Free Subunit Concentration Research Articles

Organelle Growth Control through Limiting Pools of Cytoplasmic Components

The critical importance of controlling the size and number of intracellular organelles has led to a variety of mechanisms for regulating the formation and growth of cellular structures. In this review, we explore a class of mechanisms for organelle growth control that rely primarily on the cytoplasm as a 'limiting pool' of available material. These mechanisms are based on the idea that, as organelles grow, they incorporate subunits from the cytoplasm. If this subunit pool is limited, organelle growth will lead to depletion of subunits from the cytoplasm. Free subunit concentration therefore provides a measure of the number of incorporated subunits and thus the current size of the organelle. Because organelle growth rates are typically a function of subunit concentration, cytoplasmic depletion links organelle size, free subunit concentration, and growth rates, ensuring that as the organelle grows, its rate of growth slows. Thus, a limiting cytoplasmic pool provides a powerful mechanism for size-dependent regulation of growth without recourse to active mechanisms to measure size or modulate growth rates. Variations of this general idea allow not only for size control, but also cell-size-dependent scaling of cellular structures, coordination of growth between similar structures within a cell, and the enforcement of singularity in structure formation, when only a single copy of a structure is desired. Here, we review several examples of such mechanisms in cellular processes as diverse as centriole duplication, centrosome and nuclear size control, cell polarity, and growth of flagella.

Rapid Microtubule Self-Assembly Kinetics

Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit dissociation rate from a microtubule tip is independent of free subunit concentration. Total-Internal-Reflection-Fluorescence (TIRF) microscopy experiments and data from a laser tweezers assay that measures in vitro microtubule assembly with nanometer resolution, provides evidence that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. We find that because both the association and the dissociation rates increase at higher free subunit concentrations, the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature.

Macroscopic simulations of microtubule dynamics predict two steady-state processes governing array morphology

Microtubule polymers typically function through their collective organization into a patterned array. The formation of the pattern, whether it is a relatively simple astral array or a highly complex mitotic spindle, relies on controlled microtubule nucleation and the basal dynamics parameters governing polymer growth and shortening. We have investigated the interaction between the microtubule nucleation and dynamics parameters, using macroscopic Monte Carlo simulations, to determine how these parameters contribute to the underlying microtubule array morphology (i.e. polymer density and length distribution). In addition to the well-characterized steady state achieved between free tubulin subunits and microtubule polymer, we propose that microtubule nucleation and extinction constitute a second, interdependent steady state process. Our simulation studies show that the magnitude of both nucleation and extinction additively impacts the final steady state free subunit concentration. We systematically varied individual microtubule dynamics parameters to survey the effects on array morphology and find specific sensitivity to perturbations of catastrophe frequency. Altering the cellular context for the microtubule array, we find that nucleation template number plays a defining role in shaping the microtubule length distribution and polymer density.

Kinetics of Microtubule Assembly

Understanding how microtubules (MTs) assemble and disassemble is vital to understanding fundamental cellular processes, such as mitosis and polarization, and their regulation via proteins and therapeutic drugs. Our current understanding of assembly kinetics is based on the classic one-dimensional (1D) assembly model of Oosawa, which assumes that the multiprotofilament MT can be modeled as if it were a single protofilament. In the classic 1D model the subunit off-rate is independent of free subunit concentration, an assumption that has yet to be confirmed experimentally. Using TIRF-microscopy and laser tweezers assays to measure microtubule self-assembly from GMPCPP-tubulin in vitro with nanoscale accuracy, we find that the off-rate is not constant but rather increases with increasing free subunit concentration. Consistent with this observation, we find that a simple two-dimensional (2D) model predicts the increasing off-rate with subunit concentration due to a shift in tip structure from relatively blunt at low concentrations to relatively tapered at high concentrations, which we confirmed experimentally by TIRF-microscopy. Because both the on-rate and off-rate increase with free tubulin concentration, the 2D model requires an association rate constant that is an order-of-magnitude larger than the 1D model. We tested this prediction by measuring the variability in assembly rate, and found that the on- and off-rates are consistent with the 2D model predictions and are an order-of-magnitude higher than predicted by the 1D model. In summary, we find that the kinetic rates of microtubule self-assembly have been severely underestimated in the literature, by at least an order-of-magnitude. Because net growth results from a small difference between large on-rates and off-rates, the net rate can be significantly altered by weak microtubule associated protein and therapeutic drug interactions with the microtubule.

