Cytoplasmic Filaments: Composition, Distribution and Functions

Cytoplasmic filaments are elongate, un-branched, proteinaceous strands that consist of bundles or groups of protein molecules sometimes wound into a helical shape.

Although organized arrays of cytoplasmic fila­ments were first described for muscle cells and have been most extensively studied in this tissue, it is now apparent that essentially all eukaryotic cells contain these filaments.

Cytoplasmic filaments can be subdi­vided into three major classes based on filament size; these are:

(1) Microfilaments (also called thin fila­ments), which have a diameter of about 6 n and are composed primarily of the globular protein actin;

(2) Intermediate filaments, which have diameters be­tween 7 and 11 nm and, depending on the tissue source, are formed from five different classes of pro­teins (see below); and

(3) Myosin filaments (also called thick filaments), which have diameters up to 22 nm and are rich in the fibrous protein myosin.

Muscle cells, which are especially differentiated for contrac­tion, are rich in mi­crofilaments and myosin filaments, but all cells con­tain cytoplasmic filaments and it has been shown that actin can account for as much as 20% of the total cell protein in some non-muscle cells.

Microfilaments:

Although actin molecules are globu­lar proteins (i.e., G-actin), in microfilaments the actin molecules are arranged to form two interwoven helical chains, each of which is called a proto-filament (Fig. 23-6); in this polymeric form, the protein is called F-actin. Microfilaments are intimately associated with all cellular activities that involve movement.

This is vividly demonstrated when cells are treated with cytochalasin B. In the presence of this substance, micro­filaments dissociate and the loss of the microfilaments is accompanied by a loss of certain cellular functions. Some of the more common processes sensitive to cyto­chalasin B are phagocytosis, pinocytosis, and exocytosis, cytokinesis, cytoplasmic streaming (in plant cells), movements of microvilli, cilia, and flagella, movements of the cytoskeleton, and of course muscle contraction.

Intermediate Filaments:

Five different classes of pro­teins comprise the intermediate filaments of cells and tissues; these are desmin in muscle cells (one type of protein of MW 52,000), vimentin in mesenchymal cells (one protein of MW 53,000), cytokeratins in epithelial cells (more than a dozen different proteins having MWs ranging from 40,000 to 70,000), neuro-filament proteins in nerve cells (three proteins having MWs of 65,000, 105,000, and 135,000), and glial fibrillary acidic protein in astrocytes (one protein of MW 50,000).

Although a dynamic role cannot be excluded for the ‘intermediate filaments, in most cells they ap­pear to play a structural role. For example, in epider­mal and mesenchymal cells, bundles of intermediate filaments form a basketlike weave that encircles the nucleus and is intimately associated with the nuclear envelope at the nuclear pores.

From there the fila­ments appear to radiate to the margins of the cells, where they terminate at desmosomes. Because of their intimate association with the nuclear pores, a role in mediating the transport of materials between the nucleus and the cyotplasm cannot be pre­cluded.

In nerve cells, the long axes of the intermediate fila­ments are oriented parallel to the long axes of the cells’ “long processes” (e.g., axons and dendrites) and are believed to provide these pro­cesses with a structural framework.

Myosin (Thick) Filaments:

Striated, smooth, and car­diac muscle cells contain vast numbers of cytoplasmic filaments that function during the contraction of these cells. Most of these filaments are microfila­ments (i.e., thin filaments formed primarily from F-actin but also. containing the proteins tropomyosin and troponin) and thick filaments (formed from myo­sin).

The two types of cytoplasmic filaments are ar­ranged in parallel rows and interact with each other through cross-bridges that enable the filaments to slide past one another and effect a shortening (i.e., contraction) of the cell. The contraction of striated muscle cells shortens the muscle, which then moves the skeleton. It is in striated muscle cells that the number of filaments is greatest and their geometric distribution most highly ordered.

As seen in Figure 23-7, equally spaced about each thick filament are six thin filaments. Units of several hundred thick and thin filaments are bundled together to form myofibrils, and each cell contains many hundreds of myofibrils.

