© 1999 Robert A. Freitas Jr. All Rights Reserved.

Robert A. Freitas Jr., Nanomedicine, Volume I: Basic Capabilities, Landes Bioscience, Georgetown, TX, 1999

8.5.3.11 Cytoskeleton

In addition to the membrane-enclosed organelles described above, the cytoplasm of most cells is packed with filaments of various sizes collectively called the cytoskeleton. Much as the bony skeleton occupies ~11% of the human body volume (Section 8.2.4), the cytoskeleton likewise occupies ~11% of cell volume (Table 8.17). This elaborate three-dimensional webbing of filaments and tubules forms a highly structured yet dynamic matrix that extends throughout the cytoplasm, stretching from the nuclear envelope to the plasma membrane. The cytoskeleton helps to establish and maintain shape and plays important roles in cell movement, cell division, metabolism and growth,^1426-1429 and even gene expression by accepting mechanical signals (e.g., through membrane proteins such as the integrins) originating in the extracellular matrix and transducing them into the nucleus.⁹⁴² Cellular shape appears to be maintained by an architecture known as tensional integrity or "tensegrity" -- a system that achieves mechanical stability because of the way compressive and tensional forces are distributed and balanced within the cell.^1020,1021 Forcing cells to adopt different shapes causes the cells to switch between different active genetic programs.^718,1425

The cytoskeleton also serves as a framework for positioning, anchoring, and actively moving organelles and vesicles within the cytoplasm, for muscle contraction and the beating of cilia (Section 9.3.1.1) and flagella (Section 6.3.4.2), and for chromosomal movements. It has been estimated that up to 80% of unbound cytoplasmic proteins are not freely diffusible but are associated in some way with the cytoskeleton, and up to 20-40% of cytoplasmic water may be bound to the filaments and tubules of the cytoskeleton.⁹³⁹ The most radical example of cytoskeletal dynamics may be the complete remodeling of the cellular microtubular array during replication -- from a network radiating throughout the cell in interphase to the compact bipolar mitotic spindle during mitosis.

There are five recognized classes of filaments, grouped according to their diameter and by the types of protein they contain. In order of size, starting with the thinnest, they are

1. superfine filaments,

2. microfilaments,

3. intermediate filaments,

4. muscle thick filaments, and

5. microtubules.

Microfilaments and microtubules can be rapidly assembled and disassembled, allowing a cell to modify its cytoskeletal framework according to changing requirements. The other filament types, once assembled, are less readily disassembled.

A. Superfine Filaments -- Superfine filaments are short segments measuring 2-4 nm in diameter and up to 100 nm in length. Superfine filaments such as plectin^1194,3389 serve to interconnect other filaments and microtubules, stabilize the nucleus,³³⁹¹ regulate actin dynamics,³³⁹² and also provide the means by which the membranes of organelles may be attached to stable or moving elements of the cytoskeleton.^{938,3389,3390}

B. Microfilaments -- Microfilaments are the smallest of the major cytoskeletal components, measuring 5-7 nm in diameter and up to several microns in length. They are F-actin polymers of the contractile monomer protein G-actin, a single polypeptide consisting of 375 amino acid residues with a molecular weight of ~42,000. Actin is the most abundant protein in most cells, usually comprising more than 5% of the total cellular protein.⁹³⁹ Polymerization at the "plus" end and depolymerization at the "minus" end occur spontaneously, driven by local G-actin concentration. This produces a treadmilling action, wherein a given actin monomer is incorporated at the "plus" end, transfers slowly along the microfilament from one end to the other, and finally is lost by depolymerization at the "minus" end.⁹³⁹ Assembly speed for microfilaments of the actin cytoskeleton is 0.1-1 micron/sec.⁹⁴² Tensile failure strength of a single strand is ~108 pN,³⁶² while the compressive load force or "stall force" applied during polymerization by a single actin fiber is typically ~10 pN.¹²⁰³

Microfilaments make up a major portion of all cell cytoskeletons and are best known for their role in the contractile fibrils of muscle cells. They can form connections with the plasma membrane and thereby influence locomotion, amoeboid movement, and cytoplasmic streaming. They produce the cleavage furrows that divide the cytoplasm of cells after chromosomes have been separated by the spindle fibers during mitosis. Microfilaments also help develop and maintain cell shape by acting as tensile elements in the cellular tensegrity structure, pulling the plasma membrane and all of the cell's internal constituents toward the nucleus at the core.¹⁰²¹ Microfilaments also conduct mechanical signals throughout the cell at propagation speeds of 100-1200 m/sec, allowing 0.01-2 Hz signals to cross a cell in 2-20 nanosec.¹²⁰²