Testosterone Increases TSH-β mRNA, and Modulates α-Subunit mRNA Differentially in Mouse Thyrotropic Tumor and Castrate Rat Pituitary

TSH, LH and FSH, the three pituitary glycoprotein hormones, are each composed of a common alpha-subunit and a hormone specific beta-subunit. Testosterone is known to regulate all three intact hormones differently in the rodent. However, there is only one gene encoding the common alpha-subunit. In order to elucidate the effects of testosterone on TSH subunit synthesis and its regulation of the common alpha-subunit, two in vivo models were studied: castrate rat pituitary was used as a gonadotropin-enriched tissue; and mouse thyrotropic tumor was used as a thyrotropin-enriched tissue. Male castrate rats were treated with testosterone propionate, 500 micrograms/100 g BW, sc, for 11 days. Testosterone increased plasma TSH to 131% of control values (P less than 0.02), while plasma LH fell to undetectable levels, and plasma alpha-subunit fell to 14% of control values (P less than 0.001). Testosterone increased TSH-beta mRNA to 237% of control values (P less than 0.02), while alpha-subunit mRNA fell to 20% of control values (P less than 0.001). Hypothyroid mice bearing thyrotropic tumors were treated with testosterone propionate, 150 micrograms/100 g BW, sc, for 11 days. In this model plasma TSH-beta and alpha-subunit concentrations are 1000-fold higher than in non-tumor bearing animals, and the contribution of pituitary gonadotropes to plasma subunit concentrations is negligible. "Total" TSH-beta and alpha-subunit concentrations were estimated as one-half of intact TSH plus the respective free subunit concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of changes in plasma thyrotropin β- and α-subunits, and mouse thyrotropic tumor thyrotropin β- and α-subunit mRNA concentrations after in vivo dexamethasone or T 3 administration

Dexamethasone, like T 3, inhibits the production of TSH. T 3 inhibits TSH synthesis by reducing transcription of the genes encoding TSH-β and α-subunits. Little information is available concerning the effects of dexamethasone on the individual subunits, or the combined effects of dexamethasone with T 3. In a preliminary study, hypothyroid mice bearing the thyrotropic tumor TtT 97 were treated with 25, 250, or 500 μg dexamethasone ip daily for ten days. Plasma levels of TSH and its subunits were unchanged after 25 μg of dexamethasone and maximally suppressed after 250 μg. Plasma TSH was reduced to 51% ( P < .02), free TSH-β to 54% ( P < .01), and α-subunit to 62% ( P < .001) of control values. In two similar experiments hypothyroid mice bearing TtT 97 were treated with dexamethasone (250 μg), T 3 (0.5 or 1 μg), or both dexamethasone with T 3 for 10 days. Total α-subunit and TSH-β were calculated by adding 1 2 TSH + free subunit concentrations. In the experiment using 1 μg of T 3, total plasma α-subunit was reduced by dexamethasone to 72%, by T 3 to 45% ( P < .02), and by combined treatment to 23% ( P < .01) of control values. In the experiment using 0.5 μg of T 3, total plasma α-subunit was reduced by dexamethasone to 66% ( P < .05), by T 3 to 67% ( P < .05), and by combined treatment to 46% ( P < .02) of control values. In the studies using the 1 μg or 0.5 μg dose of T 3 respectively, total plasma TSH-β was reduced by dexamethasone to 57% ( P < .05) or 48%, by T 3 to 11% ( P < .01) or 19% ( P < .05), and by combined treatment to 14% ( P < .01) or 16% ( P < .02) of control values. TSH-β and α-subunit mRNA were measured by blot hybridization of total RNA in thyrotropic tissue using cloned subunit specific cDNAs. There was no change in TSH-β or α-subunit mRNA after dexamethasone; α-subunit mRNA was unchanged after 0.5 μg T 3, however, 1 μg T 3 reduced steady-state α-subunit mRNA to 26% of controls ( P < .01). After 0.5 μg T 3 TSH-β mRNA was suppressed to 11% ( P < .01) of controls, and 1.0 μg T 3 reduced TSH-β mRNA to barely detectable levels. Although both dexamethasone and T 3 reduce plasma TSH and subunits, dexamethasone does not act like T 3 in reducing steady-state levels of TSHβ and α-subunit mRNAs in the mouse thyrotropic tumor. Additionally, at the doses studied, the suppressive effects of dexamethasone and T 3 on plasma total α-subunit were additive.

Effect of fluctuating surface structure and free energy on the growth of linear tubular aggregates

Simple linear tubular aggregates with up to eight strands are studied theoretically at equilibrium and under conditions of steady growth or shortening. The surface structure and free energy at an end of the polymer fluctuate as a consequence of the gain or loss of individual subunits. The surface free energy governs the probability distribution of surface structures at equilibrium. At steady state, on and off rate constants are crucial for this purpose; these depend on the gain or loss of neighbor interactions at the polymer end when a subunit is gained or lost. The observed on and off rate constants are averages of microscopic rate constants. A consequence of this is that the subunit flux onto the polymer end is, in general, not a linear function of the free subunit concentration, as is usually assumed. Monte Carlo calculations are needed at steady state for three or more strands. The general approach can be applied to microtubules, which have 13 strands. Actin is a special case, included here, with two strands.

