Biomedical subjects

M Caplow

Publications and source records attributed to M Caplow.

At least 19 recordsLinked to original sources

PubMed1995

Induction of microtubule catastrophe by formation of tubulin-GDP and apotubulin subunits at microtubule ends.

The recent discovery that GTP linked to latex beads binds to microtubule ends suggested that nucleotide interactions at this site may play a role in regulating microtubule (MT) dynamics. Evidence for this was sought using DIC microscopy to analyze effects of the free GTP and GDP concentration on the rates of MT elongation and phase transition to rapid shortening (catastrophe, kc). That nucleotide can dissociate and thereby destabilize the plus end by forming nucleotide-free (apotubulin) subunits was indicated by an increase in kc from 0.001 to 0.05 s-1, when the free GTP concentration was reduced from 100 to 0.5 microM, during assembly with 15 microM tubulin--GTP subunits (TuT). That nucleotide can bind to the minus end was indicated by a nearly 5-fold decrease in the rate of elongation when the free GDP concentration was increased from 1.6 to 175 microM, during assembly with a mixture of 36 microM TuT and 54 microM TuD. Further evidence that nucleotide can bind to both ends was provided by the observation that with a mixture of 36 microM TuT and 54 microM TuD, kc was increased from 0.0036 to 0.05 s-1 at the plus end, and from 0.0005 to 0.005 s-1 at the minus end, when the free GDP concentration was increased from 1.6 to 175 microM. Our evidence for destabilization of microtubules by formation of apotubulin and by nucleotide exchange to form terminal TuD subunits suggests that microtubule dynamics can be regulated in cells by an exchange factor that generates apotubulin subunits, or by a GTPase activating protein that forms TuD subunits at microtubule ends.

Animals↗

PubMed1995

What mothers say about why poor children fall behind on immunizations. A summary of focus groups in North Carolina.

OBJECTIVES: To develop a more thorough understanding of the factors that impede poor parents' utilization of health care services for their children and to refine interventions to improve immunization rates. METHODS: We conducted focus group sessions with mothers whose children received care at the health departments in five North Carolina counties. Mothers were uninsured or were receiving Medicaid. A total of 50 women participated; group size varied from three to seven mothers. RESULTS: Socially disadvantaged mothers faced barriers at multiple points in the process of obtaining preventive care for their children. Organizational barriers, such as a lack of flexibility in scheduling and long waiting times, were exacerbated by personal barriers, such as a lack of reliable transportation, chaotic home environments, and employment conflicts. Lack of knowledge regarding the timing of childhood immunizations and misperceptions about the safety of immunizations were also important obstacles. Mothers made several suggestions, such as changes in scheduling, greater assistance with transportation, improved waiting facilities, and increased health education. CONCLUSIONS: Our study suggests that even with improved financing of well-child care, many important barriers to adequate immunization will remain. Many of the changes that mothers in our focus groups advocated are not related to insurance coverage and would be simple and inexpensive to implement. To help with these changes, we developed a checklist for use by health departments to determine which organizational barriers exist at their facility and suggest strategies to overcome the problems. Organizational, personal, and attitudinal barriers pose serious problems for socioeconomically disadvantaged families. To improve vaccination rates for children, new personnel and programs are probably less important than careful strategies to maximize existing resources.

Adolescent↗

PubMed1994

How taxol modulates microtubule disassembly.

Measurement of the affinity of microtubules for the anti-cancer drug taxol is problematic, because microtubules are not stable at the very low concentrations required to detect taxol dissociation. We have circumvented this problem by using the GTP analogue GMP-CPP (guanylyl alpha, beta-methylenediphosphonate), which renders microtubules sufficiently stable to allow binding studies with nonsaturating concentrations of taxol. AKd value equal to about 10 nM was estimated from the effect of taxol concentration on the dilution-induced disassembly rate and on the binding of [3H]taxol. With GTP-microtubules the Kd value for taxol binding by tubulin-GDP subunits in the core of the microtubule appears to be comparable with that of GMPCPP-microtubules. However, the stabilizing effect of the drug bound to tubulin subunits that arrive at ends of disassembling microtubules is attenuated by a two-step reaction sequence in which taxol dissociates (k = 30 s-1), followed by rapid (k = 1000 s-1) loss of the taxol-free tubulin subunit. This sequential reaction can be disrupted by high (micromolar) concentrations of taxol, which react rapidly with tubulin subunits at the ends of microtubules (k = 2 x 10(9) M-1 s-1). The inhibitory effect of taxol on microtubule disassembly at concentrations a thousand-fold greater than the Kd value suggests the desirability of using high taxol concentrations in chemotherapy with this compound.

