Abstract. Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411--420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd ~< 1 riM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 /zM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate or extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells. rIS assembly in muscle and nonmuscle cells is regulated in part by proteins that cap the fast growing (barbed) ends of actin filaments. All of the wellknown capping proteins, gelsolin, villin, and capZ, block elongation and depolymerization at the barbed filament end, and they are also capable of nucleating actin polymerization (for reviews see Pollard and Cooper, 1986;Weeds and Maciver, 1993). These proteins play various roles in different cells at different times. For instance, capZ might provide nucleation sites in the Z band for thin filament formation during myofibril assembly in the development of muscle cells (Schafer et al., 1993). On the other hand, in some nonmuscle cells, capping proteins may play an important role in the sudden changes in the state of actin polymerization associated with stimulation of these cells (for a recent review, see Zigmond, 1993).Considerably less is known about the molecules responsible for regulating actin filament assembly at the slow growing (pointed) end. Evidence for the existence of pointed end capping proteins in muscle and nonmuscle cells comes from several observations. First, there is no elongation at the
Control of actin filament length and dynamics is important for cell motility and architecture and is regulated in part by capping proteins that block elongation and depolymerization at both the fast-growing (barbed) and slow-growing (pointed) ends. Tropomodulin is a capping protein for the pointed end of the actin filament; it is associated with the free, pointed ends of the thin filaments in striated muscle, where it is thought to bind to both tropomyosin and actin. In embryonic chick cardiac myocytes, tropomodulin assembles after the thin, as well as the thick, filaments have become organized into periodic I and A bands, suggesting that tropomodulin might be involved in maintaining actin filament length. Here we show that microinjection of an antibody that inhibits tropomodulin's pointed-end-capping activity in vitro results in a marked elongation of actin filaments from their pointed ends and a > 80% reduction in the percentage of beating cells. This demonstrates that pointed-end capping by tropomodulin is required to maintain actin filament length in vivo and that this is essential for contractile function in embryonic chick cardiac myocytes.
Quantitative measurements of the interactions of T beta 4 with muscle actin suggest that its only physiological role is monomer sequestration. T beta 4 forms a 1:1 complex with monomeric actin under physiological salt conditions. Its Kd for actin is not affected by calcium. T beta 4 binds only to actin monomers and not to filament ends or alongside the filament. T beta 4-actin complexes do not elongate actin filaments at either the barbed or the pointed end, and, unlike actobindin, T beta 4 does not specifically suppress the nucleation of polymerization. We assessed the fraction of monomeric actin that can be sequestered by T beta 4 in resting platelets. This was done on the basis of (a) its Kd of 0.4-0.7 microM for platelet actin, which had been prepared by a newly devised simpler method, and (b) the values for the concentrations of monomeric actin and of T beta 4 which we measured as 280 and 560 microM, respectively. Using the higher Kd value of 0.7 microM, the T beta 4-complexed actin is calculated to be between 70 and 240 microM, depending on the steady-state free G-actin concentration. This may vary from 0.1 to 0.5 microM, the critical concentrations for uncapped and for fully barbed-end-capped actin filaments. If the Kd in the platelet is the same as in vitro, most of the sequestered actin would be bound to T beta 4 if more than 95% of the actin filaments are capped at their barbed ends in resting platelets.
