Muscle regulation and Actin Topics: Tropomyosin and Troponin, Actin Assembly, Actin-dependent Movement

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1 1 Muscle regulation and Actin Topics: Tropomyosin and Troponin, Actin Assembly, Actin-dependent Movement In the last lecture, we saw that a repeating alternation between chemical (ATP hydrolysis) and vectorial (movement) steps is required for myosin to move forward rather than slipping back and forth along the actin filament. It turns out that this repeating mechanism is the secret behind an entire class of processes in which chemical reactions are coupled to vectorial motion. I. Regulation of muscle contraction A. Actin - tropomyosin - troponin complex Now that we ve looked at how muscles contract, the opposite question arises: given a supply of ATP and the requisite proteins, why don t muscles contract all the time? The answer has to do with a set of proteins complexed to actin that effectively turn contraction on or off depending on the presence of calcium ions. Looking at the structure of a thin filament (V&V p Fig and MBOC p. 854, Fig ), you ll notice that in addition to the double helical actin strands discussed previously, the filament contains two more proteins, tropomyosin and troponin. Tropomyosin (TM) snakes down the middle of the actin helices and consists of two alpha-helical chains (each 35kD) that form a coiled-coil about 35 nm long (V&V p Fig ). Each TM molecule comes in contact with seven actin molecules. Sitting at one end of each TM is troponin, a complex which consists of three polypeptides known as TnI, TnT, and TnC. TnI has a significant contact with both TM and actin, while TnT serves to strengthen the interactions among TnI, TM, and actin. TnC sits on TnI. B. Calcium binding by TnC TnC is the component of the actin complex that senses the local calcium concentration and thus determines whether the actin filament is active. As shown in V&V p Fig , TnC has a central alpha-helix with a globular domain (combination of alpha structure and turns) at each end. Each terminal domain contains two motifs called EF hands, and it is the EF hands that directly bind calcium ions. Thus, the EF hands are TnC s way of sensing the calcium concentration; at 100 nm calcium (the usual cellular concentration) some of the EF hands are empty, but if the local concentration rises to 1 µm, as it does when the muscle contracts, all of the EF hand binds calcium. Despite the fact that each domain has two EF hands, the domains differ in their calcium affinity; the C- terminal EF hands have high affinity while the N-terminal ones have low affinity. The high affinity sites are always bound to Ca ++, but at the resting Ca ++ concentration, the low affinity sites are empty. When the low affinity sites bind calcium, contraction can occur. Experiment demonstrating regulation by calcium As discussed in the last lecture, combining myosin, actin monomers (G actin), and ATP leads to ATP hydrolysis and Pi release at a rate of s -1 :

2 2 A + M + ATP -----> AM + ADP + Pi s -1 However, if regulated actin, composed of actin, TM, and troponin, is used instead of the actin monomers, a much slower rate is observed: A-TM-Tr + M + ATP ----> AM-TM-Tr + ADP + Pi s -1 Although the regulated actin complex does associate with myosin as before, it no longer stimulates myosin. BUT if calcium is added to the prep, the rate of the reaction goes back up to s -1. Thus it appears that 1) actin alone can stimulate myosin and Pi release, but 2) actin in its complex filament form does not unless calcium is present. Thus, a component of the actin filament - TnC - insures that muscle remains at rest unless it receives a signal in the form of a rise in local calcium concentration. C. Mechanism for coupling calcium signal and muscle activity Protein-protein interactions among the components of the filament create a relay mechanism between TnC and actin. When calcium concentration increases, there is an alteration in the conformation of tropomyosin that gets transmitted to the 7 actin molecules in contact with it, and these actin molecules can then effectively contact myosin. Textbooks usually give a simple physical blocking model for the control of muscle activity which may not be entirely true (See MBOC p. 854 Fig b and V&V p Fig ), although some physical/conformational changes in the troponin complex is probably involved. D. What causes the calcium concentration to rise during a contraction? The calcium concentration in the cytosol rises in response to an incoming nerve signal. A signal traveling down a nearby nerve causes the nerve ending to release the chemical acetylcholine, which travels the short distance from the nerve to the muscle cell. At the muscle cell membrane, acetylcholine binds to an acetylcholine receptor, which is also a gated channel. It changes its conformation to allow sodium ions to flow into the cell. The influx of sodium causes the membrane to depolarize, meaning that its electrical gradient changes from negative on the inside and positive on the outside (polarized) to equal charge potentials on the inside and outside (see MBOC p. 853 Fig ). Once one spot becomes depolarized, the depolarized state spreads along the membrane by means of voltage-dependent sodium channels. This action potential is transmitted to the T-tubules, which invaginate from the plasma membrane, and is propagated via the dihydropyridine receptor, which is a voltage dependent calcium channel located on the T-tubules. Ryanodine channels are calcium channels that are located on the sarcoplasmic reticulum. When the T-tubules depolarize, the ryanodine channels open and calcium ions that are usually bound by calsequestrin pour out of the sarcoplasmic reticulum into the cytosol. The resulting calcium wave causes the conformational changes discussed above for TnC and the actin-myosin interaction, and the muscle contracts. During repolarization, which is mediated by potassium channels and sodium/potassium ATPases, the channels shut down. Calcium pumps rapidly pump calcium back into the sarcoplasmic reticulum to prevent the contraction from continuing long after the end of the original signal.

