Date of Award

8-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physics

Advisor(s)

Martin B. Forstner

Keywords

FRET, Mechanotransduction, membrane skeleton, Spectrin, Supported lipid bilayer, Traction force

Subject Categories

Physics

Abstract

The thesis is concerned with questions regarding cellular bio-mechanics and cell surface interactions. A particular focus is thereby on the role of the spectrin membrane skeleton in transducing forces to and from non-erythroid mammalian cells. This spectrin based membrane skeleton has been the focus of much study in the context of red blood cells, as it determines their mechanical properties due to the lack of an extended actin based cytoskeleton. In stark contrast, the corresponding structure in non-erythroids is much less studied and understood, although it seems to play important roles in organization of membrane associated proteins, cellular mechanics, adhesion, traction and possibly mechanotransduction. In this work, I was able to determine the amount -down to the average copy number per cell- of the main protein components of the non-erythroid membrane skeleton, in model cell lines commonly used in cell-mechanics studies. The results of the measurements provided by combining a variety of optical microscopic and biochemical techniques, demonstrate that proteins associated with the membrane skeleton constitute a large (s10%) fraction of cellular proteins. These results are then compared with the respective quantities after mechanical stimulation of the cells. It is found that external forces result in both an up to 60% changes of the overall amounts of proteins as well as the protein composition of the membrane skeleton itself. In addition, it was established that the fraction of polyubiquitnated spectrin has significantly increased due to stimulation. The work helps to establish the fact that the spectrin based membrane skeleton, while often overlooked in non-erythroids, is indeed a verily generic and important system in mammalian cells that is also quite sensitive to external forces. Thus, the skeleton should be taken into account when studying cellular mechanics, membrane structure or composition.

Furthermore, I present my successful work on integrating several light based methods to simultaneously measure traction forces of adherent cells as well as internal strains in their membrane skeleton. For the proof of principle experiments and optimization procedures, I used NIH-3T3 fibroblast and H9c2 (2 -1) cardiomyocyte cell lines, both of which are known to be mechanically active. Using my method, I demonstrate that the internal strains in the membrane skeleton of fibroblasts are correlated with the polyacrylamide substrate stiffness. The later has also a measurable impact on the generated cellular traction forces. These findings open up a first glimpse on the questions that can now be addressed with this method and it promises to help to refine our still rudimentary understanding of the interrelated mechanisms of cellular mechanotransduction and force generation machinery.

Cells are exposed not only to mechanical forces but also to electrical, chemical and magnetic forces found in their environment. In a separate series of experiments, I observed that cellular behavior of 3T3 fibroblasts on glass supported lipid bilayers depend on the detail of lipid charge mixtures in the bilayer as well as the head group compositions. Live cell-supported lipid membrane hybrids are frequently used in dissecting membrane based inter-cellular communication. This work indicates that in the interpretation of the observed cell behavior on this hybrid system, the lipid bilayer itself and not only the protein augmentation can play an important role.

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