Abnormally shaped, lobed nuclei are often observed in cancer cells, although the mechanisms causing these misshapen nuclei are unknown. Here, the effects of chromosome organization during cell division on nuclear shape were measured as solidity (roundness). One consequence of abnormal nuclear shape is that misshapen cell nuclei may alter gene expression, dramatically shifting normal cell activity and contributing to disease. Many proteins, including the class of molecular motors, called kinesins, direct regular cell activities including cell division. The kinesin Kif18A is responsible for maintaining chromosome alignment in the middle of a dividing cell before chromosomes segregate, producing two daughter cells. Without Kif18A, chromosomes are unaligned at metaphase, causing disordered chromosome segregation. In this study, it was investigated whether proper chromosome alignment during cell division impacts the resulting nuclear shape of daughter cells. Live cells transfected with a fluorescently labeled plasmid (GFP-H2B, to label chromatin) were imaged and analyzed using the program ImageJ to observe chromosome dynamics and cell shape after division. In the absence of Kif18A, daughter cell nuclei had lower solidity measurements, meaning nuclei were more abnormally shaped (p<0.0001). Furthermore, in the absence of Kif18A, cells were more likely to have lagging chromosomes which led to the production of micronuclei (whole or fragmented chromosomes separated from the main nucleus). Additionally, population-wide, Kif18A mutants had more cells with abnormally shaped nuclei compared to the control. These results suggest that chromosome alignment during cell division impacts nuclear shape of daughter cells, and is a possible mechanism leading to abnormal nuclear shape.
How does the organization of chromosomes at interphase influence nuclear shape in
interphase? It's hypothesized that if the organization of chromosomes in cell division is disrupted, then this will affect the nuclear shape of those cells in interphase because misaligned chromosomes will cause defects
due to disrupted gene expression.
In the process of mitosis, motor proteins direct mechanisms for proper chromosome segregation (“Phases of Mitosis”). One family of these motor proteins are called kinesins. (“Cell”). Kinesins move along microtubule “tracks” within a cell during cell division. A change or absence of critical kinesins may greatly affect how daughter cells function, appear, etc., as mitosis is affected by these proteins. An important function affected by these motor proteins is the formation of the mitotic spindle, which then affects how vesicles are transported throughout the cell (Goshima). However, when there is an absence of certain important kinesins, the cell either divides improperly or encounters problems when dividing. One such kinesin, Kif18A, which is part of the group Kinesin-8, is a protein that affects the rate of movement of kinetochores, which are responsible for moving and pulling the chromatids (Stumpff). When the alignment of the chromatids is improper, it results in cells with abnormal characteristics and defects, such as the number of chromosomes (Stumpff, Jason). Furthermore, in the absence of Kif18A, chromosomes are misaligned at metaphase and experience congression defects that can be seen with live cell imaging. This can greatly alter the function of the cell, as well as being a characteristic of many diseases such as various tumors (Maiato). In breast cancer for example, this protein is overabundant and is also related to the severity and size of the tumor. This would mean that further research into this protein may help develop new treatments and cures to these issues. However, it’s currently unclear how the cells with an abnormal number of chromosomes, known as aneuploidy, will continue to divide further, and if their daughter cells will have other phenotypic defects such as abnormal nuclear shape or if the defects will correct themselves, which would be useful in understanding tumors and their growth rates. One such measurable characteristic of defects is the shape of the nucleus itself. As normal nuclei are typically more round in shape, abnormal or diseased nuclei may possess odd shapes, which can be seen with imaging and measured. Solidity is a measurement of the roundness of an object, and is measured on a scale of 0 to 1, with 1 being a perfect circle. Understanding the mechanisms which lead to abnormal nuclear shape will help provide insight into how normal cells become diseased and whether or not mistakes in the genetic code will continue to be passed on into future “generations” of cells. This research is highly relevant to many people whose lives have been affected by cancer and other diseases which are related to mutations in the cell.
The portion I completed was the computer analysis that involved measuring and counting cells to determine their solidity (roundness), and the number of micronuclei present. I ensured that the results were as accurate as possible by keeping the variables the same; variables included brightness, contrast, among others. However, before digital analysis, steps were required to grow the cells up. Below is a summary of it. Human retinal pigmented epithelial cells (RPE-1) were cultured in MEM-Alpha media (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic (penicillin/streptomycin). Cells were grown in a CO2 incubator (5% CO2) at 37 degrees Celsius. For imaging, cells were transfected via electroporation using standard Lonza manufacturer’s instructions. Transfected cells were plated on a 35 mm glass-bottomed filming dish for imaging using a temperature controlled Eclipse-Ti microscope (Nikon) equipped with a Clara CCD (Andor) camera. Cells were transferred into CO2 independent media for imaging. Random fields of view were selected to eliminate bias and imaged every 2 minutes using a 20x objective for several hours.
