Polyacrylamide (PAA) gels

PAA gels are composed of acrylamide and the cross-linker N,N’-methylene-bisacrylamide (bis). The reaction is catalysed by tetramethylethylenediamine (TEMED) and ammonium persulfate (APS). TEMED has the potential to exist in a free radical state and accelerates the free radical formation of APS, which catalyses the polymerization reaction. As a free radical donor Riboflavin can be used instead or be added to the TEMED/APS combination.

The polymerised hydrogel is chemically inert and needs an ECM coating to enable cell adhesion. To enable a coating with an ECM protein as collagen, a cross-linker needs to be applied between the gel and the final coating. Commonly used for this purpose is Sulfo-SANPAH, a water-soluble cross-linker providing a NHS ester binding site as well as an UV-activatable nitrophenyl azide. At a wavelength of 320-350 nm, the nitrophenyl

azide is covalently bound to the PAA gel. ECM ligands are then able to bind with their primary amines to the free NHS ester group. Polyacrylamide gels can be easily adjusted by varying the bis concentration ([115], [118]) and be stored for a longer time ([118]), which makes them a valuable tool.

2.5. Cytoskeleton 15

2.5 Cytoskeleton

Generally, a cell can be considered the shape of a fried egg. The so-called cell body region would be the egg yolk part, including the nucleus, cell organelles and cytoskeletal structures. The corresponding flat egg white part is called lamella and encloses the cell body. It contains cytoskeletal and cytosolic components. The comparison to a fried egg includes that parts of the lamella continue below the cell body. In contact with the substrate the cell is placed on, the lamella is able to sense the adhesive properties of the substrate and eventually starts to interact with it by building contact points. Once the contact points (focal adhesions) are established, the cell connects parts of its cytoskeleton to them and exerts traction forces to these small point-like structures. The cytoskeleton inside a cell has many different functions. Not only cell organelles and important proteins are transported throughout a cell along those filamentous structures, the cell shape and motility are orchestrated as well. As the filamentous backbone of the cell, the cytoskeleton consists of three different structure types: actin filaments, microtubules and intermediate filaments (IFs). Actin filaments (also called microfilaments) are located throughout the cell. On these thin polar structures, myosin motor proteins transport vesicles throughout the cell. Since this thesis focusses on actin stress fibres, they are explained in more detail in the following sections. Microtubules are mainly located in the cell body with extensions to the lamella. Motor proteins like kinesin and dynein use these tube-like structures for transportational purposes. Microtubules are anchored at the nucleus and are important in cell division. The intermediate filaments are responsible for maintaining the cell shape.

Unlike the other two cytoskeletal structures presented here, they lack a structural polarity.

With it, to our knowledge, no transport along intermediate filaments in terms of delivery by motor protein activity has been reported. However, intermediate filaments seem to be involved in cell signalling ([119], [120]). The intermediate filament mainly found in mesenchymal cell is vimentin [120]. It is considered to be crucial for many cellular functions and a lack thereof leads to morphological changes of even glia cells [121]. However, there is evidence that all three types of cytoskeletal structures team up at least during cell adhesion and migration ([122], [123],[124]).

2.6 Actin

Actin filaments are composed of small globular actin monomers (42 kDa - 375 amino acids), called G-actin. Each monomer contains an adenosine triphosphate (ATP) group at the ATP binding site and a hydrolytic site. These two features are located at opposing sides of the molecule. Hydrolysis of monomer one causes a dephosphorylation of the ATP-group to ADP and leads to a conformational change to which monomer two can bind. In this new formed filament, one former monomer contains an ATP-binding site and one a hydrolytic site. These structural features remain independent of the amount of bound actin monomers. The side with the ATP containing region is called barbed end

or plus end and the other hydrolytic site of the filament is called pointed end. When two or three Actin monomers bind they are called “seed” due to the fast filament growth, once the seed is established. The faster growing end of the filament is the one containing ATP and sometimes called plus end, while the other side of the filament is called minus end. However, the terms plus and minus end can be misleading, since plus and minus in combination with molecules are usually associated with electrical charges. This structural arrangement of actin filaments is utilised by motor proteins like the myosin motorprotein family in the animal and plant kingdoms alike. Myosins transport vesicles or cell organelles across the cell on the actin filament meshwork. A special arrangement of actin filaments and myosin motors called sarcomer can be found in muscle cells. Here, alternating layers of actin filaments and bundles of myosin motors cause contractions of the whole cell. In this bundle, motile myosin head groups are exposed around the bundle to be able to bind to actin filaments at at least two different sites. The motor heads are binding and moving the actin filaments in opposite directions, which causes the contraction.

2.6. Actin 17