Animals↗

PubMed1994

The free energy for hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice.

The standard free energy for hydrolysis of the GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP), which is -5.18 kcal in solution, was found to be -3.79 kcal in tubulin dimers, and only -0.90 kcal in tubulin subunits in microtubules. The near-zero change in standard free energy for GMPCPP hydrolysis in the microtubule indicates that the majority of the free energy potentially available from this reaction is stored in the microtubule lattice; this energy is available to do work, as in chromosome movement. The equilibrium constants described here were obtained from video microscopy measurements of the kinetics of assembly and disassembly of GMPCPP-microtubules and GMPCP-microtubules. It was possible to study GMPCPP-microtubules since GMPCPP is not hydrolyzed during assembly. Microtubules containing GMPCP were obtained by assembly of high concentrations of tubulin-GMPCP subunits, as well as by treating tubulin-GMPCPP-microtubules in sodium (but not potassium) Pipes buffer with glycerol, which reduced the half-time for GMPCPP hydrolysis from > 10 h to approximately 10 min. The rate for tubulin-GMPCPP and tubulin-GMPCP subunit dissociation from microtubule ends were found to be about 0.65 and 128 s-1, respectively. The much faster rate for tubulin-GMPCP subunit dissociation provides direct evidence that microtubule dynamics can be regulated by nucleotide triphosphate hydrolysis.

Animals↗

PubMed1992

Microtubule dynamics.

Although compelling evidence has been obtained for heterogeneity in the structure of subunits in microtubules, it has not been possible to prove that this results from the presence of tubulin-GDP and tubulin-GTP in polymers. There are reasons to exclude the existence of even a monolayer of tubulin-GTP subunits at microtubule ends. Dynamic behavior appears to be best accounted for by a mechanism in which tubulin-GDP in microtubules exists in two conformations. The mechanism of microtubule-associated protein binding to microtubules and the role of phosphorylation on this reaction are discussed.

Animals↗

PubMed1990

Mechanism of the microtubule GTPase reaction.

The rate of GTP hydrolysis by microtubules has been measured at tubulin subunit concentrations where microtubules undergo net disassembly. This was made possible by using microtubules stabilized against disassembly by reaction with ethylene glycol bis-(succinimidylsuccinate) (EGS) as sites for the addition of tubulin-GTP subunits. The tubulin subunit concentration was varied from 25 to 90% of the steady state concentration, and there was no net elongation of stabilized microtubule seeds. The GTPase rate with EGS microtubules was linearly proportional to the tubulin-GTP subunit concentration when this concentration was varied by dilution and by using GDP to compete with GTP for the tubulin E-site. The linear dependence of the rate is consistent with a GTP mechanism in which hydrolysis is coupled to the tubulin-GTP subunit addition to microtubule ends. It is inconsistent with reaction schemes in which: microtubules are capped by a single tubulin-GTP subunit, which hydrolyzes GTP when a tubulin-GTP subunit adds to the end; hydrolysis occurs primarily in subunits at the interface of a tubulin-GTP cap and the tubulin-GDP microtubule core; hydrolysis is not coupled to subunit addition and occurs randomly in subunits in a tubulin-GTP cap. It was also found that GDP inhibition of the microtubule GTPase rate results from GDP competition for GTP at the tubulin subunit E-site. There is no additional effect of GDP on the GTPase rate resulting from exchange into tubulin subunits at microtubule ends.

Adenylyl Imidodiphosphate↗

PubMed1990

Mechanism for oscillatory assembly of microtubules.

Dampened oscillations of microtubule assembly can accompany polymerization at high tubulin subunit concentrations. This presumably results from a synchronization of dynamic instability behavior, which generates a large population of rapidly disassembling microtubules, that liberate tubulin-GDP oligomers. Subunits in oligomers cannot assemble until they dissociate, to allow GDP-GTP exchange. To determine whether rapidly disassembling microtubules generate oligomers directly, we measured the rate of dilution-induced disassembly of tubulin-GDP microtubules and the rate of dissociation of GDP from the so-formed tubulin-GDP subunits. The rate of GDP dissociation from liberated subunits was found to correspond to that of tubulin-GDP subunits (t1/2 = 5 s), rather than tubulin-GDP oligomers. This indicates that tubulin-GDP subunits are released from microtubules undergoing rapid disassembly. Oligomers apparently form in a side reaction from the high concentration of tubulin-GDP subunits liberated from the synchronously disassembling microtubule population. The rate of subunit dissociation is 0.11 s-1 with oligomers formed by concentrating tubulin-GDP subunits and 0.045 s-1 with oligomers formed by cold-induced microtubule disassembly. This difference provides evidence that the conformation of tubulin-GDP subunits released from rapidly disassembling microtubules differs from tubulin-GDP subunits that were not recently in the microtubule lattice.