The part I did involved looking at the live imaging movies taking measurements of certain cells that fit the minimum criteria: 1) cells were expressing enough fluorescent plasmid for good visibility; 2) daughter cells remained in the field of view for at least one and a half hours post division to ensure that the cell(s) would be in interphase at the time of measurement (meaning chromatin would be fully decondensed resulting in a more accurate measure of nuclear shape ). The movie was then imported into the computer software program ImageJ. Individual cells were isolated for measurement by masking the background signal by applying a masking threshold.Then solidity, the measurement of roundness, of the unmasked cell nucleus shape was measured through ImageJ. A nuclear solidity value for the mother cell, in addition to each of the daughter cells, was obtained for fifty divisions. The standard deviation and averages were taken separately
for the mother cells of each group (control and experimental), as well as the daughter cells of each group. Then, the threshold of solidity for normal control cells was found by subtracting twice the standard deviation from the average. This value for the wild type mother cells and wild type daughter cells will be considered ‘normal’ and be used for comparison with the experimental group, whose chromosomes were unaligned due to the addition of an siRNA against Kif18A. Then the percentages of cells in each group that were considered abnormal (below the established threshold) were found. Additionally, with the experimental group, micronuclei (chromosomes, or parts of chromosomes separated from the main nucleus) were found and counted. These data were analyzed by taking the percentage of mother cells that had micronuclei, daughter cells that had micronuclei, as well as the percentage of divisions that produced a micronucleus.
It was seen that daughter cells for both RPE-1 as well as Kif18A knockdowns (Kif18A knockdowns) had lower solidity measurements (meaning that the nuclei were less round) compared to their respective mother cells (p<0.0001). Similarly, in Kif18A knockdowns cells, both mother and daughtercells, had a higher percentage of cells with abnormal nuclear shapes. Further, normally shaped mother cells in the Kif18A knockdown condition had a higher rate of producing abnormal daughter cells compared to the RPE-1 control cells (4.081% to 13.953% respectively). Additionally, Kif18A knockdown cells had a greater occurrence of micronuclei produced via abnormal cell divisions (12% of divisions produced micronuclei in Kif18A KD to 0% divisions produced micronuclei in controls). Across the general population (including both cells which divided and cells which did not divide at the beginning of each field of view), it was seen that both RPE-1 and Kif18A knockdowns cells had micronuclei, with approximately 3% of RPE-1 cells with at least one micronucleus and 6% of Kif18A knockdowns cells with at least one micronucleus. There was also similar average solidity between the control and experimental groups in the general population (including both cells that divided as well as cells that remained in interphase). However, the variation of nuclear shapes in the Kif18A knockdown group was much greater than the RPE-1 cells.
The results presented here support the hypothesis that chromosome organization during mitosis affects nuclear shape of the produced daughter cells in the next interphase. This is seen in the differences of solidity (roundness) between cells with Kif18A and cells without Kif18A (average 0.963 and 0.959 respectively for mother cells) with p < 0.05. Furthermore, approximately 94.1% of normal RPE-1 cells underwent divisions that produced two normal daughters , yet only 68% of divisions in normal Kif18A knockdown cells underwent divisions that produced two normal daughters. Taken together, this data supports the idea that an abnormal chromosome organization during chromosome segregation contributes to an abnormal nuclear shape for nuclei of daughter cells.
Another result was that cells with round, normally shaped nuclei (defined here as a solidity value at least 0.948) from both groups produced a certain percentage of abnormal daughter cells. This percentage is most likely higher than the percentages found in cells that have not been transfected with the GFP protein, as cell shape may be altered due to such transfection. However, both the control and the experimental groups have been transfected with GFP proteins, thus comparisons between the two are still informative. Out of all the divisions recorded, 4.08% of control group cells underwent a division that created an abnormal cell, and 25.58% of experimental group cells created at least one abnormal cell. However, only 19% of all knockdown daughter cells were abnormal, and an even smaller percentage, 2.94% of RPE-1 cells were abnormal. Furthermore, this is also supported with data from the general population. The average solidity of nuclei for both groups in the general population were 0.948 for RPE-1, and 0.943 for knockdown cells. However, as mentioned previously, the averages for dividing cells were higher. Additionally, the range for the general population is much greater than the range for the dividing cells. This suggests that certain abnormal cells do not continue to divide, instead undergoing apoptosis or some other mechanisms that prevent cells with defects to divide.
