Quantitative analysis of coordinated epithelial rotation on a two-dimensional discoidal pattern

概要

(Form 1)
Kyoto
Doctor of Philosophy in
Name
LUO, Shuangyu
University
Life Sciences
Thesis
Quantitative analysis of coordinated epithelial rotation
Title
on a two-dimensional discoidal pattern
(Thesis Summary)
Among a number of dynamic cell behaviors during animal life, collective cell migration covers
a variety of coordinated movements that are exhibited by tightly interconnected cells. One unique
type of such collective cell migration in vivo is coordinated epithelial rotation in development,
which has been modeled in vitro under spatially fine-tuned conditions. Despite the wellestablished role of cell-cell interactions in such coordinated rotational motion, the contribution of
E-cadherin, a major homophilic cell-cell adhesion molecule in epithelia, has not been directly
addressed on two-dimensional (2D) confined environments.
Benefiting from the microcontact printing technique, the applicant used MDCK (Madin-Darby
canine kidney) II cells, one of the popular model mammalian epithelial cell lines, generated 2D
monolayers of either strip or discoidal shape, and addressed the contribution of E-cadherin on
coordinated rotational motion. The applicant tracked movements of wild-type or E-cadherin
knockout (KO) cells under two distinct boundary conditions, the strip and the disc, and analyzed
their migrations. As far as the strip pattern was concerned, the KO cells migrated towards free
space as fast as the wild-type cells. In contrast, on the disc, the wild-type cells exhibited persistent
and large-scale rotations, while the E-cadherin KO cells migrated in a less coordinated manner
and showed only transient local rotations. Direct quantitative comparison between these two cell
types using particle image velocimetry (PIV) unveiled that the E-cadherin KO cells exhibited the
following changes in migratory behaviors: (1) decreased global migration speed, (2) enhanced
irregularity in quantified coordination and (3) increased average number of the topological defects.
Collectively, under 2D discoidal confinements, these findings show that the emergence of largescale coordinated migration of epithelia depends on E-cadherin.
It has not been addressed how the collective motion is affected when E-cadherin-deficient cells
appear in epithelial monolayers from biophysical and pathological points of view. On the basis of
the above results, the applicant attempted to conduct cell migration analysis by mixing wild-type
and E-cadherin KO cells on the disc. Because the applicant intended to monitor intercellular
dynamics at both homo- and hetero-interfaces in the cell mixture, visualization of the plasma
membrane of individual cells became particularly important. To this end, the applicant established
EGFP-CAAX or TagRFP-T-CAAX-expressing wild-type MDCK II clones. Unexpectedly, in contrast
to the original wild-type cells, those plasma membrane-labeled wild-type cells did not initiate
large-scale coordinated movements when cultured on the disc. The applicant discussed possible
effects of the expression of the CAAX-fused fluorescent proteins on collective behaviors of cells.

(Form 2)
（Thesis Evaluation Summary）
The applicant reported that migratory manners between the two cell types (wild-type
and E-cadherin KO MDCK II cells) became profoundly different on the discoidal
pattern, which showed that E-cadherin is required for the rapid collective rotation, and
discussed the experimental results in satisfactory manners. In the E-cadherin KO cells,
it is likely that the decrease in the total cadherin activity in each cell does not allow
the long-range intercellular force transmission. This possibility may be strengthened
by visualizing distributions of the actin cytoskeleton and vinculin. Alternatively, Ecadherin may have a unique role in collective epithelial migration, which cannot be
compensated by other cadherin subclasses including cadherin-6 expressed in MDCK II
cells. Further comparative studies using wild-type cells, E-cadherin KO cells, and
cadherin-6 KO cells would resolve these possibilities.
One feature of the adhesive disc in the applicant’s study was its large size, compared
to the discs employed in other studies and the intrinsic correlation length of MDCK
cells. It can be addressed in future how differently E-cadherin KO cells behave on
smaller discs where the applicant could reproduce clear rotational migration of wildtype cells. Another point of discussion concerning the experimental design was the
broader definition of “collective migration” of cells on discs under the confluent
proliferation-inhibited condition, in contrast to a classic view of collective cell migration
where leader cells exert traction on the follower cells towards free space. Finally, effects
of the CAAX-fused fluorescent proteins on the collective rotation was discussed in the
context of additional experiments to further verify the applicant’s hypothesis.
This thesis substantiates the candidate’s extensive and wide knowledge of life
sciences, demonstrates expert research capability in the field of cell biology, and presents
new discoveries that contribute to the profound understanding and further development
of the candidate’s research field. Moreover, the thesis is written logically and coherently,
which satisfies the degree requirement that the thesis shall serve as a valuable
document for future reference. On April 13th, 2023, the PhD thesis oral examination was
held. Pursuant to this oral examination, the thesis examination committee hereby
concludes that the candidate has passed all of the requirements for the degree of Doctor
of Philosophy in Life Sciences.
The thesis, thesis summary (Form 1), and thesis evaluation summary (Form 2) will be
published through the Kyoto University Research Information Repository. If the thesis
cannot be published on the website immediately after the degree is awarded, due to patent
application, journal publication constraints, or other reasons, please indicate the earliest date
below that the thesis can be published. ...