Most cells have a dense network of subsurface microfilaments called the cell cortex, which includes actin-binding peripheral proteins such as spectrin (in erythrocytes) or filamin and vinculin (in fibroblasts) just below the plasma membrane. In the case of the red cell, the membrane skeleton (Fig. 8.43) is a highly organized two-dimensional triangulated network of actin oligomers tethered to spectrin tetramer filaments interconnecting ~70,000 nodes¹¹³⁶ or microfilament junctional complexes comprising an equal number of triangular ~50 nm-wide meshes¹¹²⁶ (mean mesh size 3000-4800 nm²).³⁶¹²

The cell cortex confers structural rigidity on the cell surface and facilitates shape changes and bulk movement. In some cells, microfilaments are ordered into long parallel bundles called stress fibers, which may span the entire length of the cell. Stress fibers also comprise the core of microvilli. Each cell has its own arrangement of microfilaments, thus no two cells are exactly alike.⁹⁴⁰ Indeed, the microfilament network itself may form a complete tensegrity substructure within the larger cytoskeletal network.¹⁰²¹

C. Intermediate Filaments -- Tough, insoluble intermediate filaments (IFs) have a diameter of 8-12 nm with a mean intertubule spacing of perhaps ~50-100 nm. They are the most stable of the cytoskeletal elements, not being subject to constant de/repolymerization, with a tensile failure strength of ~20 nN per fiber (Table 9.3). With their high tensile strength and comparative positional stability, IFs act as internal guy wires to resist mechanical stress on the cell⁹⁴⁰ and thus are regarded as a scaffold supporting the entire cytoskeletal framework, with polysomal ribosomes located at IF junction control nodes (Fig. 8.44). IFs also have a tension-bearing role in some cells, as intermediate filaments are most extensively developed in those regions of cells most subject to mechanical stress. Another specific function of IFs is to maintain the position of the nucleus within the cell. Intermediate filaments form a ring around the nucleus with branches extending outward through the cytoplasm, and possibly extending downward into the pores of the nuclear envelope (Section 8.5.4.2) to connect with the nuclear cortex (Section 8.5.4.3). Intermediate filaments constitute ~1% of total protein, although in some cells (e.g., epidermal keratinocytes, neurons) IFs may represent up to 85% of the total protein of fully differentiated cells.¹⁵⁴³

In contrast to microtubules and microfilaments, intermediate filaments differ in their composition from one tissue to another. The known classes of IF protein (based on biochemical and immunological criteria) include the keratins of which there are at least 15 different varieties in each of the acidic and basic/neutral subclasses (in the tonofilaments of epithelial cells); vimentin, which is found in fibroblasts, connective tissue and other cells of mesenchymal origin; desmin, in muscle cells; glial fibrillary acidic (GFA) protein, in glial cells; neurofilament (NF) protein, present in the neurofilaments of nerve cells; and nuclear lamins A, B and C, which are found in the nuclear cortex of all cells.⁹³⁹ Most cells contain at least two different types of IF proteins: the three lamins in the nucleus, plus the cytoplasmic IF protein appropriate to the specific cell type. Because of this tissue specificity, cells from different tissues can be distinguished almost solely on the basis of the IF proteins present³³⁹³ -- intermediate filament typing via immunofluorescence microscopy is a useful diagnostic tool in detecting cancer and prenatal birth defects.^3394-3397 In cyto nanorobots can use this same information to continuously verify the cell type in which they are resident.

D. Muscle Thick Filaments -- Muscle thick filaments, ~15 nm in diameter and composed of the very large contractile protein myosin, are found mainly in striated muscle cells and occasionally in the cortex of nonmuscle cells.^3398-3400 Myosin molecules not in filament form are also present in many, if not most, other cells where they interact with microfilaments to produce local forces and movements.

