Introduction and Goals
Cells are inherently dynamic; there is movement of vesicles and organelles within the cell, cell shapes can be modulated and altered, and some cells actually crawl from one location to another. In this tutorial, the proteins that mediate this movement and the proteins that protect cells from mechanical stresses will be described. These are the proteins that make up the cytoskeleton, the large and complex network of biological filaments that traverse a cell, support and shape the plasma membrane, and guide the
movement of material in the cell (and sometimes even the cell itself). By the end of this tutorial, you should know:
- The organization and regulation of microtubules, including
- the process of dynamic instability,
- How motor proteins travel along microtubules,
- The organization and regulation of actin filaments, including
- the role of actin-binding proteins,
- How myosin interacts with actin,
- How actin directs cell crawling,
- How actin/myosin interactions generate muscle contractions and
- The organization and role of intermediate filaments.
Subcellular Structures Move on Microtubules
Microtubules are the components of the cytoskeleton that are responsible for moving vesicles, organelles and other subcellular structures throughout the cell. Cytoplasmic microtubules form tracks along which vesicles are transported throughout the cell. For example, microtubule tracks carry the secretory vesicles derived from the Golgi complex and destined to be delivered to the plasma membrane. Microtubules in the mitotic spindle provide the means of separating the replicated chromosomes into the two daughter cells. During plant cell division, microtubules are arranged in the middle of the mitotic spindle and recruit vesicles that will form the new cell wall between the two daughter cells. Finally, cilia and flagella, specialized projections of the plasma membrane on many eukaryotic cells, are formed by microtubules.
Microtubule Organization and Growth
Microtubules are composed of two tubulin subunits (alpha- tubulin and beta-tubulin). Dimers of alpha- tubulin and beta-tubulin subunits are added to the end of a growing microtubule. The tubulins are organized to form a hollow tube of 11-15 parallel strands of tubulin dimers, with 13 being the most common. Microtubules have an inherent polarity; the plus end is the end with the exposed beta-tubulin and the minus end is the end with the exposed alpha-tubulin (see Figure 1). In general, tubulin dimers are added more rapidly to the plus end and lost from the minus end. The addition of tubulin subunits is termed polymerization, and the loss of subunits is termed depolymerization.
Microtubules originate from specialized structures called microtubule organizing centers (MTOC), typically a centrosome, which contain rings of gamma-tubulin that are the nucleation site (starting point) of microtubule polymerization. The minus end of a microtubule is associated with a gamma-tubulin ring, and it grows by radiating out from the centrosome. Microtubules are very dynamic, growing and shrinking at either end. The polymerization of a microtubule requires tubulin subunits bound to GTP. The rate of
growth (or shrinkage) of the plus and minus ends of a microtubule is a function of the free tubulin-GTP concentration. The critical concentration of a microtubule end is the threshold concentration of free tubulin-GTP that determines if the end will grow or shrink. At concentrations of tubulin-GTP higher than the critical concentration, the end will grow by the addition of new subunits. At concentrations of tubulin-GTP lower than the critical concentration, the end will shrink due to depolymerization. Generally, the critical concentration of the plus end is lower than that of the minus end. Therefore, when the concentration of monomeric tubulin is between the critical concentrations of the two ends, the plus end grows while the minus end shrinks. This phenomenon is referred to as microtubule treadmilling.
As microtubules grow, they sometimes undergo a rapid and catastrophic loss of tubulin subunits that result in a greatly shortened microtubule. This behavior is referred to as dynamic instability (illustrated in Figure 2). Free tubulin subunits bound to GTP are added to the plus end of a growing microtubule, resulting in the so-called GTP cap. Once incorporated in the microtubule, the GTP will be hydrolyzed to GDP. Tubulin bound to GDP is much less stable in the microtubule than tubulin-GTP. If polymerization occurs at a rapid rate, then the plus end will always have the GTP cap and new tubulin subunits will continue to be added. Dynamic instability occurs when GTP hydrolysis is more rapid than the addition of new tubulin subunits. This results in a plus end composed of tubulin-GDP, which is less stable in the microtubule, and it will begin to depolymerize rapidly, dramatically reducing the length of the microtubule.