Cytokinesis:

Cell division in animal cells involves two separate mechanisms mitosis and cytoplas­mic cleavage, or cytokinesis. In animal cells, cytoki­nesis begins toward the end of anaphase and is charac­terized by the appearance of a constriction about the midline of the spindle. The constriction deepens as the plasma membrane moves inward at the cleavage fur­row. Material collecting at the midline of the spindle becomes quite dense, forming a structure known as the mid-body (Fig. 23-8). Just before the in-folding edges of the plasma membrane meet and fuse, the mid-body disappears.

The furrowing or pinching-in of the plasma mem­brane at the cleavage furrow is reminiscent of the action of a purse string or of a rubber band tightening about a soft object. The process clearly involves the action of microfilaments and these are seen in abun­dance in the area of the cleavage furrow. Microfila­ment involvement is also supported by the observation that cytochalasin B inhibits the process.

The presence of a band of microfilaments, the contractile ring, just underneath the plasma membrane in the area of con­striction can be seen in the electron photomicrographs of Figure 23-9. Here Arbacia sea urchin eggs are ob­served at various stages of cleavage. Prior to the onset of cleavage, no microfilaments are seen in the area that is about to constrict (Figs. 23-9a and 23-9d).

However, once cleavage begins, microfilaments quickly appear about the area of constriction, forming the contractile ring (compare Figs. 23-9d and 23-9e). Because the bundles of microfilaments are arranged in a circle just below the cleavage furrow, their long axes run perpendicular to the plane of the sections seen in Figures 23-9b and 23-9e.

As cleavage is com­pleted, the spindle fibers and mid-body fade and the contractile ring and microfilaments disappear (Fig. 23-9c). The molecular basis of the constriction that characterizes cytokinesis in animal cells remains un­certain, but the clear presence of actin and the sus­pected presence of myosin has led to the speculation that the mechanism involves filament sliding similar to that which occurs during muscle contraction.

Plasma Membrane Movement:

Intestinal epithelial cells have thousands of small fin­gerlike projections called microvilli that cyclically shorten and extend into the lumen of the intestine. This action facilitates the absorption of digested food by greatly increasing the surface area of the cells.

Microfilaments in the intestinal microvilli are bundled to­gether to form a core by the interaction of F-actin with the two proteins villin and fimbrin. The micro­filament bundle runs parallel to the length of the mi­crovillus and is anchored to the plasma membrane at the microvillus’ apical tip.

No myosin appears to be present in the microvilli. At the base of the microvilli is another microfilament network called the terminal web. Myosin has been localized in the terminal web and its interaction there with actin is believed to sup­port the microvilli and perhaps also create a contract­ile mechanism that lowers the microvilli into the cell. A second possibility is that in the presence of high Ca²⁺, villin can act to cleave F-actin into short frag­ments. This would cause the microvilli to collapse into the cell in a mechanism that is independent of myosin. Calmodulin has also been implicated in the regulation of movements of microvilli.

Amoeboid Movement:

The locomotion of amoebae, slime molds, white blood cells, and a number of other cells involves the forma­tion of pseudopodia—large cytoplasmic extensions from the main body of the cell and into which the re­maining cytoplasm subsequently streams. Most amoe­boid cells from more than one pseudopod at a time, but continued locomotion in one direction require the reversal of the process in the non-dominant pseudopo­dia.

In pseudopodia-forming cells, the outer margin of the cytoplasm, called ectoplasm, is very viscous or gel-like and is generally free of granules and other cy­toplasmic inclusions; the remaining cytoplasm, called endoplasm, is more fluid or sol-like. During locomo­tion, the fluid endoplasm flows forward into the ad­vancing pseudopod, and as it reaches the anterior end of the pseudopod, it flows laterally and is converted into gel, thereby forming new ectoplasm. At the rear of the moving cell, in a region called the uroid, the ectoplasm solates, streams deeper into the cell, and becomes endoplasm (Fig. 23-10).

