9 Cell dynamics and the actin cytoskeleton

The many different cellular states that occur can be examined, with varying degrees of difficulty. The most difficult state is when the cells of interest are embedded in a tissue matrix in a living animal, such as chondrocytes within the matrix of joint cartilage. Some cells, such as the endothelial cells that line the cardiovascular tree of mammals, are more accessible because they lie closely packed along the surface of blood vessels. This configuration is called the endothelial monolayer because the equilibrium condition for these cells is to form a single-cell-thick layer in which all cells are in contact with their neighbors. Much study has been given to endothelial cells because of their putative role in controlling the events that can cause atherosclerosis and subsequent heart disease. An additional attractiveness of studying endothelial cells is the ease with which they can be grown in culture (Gimbrone, 1976). One can study the influence of many physical and chemical properties in a setting having strong similarities to the in vivo conditions, while varying the environment one parameter at a time. Many of the data in this chapter were derived from experiments using endothelial cells in culture.

The interior of the cell between the cytoskeletal members is filled with cytoplasm, a water-based slurry composed primarily of electrolytes and small monomeric proteins. The small proteins diffuse through the viscous cytoplasm with a diffusion coefficient that can be measured with modern fluorescence techniques. For actin monomer in the cytoplasm of a vascular endothelial cell, the diffusion coefficient D is about 3 − 6 × 10−8 cm2/s (Giuliano and Taylor, 1994; McGrath et al., 1998b). By contrast, water molecules in the liquid have a self-diffusion coefficient three orders of magnitude larger.

Of the three types of cytoskeletal polymers – actin filaments, intermediate filaments, and microtubules – that determine endothelial cell shape, actin filaments are the most abundant and are located in closest proximity to the cell membrane. Confluent endothelial cells assemble 70 percent of their 100 µM total actin into a rich meshwork of just over 50,000 actin filaments that are on average 3 µm long (McGrath et al., 2000b). Cross-linking proteins organize actin filaments into viscoelastic gels that connect to transmembrane proteins and signaling complexes located at intercellular attachment sites and extracellular matrix adhesion sites. Of particular importance are the direct connections of actin filaments to β integrin tails by talin (Calderwood and Ginsberg, 2003) and filamins (Stossel et al., 2001), and to cadherins by vinculin and catenins (Wheelock and Knudsen, 1991). During cell locomotion and shape change events, the actin cytoskeleton is extensively remodeled (Satcher et al., 1997; Theriot and Mitchison, 1991), primarily by adding and subtracting subunits at free filament ends.

Monomeric actin has a molecular weight of 42.8 KDaltons and is found in all eukaryotic cells. It is the major contributor to the mechanical structure of the cell, and the degree to which it is polymerized into long filaments changes dramatically depending on the conditions in which the cell finds itself. The properties of endothelial cells, those that line blood vessels, are illustrated by the typical values for the actin polymerization and other mechanical properties summarized in Table 9-1. Similar numbers have been found for many eukaryotic cells (see Stossel et al. (2001)).

Interaction of the cell cytoskeleton with the outside environment

Eukaryotic cells are composed of a semistructured interior and an enclosing membrane that separates the interior from the environment. In Fig. 9-1A, it can be seen how the external membrane covers the cell. Removing the membrane and the interior cytoplasm with a suitable solvent reveals the actin cytoskeleton as seen in Fig. 9-1B. These views are from the apical side of cultured endothelial cells. One can see remnants of the membrane at junctions where the bounding bilayer membrane shell was attached to the underlying structure with large protein complexes. A diagram of the current view of these complicated attachments is given in Fig. 9-2. These complexes figure prominently in transducing mechanical signals from the outside, responding to external forces on the membrane such as the shear stress produced by flow.

Endothelial cells are found to be strongly attached to a substrate. In vivo, the cells attach to the artery wall, which is covered with a basement membrane of collagen and other proteins. The attachment consists of large protein complexes that connect the substrate to the internal actin cytoskeleton through the intermediary of transmembrane proteins. The transmembrane proteins attach to ligands in the substrate. In equilibrium, the cell is pulled into a flat configuration varying in thickness from about 2–3 µm in the cytoplasmic periphery to around 4–6 µm over the nucleus.