E. Microtubules -- Microtubules are straight hollow cylinders with an outer diameter of 25 nm and an inner diameter of 15 nm. They vary greatly in length. Some are less than 200 nm long, while others, particularly in nerve cells (where they provide the framework that maintains the cell's cylindrical shape), can be as long as 25 microns. Mean intertubule spacing is at least 200-300 nm, with a minimum radius of curvature of ~0.2 microns.¹⁴³⁷ Each microtubule is composed of a helical arrangement of the ab-tubulin heterodimer protein, with 13 tubulin subunits in each rotation of the tight helix.¹⁰⁹² The uniform orientation of the tubulin molecules confers an inherent polarity on the microtubule. Placed in a tubulin-rich environment, the "plus" end of the tubulin polymer (b-tubulin) will elongate much more rapidly than the "minus" end (a-tubulin).¹¹²⁴ In the living cell, the ends of the microtubules farthest away from the center of the cell are always the "plus" ends. Microtubules radiate out as lacelike threads toward the periphery of the cell from a microtubule-organizing center (MTOC) near the nucleus.⁹³⁹ The best-known MTOC is the centrosome, which consists of granular material surrounding two centrioles (Fig. 8.36). Other examples of MTOCs include the kinetochore and the poles of the mitotic spindle.

Microtubules are the most rigid of the cytoskeletal filaments and thus often serve as the principal cytoskeletal organizers and backbone elements (Fig. 8.44), providing (along with the ECM) the compressive elements of the cellular tensegrity structure. Tensile failure strength is ~1000 pN per fiber (Table 9.3). The flexural rigidity of individual microtubules has been directly measured as k_rigid = 3.4 x 10^-23 Nm²,¹⁴⁶⁸ which is higher than the rigidity of actin filaments. What does this mean? For an elastic rod of length L attached to a wall by a pivot about which the rod is completely free to rotate, the magnitude of the critical buckling force of the rod is:^642,1468

{Eqn. 8.6}

Hence a 1-micron long microtubule will not begin to buckle until an axial load of F_buckle >~ 340 pN is applied.

Microtubules define and maintain the overall shape and architecture of the cell, confer polarity on the cell, and determine the distribution of the microfilaments and intermediate filaments. By providing a radiating system of fibers to guide the movement of vesicles and other organelles, microtubules also contribute to the spatial disposition and directional movement of subcellular structures. For example, microtubules serve as "tracks" for the outward movement of the endoplasmic reticulum in growing cells, and microtubules transport membrane-bound vesicles in both directions along the axons connecting the body of the nerve cell with the synaptic knobs (axonal transport) at a typical speed of ~2 microns/sec.⁹³⁹ Particles easily switch from one microtubule to another intersecting one during transport.¹⁴⁴⁸

The structure immediately responsible for the separation of the chromosomes, the spindle fibers, is composed of microtubules, as are the organelles (the centrioles) that generate the spindle fibers at the time of cell division. (Each cell has two centrioles, solid structures composed of microtubules. Prior to cell division, the centrioles replicate and then produce the intracellular architectural skeleton called the mitotic apparatus that guides the cell through mitosis.) Spontaneous spatial pattern formation from oscillating microtubules has been observed,^1071,1073 and cytoskeletal rearrangements appear to be biochemically regulated.¹⁰⁸³

The microtubule elongation rate in cultured fibroblasts has been measured as ~0.06 microns/sec.¹⁴²³ The turnover time for the entire microtubular apparatus has been estimated as ~900 sec (0.25 hour),¹⁴²³ with a specific power output of ~0.6 watts/m³ averaged over the entire array.¹⁴²⁴ Not all cytoplasmic microtubules immediately participate in this rapid turnover during interphase -- about 10% of the population seems to remain stable for at least 2 hours.¹⁴²³ Dynamic instability causes microtubule shortening in human monocytes, in the range of ~260 dimers/sec¹⁴⁴⁶ or ~0.15 microns/sec.¹⁴⁴⁷ Depolymerizing microtubules can drive kinetochore movements towards the minus end at ~0.5 microns/sec, exerting forces of up to ~100 pN.¹⁵⁹³

Superfine filaments connect microtubules with each other, with intermediate filaments, and with adjacent organelles.^1439,1440 The filaments extend from the microtubular surface and prevent direct contact with other cellular structures.¹⁴⁴¹ This forms a narrow exclusion zone around microtubules that is devoid of other structures, and often appears as a clear halo¹⁴⁴² with a radius from ~10 nm in insect cells¹⁴⁴³ up to ~50 nm in neurons.¹⁴⁴⁴ High molecular weight microtubule-associated proteins (MAPs) define zones of exclusion around microtubules and may help maintain the observed spacing between microtubules and cell organelles.¹⁴⁴⁵

Last updated on 20 February 2003