Two different views have dominated attempts to ex­plain amoeboid movement. According to the “front- zone contraction” theory of R. D. Allen and others, the endoplasm in the anterior region of a pseudopod undergoes contraction as it is transformed into ecto­plasm. In effect, this contraction pulls the endoplasm forward and into the pseudopod. An older view ad­vanced in the 1920s by S.O. Mast and called the “ectoplasmic tube contraction” theory suggests that the ectoplasm toward the rear of the cell contracts, push­ing the endoplasm forward.

Regardless of the site of origin of the force that causes the cytoplasm to stream forward, overwhelm­ing evidence implicates actin microfilaments in the process. For example, the actin-binding drug cytochalasin B inhibits amoeboid movement.

The pres­ence of myosin filaments in amoeboid cells has also been reported as has an ATPase similar to one known to be important in the chemistry of muscle contrac­tion. Contractions of the ectoplasm of amoeboid cells can be induced by adding ATP and Ca^{2 +} to the cells. Although not fully understood, amoeboid movement appears to use an actinmyosin contractile system similar to that of muscle cells.

An amoeboid like but much slower form of locomo­tion is also exhibited by mammalian connective tissue cells (i.e., fibroblasts) grown in culture. As a fibroblast moves across a surface (e.g., the culture dish), the cell continuously forms sheetlike extensions, called lamellipodia, and micro spikes at its advancing edge (Figs. 23-11 and 23-12). Some of the lamellipodia attach to the surface (at anchor points called adhesion plaques) and others sweep backward over the cell’s upper surface in a wavelike motion called surface ruffling.

Electron-microscopic studies reveal that the lamellipodia contain large numbers of actin microfilaments, and like true amoeboid motion, the movements of fibroblasts are inhibited by cyto­chalasin B. As the leading edge of the cell stretches forward, the trailing edge is drawn out into long re­traction fibers that are eventually pulled free from their points of attachment to the underlying surface. The latter action may leave behind small pieces of plasma membrane and cytoplasm.

Capping, Membrane Flow, and Cell Locomotion:

Substances bound to mo­bile plasma membrane receptors migrate laterally within the membrane and are concentrated in clathrin-coated pits prior to endocytosis. During endocytosis, these receptors and the ligands that they have bound are internalized along with small pieces of the plasma membrane. The receptors are eventually returned to the plasma membrane during exocytosis, which also restores membrane surface area lost dur­ing endocytosis. In non-motile cells, endocytosis and exocytosis occur at positions scattered over much of the cell surface.

Many membrane proteins avoid internalization during endocytosis because their rates of diffusion within the membrane preclude their concentration within the coated pits. These membrane proteins are referred to antibodies against non-cycling membrane proteins, the proteins are cross-linked to form large aggregates. Because the diffusion rates of cross-linked proteins are so much lower than the diffusion rates of individ­ual proteins, the aggregate can be clustered together.

In motile cells, cross-linked proteins cluster at one point on the cell surface called a cap, and the process is called capping. Capping is a general phenomenon displayed by aggregates in the plasma membrane and is not peculiar to certain proteins; non-motile cells, however, do not cap cross-linked surface proteins. Cy­tochalasin B and another drug called colchicine pre­vent capping, suggesting that membrane flow in­volves the actions of microfilaments (and perhaps also microtubules).

When fibroblasts are allowed to migrate over a sub­strate littered with tiny carbon particles, the particles become attached to the upper surface of extended la­mellipodia and are slowly swept backward toward the trailing edge of the cell. This implies that the mem­brane as a whole is flowing backward. It has therefore been suggested that a moving fibroblast internalizes portions of the plasma membrane-at the rear of the cell and reinserts this membrane at the leading edge of the cell (Fig. 23-13).

This would provide a mecha­nism by which the cell could move itself forward along a substrate, for if the cell forms transient connections with the substrate at its anterior end, the forces that cause the membrane to flow backward would simulta­neously propel the cell forward. The action would be similar to that of a tractor or tank track.