Mechanics of leukemic T-cell

Cell mechanics is a factor that determines cell growth, migration, proliferation, or differentiation, as well as trafficking inside the cytoplasm and organization of organelles. Knowledge about cell mechanics is critical to gaining insight into these biological processes. Here, we used atomic force microscopy to examine the elasticity, an important parameter of cell mechanics, of non-adherent Jurkat leukemic T-cells in both interphase and mitotic phases. We found that the elasticity of an individual cell does not significantly change at interphase. When a cell starts to divide, its elasticity increases in the transition from metaphase to telophase during normal division while the cell is stiffened right after it enters mitosis during abnormal division. At the end of the division, the cell elasticity gradually returned to the value of the mother cell. These changes may originate from the changes in cell surface tension during modu-lating actomyosin at the cleavage furrow, redistributing cell organelles, and constrict-ing the contractile ring to sever mother cell to form daughters. The difference in elasticity patterns suggests that there is a discrepancy in the redistribution of the cell organelles during normal and abnormal division


| INTRODUCTION
Cell mechanics defines the response of cells to the mechanical forces exercised by the surrounding microenvironment, including the extracellular matrix and other cells. These forces exert continuously compressive, extensional, and shear forces on cells in vivo. 1 The deformability in response to mechanical forces of cells plays a pivotal role in the homeostasis of tissues and organs and the development of proper embryonic. The ability to resist deformation, transport intracellular cargo, and change shape during movement depends on the cytoskeleton. This is an interconnected network of various regulatory cytoplasmic proteins including myosin motors, microtubules, and actin filaments. It involves in upstream and downstream signaling pathways, acts as an interface for cellular processes, determines elasticity and local behavior of cells, and plays an important role in providing structural support to maintain cell shape and facilitate cell movement. 2,3 As a cell enters mitosis, the creation of the mitotic spindle and the contractile ring during nuclear and cytoplasmic division requires the cooperation of cytoskeletal polymers and motors to generate the necessary forces for the division process. The dynamic reorganization of the cytoskeleton, cell organelles, and membrane molecules will lead to changes in cell elasticity. [4][5][6][7] This is an important biophysical property of the cell since it can be used to differentiate cancerous from normal cells 8 and can reflect changes in the chemical environment 9,10 or in response to genetic mutations. 11 The cell cycle is separated into the interphase and mitotic phases.
Interphase includes DNA synthesis and gap phases while the mitotic phase leads up to cell division, 12 which plays a vital role in cell proliferation and differentiation. The mitotic phase is managed by a complex and coordinated sequence of the membrane, cell organelle, and and abnormal divisions remains unclear.
In this study, the nanoindentation-based AFM technique was used to continuously measure the elasticity of individual Jurkat T-cells in both synthesis and mitotic phases. This cell line is an immortalized line of human T lymphocytes that causes acute lymphoid leukemia, a disease that is characterized by the dysfunction of normal immune surveillance function in the body. 19,20 The results showed that the cell elasticity does not change during DNA synthesis phase while it significantly changes during division and gradually returns to the elasticity of the mother cell at the end of cytokinesis.

| Cell preparation
Jurkat cells, clone E6-1, (ATCC, TIB-152) were cultured in suspension in RPMI-1640 medium supplemented with 1% penicillin/streptomycin, 1% L-glutamine, and 10% heat-inactivated fetal calf serum (Invitrogen, Darmstadt Germany) at 37 C under a 5% CO 2 humidified atmosphere. An aliquot of the cell suspension was dropped onto a glass coverslip pre-coated with poly-L-lysine (PLL), which was already mounted onto the AFM biocell, for AFM measurements. The PLL coating glass was done as follows: 24-mm round glass coverslips (Plano GmbH, Wetzlar, Germany) were sonicated in turn in acetone, ethanol, and ultrapure water for 5 min followed by immersing into PLL solution 0.02% (Sigma Aldrich, Darmstadt, Germany) for 30 min.
The coated substrates were dried with a N 2 blow.

| AFM measurements and data analysis
where ν is Poisson's ratio (ν = 0.5 for a soft sample), R is the radius of the bead, δ is the indentation depth, and E is cell elasticity. These were done with JPK data processing software version 4.4.18+. Origin software (version 9.1) was used for data analysis. The mean elasticity values and their corresponding SE of the mean were determined by applying Gaussian fits to the data. The statistic was determined using a one-way ANOVA test integrated in the origin software.

| Elasticity of nondividing cell
Before investigating the mechanical characteristics of the cell during division, the elasticity of nondividing was examined by using nanoindentation-based AFM ( Figure 1). Figure 1A shows a schematic model of nanoindentation measurement on a non-adherent cell adhered to PLL-coated glass surface. The AFM probe is positioned on top of the cell. Applying a force F to the cantilever, the cell will be indented and the force vs displacement (FD) curves will be measured. The approach force curves are then converted to force-indentation curves followed by fitting the resulting curves with the Hertz model to estimate the elasticity of the cell. Figure 1B shows an overlay of the optical and fluorescence images, which shows the silhouette of an AFM cantilever end placed on top of a cell (nuclear region, blue) for force measurements. Figure  When the sample surface is far away from the tip, the cantilever is not bent (1) and the force is equal to zero. Approaching the sample surface until it touches the tip (2), the cantilever starts to bend, and the force starts to increase. If the substrate is a hard glass surface, the cantilever (3) will proportionally bend with the extension of the AFM piezo stage, while if the substrate is a soft cell surface, the cantilever will bend (4) following an inward course. The force curve difference recorded on these two surfaces is the indentation depth (δ) of the cell. which was used for whole nanoindentation experiments in this study. Approaching the cell with a loading force F to the bead, the cell will be indented upon contact and the cantilever will be bent following an inward course of the indentation. The AFM will record this bending of the cantilever as input parameters of the F-D curve, which is schematically outlined as a solid curve ( Figure 1D). The difference between force curves measured on soft cells ( Figure 1D Figure 1F shows the elasticity histogram with a mean value ± SE of 134.5 ± 2.8 Pa.