It was also observed that chromosome organization affects the production of micronuclei, which are chromosomes, or parts of chromosomes separated from the main nucleus. Just like with abnormal solidity, a lack of Kif18A increases the frequency of lagging chromosomes which lead to micronucleus formation, as 6% of divisions occurring in these knockdown cells result in the formation of at least one micronucleus in a single daughter cell. Moreover, this relationship also holds in the general population, with 6% of cells in the knockdown population having at least one micronucleus but only 3% of cells in the RPE-1 population having at least one micronucleus. This also further confirms the idea that improper chromosome alignment during metaphase is correlated to the production of micronuclei.
Possible sources of error in this experiment include a variation between cells in expressing fluorescently modified proteins, which affects imaging. Another possible source of error was brightness of images, which could affect thresholding effectiveness. Further experiments that could be done to better understand this topic could be to investigate gene expression profiles of these abnormally shaped cells. As chromosomes occupy a certain space within the nucleus of normal cells, known as chromosome territories, a misshapen nucleus may cause changes in both placement of chromosomes and consequently, gene expression. Analyzing this information would assist in the understanding of how certain disease that result from abnormal gene expression arise.
From a young age, I've always been interested in the field of medicine and healthcare. This interest was driven by a curiosity of the human body, and the ailments that plagued it. For me, there were so many answers waiting to be learned and discovered. For a long time, I'd been mainly interested in the system of diseases as whole, but it wasn't until high school that I really developed an interest in genetics and cellular biology. This new revelation came when I realized just how large of a role genes and cells play in human disease. I realized that in order to solve big problems, one must start small. Consequently, I signed up for a biology class that had a research project to go along with it, which is this one. Ever since, I've been immensely curious about these tiny, tiny portions of our biological system.
Outside of academics, I love to run and be active. I also enjoy reading, hanging out with friends, and playing music. I'm an active member of the neuroscience club at our school, and I founded the Endangered Animals Club at our school as well. These two clubs have allowed me to pursue science in a greater depth, as well as allowing me to show a more fun side.
Will follow UVM safety precautions and methods of disposal. This includes safety glasses, lab coat, and nitrile gloves. Cells are cultured in Class II Biosafety cabinet. Additionally, a 10% bleach solution is added to human cell liquid waste and benches/safety cabinets disinfected with 70% ethanol. Solid waste will be disposed of using a boxed waste system for biological hazards. Risk assessment determined by UVM institutional biosafety committee. Will follow UVM safety precautions and methods of disposal. This includes safety glasses, lab coat, and nitrile gloves. Cells are cultured in Class II Biosafety cabinet. Additionally, a 10% bleach solution is added to human cell liquid waste and benches/safety cabinets disinfected with 70% ethanol. Solid waste will be disposed of using a boxed waste system for biological hazards.
Human cell line - RPE1 from ATCC, catalog number CRL-4000
Recombinant DNA - H2B-GFP to label DNA of cells. Grown in E.coli.
RNAi - Purchased from Fischer Scientific/Life Technologies
Note: I mostly did computer based work, which is very safe.
BSL-2, determined by the UVM institutional biosafety committee.
Mentor whom I worked with frequently: Leslie Sepaniac ( Leslie.Sepaniac@uvm.edu)
Primary Investigator: Jason Stumpff (Jason.Stumpff@uvm.edu)
UVM main laboratory safety page: http://www.uvm.edu/safety/lab
Specific lab safety: http://www.uvm.edu/policies/riskmgm/labsafety.pdf
Lab safety plan: http://www.uvm.edu/safety/lab/laboratory-safety-plan
I would like to express my gratitude to my mentors, Jason Stumpff and Leslie Sepaniac for significantly assisting with this project, from picking a topic to generously donating their time to help. This couldn’t have been done without them. I would also like to thank the entire Stumpff lab for graciously allowing me to use the equipment, as well as UVM for fostering an environment where high school students are supported in their research endeavors.
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