Mechanism For Length Regulation Research Articles

TGF-β controls stromal telomere length through epigenetic modifications.

The online version contains supplementary material available at 10.1007/s13205-022-03346-5.

The Bur1 cyclin-dependent kinase regulates telomere length in Saccharomyces cerevisiae.

Telomere length regulation is essential for cell viability in eukaryotes. While many pathways that affect telomere length are known, we do not yet have a complete understanding of the mechanism of length regulation. To identify new pathways that might regulate telomere length, we carried out a genetic screen in yeast and identified the cyclin‐dependent kinase complex Bur1/2 as a regulator of telomere length. Mutations in either BUR1 cyclin‐dependent kinase or the associated BUR2 cyclin resulted in short telomeres. This regulation did not function through the known role of BUR1 in regulating histone modification as bur1∆ set2∆ and bur2∆ set2∆ double mutants rescued cell growth but did not rescue the telomere shortening effects. We found that both bur1∆ and bur2∆ set2∆ were also defective in de novo telomere addition, and deletion of SET2 did also not rescue this elongation defect. The Bur1/2 cyclin‐dependent kinase regulates transcription of many genes. We found that TLC1 RNA levels were reduced in bur2∆ set2∆ mutants; however, overexpression of TLC1 restored the transcript levels but did not restore de novo telomere elongation or telomere length. These data suggest that the Bur1/2 kinase plays a role in telomere elongation separate from its role in transcription of telomerase components. Dissecting the role of the Bur1/2 kinase pathway at telomeres will help complete our understanding of the complex network of telomere length regulation.

Design and Optimization of Tropomyosin Fragments for Tropomodulin Interaction Studies

Monomeric actin, or G-actin, polymerizes into filamentous actin, or F-actin which is an important component of cellular structures controlling many processes including vesicle trafficking, cell motility, inter and intracellular transport, and muscle contraction. Actin filaments are composed of a pointed end (slow growing) and a barbed end (fast growing). Interactions at each end enables control of the length of actin filaments. Tropomodulin (Tmod1-4) modulates actin filament length by interacting with tropomyosin (Tpm) and actin at the pointed end of the filament. Tmod binds two Tpm molecules through two distinct Tpm binding sites. Establishing the structure of each Tmod-Tpm binding interface is critical to understand the thin filament length regulation mechanism of Tmod. To facilitate NMR structure determination, we have established the optimal size of Tpm fragment necessary to form respective stable complexes. The four fragments we designed were residues 1-43, 1-33, 1-26 or 1-19 of Tpm1.8 fused with a GCN4 sequence promoting coiled-coil formation. We used NMR to determine and compare spectral effects between the optimized Tpm fragments and Tpm-binding sites of Tmod2. A significant change in spectra occurred when length of Tpm sequence changed from 19 to 26 residues and with almost no change with further increase in Tpm sequence length. Stability of the designed Tpm fragments and their affinity for Tpm-binding sites on Tmod2 were evaluated by CD spectroscopy. Our results revealed that Tpm1.8 fragments containing residues 1-19 and 1-43 were more stable with a melting temperature of 44 and 52oC, respectively, however, the 1-19 fragment was not long enough to represent the whole binding interface. The calculated dissociation constants showed strong affinity of Tmod2 for the Tpm1.8 1-43. Therefore, Tpm1.8-1-43 fragment is the most optimal for use in NMR studies to further study its interaction with Tmod.

Titin and Nebulin in Thick and Thin Filament Length Regulation.

In this review we discuss the history and the current state of ideas related to the mechanism of size regulation of the thick (myosin) and thin (actin) filaments in vertebrate striated muscles. Various hypotheses have been considered during of more than half century of research, recently mostly involving titin and nebulin acting as templates or 'molecular rulers', terminating exact assembly. These two giant, single-polypeptide, filamentous proteins are bound in situ along the thick and thin filaments, respectively, with an almost perfect match in the respective lengths and structural periodicities. However, evidence still questions the possibility that the proteins function as templates, or scaffolds, on which the thin and thick filaments could be assembled. In addition, the progress in muscle research during the last decades highlighted a number of other factors that could potentially be involved in the mechanism of length regulation: molecular chaperones that may guide folding and assembly of actin and myosin; capping proteins that can influence the rates of assembly-disassembly of the myofilaments; Ca2+ transients that can activate or deactivate protein interactions, etc. The entire mechanism of sarcomere assembly appears complex and highly dynamic. This mechanism is also capable of producing filaments of about the correct size without titin and nebulin. What then is the role of these proteins? Evidence points to titin and nebulin stabilizing structures of the respective filaments. This stabilizing effect, based on linear proteins of a fixed size, implies that titin and nebulin are indeed molecular rulers of the filaments. Although the proteins may not function as templates in the assembly of the filaments, they measure and stabilize exactly the same size of the functionally important for the muscles segments in each of the respective filaments.