Animals↗

PubMed1989

Stabilization of microtubules by tubulin-GDP-Pi subunits.

Microtubule dynamic instability has been accounted for by assuming that tubulin subunits at microtubule ends differ from the tubulin-GDP subunits that constitute the bulk of the microtubule. It has been suggested that this heterogeneity results because ends contain tubulin subunits that have not yet hydrolyzed an associated GTP molecule. Alternatively, in a recent model it was proposed that ends contain tubulin-GDP-Pi subunits from which Pi has not yet dissociated. The models differ in their predicted response to added ligands: because GDP in subunits in microtubules does not exchange with nucleotide in solution, the heterogeneity from a tubulin-GTP cap will not be eliminated by added GTP; however, the dissociability of Pi in tubulin-GDP-Pi subunits will allow a heterogeneity resulting from a tubulin-GDP-Pi cap to be eliminated by added excess Pi. Elimination of the heterogeneity is expected to be manifested by an elimination of dynamic instability behavior. Using video microscopy to study the kinetic behavior of individual microtubules under reaction conditions where dynamic instability is the dominant mechanism for microtubule length changes, we have determined the effects of 0.167 M Pi on the rate of subunit addition in the elongation phase, the rate of subunit dissociation in the rapid shortening phase, and the rates of the phase transitions from elongation to rapid shortening and from rapid shortening to growing. Since 0.167 M Pi did not decrease the subunit dissociation rate in the rapid shortening phase or the rate of the phase transition from growing to rapid shortening, our results provide no support for the hypothesis that tubulin-GDP-Pi subunits are responsible for dynamic instability behavior of microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Kinetics and mechanism of microtubule length changes by dynamic instability.

Microtubules at steady state were found to undergo dramatic changes in length, with only very little change in number concentration and mean length. This result is accounted for by a mechanism in which microtubules are capped at ends by tubulin-GTP subunits; loss of the tubulin-GTP cap at one end results in disassembly of all the tubulin-GDP subunits, so that the medial edge of the distal tubulin-GTP cap is exposed; the exposed tubulin-GTP cap is sufficiently stable, so that microtubule regrowth from the cap rather than loss of the cap occurs. This mechanism predicts that a bell-shaped length distribution of sheared microtubules will be transiently bimodal, with peaks of short and moderate length microtubules, in rearranging to an exponential length distribution. We have observed the predicted transient bimodal length distribution experimentally and in a Monte Carlo simulation. Dynamic instability has recently been accounted for by assuming that microtubule ends are capped with only a single tubulin-GTP subunit at each end of the five helices that serve as elongation sites. Such a minimal tubulin-GTP cap is apparently ruled out by our observations, which require that the remnant tubulin-GTP cap generated from disassembly be able to serve as nucleating site; we do not expect that a stable nucleating site can be generated from five tubulin-GTP subunits, oriented as the five helices that serve as elongation sites.

Animals↗

PubMed1988

Temperature-jump studies of microtubule dynamic instability.

Evidence for a slowly dissociating tubulin-GTP cap at microtubule ends was derived from observation of a delay for attaining a maximum disassembly rate, after the temperature of steady state microtubules was rapidly decreased from 36 to 34 degrees C. The possibility that the microtubules were capped by a single tubulin-GTP subunit on each subhelix was ruled out, by comparison of the disassembly kinetics following a temperature decrease and dilution. The existence of a subpopulation of microtubules that underwent irreversible or near irreversible disassembly was demonstrated by a 30-s lag for attainment of a maximum assembly rate, after steady state microtubules were shifted from 34 to 36 degrees C. A dynamic instability model predicts that a maximum assembly rate will be delayed until disappearance of a subpopulation of microtubules that disassemble before being recapped. Analysis indicates that the 30-s lag resulted because approximately 2% of the mass in the steady state microtubule population was uncapped and disassembling and not readily recapped. The half-time for recapping of disassembling microtubules, by addition of tubulin-GTP subunits to ends, was equal to or greater than 20 s. Since tubulin-GDP dissociated from microtubules at a rate of about 4500 s-1, slow recapping resulted in dramatic shortening of disassembling microtubules.

Animals↗

PubMed1986

Differentiation between dynamic instability and end-to-end annealing models for length changes of steady-state microtubules.

