Figure1. 7 SEM image of tantalum oxide nanodots arrays [1].
1.3 Cytoskeleton and focal adhesions
Cells always explore and react with environment by filopodia. Once they detected a proper site, focal adhesions would be formed and signals related to cellular differentiation would be transduced. Then the cells started to enter their life cycle [2]. The current study also suggests
that development of cytoskeleton would be influenced by nanotopography [10]. So we believed that actin filament and focal adhesion are important clues to figure out the cell-matrix interaction.
1.3.1 Actin filaments
Actin is the most major cytoskeletal protein of most cells and able to polymerize to from actin filaments-thin, flexible fibers with naoscale in diameter and microscale in length. Within the cell, actin filaments (also called “microfilaments”) are organized into high-order structures, forming bundles or three-dimensional networks with properties of semisolid gels. The assembly and disassembly of actin filaments are regulated by a variety of actin-binding protein, which are critical components of the actin cytoskeleton. The network formed by actin provides mechanical support, determines cell shape, and allows movement of the cell surface;
thereby enabling cells to migrate, engulf particles, and divide.
Individual actin molecules are globular proteins 375 amino acids (43kd). Each actin monomer (globular G actin) has tight binding sites that mediate head-to-tail interactions with two other actin monomers, so actin monomers polymerize to form filaments (filamentous F actin). The first step in actin polymerization (called nucleation) is the formation of a small aggregate consisting of three actin monomers. Actin filaments are then able to grow by the reversible addition of monomers to both ends, but one end (the plus end) elongates five to ten times faster than the minus end. Because actin polymerization is reversible, filaments can depolymerizate by the dissociation of actin subunits, allowing actin filaments to be broken down when necessary. Thus an apparent equilibrium exists between actin monomers and filaments. Moreover the equilibrium is dependent on the concentration of free monomers [13].
(A)
(B)
Figure1. 8 Assembly of actin filaments. (A) Actin monomers (G actin) polymerize to form actin filaments (F actin). (B) The minus ends grow less rapidly than the plus ends of actin filaments. This difference in growth rate is reflected in a difference in the critical concentration for addition of monomers to the two ends of the filament [13].
1.3.2 Effect of cytochalasin D
Cytochalasin D is a well-characterized agent and cell-permeable fungal toxin which binds to the plus ends of actin filaments inhibiting both the association and dissociation of subunits [14]. This causes the disruption of actin filaments and inhibition of actin polymerization.
Cytochalasin D alone induces a dose-dependent cytoskeletal collapse that causes apoptosis [15-18].
1.3.3 Focal adhesion
Most cells have specialized regions of the plasma membrane that form contacts with adjacent cells, tissue components, or substrates. These regions also serve as attachment sites for bundles of actin filaments that anchor the cytoskeleton to areas of cell contact. These discrete sites of attachment are called focal adhesions and particularly evident in fibroblasts maintained in tissue culture. Such cultured fibroblasts then attach to the culture dish via the binding of transmembrane proteins (called integrins) to the extracellular matrix. The associations, which are complex and not well understood, are mediate by several other proteins, including talin and vinculin. Vinculin is a prominent component of focal complexes and focal adhesions [19, 20]. Fig 1.9 shows the schematic of attachment of stress fibers to the plasma membrane at focal adhesions.
(A) (B)
Figure1. 9 Junctions between cells and the extracellular matrix. (A) Integrins mediate junction in which the cytoskeleton is linked to the ECM. B) Stress fiber s (bundles of actin filaments crosslinked by α-actinin) are then bound to the cytoplasmic domain of intergins by complex associations involving a number proteins. Two possible associations are illustrated: 1) talin binds to both intergin and vinculin, which in turn binds to actin, and 2) intergin binds to α-actinin [13].
1.3.4 Function and elements of ECM
In vivo, cells are immobilized within tissue, embedded in the diverse array of scaffoldings known as the extracellular matrix (ECM). The individual components of the ECM exist in the nanometer length scale and thus many tools from nanotechnology are appropriate to mimic their features. The ECM consists predominantly of interwoven protein fibers such as collagen or elastin that have 10–300 nm diameters. Extracted basement membranes imaged with electron microscopy show that its three-dimensional architecture consists of nanopores, roughly 70 nm in diameter, and intertwined fibrils that form a felt-like landscape with peaks and valleys that are approximately 100 nm in height and depth. The meshwork of ECM can be organized randomly or with semi-alignment, and the size of fibrils and pores differ, depending on the source tissue. It is now clear that cells detect and respond to numerous features of the ECM, including the composition and availability of adhesive ligands, mechanical stiffness, and spatial and topological organization of these scaffolds, through surface receptors known as integrins [2]. Here we introduce two major components of ECM related to cell adhesion.
One is collagen, which is the single most abundant protein in animal tissues and constitutes the structure of ECM. The collagens are a large family of proteins, containing at least 19 different members. They are characterized by the formation of triple helices in which three polypeptide chains are wound tightly around one another in a ropelike structure (Fig 1.10A).
The most abundant type of collagen (type I collagen) is one of the fibril-forming collagens that are the basic structural components of connective tissues. After being secreted from the cell, these collagens assemble into collagen fibrils in which the triple helical molecules are associated in regular staggered arrays [13].
The other is fibronectin, which plays a crucial role in a wide variety of developmental and cellular processes. Fibronectin is a dimeric glycoprotein consisting of two polypeptide chains, each containing nearly 2500 amino acids (Fig 1.10B). At the molecular level, cell movement
and behavior are mediated by FN fibrils extending between cells and to the substratum. In the extracellular matrix, fibronectin is further crosslinked into fibrils by disulfide bonds.
Fibronectin has binding sites for both collagen and GAGs (glycosaminoglycans), so it crosslinks these matrix components. A distinct site on the fibronectin molecule is recognized by cell surface receptors and is thus responsible for the attachment of cells to the extracellular matrix [13, 21].
(A) (B)
Figure1. 10 Structure of collagen and fibronectin. (A) Three polypeptide chains coil around one another in a characteristic triple helix structure. (B) Fibronectin is a dimmer of similar polypeptide chains joined by disulfide bonds near the C terminus. Sites for binding to proteoglycans, cells, and collagen are indicated [13].