Theoretical study of a model for the ATP cap at the end of an actin filament

The model used successfully by Pantaloni et al. to fit experimental data on steady-state actin polymerization is investigated theoretically. Many properties are deduced, as functions of the free subunit concentration. The model is simple enough so that one can examine analytically the question of whether actin shows the same dramatic phase changes associated with the GTP cap in microtubules. The answer is negative, judging from this model. However, it is possible to obtain such phase changes using the same model but with quite different, hypothetical choices of parameters. Thus, aside from its application to actin, this model is useful pedagogically to illustrate the nature of phase changes that may occur at the end of a steady-state polymer.

On the nonlinear relationship between the initial rates of dilution-induced microtubule disassembly and the initial free subunit concentration.

We have examined the dilution-induced in vitro disassembly kinetics of bovine brain microtubules, initially at steady state, using a wider range of dilutions (2-100-fold) than previously employed. In contrast to earlier results, as well as to the simple nucleation-condensation model for microtubule formation, the initial rate of dimer loss from microtubule ends was not a linear function of the initial concentration of unpolymerized tubulin. Over a 2-20-fold dilution range, plots of the initial rate of dimer loss versus the initial unpolymerized tubulin concentration were approximately linear. However, at greater dilutions, rates of microtubule depolymerization increased nonlinearly. For example, between a 10-fold dilution and a 100-fold dilution, the initial rate of dimer loss for microtubule-associated protein-containing microtubules increased by 300%, rather than a maximum of 11% expected on the basis of a linear rate plot. The nonlinear response was observed for dimer loss from opposite microtubule ends separately and with microtubules containing and lacking associated proteins. Qualitatively similar results were obtained using a wide range of experimental protocols, from which we can reasonably exclude methodological artifact as a basis for the data. We can also reasonably exclude the dissociation of the high molecular weight microtubule-associated proteins 1 and 2 from the microtubules as an explanation for the nonlinearity of the rate plots. The nonlinearity of the rate plots indicates that kinetic constants obtained under nonsteady state conditions of extreme microtubule dilution may not describe the steady state condition accurately.

Elevation of tubulin levels by microinjection suppresses new tubulin synthesis.

Most eukaryotic cells rapidly and specifically depress synthesis of alpha- and beta-tubulin polypeptides in response to microtubule inhibitors which cause microtubule depolymerization and presumably increase the intracellular concentration of free subunits. Other drugs which interfere with microtubule function but which lead to a decrease in the subunit pool size have little effect on the rate of new tubulin synthesis. These findings have previously been interpreted to indicate that cultured cells synthesize tubulin constitutively unless the subunit pool rises above a specified level. At this point an autoregulatory control mechanism is triggered which suppresses new tubulin synthesis through specific loss of tubulin mRNAs. That tubulin RNA levels are dramatically lowered by microtubule depolymerizing drugs is unquestionably correct; that fluctuations in the depolymerized tubulin pool size are responsible for altered RNA levels rests, however, entirely on the presumptive effects of different microtubule drugs. This caveat is not trivial, as these drugs induce gross morphological alterations, and the specificities and detailed mechanisms of action of such drugs remain poorly understood. To investigate the effect of altered levels of tubulin subunits on the rate of new tubulin synthesis in mammalian cells, we have microinjected purified tubulin subunits into cells in culture and analysed the synthesized proteins. We report here that tubulin synthesis is rapidly and specifically suppressed by injection of an amount of tubulin roughly equivalent to 25-50% of the amount initially present in the cell, thus indicating the presence of an eukaryotic, autoregulatory control mechanism which specifies tubulin content in a cultured mammalian cell line.

The role of subunit entropy in cooperative assembly. Nucleation of microtubules and other two-dimensional polymers

The self-assembly and nucleation of two-dimensional polymers is described by a theory based on a model of rigid subunits and bonds and simple principles of thermodynamics. The key point in the theory is to separate as an explicit parameter the free energy, primarily attributed to the entropy of the free subunit, that is required to immobilize a subunit in the polymer. Quantitative relations for the association of a subunit forming a longitudinal bond, a lateral bone, or both together are obtained, which demonstrate the basis and magnitude of cooperativity. The same formalism leads to a quantitative estimate for th concentration of the small polymers that are important intermediates in nucleation. It is shown that, if the concentration of free subunits is below a certain "critical supersaturation," the concentration of some essential intermediates is too low to support any significant assembly and nucleation is blocked. If the subunit concentration is above the critical supersaturation, all of the small intermediates are sufficiently stable to form and grow spontaneously. The theory predicts a critical supersaturation of 3.5 to 7 (the ratio of subunit concentration to the equilibrium solubility) for parameters appropriate to assembly of the microtubule wall. Experimentally, nucleation and assembly of microtubules is obtained at somewhat lower concentrations, 1.5 to 3 times the equilibrium solubility. Special mechanisms that could stabilize small polymers and facilitate nucleation of microtubule assembly are suggested.