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Modulation of cell-matrix adhesions to affect mechanotransduction

Lutz, Roman. Modulation of cell-matrix adhesions to affect mechanotransduction. 2010, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_9136

Abstract

In cultured fibroblasts three types of cell-matrix adhesion with specific cellular localization
and protein composition can be distinguished: focal complexes, focal adhesions and fibrillar
adhesions. The formation of fibrillar adhesions is known to be required for fibronectin
fibrillogenesis. However, little is known about specific signaling from these adhesion sites.
Previous experiments showed that ILK (integrin-linked kinase) knockout fibroblasts are
defective in RhoA-dependent responses to cyclic strain. In addition these cells did not form
fibrillar adhesions and did not assemble fibronectin. We therefore speculated that pericellular
fibronectin and the fibrillar adhesions are important to activate the RhoA/ROCK pathway by
mechanical stress. To test this hypothesis, we generated fibronectin knockdown fibroblasts
and analyzed their ability to activate specific RhoA dependent responses to cyclic strain in
the absence and presence of exogenous fibronectin. Normal fibroblasts seeded on vitronectin
in fibronectin-depleted medium deposited their own fibronectin matrix and in response to
cyclic strain, activated RhoA, formed stress fibers, translocated MAL (megakaryocytic
leukemia protein) to the nucleus, and induced tenascin-C. By contrast, these responses were
suppressed in fibronectin knockdown or knockout cells grown under identical conditions.
Interestingly, on vitronectin substrate, fibronectin-deficient cells lacked integrin á5â1-positive
fibrillar adhesions. However, when fibronectin-deficient fibroblasts were plated on
exogenous fibronectin, their defects in adhesions and mechanotransduction were restored.
Studies with fibronectin fragments indicated that both, the RGD-synergy site and the adjacent
heparin-binding region were required for full activity in mechanotransduction, but not its
ability to self-assemble. In contrast to RhoA-mediated responses, activation of Erk1/2 and
PKB/Akt by cyclic strain was not affected in fibronectin-deficient cells. Our results indicate
that activation of the RhoA/ROCK pathway by mechanical stress originates from fibrillar adhesions connected to fibronectin in the extracellular matrix.
In a second project we tried to get more insight in signaling by focal complexes. These cellmatrix
adhesions are the first to be formed by adhering fibroblasts. They are clearly smaller
than more mature focal adhesions and are uniquely found at the cell border of lamellipodia.
Using patterns with square-shaped, RGD-coupled gold dots of a limited size, we attempted to
inhibit maturation of focal complexes into focal adhesions. Indeed, on RGD-coated goldsquares
smaller than 1ìm, cells uniquely formed focal complexes indicated by low
recruitment of á5 integrin. Cells formed excessive amounts of lamellipodia and assembled
actin only into a fine meshwork. However, on squares equal or larger than 1 ìm cells
exhibited focal adhesions, spread normally and assembled actin into thick fibers. Our results
show that at the level of focal complexes cell adhesion maturation can be inhibited by
restricting size. In addition, according to the lamellipodia which are seen with cells on
patterns smaller than 1 ìm, let suggest increased Rac signaling deriving from these focal
complexes.
Advisors:Chiquet, Matthias
Committee Members:Lütolf, Matthias and Rüegg, Markus A.
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI
Item Type:Thesis
Thesis no:9136
Bibsysno:Link to catalogue
Number of Pages:129 Bl.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:41
Deposited On:25 Aug 2010 13:00

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