# Tracking of Endothelial Cell Migration and Stiffness Measurements Reveal the Role of Cytoskeletal Dynamics

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## Abstract

**:**

## 1. Introduction

## 2. Biased Persistent Random Walk Model of Cell Migration

## 3. Results

#### 3.1. Cytoskeletal Dynamics Contributes to Wound Healing and Gap Closing Speed

`X`zoom-in, qualitative differences in cell morphology can be identified (Figure 2). Control HUVECs appear oblate with clear definition in their stress fibers. Notably, the control monolayers appear simply connected, meaning that no holes are found in the gap-separated monolayers (Figure 2). After disrupting actin polymerization, irregular morphology can be identified, and the existing actin appears concentrated at the cell periphery (Figure 2). At the 10-h mark, the actin-disrupted monolayer is no longer simply connected—numerous instances of cell–cell connectivity loss are identified (Figure 2). Tubulin-disrupted cells appear with more subtle changes. Firstly, the individual ECs appear less oblate, and instances of cell connectivity loss are identified (Figure 2).

#### 3.2. ECs Organize Speed and Persistence in a Directionally Dependent Manner

#### 3.3. Cytoskeletal Dynamics and Cell Morphology

#### 3.4. Cytoskeletal Dynamics and Cell Stiffness

## 4. Discussion

## 5. Materials and Methods

#### 5.1. Cell Culture

_{2}and 5% O

_{2}on tissue culture flasks containing one of three types of media: Endothelial Medium (EM), Reduced Serum Medium (RSM), and Serum-Free Medium (SFM).

#### 5.2. Media Selection

#### 5.3. Pharmacological Disruptions

#### 5.4. Wound Healing Assay

^{4}cells/cm

^{2}were seeded onto a gelatin-coated polystyrene six-well plate containing RSM. Cells were incubated for at least 24 h at standard culture conditions before the experiment. The monolayers were then scratched with a 200 μL micropipette tip. The samples were mounted onto the incubating stage of the Zeiss AxioObserver and let sit in the incubating unit for three hours prior to time-lapse imaging.

#### 5.5. Time-Lapse Microscopy

#### 5.6. Track Quantification

#### 5.7. Atomic Force Microscopy

#### 5.8. Topography Measurement

#### 5.9. Elasticity Measurement

^{2})]R

^{1/2}δ

^{3/2}, for the force (F), indentation ($\delta $), given indentation curvature radius (R), and estimated Poisson’s ratio ($\nu $ = 0.45). We restricted the stiffness analysis to the cell body, thus excluding the substrate contributions in the cell periphery, in accordance with previous studies [34].

## 6. Conclusions

## Supplementary Materials

## Author Contributions

## Funding

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

## References

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**Figure 1.**(

**a**) Simple schematic detailing endothelial cell migration at the cellular level; (

**b**) Visual representation of the migration zone convention is denoted by: Transverse (across the gap), lateral (generally parallel to the leading edge), and reverse (opposite to the gap) directions in the context of the leading edge of a wound. In healing, cell transverse velocities are ideal (moving across the gap), the reverse region is antithetical to wound healing, and velocities in the lateral region are neither as bad as the reverse region nor as good as the transverse region; (

**c**) Migration zones in the context of a wound healing assay; (

**d**) A simplified model of a wound healing assay. In this view, one may more clearly see the transverse, lateral, and reverse zones in relation to the leading edge. A representative single EC motion is identified, and we denote the equation for the turning angle $\theta $. Note that the sign of the angle is important (counterclockwise form the horizontal is positive). Lastly, we show the representation of a two-dimensional vector as a polar complex number, which was used for brevity throughout the computations.

**Figure 2.**Wound healing assays for cytoskeletal disruptions and control. Stained actin is presented in purple, tubulin stains are shown in green, and then nuclear counterstain is provided in yellow. To better view single cell morphology, 4

`X`zoom-in of the field of view is provided for each condition.