Short microtubules can be formed by shearing a sample at polymerization steady state of microtubules formed by glycerol-induced assembly of pure tubulin dimer. Such short microtubules show a rapid increase in mean length. The rate of this increase is too fast to be accounted for by statistical redistribution of subunits between microtubules. We propose that the fast length changes are a result of the end-to-end annealing of microtubules demonstrated by Rothwell et al. (Rothwell, S. W., Grasser, W. A., and Murphy, D. B. (1986) J. Cell Biol. 102, 619-627). This proposal has been tested by measuring the rate of annealing of free microtubules to Tetrahymena axonemes under conditions identical to those used for the lengthening of sheared microtubules. That free microtubules anneal to axonemal microtubules is indicated by the following observations. Axonemes elongate at both ends in the presence of steady state microtubules, as predicted for a symmetrical annealing process; under conditions where the microtubule number concentration is greater than that for axonemes, the initial rate of axoneme elongation is more rapid with a low concentration of long microtubules at steady state than with a high number concentration of short microtubules at steady state. These observations are inconsistent with the predictions of a model based on microtubule dynamic instability (Mitchison, T., and Kirschner, M. (1984) Nature 312, 237-242). The annealing rate observed with axonemes can account for the rate of elongation of sheared steady state microtubules.

Animals↗

PubMed1986

Location of the guanosine triphosphate (GTP) hydrolysis site in microtubules.

The rate for GTP hydrolysis remains approximately constant during microtubule assembly from microtubular protein. This indicates that GTP hydrolysis does not accompany tubulin-GTP subunit addition to microtubule ends. We suggest that GTP, within tubulin-GTP subunits that are incorporated into microtubules, is hydrolyzed predominantly at one or both microtubule ends at an interface of a cap of tubulin-GTP subunits and a core of tubulin-GDP subunits.

Animals↗

PubMed1985

Concerning the anomalous kinetic behavior of microtubules.

We have demonstrated that tubulin-GTP subunits can react with microtubule ends containing subunits with E-site-bound GDP. This observation can be taken to rule out a previous interpretation of a biphasic dependence of the rate for subunit flux into microtubules on the subunit concentration, which is based upon an assumption that GTP is required to be present in subunits at microtubule ends in order to allow addition of tubulin-GTP subunits. The nullified mechanism had been suggested to be the basis of the observation that growing and shrinking microtubules coexist as independent species. We have also confirmed previous studies indicating that the flux rate is nonlinearly dependent on the subunit concentration and account for this behavior by assuming that tubulin-GTP subunits reversibly add to microtubule ends by two paths. In one, tubulin-GTP subunits add nonproductively to generate an end which is unable to undergo further net microtubule elongation; however, this reaction can retard the rate for microtubule disassembly under conditions where the disassembly reaction predominates. In the other, tubulin-GTP subunits add productively to microtubule ends to generate ends which can undergo subsequent net elongation.

Animals↗

PubMed1985

Directed elongation model for microtubule GTP hydrolysis.

We propose a role for GTP hydrolysis in microtubule assembly in which the GTPase reaction serves to stabilize tubulin subunits in the microtubule. The GTPase reaction in tubulin subunits containing GTP at microtubule ends is presumed to occur predominately in subunits at one of the interfaces between a cap of GTP-containing tubulin subunit and a core of GDP-containing tubulin subunit in the microtubule, resulting in elongation of the core. The proposed model interprets the effects of GDP on microtubule assembly, using a reaction scheme in which GDP-containing tubulin subunits are able to add to microtubule ends. The model can account for the GTP requirement for microtubule assembly, the GDP inhibition of the rate for microtubule elongation, and the fact that a metastable state exists after the enzymic conversion of GTP to GDP, with microtubules which are at steady state. To account for the fact that the microtubule assembly and disassembly rates are nonlinearly dependent upon the tubulin subunit concentration and for the effects of GDP-containing tubulin subunits on the kinetic properties of microtubules, our scheme includes nonproductive as well as productive binding of GTP- and GDP-containing tubulin subunits. We compare our model with an alternative scheme [Hill, T. L. & Carlier, M. F. (1983) Proc. Natl. Acad. Sci. USA 80, 7234-7238], which interprets the effects of GDP on microtubule assembly using a reaction scheme in which GDP is able to exchange with GTP in GTP-containing tubulin subunits in the microtubule and in which the principal GTPase occurs in GTP-containing tubulin subunits at the microtubule/solution interface.

Guanosine Diphosphate↗

PubMed1985

Concerning the location of the GTP hydrolysis site on microtubules.