Uncorrelated changes of subunit stability can generate length-dependent disassembly of treadmilling filaments

Unregulated polymer assembly typically leads to exponential length distributions. In living cells, though, the length distribution of cytoskeletal filaments often shows a sharp maximum at a finite value. We discuss a simple mechanism for length regulation that is based on generating an effectively length-dependent disassembly rate by locally acting cytoskeletal proteins. We analyze steady-state distributions by stochastic simulations and develop a method to analytically characterize the typical filament length. We find three different types of behavior: (1) unbounded filament growth, (2) exponential distributions and (3) unimodal distributions, which can be very sharp with a standard deviation much smaller than the mean filament length. We discuss possible implications of our results for subcellular processes.

The Wall Teichoic Acid Polymerase TagF Is Non-processive in Vitro and Amenable to Study Using Steady State Kinetic Analysis

Wall teichoic acids are a chemically diverse group of anionic polymers that constitute up to 50% of the Gram-positive cell wall. These polymers play a pivotal role in virulence and have been implicated in a diverse range of physiological functions. The TagF-like family of enzymes has been shown to be responsible for wall teichoic acid priming and polymerization events. Although many such enzymes are well validated therapeutic targets, a mechanistic understanding of this enzyme family has remained elusive. TagF is the prototypical teichoic acid polymerase and uses CDP-glycerol to catalyze synthesis of the linear (1,3)-linked poly(glycerol phosphate) teichoic acid in Bacillus subtilis 168. Here we used a synthetic soluble analog of the natural substrate of the enzyme, Lipid , to conduct the first detailed mechanistic investigation of teichoic acid polymerization. Through the use of a new high pressure liquid chromatography-based assay to monitor single glycerol phosphate incorporations into the Lipid analog, we conducted a detailed analysis of reaction product formation patterns and unequivocally showed TagF to be non-processive in vitro. Furthermore by monitoring the kinetics of polymerization, we showed that Lipid analog species varying in size have the same K(m) value of 2.6 microm and validated use of Bi Bi velocity expressions to model the TagF enzyme system. Initial rate analysis showed that TagF catalyzes a sequential Bi Bi mechanism where both substrates are added to the enzyme prior to product release consistent with a single displacement chemical mechanism.

Uncoupling of Longevity and Telomere Length in C. elegans

The nematode Caenorhabditis elegans, after completing its developmental stages and a brief reproductive period, spends the remainder of its adult life as an organism consisting exclusively of post-mitotic cells. Here we show that telomere length varies considerably in clonal populations of wild-type worms, and that these length differences are conserved over at least ten generations, suggesting a length regulation mechanism in cis. This observation is strengthened by the finding that the bulk telomere length in different worm strains varies considerably. Despite the close correlation of telomere length and clonal cellular senescence in mammalian cells, nematodes with long telomeres were neither long lived, nor did worm populations with comparably short telomeres exhibit a shorter life span. Conversely, long-lived daf-2 and short-lived daf-16 mutant animals can have either long or short telomeres. Telomere length of post-mitotic cells did not change during the aging process, and the response of animals to stress was found independent of telomere length. Collectively, our data indicate that telomere length and life span can be uncoupled in a post-mitotic setting, suggesting separate pathways for replication-dependent and -independent aging.

Developmental control of telomere lengths and telomerase activity in plants.

Telomere lengths and telomerase activity were studied during the development of a model dioecious plant, Melandrium album (syn Silene latifolia). Telomeric DNA consisted of Arabidopsis-type TTTAGGG tandem repeats. The terminal positions of these repeats were confirmed by both Bal31 exonuclease degradation and in situ hybridization. Analysis of terminal restriction fragments in different tissues and ontogenetic stages showed that telomere lengths are stabilized precisely and do not change during plant growth and development. Telomerase activity tested by using a semiquantitative telomerase repeat amplification protocol correlated with cell proliferation in the tissues analyzed. Highest activity was found in germinating seedlings and root tips, whereas we observed a 100-fold decrease in telomerase activity in leaves and no activity in quiescent seeds. Telomerase also was found in mature pollen grains. Telomerase activity in tissues containing dividing cells and telomere length stability during development suggest their precise control during plant ontogenesis; however, the telomere length regulation mechanism could be unbalanced during in vitro dedifferentiation.

