dynactin complexes to
cells in culture would be beneficial. As Arp1 and p150Glued are both subunits of a full
complex, over expression of those genes would not produce any more dynactin than extra
tires and windshields would an automobile. As such, microinjection of whole dynactin
complexes to the index lagging chromosome occurrences would be beneficial.
Furthermore, inducing lagging chromosomes via some instigating chemical and then
introducing the dynactin complexes may provide for more opportunities in which
dynactin function may be observed in relation to rescuing lagging chromosomes.
And lastly, further experimentation to reproduce the results found in this thesis is
needed in order to justify the assumptions made previously. Providing more data from
which to draw from and with which to make a sound characterization of the mitotic
defects of UPCI:SCC103, further trials may help to determine whether the data gathered
to be the norm or an anomaly.
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