The kinetics for GTP hydrolysis associated with microtubule assembly with microtubular protein has been analyzed under reaction conditions where tubulin-GDP does not readily assemble into microtubules. The GTPase rate is only slightly faster during the time when net microtubule assembly occurs, as compared with steady state. The slightly slower steady-state GTPase rate apparently results from GDP product inhibition, since the progressive decrease in the rate can be quantitatively accounted for using the previously determined GTP dissociation constant and the Ki value for GDP. Since the GTPase rate is not a function of the rate for net microtubule assembly, it is concluded that GTP hydrolysis is not required for tubulin subunit incorporation into microtubules. The constancy of the rate indicates that the GTPase reaction occurs at a site, the concentration of which does not change during the assembly process. This result is consistent with a reaction scheme in which GTP hydrolysis occurs primarily at microtubule ends. We propose that hydrolysis occurs at microtubule ends, at the interface between a long core of tubulin-GDP subunits and a short cap of tubulin-GTP subunits.

GTP Phosphohydrolases↗

PubMed1984

Mechanism for nucleotide incorporation into steady-state microtubules.

We have extended our previous theoretical analysis of the kinetics for radioactive GTP incorporation into steady-state microtubules [Zeeberg, B., Reid, R., & Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899] to include the effects of a kinetic barrier for equilibration of labeled GTP with the tubulin E site. This binding has been found to be relatively slow; the half-time for GTP dissociation is approximately 25 s (k = 0.028 s-1). The slow binding of radioactive GTP apparently accounts for the following observations: (a) more radioactive nucleotide is incorporated into steady-state microtubules in the first 20 s when tubulin-[3H]GTP is used in a pulse than when [3H]GTP is used; (b) when steady-state microtubules are pulsed for 20 s with tubulin-[3H]GTP and then chased with excess nonradioactive GTP, radioactive nucleotide incorporation is not stopped immediately. Quantitative analysis of these results indicates that our steady-state microtubules do not contain significant amounts (greater than 1%) of GDP or GTP which can exchange with added GTP. The principal route for labeled nucleotide incorporation appears to be from tubulin-[3H]GTP subunit uptake, by diffusional and treadmilling processes.

Animals↗

PubMed1983

Rate for nucleotide release from tubulin.

The lower limit for the first order rate constant for dissociation of GDP from the tubulin E-site has been determined to be 0.14 s-1; this corresponds to a reaction with a half-life of 5 s. Using this rate constant and the previously determined equilibrium constant for GDP dissociation, equal to 6.1 X 10(-8) M (Zeeberg, B., and Caplow, M. (1979) Biochemistry 18, 3880-3886), the calculated association rate constant is 2.2 X 10(6) M-1 s-1. The tubulin E-site is highly reactive and it is calculated that: the half-life is 5 s for quantitative displacement of E-site bound radioactive GDP, by added excess nonradioactive GDP; the half-life is about 260 ms for isotopic equilibration when a trace amount of radioactive GDP is added to 20 microM tubulin-GDP; the half-life is about 850 ms for re-establishing the equilibrium for GDP binding, when 20 microM tubulin is diluted 20-fold. Thus, tubulin-GDP nucleotide exchange is rapid, so that added radioactive guanine nucleotides can be used in studies of relatively rapid reactions involving the tubulin subunit.

Guanine Nucleotides↗

PubMed1982

Concerning the efficiency of the treadmilling phenomenon with microtubules.

Radioactive tubulin subunit incorporation into porcine and dogfish shark brain microtubules which are at steady state has been found to result primarily from a diffusional reaction, in which subunits are incorporated although there are an equal number of tubulin subunit additions to and losses from each of the two microtubule ends in a unit of time. Treadmilling is very inefficient, and the Wegner s-values are equal to 0.0005-0.001. At steady state there are approximately 5000 (pig brain) or 2500 (dogfish brain) tubulin subunits lost from the two microtubule ends/s; an equivalent number of subunit addition reactions maintains a constant microtubule mass. The rate constants for subunit loss and addition with porcine brain microtubules, determined from analysis of the steady state rate for radioactive subunit incorporation, are much larger than those measured previously, when the rate constants were determined from the disassembly rate following perturbation of the steady state by dilution (Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899). To account for this discrepancy it is suggested that at steady state the microtubule is capped by a short finite length of tubulin-GTP subunits, which undergo extremely facile association and dissociation (2500-5000 subunits/microtubule/s). This cap would be rapidly lost following dilution so that the observed rate only measures the relatively slow loss of tubulin-GDP subunits (120 subunits/microtubule/s) which had been in the interior of the microtubule; this is not equal to the rate constant for subunit loss (and addition) from the ends at steady state. Because of this, previous estimates of the Wegner s-value for treadmilling which utilized dilution for determining the steady state molecular rate constants for subunit loss are believed to be too high.

Animals↗