| Cellular elasticity during normal division
During division, a massive rearrangement of the cell organelles causes changes in its biophysical characteristics. Especially the elasticity at the furrow of the adherent cell will be significantly changed due to the accumulation of actin and myosin. 6 To verify whether this characteristic will also change for non-adherent cells, the elasticity of the dividing Jurkat T-cells was investigated. In this work, the colloid probe was positioned on top of the mother cell and the force curves were continuously measured. During cell division, the colloid probe was kept close to the cleavage furrow.

| Cell elasticity during abnormal division
In some cases, a mother cell can divide into more than two daughters.
It has been proved that extra-centrosome mediates multipolar spindle assembly and the cell undergoes multipolar anaphase that produces three or more mononucleated daughter cells. 24,25 Figure 3A shows optical images of a mother cell dividing into three daughters. In this abnormal division, the time required for the cell to complete the division process was $1 h. The cell elasticity also increased after the cell entered mitosis and gradually returned to the value of the mother cell at the end of the division ( Figure 3B). The mean elasticity of daughter and mother cells changed very much during the division.

| DISCUSSION
In this article, the elasticity of Jurkat T-cells was measured using a constant loading force. The resulting force-distance curves were converted to force-indentation curves and were fitted with the Hertz model. Dimitriadis and co-workers 26 showed, when estimating cell elasticity by the Hertz model, that the complication of the underlying hard substrate should be considered. This effect is less prominent for the pyramidal tip if local strains created by this tip do not exceed the linear-elastic assumption. However, this type of tip will provide a cell elasticity value of 60% higher than that measured by a spherical tip. 26 Applying this modified Hertz model to estimate cell elasticity showed that when indenting cells with a cell diameter of 12 μm by a bead of 4.3 μm, the apparent cell elasticity is overestimated by less than 1% Cell elasticity is an integrative parameter reflecting cell mechanics and is oscillating in standard physiological conditions. The oscillation is self-induced (spontaneous) 24,25 and the magnitude is different depending on cell state, cell type, or surrounding microenvironment.
The cell elasticity oscillation can also be measured by applying a repetitive indentation technique. This approach allows not only timeresolving sequences of cell elasticity but also quantifying the magnitude of the oscillations. 30 In this study, this approach was applied to investigate the elasticity of both nondividing (interphase) and dividing cells. In interphase, the cell elasticity oscillates slightly, and this oscilla- In normal cell division process, kinetochore microtubules attach to the chromosomes, which align at the center of the cell at metaphase (1), separate them into sister chromatids, and pull them to the poles at anaphase (2). The contractile ring formed at the cell equator at telophase (3) will constrict to separate the mother cell into two daughters. (B) In the abnormal division process, chromosomes align in three or more directions at the cell center at metaphase (1 0 ) and microtubules will drag them to the poles at anaphase (2 0 ). At telophase (3 0 ), the newly formed contractile ring will constrict to sever the mother cell into daughters.
tumor initiation and proliferation. The chromosomal instability is a major source of aneuploidy and tumor initiation and proliferation. [33][34][35][36] In most cases, a mother cell will undergo two-polar cell divisions and only a minor fraction of cells will undergo multipolar cell division. This means that in an abnormal division process, the chromosome of the mother cell will incorrectly segregate as schematically outlined in Figure 4B. It will segregate into three directions instead of lining up at the center of the mother cell at metaphase (1 0 ) like in the case of normal cell division, and at anaphase (2 0 ), the mother chromosomes will be separated into three sets of sister chromatids. After that, the contractile ring formed at telophase (3 0 ) will continuously constrict to separate the mother cell into three daughters. In this abnormal division process, daughter cells will not inherit the complete set of chromosomes of the mother cell. This may explain the reason why daughter cells often die after division or before the second round of mitosis. 5 The change in cell elasticity, in this case, is also different from that of normal cell division. This may be because the cell may require more force to rearrange the cell organelles before the spindles pull the sister chromatids to the poles. This force creates tension over the cell surface, leading to an increase in cell elasticity right after the cell enters mitosis. Further studies may be necessary for a precise conclusion for this phenomenon.
In summary, the approach of repetitively measuring the elasticity as a model for non-adherent Jurkat T-cell allows real-time monitoring of the changes in cell elasticity during division with high time resolution. Together with the accumulation of the actomyosin at the furrow to form a contractile ring, the force required to rearrange cytoplasmic and surface molecules and to pinch the contractile ring to separate mother cell into daughters are also important factors that contribute to the change in cell elasticity. Thus, a direct, high time resolution measurement of the cell elasticity can provide insight into involved molecules and the mechanism of cell division processes.

AUTHOR CONTRIBUTIONS
Van-Chien Bui designed the research study, performed the experiments, analyzed the data, and wrote the manuscript, Thi-Huong Nguyen discussed the data and wrote the manuscript. All authors have read and approved the final manuscript.