Numerical homogenization of subcellular cytoskeletal processes to

Diss. ETH No. 20533
Numerical homogenization of subcellular cytoskeletal
processes to reveal novel mechanisms of Rho/Rac
dependent adhesion
A dissertation submitted to the
ETH Zürich
For the degree of
Doctor of Science
Presented by
Yannick Loosli
Dipl. Masch-Ing. ETH
Born 31th August, 1979
Citizen of Sumiswald, BE
Accepted on the recommendation of
Prof. Dr. Jess G. Snedeker
Dr. Alexander Verkhovsky
Dr. Reto Luginbühl
2012

Summary
Summary
From the early beginning of humanity, humans have had to fight against injuries and diseases.
Consequently a wide range of treatment strategies have been empirically developed during these
past three thousand years. Without any knowledge of the existence of cells, five centuries B.C.,
Hyppocrates and his contemporaries have successfully established mechanical and chemical
methods that modify cellular behaviors to achieve healing. For instance, they resorted to plant
based medications to alter biochemical pathways, which today are known as intra- and extra-cellular
communication means. Cells respond to these stimuli and produce proteins, which, for instance,
inhibit pain receptors or act against inflammation. Similarly, local mechanical constraining of limbs
favors fracture healing by providing cells with an ideal environment for healing.
Current medical science aims at more specificity and efficiency by triggering a targeted adaptation of
the cellular behavior. This is only achievable by enhancing our knowledge of the interactions both
within cells and between cells and their environment. Although long underestimated, the ability of
cells to dynamically sense their mechanical environment has emerged as a major vector that
influences cellular behavior. For instance, cells respond to periodic deformations of their
environment by adapting their internal structural organization. Another illustration is how
mesenchymal stem cells differentiate toward bone-like or neuron-like cells depending on the
stiffness of their surroundings. These are examples of mechanobiology, an interdisciplinary field
dedicated to the survey of the processes employed by cells to sense, transduce and response to
mechanical stimuli. The coming section gives a broad overview of the some underpinnings of
mechanobiology, in which adhesions sites and actin cytoskeleton plays a central role.
Adhesion sites are clusters of proteins organized around a transmembrane protein connected to the
cell “muscle”. Cells generate contractile and protruding forces with their actin cytoskeleton (a
network of filamentous bio-polymers) and myosin (a molecular motor). Besides force generation,
contractility plays a second role. According to recent investigations, contractility is a major
mechanism for cells to sense their environment. This occurs by a subtle dynamic balance of the cell
endogenous forces and the extra-cellular forces at adhesion sites. A consequence, on a short time
scale (hour), is the maturation of the adhesion sites along with their anchoring actin bundles. While
maturating, adhesion sites grow by recruiting further proteins. Consequently mechanical stability
increases, endogenous force rises and an adhesion signaling is modified. Adhesion sites that have
undergone the whole maturation process (focal adhesions) last for hours, whereas small nascent
adhesions lifetime only spans minutes. Similarly actin bundles thicken, while maturating, by
recruiting further actin filaments and certainly cross-linking proteins. In their final stage myosin
colocalizes and enables high contraction forces. How adhesions sites and actin bundles mature is of
paramount importance for cellular behavior such as adhesion, motility or modification of cells fate. A
deeper understanding of adhesion site and actin bundle maturation processes will certainly bring
essential clues on cancer circumvention, wound healing promotion, orthopedic implant design, etc.
As established by recent studies, cellular behavior is the result of the coordination of numerous
meso-cellular processes (process occurring at an intermediate scale between cell and molecular
length scale), which themselves involve a multitude of molecular events. Current methods of cell
biology have revealed key aspects of all these underlying subcellular processes. However,
deciphering how these processes interact one with each other on various time and length scales is
extremely challenging with classical techniques.

ix
Preface
In the first part of this thesis, we elaborate a method to alleviate the aforementioned limitation. We
develop a stochastic numerical top-down framework beyond current state of the art, where meso-
scale processes are, in contrast to the classic analytical approach, geometrically modeled to
principally focus on their interactions and not on their own underlying mechanisms. We resort to the
top-down approach to decipher how motility functions, the principal meso-scale processes driving
cell motion, interact with actin bundles and adhesion sites to achieve cell spreading. Cell spreading is
common behavior for cells that contact a 2D substrate (surface) they can interact with. During
spreading, cells initiate specific anchorage locations (adhesion sites) and heavily reorganize their
actin cytoskeleton. For the numerical outcomes to reproduce with fidelity published data of cell
spreading on geometrically constraining substrates (substrate with well-defined regions on which
cells can attach to), we had to elaborate an additional interaction rule to complete the one derived
from the literature. This novel rule describes how lamellar contractility (centripetal forces generated
within the cell body) is gathered by cells to trigger the stabilization of the adhesion sites and actin
bundles. To support the existence of this innovative mechanism called “the length threshold
maturation process”, we subjected it to tailored experiments of cell spreading on constraining
adhesive regions. The aim is to provide the cells with the ideal boundary conditions supposed to
trigger the length threshold maturation process. In agreement with our expectations we observed
actin bundles having the spatial characteristics inherent to the length threshold maturation process,
which strongly supports the existence of the length threshold maturation process. We were
furthermore able to obtain quantitative insight that corroborated in-silico predictions.
In the second part of the thesis, we successfully attempt to explore the mechanism by which cells
integrate intra-cellular biomechemical signaling into morphology adaptation with our thoroughly
validated top-down numerical approach. It was experimentally established that cells adopt either
circular or polygonal shapes depending of the constituents of their culture media. The numerical
top-down approach successfully reproduces this observation by adequately varying the parameters
describing the motility function dynamics and the length threshold maturation process. We were
able to relate this parameter variation to the activation of two signaling proteins, Rho and Rac.
Scientists already suspected Rho and Rac to be upstream from cell morphology regulation. However
the outcomes presented in this thesis go one step further by describing a mechanism by which cells
transform a molecular biochemical signal (Rho/Rac activity) into an alteration of the global cell
mechanical and structural behavior (cell morphology).
Refocusing on medical science, this thesis brings interesting pieces to the picture though the puzzle
is far from completion. Knowing how to regulate these adhesion sites and actin bundles during
spreading, which are essential for cellular contractility, provides critical insight to design cell-
instructive biomaterials with enhanced cellular adherence and optimized controlled on cell fate.
Orthopaedic devices manufactured out of such biomaterials are expected to perform better than
current ones. The second major finding is the mechanism by which cell morphology responds to the
variation of the Rho/Rac balance. This finding could eventually assist to find a point of attack to fight
against metastatic invasion, which resorts to shape and actin cytoskeleton alteration to invade the
organism.
In conclusion, in this thesis, we elaborated and validated an efficient novel innovative numerical
approach to investigate how cells orchestrate sub-cellular processes. By applying this strategy to cell
spreading, we predicted the downstream consequences of geometrically constraining a cell