In vitro cell attachment in culture medium is similar, except that there is no basement membrane; the substrate is normally covered with fibrinogen or collagen and the transmembrane proteins attach directly to this layer. Within twenty-four hours in culture, the cells begin to excrete their own substrate proteins, and this forms a surface to which the cells stick tenaciously. Fluid shear stresses up to 40 dynes/cm2 have no ability to detach the cells. On the other hand, the individual attachment complexes turn over continuously, as shown by exquisite confocal microscopy experiments, with a time scale on the order of fifteen minutes (Davies et al., 1993; Davies et al., 1994).

Although the mechanosensitive molecular mechanisms that determine shear-stress- mediated endothelial shape change are poorly defined, a growing body of evidence supports a decentralized, integrated signaling network in which force-bearing cytoskeletal polymers attach to transmembrane proteins where conformational

174J.L. McGrath and C.F. Dewey, Jr.

Clearly, the nature of the cytoskeleton and its ability to transmit these forces plays a key role in cell function.

Properties of actin filaments

Eukaryotic cells exhibit three distinct types of internal polymerized actin structures. The first and arguably most important is the distributed actin lattice that supports most cells and appears to be very well distributed throughout the cell. That configuration is shown in Fig. 9-1B. The individual filaments are too small to be resolved with optical microscopy, and staining for fluorescence of actin can at most show a dull glow throughout the cell when staining this component.

The second type of polymerized actin is found in filament bundles called stress fibers. These stress fibers are often seen to have ends coincident with the location of attachment complexes at the cell-substrate boundary, and also at cell-cell junction complexes. These stress fibers are collections of five to twenty individual actin filaments tightly bound together by other proteins. Their large size and high density makes them prominently visible in actin fluorescence experiments. Because the filaments are visible and also change with the stress conditions of the cell, it is often believed that these stress fibers reflect the main role of actin in a cell. Careful estimates of the fraction of total cellular actin associated with stress fibers, however, suggest that they only play a small role in maintaining cell shape and providing the cytoskeleton of the cell (Satcher and Dewey, 1996). The distributed actin lattice is much more important to cell shape and mechanical properties.

A third type of actin structure is a lattice similar in appearance to Fig. 9-1B that is confined to a small region of the cell just under the surface membrane. This is termed cortical actin, and is found for example in red blood cells. Red blood cells are devoid of any distributed lattice within the rest of the cell interior, and their reliance on cortical structure is not typical of most other cells, including endothelium (Hartwig and DeSisto, 1991).

Single actin filaments have been studied intensively for over twenty-five years. Reviews of their properties can be found in the excellent treatises by Boal (2002) and Preston et al. (1990). An actin filament in a thermally active bath of surrounding molecules will randomly deform from a straight rod into a curved shape. The characteristic length, ξp, over which the curvature of an isolated actin filament can become significant is very long, about 10–20 µm at 37 C (Boal, 2002; Janmey et al., 1994; MacKintosh et al., 1995). This is comparable to the dimensions of the cell, whereas the distance between points where the filaments are in contact is much shorter. Typical actin filament lengths are less than 1 µm, so that the cytoskeleton appears to be a tangle of fairly stiff rods. A most important feature is the fact that the small actin filaments are bound to one another by special actin-binding proteins. The most prominent of this class is Filamin A, and its characteristics are described in the next two paragraphs. For the purposes of classifying the mechanical properties of this mixture, one can visualize a relatively dense packing of filaments bound together at relatively large angles by attached protein bridges. An examination of Fig. 9-1B suggests that the filament spacing is typically 100 nm, the intersection angles of the filaments vary

Fig. 9-3. Functional properties of filamin. The bar is 200 nm. Text and drawing courtesy of J.H. Hartwig; micrographs courtesy of C.A. Hartemink, unpublished, 2004.

significantly but are most often closer to 90 degrees than to 45 degrees, and that the spacing will vary with the density of filaments.

The role of filamin A (FLNa)

Cellular actin structure is controlled at different levels. Of particular importance are proteins that regulate actin filament assembly/disassembly reactions and those that regulate the architecture of F-actin and/or attach it to the plasma membrane. One such protein is filamin A (FLNa), a product of the X-chromosome (Gorlin et al., 1993; Gorlin et al., 1990; Stossel et al., 2001). Filamin A was initially isolated and characterized in 1975 (Stossel and Hartwig, 1976) and subsequent research has continued to find important new functions for the protein (Nakamura et al., 2002). As shown in Fig. 9-3, this large protein binds actin filaments, thus defining the cytoskeletal architecture, and attaches them to membrane by also binding to a number