Single-molecule cohesion and adhesion in muscle cells

133 Pages·2017·16.34 MB·English

marco grison
SINGLE-MOLECULE
COHESION AND ADHESION
IN MUSCLE CELLS
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Dissertation at Physics Department E
Technische Universität München

technische universität münchen
physics department
SINGLE-MOLECULE COHESION AND ADHESION IN
MUSCLE CELLS
marco grison
Vollständiger Abdruck der von der Fakultät für Physik der Technischen
Universität München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
genehmigten Dissertation.
Vorsitzender: Prof. Dr. Martin Zacharias
1
Prüfer der Dissertation: . Prof. Dr. Matthias Rief
2
. Prof. Dr. Andreas Bausch
05042017
Die Dissertation wurde am . . bei der
Technischen Universität München eingereicht und durch die
17052017
Fakultät für Physik am . . angenommen.
Marco Grison: Single-molecule cohesion and adhesion in muscle cells, ©
2017
April
ABSTRACT
Muscle is the tissue responsible for the locomotion of the body and
for the beating of the heart, thanks to its ability to generate active
forces upon contraction. A large number of protein/protein inter-
actions are involved in the stabilisation of the muscular tissue. Sur-
prisingly,manyofthoseinteractionsehibitlowaffinities.Hence,the
questionariseshowweakbondscanprovidelong-termmyocyteco-
hesion and adhesion. The work presented in this thesis deals with
two weak protein/protein interactions, the first involving α-actinin
and titin, the other talin and integrin. To mechanically probe the
strength of the bonds between single protein pairs, optical tweez-
ers were employed, since they allow high resolution force measure-
ments in the low-piconewton range.
Titin acts as a molecular spring, restoring the muscle resting
length after the tissue is streched, but how it is connected to the
cytoskeleton is still a matter of debate. The interaction of titin with
the actin crosslinker α-actinin has been proposed as a candidate
for titin anchoring. Upon force application with optical tweezers,
we found that the bond between α-actinin and titin broke apart at
forces lower than those which are physiologically relevant. The un-
binding forces were strongly dependent on the pulling geometry,
2
but the lifetime of a single bond never exceeded s. Since titin has
seven similar α-actinin binding motifs, we probed different interac-
tions and observed that only three motifs bind α-actinin with sig-
nificant mechanical stability. We propose a model explaining how
the concerted action of many parallel bonds can provide long-term
anchoring of titin.
With similar methods we studied the interaction between talin
and integrin, required for the lateral adhesion of parallel myocytes
and for the connection with tendon cells. We found talin and in-
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tegrin to detach at low forces, with lifetimes lower than ms
even in the absence of load. Such fast kinetics suggest that a sta-
bleconnectioncanbeachievedonlywithmanyparallelbonds,orif
other interactions are involved. We further investigated the affinity
of the bond using the competitive binding of an integrin molecule
tethered to talin versus one free in solution, and measured values
comparable to those found in literature. These experiments will fa-
cilitate the further investigation of the coordinated role of the cell
membrane and different integrin substrates in connecting together
the cytoskeleton and the extracellular matrix.
v

PUBLICATIONS
Some ideas and figures have appeared previously in the following
publication:
Grison M, Merkel U, Kostan J, Djinovic´-Carugo K, Rief M, α-
Actinin/titin interaction: A dynamic and mechanically stable clus-
ter of bonds in the muscle Z-disk. PNAS, 114(5):1015–1020, January
2017
.
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And the eighth and final rule:
If this is your first night at Fight Club, you have to fight.
Tyler Durden
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ACKNOWLEDGMENTS
First and foremost I want to express my gratitude to my advisor,
Prof. Matthias Rief. He taught me the importance of solid scientific
method, encouraged my research and supported me throughout
failures. Moreover, he is a source of inspiration because of his pro-
motionofanincomparablyproductivebutcomfortableatmosphere
in the lab.
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I would like to acknowledge all the members of the E , E
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and E chairs for their cooperation, feedback and friendship. In
particular, my thanks go to Lorenz Rognoni, who introduced me
to the techniques and the methods. To my (c)office mates Markus
Jahn and PhilippWortmann, who gracefully put up with my endless
complaints. To the lunchy-lunchy crew, who prolonged my life
expectancy by keeping me away from the mensa. Especially Leone
Rossetti, Francesco Dighera, Benni Pelz, Kasia Tych and Katharina
Henneberg. Kasia deserves a special mention also for editing this
text and making it readable to the rest of the world.
I thank Thomas Suren and Fabian Schneider for being a precious
source of friendship, and Florian Praetorius and Matthias Schickinger
for the comments on the obscene Italian comics of the Alter Ofen.
Forfruitfuldiscussions,IwanttoacknowledgeAlexMehlich,Fabian
Ziegler, Ulrich Merkel, Ziad Ganim, Gabriel Zoldak, Daniela Bauer,
Soumit Mandal and Andreas Weißl.
For assisting me to the research facilities I thank Gabriele Chmel,
Karin Vogt, Monica Rusp and Rudi Lehrhuber. For helping me in
the labyrinthine depths of bureaucracy, Elke Fehlsenfeld, Nicole
Mittermüller and Anne Ploss.
I am also thankful to our collaborators: Kristina Djinovic´-Carugo
and her group—together with Andrea Ghisleni—for sharing their
knowledge about muscle; Reinhard Fässler and his group for
helping with comments and ideas; Lukas Miller for providing me
the precious sequence of a long protein linker, and Magnus Bauer
for the assistance in the ybbR-tag conjugation. I also want to thank
three of the students I supervised, Stefan Ernst, Maximilian Zwiebel
and Julia Hirschmann, for their contribution to the α-actinin project.
A very special gratitude goes to my mother, my father and my
sister. They sustained me in my studies and made sacrifices on my
behalf.
My last sincere thanks go to Paola Pecci for the support and the
affection along the years I spent in Munich. Words cannot express
how immensely grateful I am to her.
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