Microtubules can extend through the cytoplasm of a cell as they grow; however, the growth of a microtubule does not explain the speed with which a microtubule track can be used to transport a vesicle. The movement of vesicles and organelles (i.e., cargo) are controlled by motor proteins, which bind to microtubules and "walk" along them to deliver their cargo. Motor proteins bind to the microtubule, as well as their cargo, and move in an ATP-dependent fashion along the microtubule. Typically, kinesins are motor proteins that move toward the plus end, whereas dyneins move toward the minus end (illustrated in Figure 3). The two globular head domains of a motor protein bind to the microtubule and movement is generated by successive binding and release of the microtubule, all the while moving along the length of the microtubule.
Actin Filaments Move Cells
Actin filaments (also called microfilaments) allow the cell to move, maintain, or change the shape of the plasma membrane. In animal cells, actin filaments are used to provide force as they crawl along a substrate. Actin makes up the contractile ring in a dividing animal cell, pinching the plasma membrane to segregate the cytoplasm into the two daughter cells. Stable bundles of actin underneath the plasma membrane of the apical surface of intestinal cells generate the microvilli, which project into the lumen of the intestine. Finally, parallel, but mixed orientation, arrays of actin and the motor protein myosin power muscle contractions.
Actin Organization and Growth
Actin filaments are twisted chains of G-actin subunits (illustrated in Figure 4). An actin filament has an inherent direction because all the subunits point in one direction. Both ends can grow, but the plus end grows more rapidly than the minus end. Actin monomers are bound to ATP and added to the filament, and shortly after polymerization the ATP is hydrolyzed to ADP and the actin-ADP subunit is much less stable in the filament. Analogous to a microtubule, the ends of an actin filament have a critical concentration; the threshold concentration of free actin-ATP above which it will grow and below which it will shrink. Similar to microtubules, actin filaments can exhibit treadmilling simultaneous depolymerization at the minus ends and polymerization at the plus ends. However, actin filaments do not exhibit dynamic instability.
Actin-Binding Proteins and Assembly of Actin Filaments
The dynamic assembly and organization of actin filaments is regulated by a variety of proteins collectively termed actin-binding proteins. These include proteins that bind and sequester actin monomers, proteins that cap and block either growth or shrinkage of filament ends, and proteins that sever filaments. Actin filaments can be organized in precise parallel arrays by bundling proteins, or they can be cross-linked into large networks.
Once these actin-binding proteins have established a pattern of actin filaments, it is by no means permanent. Often, in response to extracellular signals, the actin network is reorganized. One of the targets of many signaling pathways is a specific family of monomeric GTP-binding proteins (G-proteins) referred to as Rho proteins (Rho, Rac and Cdc42), which when active can trigger dramatic and rapid changes in the actin filaments through the regulation of different actin-binding proteins. Remember, G-proteins are active when bound to GTP and inactive when bound to GDP. Upon receptor activation, exchanging GDP for GTP activates a Rho G-protein. Rho-GTP will, in turn, activate a variety of actin-binding proteins either directly or via protein kinases. Rho-GTP will rapidly hydrolyze the GTP to GDP and become inactive. Thus, the actin network has been reconfigured in response to an extracellular signal.
The motor proteins that travel along actin filaments are termed myosins, and there are many different types in vertebrate cells. They all travel toward the plus end of the filament in an ATP-dependent fashion. The Myosin I class has a single head and tail region, and is found in all cell types. Myosin I can attach to a vesicle or organelle and move it along the actin filament (illustrated in Figure 5A). Alternatively, myosin I can attach to the plasma membrane and move along the actin filament, thus pulling and contracting the plasma membrane (Figure 5B). Myosin II is a unique form of myosin, composed of two head regions and a single tail (Figure 5C). Myosin II plays an important role in muscle contractions, as described below.
Some animal cells move by crawling along a surface, usually along a surface composed of a complex of macromolecules. Actin mediates the movements necessary for cell crawling (illustrated in Figure 6). As the cell crawls, it extends a thin protrusion termed a lamellipodium. This leading edge of the cell is created by a branched array of actin filaments, polymerizing and pushing the plasma membrane forwards. The lamiellipodium makes focal contacts with the surface it is crawling to anchor the cell and provide traction. This occurs through the transmembrane protein integrin, which binds to extracellular proteins and to intracellular actin filaments. Finally, the back end of the cell is pulled forward due to contractions caused by myosin II interacting with actin filaments (see below).