A model for length-regulation in thick filaments of vertebrate skeletal myosin

A mechanism for length regulation in the parallel-packed section of the thick filament is proposed. It is based on experiments done on synthetic, mini- and native filaments, and its primary purpose is to explain the physical basis of the kinetic mechanism for the assembly of synthetic thick filaments from myosin alone. Kinetically, length is regulated by a dissociation rate constant that increases exponentially as the filament grows bi-directionally from its center. Growth ceases at the point of equilibrium between invariant on and length-dependent off rates. The three subfilaments structure of the parallel-packed region of the thick filament is fundamental to the proposed scheme. The intra-subfilament bonding is strong and predominantly ionic in character, whereas the inter-subfilament bonding is relatively weak. These strong and weak interactions participate directly in the strictly sequential mechanism of assembly of dimer subunit observed in the kinetics. A third domain, independent of the sequential mechanism, consists of opposing negative charges on the subfilament surface, juxtaposed at or close to the thick filament axis. The weak and repulsive domains are additively coupled to each other through the rigidity in the subfilaments. Length regulation occurs through the repulsive component rising in intensity more rapidly with length than the initially stronger positive interactions. Growth ceases at the point where the repulsive interactions weaken the attractive interactions to the extent that equilibrium is established between head-to-tail dimer subunit and its binding sites at the tips of the arms of thick filament. This myosin-mediated mechanism, which gives rise to a narrow length distribution, is considered to be fine-tuned by co-polymerizing proteins to give the precise length of the native filament.

Kinetics and thermodynamics of the assembly of the parallel- and antiparallel-packed sections of synthetic thick filaments of skeletal myosin: a pressure-jump study.

Earlier work on the length regulation mechanism of synthetic myosin filaments generated at pH 8.2 showed the process to be mediated through the dissociation rate constant which had an increasing and apparently monophasic exponential dependence on filament length and an association rate constant that was length independent, filament growth ceasing at the point of equilibrium [Davis, J.S. (1981) Biochem. J. 197, 309-314]. In this work, the exponential dependence of the dissociation rate constant on thick filament length was shown to be more complex than originally thought. Two phases were resolved, one of which correlated with the dissociation of parallel-packed myosin and the other with that of antiparallel-packed material. The pressure dependence of the dissociation reaction for the parallel-packed material showed that the activation volume decreased linearly with length while the Gibbs energy increased. This was interpreted as indicating that the weakening of the interaction between dimer and filament with length was accompanied by a decrease in the extent of ionic bonding. The case in the antiparallel-packed region was quite different, with the activation volume and the Gibbs energy both increasing linearly. The contribution from ionic bonding thus rises counter to the change in Gibbs energy, presumably at the expense of other noncovalent interactions. The relationship between the synthetic thick filaments and their in vivo counterparts is also considered in some detail.

Evidence for an internal component of the bacteriophage T4D tail core: a possible length-determining template.

The length of the T4 tail is precisely regulated in vivo at the time of polymerization of the tail core protein onto the baseplate. Since no mutations which alter tail length have been identified, a study of in vivo-assembled tail cores was begun to determine whether the structural properties of assembled cores would reveal the mechanism of length regulation. An assembly intermediate consisting of a core attached to a baseplate (core-baseplate) was purified from cells infected with a T4 mutant in gene 15. When core-base plates were treated with guanidine hydrochloride, cores were released from baseplates. The released cores had the same mean length as cores attached to baseplates. Electron micrographs of these cores showed partial penetration of negative stain into one end, and, at the opposite end, a modified tip which often appeared as a short fiber projecting from the core. When cores were purified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two minor proteins and the major core protein were detected. One minor protein, the product of gene 48 (gp48), was present in at least 72% of the amount found in core-baseplates, relative to the amount of the major core protein. These findings suggest that cores contain a fibrous structure, possibly composed of gp48, which may form a "ruler" that specifies the length of the T4 tail.

The influence of pressure on the self-assembly of the thick filament from the myosin of vertebrate skeletal muscle.

The thick-filament-monomeric-myosin equilibrium was prepared from pure myosin at pH 8.1. The application of hydrostatic pressure to the self-assembly equilibrium resulted in a biphasic dissociation curve in which a linear decrease in turbidity (a measure of weight added to or lost from the filament) was followed by a transition to a second pressure-insensitive phase. The first phase represents the effect of hydrostatic pressure on the growth or propagation phase of filament assembly. Here is was shown that hydrostatic pressure served to shorten the filaments in concert towards the bare zone whilst maintaining the narrow length distribution seen at atmospheric pressure; the filament concentration remained constant during the experiment. A more precise definition of the delta-v for the assembly of monomer into filament was obtained than had hitherto been possible. The positioning of the bare zone at the centre of the filament seems to be one of the more obvious functions of the length-regulation mechanism. It also appears that all the basic structural elements of the native thick filament are potentially present in the pH 8.1 homopolymer; its length can be increased by physiological concentrations of MgCl2 and decreased by pressure. The monodisperse native filament could then be formed by a fine tuning of the basic length-regulation mechanism of the homopolymer by the co-polymerization of the other thick-filament proteins.