Figure 239

Staining of microtubules with fluorescent dye. This confocal im-munofiuorescent image shows the organization of the microtubules within an epithelial cell in tissue culture. In this example, the specimen was immunostained with three primary antibodies against tubulin (green), centrin (red), and kinetochores (light blue) and then Incubated in a mixture of three different fluorescently tagged secondary antibodies that recognized the primary antibodies. Nuclei were stained (dark blue) with a fluorescent molecule that Intercalates into the DNA double helix. Note that the microtubules are focused at the MTOC or centrosome (red) located adjacent to the nucleus. The cell is in the S phase of the cell cycle, as indicated by the presence of both large unduplicated kinetochores and smaller pairs of duplicated kinetochores. x3,000. (Courtesy of Dr. Wilma L. Lingle and Ms. Vivian A. Negron.)

The GTP-tubulin complex is then polymerized, and at some point GTP is hydrolyzed to guanosine diphosphate (GDP). As a result of this polymerization pattern, each microtubule possesses a minus (nongrowing) end embedded in the MTOC and a plus (growing) end elongating toward the cell periphery. Tubulin dimers dissociate from microtubules in the steady state, which adds a pool of free tubulin dimers to the cytoplasm. This pool is in equilibrium with the polymerized tubulin in the microtubules; therefore, polymerization and depolymerization are in equilibrium. The equilibrium can be shifted in the direction of de-polymerization by exposing the cell or isolated microtubules to low temperatures or high pressure. Repeated exposure to alternating low and high temperature is the basis of the purification technique for tubulin and microtubules. The speed of polymerization or depolymerization can also be modified by interaction with specific microtubule-associated proteins (MAPs). These proteins, such as MAP-1, 2, 3, and 4, MAP-t, and TOGp, regulate microtubule assembly and anchor the microtubules to specific organelles. MAPs are also responsible for the existence of stable populations of nondepolymerizing microtubes in the cell, such as those found in cilia and flagella.

The length of microtubules changes dynamically as tubulin dimers are added or removed in a process of dynamic instability

Microtubules observed in cultured cells with real-time video microscopy appear to be constantly growing toward the cell periphery (by addition of tubulin dimers) and then suddenly shrinking in the direction of the MTOC (by removal of tubulin dimers). This constant remodeling process, known as dynamic instability, is linked to a pattern of GTP hydrolysis during the microtubule assembly and disassembly process. The MTOC can be compared to a feeding chameleon, which fires its long, projectile tongue to make contact with potential food. The chameleon then retracts its tongue back into its mouth and repeats this process until it is successful in obtaining food. The same strategy of "firing" microtubules from the MTOC toward the cell periphery and subsequently retracting them enables the cell to establish an organized system of microtubules linking peripheral structures and organelles with the MTOC. As mentioned above, association of a microtubule with MAPs, such as occurs within the axoneme of a cilium or flagellum, effectively blocks this dynamic instability and stabilizes the microtubules.

The structure and function of microtubules in mitosis and in cilia and flagella are discussed later in this chapter and in Chapter 4.

Microtubules can be visualized in the light microscope and are involved in intracellular transport and cell motility

Microtubules may be seen in the light microscope by using special stains, polarization, or phase contrast optics.

Because of the limited resolution of the light microscope, in the past microtubules were erroneously called fibers, such as the "fibers" of the mitotic spindle. Microtubules may now be distinguished from filamentous and fibrillar cytoplasmic components even at the light microscopic level by using antibodies to tubulin, the primary protein component of microtubules, conjugated with fluorescent dyes (Fig. 2.39).

In general, microtubules are found in the cytoplasm, where they originate from the MTOC; in cilia and flagella, where they form the axoneme and its anchoring basal body; in centrioles and the mitotic spindle; and in elongating processes of the cell, such as those in growing axons.

Microtubules are involved in numerous essential cellular functions:

• Intracellular vesicular transport (e.g., movement of secretory vesicles, endosomes, lysosomes)

• Movement of cilia and flagella

• Attachment of chromosomes to the mitotic spindle and their movement during mitosis and ineiosis

• Cell elongation and movement (migration)

• Maintenance of cell shape, particularly its asymmetry

Movement of intracellular organelles is generated by molecular motor proteins associated with microtubules

In cellular activities that involve movement of organelles and other cytoplasmic structures, such as transport vesicles, mitochondria, and lysosomes, microtubules serve as guides to the appropriate destinations. Molecular motor proteins attach to these organelles or structures and ratchet along the microtubule track (Fig. 2.40). The energy required for the ratcheting movement is derived from ATP hydrolysis. Two families of molecular motors have been identified that allow for unidirectional movement:

• Dyneins constitute one family of molecular motors. They move along the microtubules toward the minus end of the tubule. Therefore, cytoplasmic dyneins are capable of transporting organelles from the cell periphery toward the MTOC. One member of the dynein family, axonemal dynein, is present in cilia and flagella. It is responsible for the sliding of one microtubule against an adjacent microtubule of the axoneme that effects their movement.

• Kinesins, members of the other family, move along the microtubules toward the plus end; therefore, they are capable of moving organelles from the cell center toward the cell periphery.

Both dyneins and kinesins are involved in mitosis and meiosis. In these activities, dyneins move the chromosomes along the kinetochore microtubules toward the spindle pole. Kinesins are simultaneously involved in movement of polar microtubules. These microtubules extend from one spindle pole past the metaphase plate and overlap with microtubules extending from the opposite spindle pole. Kinesins located between these microtubules generate a sliding movement that reduces the overlap, thereby pushing the two spindle poles apart to each daughter cell (Fig. 2.41).

Actin Filaments

Actin filaments are present in virtually all cell types

Actin molecules (42 kDa) are abundant and may constitute up to 20% of the total protein of some nonmuscle cells (Fig. 2.42). Similar to the tubulin in microtubules, actin molecules also assemble spontaneously by polymerization into a linear helical array to form filaments 6 to 8 nm in diameter. They are thinner, shorter, and more flexible than microtubules. Free actin molecules in the cytoplasm are referred to as G-actin (globular actin) in contrast to the polymerized actin of the filament, called F-actin (filamentous actin). Actin filaments are polarized structures; their fast-growing end is referred to as the plus or barbed end, and their slow-growing end is referred to as the minus or pointed end. The dynamic process of actin polymerization requires the presence of K+, Mg2+, and ATP, which is hydrolyzed to ADP after each G-actin molecule is incorporated into the filament (Fig. 2.43). The control and regulation of the polymerization process depends on the local concentration of G-actin and the interaction of actin-binding proteins (ABPs), which can prevent or enhance polymerization.

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