Summary
regarding to adhesion sites and actin bundle layout. We further revealed how cells integrate
changing molecular signaling in cell morphology modification via lamellar contraction and the length
threshold maturation process. Conjugating multi-scale aspects and detailed explanation of the
underpinnings render this thesis complete.

xi
Preface
Résumé
Depuis ses origines, l’humanité essaie de se soigner. L’humain a appris à se battre contre différentes
maladies et blessures en développant diverses solutions empiriques durant plus de trois mille ans.
Cinq siècles avant J.C., Hyppocrate et ses contemporains ont posé les fondements de la médecine
moderne sans même connaitre l’existence des cellules. Ils ont réussi à modifier le comportement
cellulaire pour permettre la guérison. Pour ce faire, ils ont altéré les voies de communications des
cellules par des moyens chimiques, grâces à des médicaments dérivés de plantes. Les cellules ont
répondu en conséquence en produisant des protéines qui inhibent la douleur ou agissent contre
l’inflammation. De manière similaire, une immobilisation d’un membre cassé favorise la
régénération osseuse en fournissant aux cellules un environnement idéal pour permettre la
guérison.
De nos jours, les techniques médicales tendent vers l’efficacité et la spécificité ce qui est
envisageable en induisant des changements ciblés du comportement cellulaire. La meilleure
stratégie pour atteindre cet objectif est d’approfondir notre compréhension actuelle des interactions
entre les cellules et leur environnement. Longtemps négligée, la capacité des cellules à percevoir
dynamiquement leur environnement mécanique est acceptée, aujourd’hui, comme un des
principaux vecteurs influençant le comportement cellulaire. Un exemple est la réorganisation des
composants internes de la cellule lorsque son environnement est sujet à des déformations
périodiques. De la même manière, les cellules souches se différencient en cellules osseuses ou en
neurones suivant la rigidité de leur environnement. Ces deux exemples sont tirés de la mécano-
biologie, une science interdisciplinaire dédiée aux processus utilisés par les cellules pour percevoir,
convertir le signal et réagir aux stimuli mécaniques. Le paragraphe suivant décrit quelques
mécanismes de mécano-biologie dans lesquels les sites d’adhésion entre une cellule et son substrat
(2D environnement) ainsi que le cytosquelette jouent un rôle primordial.
Les sites d’adhésion sont des agrégats de protéines organisés autour d’une protéine
transmembranaire. Ces régions adhésives lient le cytosquelette de la cellule à l’extérieur. Ce
cytosquelette, qui est un réseau de bio-polymère, permet aux cellules de générer des forces dites de
contraction et de protrusion. La contractilité n’a pas que des effets mécaniques. En effet, de
récentes études ont établi que la contractilité est au centre du processus de perception mécanique
de la cellule. Ceci est possible par un subtil changement de l’équilibre entre les forces intra- et extra-
cellulaires. A court terme (quelques heures), les cellules intègrent ces stimuli en déclenchant la
maturation des sites d’adhésion ainsi que des câbles d’actine pour les stabiliser. La maturation des
sites d’adhésion est caractérisée par un recrutement de nouvelles protéines ce qui permet
d’augmenter la stabilité mécanique des adhésions. En conséquence, de plus grandes forces intra-
cellulaires peuvent agir dessus ce qui modifie les signaux émis par les sites d’adhésion. Les sites
d’adhésion qui ont entièrement subi le processus de maturation, les adhésion focales, sont stables
pour plusieurs heures. En revanche les sites d’adhésion qui ne maturent pas ont une espérance de
vie de quelques minutes. De manière similaire, les câbles d’actine s’épaississent pendant le
processus de maturation en recrutant de nouveaux filaments d’actine qui sont interconnectés par
des protéines dites de «réticulation». A la fin de leur maturation, les câbles d’actine se lient à des
myosines. Ces complexes peuvent générer d’importantes forces contractiles. L’importance des sites
d’adhésion et des câbles d’actine pour la détermination de comportements cellulaires tels que
l’adhésion, la motilité et même, en fin de compte, le sort de cellules est aujourd’hui clairement