3 3 E. Dystrophin: integrity of the sarcolemma The plasma membrane of skeletal cells, or the sarcolemma, is supported by a network of proteins. Dystrophin, a 400 kd protein, is situated right underneath the plasma membrane, and is part of a glycoprotein complex that is in contact with the basal membrane, or extracellular cell matrix. Dystrophin also interacts with F-actin, and these interactions provides structural integrity and resistance to the sarcolemma. Duchenne's muscular dystrophy is an X-linked disease in which the dystrophin gene is mutated. Onset of disease occurs when the affected individual reaches 3-4 yrs of age. Apparently, this disease affects muscles which have attained a certain size, since knocking out this gene in mice appears to have no effect. II. Actin polymerization Although up to this point we ve seen that movement is produced by an interaction between actin and myosin, organisms that lack myosin still manage to move. How? The answer is via the dynamic process of actin polymerization. Actin is a highly conserved molecule in all eukaryotic cells and it is complexed to a nucleotide (either ATP or ADP). Actin can be induced to polymerize by first preparing monomers (G-actin, or globular actin) and then adding Mg, KCl, and ATP to form the polymer F-actin, in which each monomer contacts four other monomers. The monomers polymerize in a head to tail direction. A graph of the formation of F-actin vs. time (see MBOC p. 824) reveals that polymerization occurs in three distinct phases, an initial lag time, a growth phase, and a steady-state. The lag time, during which polymerization is slow, represents the time it takes for three monomers to come together - that is, to nucleate. Before nucleation, the individual interactions between monomers are weak, but after three come together the interaction of an additional monomer becomes more stable, so that the polymer can start to form. Polymerization continues until the monomer concentration falls to a level where the actin filaments reach a state of equilibrium with the monomers. In this condition of steady state, the rate of monomer addition equals the rate off monomers falling off of the polymer. As suggested by the head to tail arrangement of monomers, the two ends of the filament have different properties. As mentioned in the previous lecture, when S1 myosin heads are added to the filament, they all point in the same direction so that the decorated actin takes on an arrow-like shape: barbed end (+) pointed end (-) It turns out that if myosin-decorated actin is used as a nucleating site and the polymerization ingredients actin, Mg, KCl, and ATP are added, a long filament grows from the barbed end while only a short filament grows from the pointed end. Thus, actin seems to add more rapidly to the barbed (+) end.

4 4 Why does this happen? For simple monomer addition, G has to be the same at both ends, since the same interactions between monomer and filament must be broken or formed. Thus, even if the specific rate constants differ at the two ends, their ratio would be expected to be the same, so that if one end grew faster it would also shrink faster and the overall growth rates of the two ends would be equal. However, the involvement of ATP hydrolysis changes the picture and causes the disparity between the ends. For both ends, the actin monomer that adds to the filament is ATP-bound, since the addition rate is faster for ATP-actin than for ADP-actin. The corresponding monomer loss rate for ATP-actin is slow, but once the monomer is on the filament its rate of ATP hydrolysis goes up, so that monomer dissociation involves loss of ADP-actin, which falls off faster than ATP-actin. Since the on and off reactions are no longer simply the reverses of each other under these conditions, the constraint that if one end grows faster it must also shrink faster no longer applies. Steady state and critical concentration A related consequence of the loss of the requirement for a constant ratio between the on and off rates is that the ends reach the steady state (at which monomer addition equals monomer loss, or kon [A] = koff) at different concentrations of monomer. This concentration, which is also the steady state dissociation constant (Kd) for monomer addition, is known as the critical concentration. The critical concentration can also be defined as the concentration of actin monomer above which monomer adds to the filament faster than it falls off and therefore above which that end of the filament grows. The critical concentration for the plus end is lower than for the minus end, so that the plus end can grow with fewer monomers around. Consequently, if the monomer concentration is between the critical concentrations for the plus and minus ends, the plus end will grow while the minus end shrinks. At some concentration the rate of addition to the plus end will equal the rate of loss at the minus end, so that the filament will appear to move in the direction of the plus end at a constant length. This is called treadmilling. III. Actin-dependent movement Although an actin filament at the steady state doesn t grow, the fact that a monomer adds to the plus end at the same time another one comes off the minus end leads to an overall displacement of the filament toward the plus end. Some types of movement in the cell are thought to occur by this mechanism. For example, the listeria bacterium, which enters cells by phagocytosis and zooms across the cell to the other side where it makes a protrusion in the membrane en route to the next cell in order to avoid being exposed to antibodies outside the cells, may use this feature of actin to cross the cell (see MBOC p. 830 Fig ). Observed in the act of swimming, the bacterium appears to carry an actin tail of constant length behind it. However, if one of the monomers in the filament is tagged, it turns out that this monomer stays put while the bacterium moves. This paradox is explained by the observation that as the bacterium moves forward, a space between it and its

5 actin tail opens up, and subsequently an actin monomer slips in, adds to the filament, and allows the filament end to catch up with the bacterium and force it forward further. Since depolymerization is occurring at the other end of the filament, the overall appearance is that of a constant length jet engine tail pushing the bacterium along. Thus, one way actin can cause movement in the absence of myosin is by this process of polymerizing (or depolymerizing) at different rates at its two ends. 5