Actin and Myosin in Muscle Contractions
The organization of actin filaments and myosin II in skeletal muscles is very distinct and regulates the contraction and relaxation of the muscle. This is illustrated in Figure 7. The unit of contraction is termed a sarcomere, and repeating sarcomeres in a muscle fiber give it a characteristic striated appearance. The sarcomere is composed of two types of filaments: parallel arrays of actin filaments (thin filaments) and interdigitating bundles of myosin II (thick filaments). The thin filaments of actin are anchored at their plus ends at the Z line. The thick filaments are bipolar bundles of myosin II (arranged with overlapping tails and heads pointing right and left). Contraction occurs as bipolar myosin IIs move along the actin filaments on either side, where they pull the actin filaments closer together and lead to a shortening of the sarcomere. ATP powers this movement, as the myosin head successively attaches and detaches from the actin filament.
Intermediate Filaments Withstand Mechanical Stresses
Intermediate filaments are a diverse group of cytoskeletal proteins that enable animal cells to withstand mechanical stresses. They are found in many cell types, and most form a network throughout the cytoplasm. There are distinct types of intermediate filaments in different cell types. Keratin is an intermediate filament found in epithelial cells, which are subjected to a great deal of stress (consider how much you can stretch your skin without the tissue tearing). Different intermediate filaments are found in nerve cells (i.e. neurofilaments) and in connective tissues and muscles (i.e. vimentin). A distinct intermediate filament (i.e. lamina) is found in the nucleus and makes up a complex of filaments underlying the nuclear envelope.
Intermediate Filament Organization
Intermediate filaments are long, rope-like molecules composed of many monomers (Figure 8). The monomer is an extended protein composed largely of an alpha helix. Two monomers twist around each other to form a dimer, and two dimers align laterally to form a tetrameric protofilament. The protofilaments then associate in a staggered fashion to form a long protofilament. Finally, the protofilaments are arranged in parallel to form the intermediate filament.
The cytoskeleton of eukaryotic cells is composed of three distinct types of microfilaments: microtubules, actin filaments and intermediate filaments. Microtubules are composed of dimers of alpha and beta-tubulin subunits, forming a hollow tube. Polymerization usually occurs at the plus end when the cytoplasmic concentration of tubulin dimers exceeds the critical concentration of that end. Depolymerization occurs at the minus end when the cytoplasmic concentration of tubulin dimers is below the critical concentration for that end. Treadmilling occurs between these two critical concentrations, when there is concurrent polymerization at the plus end and depolymerization at the minus end. Dynamic instability is the rapid and catastrophic depolymerization at the plus end of microtubules, due to the loss of the GTP cap. Microtubule filaments act as tracks that are used by motor molecules to transport vesicles and organelles throughout the cell. Motor proteins bind to their cargo (e.g. a vesicle) and travel with it along the microtubule filament. Kinesins are motors that travel toward the plus end and dyneins travel toward the minus end. Actin filaments polymerize by addition of actin monomers bound to ATP at the plus end and depolymerize by the loss of actin-ADP monomers at the minus end. Actin filament exhibit treadmilling, similar to microtubules, but do not exhibit dynamic instability. The assembly and organization of the actin filaments are regulated by actin binding proteins. The Rho family of GTP-binding proteins, which are activated in response to extracellular signals, and regulate the activity of actin-binding proteins, can rapidly remodel this network. Myosin proteins are plus-end directed motor proteins that travel along actin filaments in an ATP-dependent fashion. Cell crawling is mediated by actin polymerization at the leading edge of the cell and actin/myosin mediated contraction at the back of the cell. Muscle contractions are powered by actin/myosin interactions in the sarcomere. Parallel arrays of actin filaments are anchored to opposite Z lines and are separated by bundles of bipolar myosins. During a muscle contraction, the mysosin pulls the opposite actin filaments toward each other, reducing the distance between the Z lines. Intermediate filaments are a diverse group of proteins that vary with cell types and provide resistance to mechanical stress