The pointed end capping protein, tropomodulin, increases the critical concentration of barbed end capped actin, i.e. it lowers the apparent affinity of pointed ends for actin monomers. We show here that this is due to the conversion of pointed end ADP⅐P i -actin (low critical concentration) to ADP-actin (high critical concentration) when 70 -98% of the ends are capped by tropomodulin. We propose that this is due to the low affinity of tropomodulin for pointed ends (K d ϳ 0.3 M), which allows tropomodulin to rapidly exchange binding sites and transiently block access of actin monomers to all pointed ends. This leaves time for ATP hydrolysis and phosphate release to go to completion between successive monomer additions to the pointed end. When the affinity of tropomodulin for pointed ends was increased about 1000-fold by the presence of tropomyosin (K d < 0.05 nM), capping of 95% of the ends by tropomodulin did not alter the critical concentration. However, the critical concentration did increase when the tropomodulin concentration was raised to the high values effective in the absence of tropomyosin. This may reflect transient tropomodulin binding to tropomyosin-free actin molecules at the pointed ends of the tropomyosin-actin filaments without a high affinity tropomodulin cap, i.e. the ends that determine the value of the actin critical concentration.Tropomodulin is a ϳ40-kDa actin-and tropomyosin-binding protein that caps the pointed ends of actin filaments (1). Tropomodulin isoforms are associated with the actin cytoskeleton in a variety of post-mitotic, differentiated cell types in vertebrates, including striated muscle, erythrocytes, lens fiber cells, and neurons (2, 3). Recently, tropomodulin homologs have also been identified in flies (4) and in worms (2, 3) but not in yeast or fungi. In vertebrate striated muscle, tropomodulin is tightly bound to both tropomyosin and actin at the pointed ends of the thin filaments where it is believed to function to maintain thin filament length, sarcomere organization, and contractile function (1, 5). Although tropomodulin function has generally been considered in the context of its tight association with the stable, tropomyosin-actin filaments in striated muscle and erythrocytes, it is an open question as to whether tropomodulin could also regulate the assembly of more dynamic actin filaments in other contexts (6).This possibility is suggested by the observation that tropomodulin, in addition to inhibiting elongation, increases the steady state monomer concentration of barbed end-capped actin 2-fold, close to the value for ADP-actin (7). This is not the result of monomer sequestration by tropomodulin, because tropomodulin binds exclusively to the pointed filament ends and not to actin monomers or alongside actin filaments (7,8). Thus, tropomodulin must have increased the pointed end critical concentration (the critical concentration of barbed end-capped actin filaments).The critical concentration of barbed end capped actin filaments represents the monomer concentration ...
An increasing number of neurodegenerative diseases are being linked to mutations in genes encoding proteins required for axonal transport and intracellular trafficking. A mutation in p150(Glued), a component of the cytoplasmic dynein/dynactin microtubule motor complex, results in the human neurodegenerative disease distal spinal and bulbar muscular atrophy (dSBMA). We have developed a transgenic mouse model of dSBMA; these mice exhibit late-onset, slowly progressive muscle weakness but do not have a shortened lifespan, consistent with the human phenotype. Examination of motor neurons from the transgenic model reveals the proliferation of enlarged tertiary lysosomes and lipofuscin granules, indicating significant alterations in the cellular degradative pathway. In addition, we observe deficits in axonal caliber and neuromuscular junction (NMJ) integrity, indicating distal degeneration of motor neurons. However, sciatic nerve ligation studies reveal that inhibition of axonal transport is not evident in this model. Together, these data suggest that mutant p150(Glued) causes neurodegeneration in the absence of significant changes in axonal transport, and therefore other functions of dynein/dynactin, such as trafficking in the degradative pathway and stabilization of the NMJ are likely to be critical in maintaining the health of motor neurons.
We show here that DNase is distinguished from other known actin-binding proteins by its unique ability to increase the depolymerization rate constant of actin at the pointed filament end, thereby speeding up depolymerization of filaments capped at their barbed ends. This action requires relatively high DNase concentrations, 3 orders of magnitude higher than those needed to block elongation, although 10 times lower than those needed for DNase binding to the side of the filament. We propose that a high DNase concentrations, steric interference between the two DNase molecules, bound to the ends of both strands of the two-start actin helix, destabilizes actin binding to the filament.
Polyribosomes, mRNA and other elements of translational machinery have been reported in peripheral nerves and in elongating injured axons of sensory neurons in vitro, primarily in growth cones. Evidence for involvement of local protein synthesis in regenerating CNS axons is less extensive. We monitored regeneration of back-labeled lamprey spinal axons after spinal cord transection and detected mRNA in axon tips by in situ hybridization and micro-aspiration of their axoplasm. Poly(A)+mRNA was present in the axon tips, and was more abundant in actively regenerating tips than in static or retracting ones. Target-specific PCR and in situ hybridization revealed plentiful mRNA for the low molecular neurofilament subunit and β-tubulin, but very little for β-actin, consistent with the morphology of their tips, which lack filopodia and lamellipodia. Electron microscopy showed ribosomes/polyribosomes in the distal parts of axon tips and in association with vesicle-like membranes, primarily in the tip. In one instance, there were structures with the appearance of rough endoplasmic reticulum. Immunohistochemistry showed patches of ribosomal protein S6 positivity in a similar distribution. The results suggest that local protein synthesis might be involved in the mechanism of axon regeneration in the lamprey spinal cord.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.