**Figure 3.**(

**a**) Linear regressions of collected gap distance data at increasing time points for cytoskeletal disruptions (Ctrl—control; CD—50 ng/mL cytochalasin D; Noc—50 ng/mL nocodazole). The slope of the linear regression is the gap closing speed; (

**b**) Gap closing speeds comparing cytoskeletal disruptions, with the bars representing the respective 95% confidence interval; (

**c**) Violin plot of EC 10-h displacements under different cytoskeletal disruptions in a wound healing assay; (

**d**) Mean displacement in 10 h. The error bars correspond to the 95% confidence interval.

**Figure 4.**(

**a**) Persistence parameter for each direction group; (

**b**) Mean speed calculated for motion in each direction group; (

**c**) Mean coordinate displacement in 10 h by direction grouping. The reverse direction was excluded from the visualization due to few samples (see Supplementary Materials); (

**d**) Mean total displacement in 10 h by initial proximity to the wound gap; (

**e**) Mean transversal (orthogonal to the leading edge) displacement in 10 h by initial proximity to the wound gap. As described in Figure 1, the ideal motion for wound healing is positive coordinate displacement along the transverse axis; (

**f**) Mean coordinate displacement of the lateral component (parallel to the leading edge) in 10 h by initial proximity to the wound gap. The error bars represent the 95% confidence interval. Definitions for the displacement and total displacement can be found in the framework section.

**Figure 5.**(

**a**) Plot of the mean squared displacement (MSD) over time for an ensemble of tracked ECs (n = 269). The same plot is transformed into log-log coordinates to estimate the exponent $\mu $ in $MSD=a{t}^{\mu}$ with a linear regression. Note that the log(x) function in these calculations refers to the natural logarithm (base $e$). Note that the standard linear regression problem utilizes the line slope and y-intercept to minimize the error in the dataset; (

**b**) Normalized histogram of empirically measured turning angles and the wrapped Cauchy probability density. This was performed on the previously denoted directional regions for cytoskeletal disruptions (Ctrl—control; CD—50 ng/mL cytochalasin D; Noc—50 ng/mL nocodazole). The number on the top left-hand corner represents the maximal likelihood estimate of the concentration around ${0}^{\xb0}$ parameter $\rho $ to produce the observed turning angle distributions; (

**c**) Histograms from (

**b**) represented radially. Positive angles correspond to counter-clockwise rotations from the 0 axis, consistent with the convention used in (

**b**); (

**d**) Estimated exponent $\mu $ in $\mathit{MSD}~{t}^{\mu}$ comparison by biological replicate (error bars correspond to the 95% confidence interval). (

**e**) Relative sizes direction groups for each wound healing assay and cytoskeletal condition.

**Figure 6.**(

**a**) Topographical images of cells taken with the Atomic Force Microscope. Cytochalasin D and nocodazole treatments were administered to cells four hours before imaging. (

**b**) Measurements of length, width, and height from topographical images. (

**c**) Aspect Ratio ($\mathrm{width}/\mathrm{length}$) calculations from width and length measurements of HUVECs. Within each box in the box and whisker plots, the horizontal lines denote median values; boxes extend from the 25th to the 75th percentile of each group’s distribution of values. Comparisons were carried out with a one-way ANOVA test. p-values were then calculated with a multi-comparison test (** p < 0.01).

**Figure 7.**(

**a**) Schematic of live cell indentation via AFM. (

**b**) Force curve obtained over the duration of indentation. (

**c**–

**f**) The middle bar represents the median value of the distribution, and the notch represents the 95% confidence interval. (

**c**) Compressive Modulus of ECs under varying cytoskeletal disruptions. (

**d**) Measured compressive moduli of HUVECs 1 h after wound. (

**e**) Compressive moduli of cytoskeletal disruptions normalized to the compressive modulus of the control. (

**f**) Measured Compressive moduli 16 h after wound (* p < 0.05).

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**MDPI and ACS Style**

Romano, D.J.; Gomez-Salinero, J.M.; Šunić, Z.; Checco, A.; Rabbany, S.Y.
Tracking of Endothelial Cell Migration and Stiffness Measurements Reveal the Role of Cytoskeletal Dynamics. *Int. J. Mol. Sci.* **2022**, *23*, 568.
https://doi.org/10.3390/ijms23010568

**AMA Style**

Romano DJ, Gomez-Salinero JM, Šunić Z, Checco A, Rabbany SY.
Tracking of Endothelial Cell Migration and Stiffness Measurements Reveal the Role of Cytoskeletal Dynamics. *International Journal of Molecular Sciences*. 2022; 23(1):568.
https://doi.org/10.3390/ijms23010568

**Chicago/Turabian Style**

Romano, Dominick J., Jesus M. Gomez-Salinero, Zoran Šunić, Antonio Checco, and Sina Y. Rabbany.
2022. "Tracking of Endothelial Cell Migration and Stiffness Measurements Reveal the Role of Cytoskeletal Dynamics" *International Journal of Molecular Sciences* 23, no. 1: 568.
https://doi.org/10.3390/ijms23010568