Cell Migration and Extracellular Matrix

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Chapter 3. Parallel Cell CIL

Leading-Leading collisions without cell division

In addition to spindle cell CIL, we also qualitatively and quantitatively studied parallel cell CIL between two approaching cells. In cases where there was no cell-division, out of 26 collisions, we report that 100% of the time the cell pair will come into contact and result in one cell repolarizing.We believe that this occurs due to fiber spacing constraint that induces a parallel shape. Unlike spindles, parallel cells do not have the ability to maneuver past and walk by one another as easily because they are adhered to two fibers instead of a single like spindle cells. Like the behavior reported in spindle cases, we term this non-mutual CIL as both the cells do not switch their migration direction. The speeds of both the parallel and repolarizing remain consistent pre and
during contact without any significance which is consistent with our previous findings as no cell division is occurring.

Leading-Leading collisions with cell division

With the presence of cell division, out of 61 random collisions observed, we report that 62% of time the dividing cell will approach the non-dividing parallel cell and they will walk-by one another without repolarization (Figure 3.3). This behavior is similar to that of two approaching spindle cells as well. However, this walk-past is only possible if during the contact period, the parallel changes its shape to that of a spindle in order to surpass and then it regains its morphology post contact and continues to migrate. With respect to cell speeds, we report that the daughter cell loses speed during this contact period and further decreases in speed post-contact. Similarly, the non-dividing parallel also loses speed during the contact period and picks it up once it detaches from the daughter cell (Figure 3.4). A rare (7 collisions/ 15%) yet extremely intriguing phenomenon seen in parallel cell CIL is that of a cell ‘push’. In cell push cases, two parallel cells approached one another and prior to contact, one cell divided. The newly formed daughter cell initiated contact with the non-daughter and rapidly caused the non-daughter cell to repolarize and begin migrating in the opposite direction. In this scenario, one is able to visualize a “jamming plane” where both cells remained in contact and jammed against one another before cell repolarization took place (Figure 3.5) followed by the push behavior. We believe that this is partly attributed to the significantly faster daughter cell speed post-division in comparison to that of a non-dividing parallel cell (Figure 3.6a). Furthermore, we report that the daughter cell during contact significantly loses speed as it is jamming with the subsequent cell. In a similar scenario, we report two parallel cells approaching, however as in this case, the cell jam followed by a push is not visible. For this behavior, we observe 23% of the time, a daughter cell post cell division approaches a parallel cell and the collision results in the parallel cell repolarizing, similar to a spindle CIL case described previously (Figure 3.7a). Here, we conclude that the daughter cell significantly loses speed during contact with the approaching parallel cell whereas the parallel cell gains speed during the collision period.
In addition to cell speeds for non-division and division cases, we wanted to understand whether the starting distances between the cell centroids differed in order to define a criterion for cell push. We thus concluded that the starting distance between the centroids for two cells approaching without division involved was significantly larger than that leading to a cell push (Figure 3.8a). This allows us to infer that for a push to occur, the post division, the daughter cell immediately contacts the non-dividing parallel resulting in it being pushed and repolarized. Moreover, the duration for repolarization for a non-division approaching case is longer than that seen when a cell push occurs, where the parallel repolarizes faster.

Leading-Trailing collisions

Through our studies of parallel cell CIL, we also report the occurrence of leading-trailing (headtail) collisions with and without cell division (Figure 3.9a). Once again, like spindle leadingtrailing collisions, in parallel cells without cell division, we report that the trailing cell is significantly faster than the leading cell pre contact, which allows for it to make contact  During the contact duration, the leading cell gains speed and both cells continue to migrate as a cohort in the same direction. Likewise, with the occurrence of cell division, the trailing cell has a higher average speed before the contact period in order to reach the leading cell and induce its speed.

Chapter 4. Spindle-Parallel CIL

Leading-Leading collisions without cell division

We also wanted to inquire about the outcome following an interaction between a spindle and parallel cell without the influence of cell division. Although the occurrence of such collisions is rare, we were able to identify certain patterns and rules for these interactions as well. When a parallel and spindle cell approach one another, the spindle will repolarize and continue migrating as a unit with the parallel cell. However, upon contact, the spindle both maintains its shape and proceeds with the migration or it alters its morphology to that of a parallel cell .
We quantified speeds for both cells pre and during contact and show that both the spindle and parallel cells experience a slight decrease in speed during contact (Figure 4.2). However, we were only able to analyze 3 such collisions thus the sample size will need to increase for more conclusive
results.
Another shared behavior among these cell-cell collisions was the walk-past. Similar to the walk past described with two approaching parallel cells, in this scenario, the parallel cell has to alter its morphology to that of a spindle shape in order to walk-by and continue in its original migration direction.Out of 4 analyzed collisions, we see that the spindle cell loses speed during the contact period and regains speed post contact with the parallel cell. Similarly, the parallel cell also experiences a decrease in speed during contact but unlike the spindle cell, it further loses speed during the post contact period Once again, we report these speeds based off of 4 cell collisions and aim to increase this sample size with more experimentation.

Leading-Leading collisions with cell division

Spindle-parallel cell collisions also occur post mitotic cell division. When approaching, the spindle cell divides, repolarizes and alters its shape to that of a parallel cell before continuing to migrate as a cohesive with the parallel cell. However, in this case, the spindle is not always the one to divide. We report parallel cell division as well which results in contact with the spindle leading to its repolarization and new migration direction either maintaining its spindle shape or altering it to that of a parallel .

Table of Contents 
Acknowledgments
Table of Contents
List of Figures
Chapter 1.  Introduction
1.1 Cell Migration and Extracellular Matrix
1.2 Importance of Contact Inhibition of Locomotion
1.3 Effect of chemical gradients on CIL
1.4 Previous methods to study CIL
1.5 Step platform and materials and methods
1.6 Objectives of Study
Chapter 2. Spindle cell CIL
2.1 Leading-Leading collisions without cell division
2.2 Leading-Leading collisions with cell division
2.3 Leading-Trailing collisions
Chapter 3. Parallel cell CIL
3.1 Leading-Leading collisions without cell division
3.2 Leading-Leading collisions with cell division
3.3 Leading-Trailing collisions
Chapter 4. Spindle-Parallel CIL
4.1 Leading-Leading collisions without cell division
4.2 Leading-Leading collisions with cell division
4.3 Leading-Trailing collisions
Chapter 5. Geometric Rules in multi-cell interactions
Chapter 6. Discussions and Conclusions
Chapter 7. Future Directions
7.1 Effect of fiber diameter on CIL outcome
7.2 Influence of biological inhibitors on CIL behavior
7.3 Heterotypic CIL
References
Appendix A: Speed box and whisker plot description

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Rules of Contact Inhibition of Locomotion for Cell-pairs Migrating on Aligned and Suspended Nanofibers