Microtubule dynamic instability does not result from stabilization of microtubules by tubulin-GDP-Pi subunits.

The proposal that microtubule dynamic instability results from stabilization of microtubule ends by tubulin-GDP-Pi subunits (where Pi is inorganic phosphate) [Melki et al. (1996) Biochemistry 35, 12038] was based on studies of GTP hydrolysis and microtubule assembly that showed that tubulin-GDP-Pi subunits can transiently accumulate at microtubule ends. There is no direct evidence that GDP-Pi-subunits can stabilize microtubules under conditions where dynamic instability is observed and this has been inferred from the observation that tubulin-GDP-BeFn subunits stabilize microtubules. To test if tubulin-GDP-Pi stabilizes microtubules we sought evidence for a synergism between the effect of Pi and BeFn. We found, however, that Pi antagonizes the effect of BeFn by displacing it from tubulin subunits. The alternate mechanism in which Pi inhibits BeFn stabilization of microtubules by displacing fluoride from beryllium was ruled out from the 9Be and 19F NMR spectra in the presence and absence of Pi. Further evidence that tubulin-GDP-BeFn is not an analogue of tubulin-GDP-Pi and that tubulin-GDP-Pi is not responsible for maintaining the growth phase in microtubules manifesting dynamic instability was provided by our observation that Pi did not decrease the disassembly rate under conditions where tubulin-GDP-Pi subunits are expected to have formed. Results showing that BeFn binds randomly to subunits in microtubules provided evidence that Pi dissociation from the tubulin-GDP-Pi intermediate formed during GTP hydrolysis occurs randomly rather than processively starting at the growing microtubule tip.

A training program to improve domestic violence identification and management in primary care: preliminary results.

Domestic violence as encountered in day-to-day practice is greatly underidentified. It is estimated that only 3% of cases are presently being identified, and practitioners are uncertain of what to do if a case is discovered. In this paper, a training program to improve identification and management of domestic violence (DV) in primary care and the providers' responses to the program are described. A multimodal training program was undertaken to demonstrate and practice the incorporation of didactic content into practice for the health care teams. Two medical centers from a large staff-model HMO were chosen at random from five volunteering for training. The entire adult health care medical center teams, including physicians, physician assistants, RNs, LPNs, medical assistants, and receptionists, were the recipients of the training. Assessment of provider valuation of the components of the training program was performed by administering a standardized 5-point Likert-scaled questionnaire 9 months after the training. This time interval was chosen because we were interested in lasting program effects. Core didactic content, such as the epidemiology of DV, identification and management of victims and batterers, and legal issues, was highly rated. Delivery of the content through role-playing, start-stop videos and presentations by former victims received lesser but solid support. Follow-up assessment 9 months post training demonstrates solid support for many components of the program: highest for specific information content areas, but strong for techniques and processes. The training program appears to be a promising method to improve provider skills in DV management.

Evidence that a single monolayer tubulin-GTP cap is both necessary and sufficient to stabilize microtubules.

Evidence that 13 or 14 contiguous tubulin-GTP subunits are sufficient to cap and stabilize a microtubule end and that loss of only one of these subunits results in the transition to rapid disassembly(catastrophe) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules assembled with GTP was transiently stabilized against dilution-induced disassembly by reaction with tubulin-GMPCPP subunits for a time sufficient to cap the end with an average 40 subunits. The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated from an observed 25- to 2000-s lifetime of the GMPCPP-stabilized microtubules following dilution with buffer and from the time required for loss of a single tubulin-GMPCPP subunit from the microtubule end (found to be 15 s). Rather than assuming that the 25- to 2000-s dispersion in cap lifetime results from an unlikely 80-fold range in the number of tubulin-GMPCpP subunits added in the 25-s incubation, it is proposed that this results because the minimum stable cap contains 13 to 14 tubulin-GMPCPP subunits. As a consequence, a microtubule capped with 13-14 tubulin-GMPCPP subunits switches to disassembly after only one dissociation event (in about 15 s), whereas the time required for catastrophe of a microtubule with only six times as many subunits (84 subunits) corresponds to 71 dissociation events (84-13). The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was also estimated with microtubules in which a GMPCPP-cap was formed by allowing chance to result in the accumulation of multiple contiguous tubulin-GMPCPP subunits at the end, during the disassembly of microtubules containing both GDP and GMPCPP. Our observation that the disassembly rate was inhibited in proportion to the 13-14th power of the fraction of subunits containing GMPCPP again suggests that a minimum cap contains 13-14 tubulin-GMPCPP subunits. A remeasurement of the rate constant for dissociation of a tubulin-GMPCPP subunit from the plus-end of GMPCPP microtubules, now found to be 0.118 s-1, has allowed a better estimate of the standard free energy for hydrolysis of GMPCPP in a microtubule and release of Pi: this is +0.7 kcal/mol, rather than -0.9 kcal/mol, as previously reported.

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.

Parental responses to a child bicycle helmet ordinance.

Head injury associated with bicycle-related crashes is experienced disproportionately by children under age 20. Helmets are effective, but usage of helmets by children is minimal. This descriptive study, conducted in a small academic community with an ordinance requiring helmet use by children under 16, examined the views of parents and guardians about a bicycle helmet law and reports of helmet use in their families. Telephone interviews were conducted with a random sample of parents/guardians of third-, fourth-, and fifth-grade children (n = 142, 73% response rate). Sixty-five percent of those surveyed were aware of the ordinance. The vast majority (90%) agreed that bicycle helmet laws are "a good idea." Since the community helmet law was implemented, reported child helmet use increased, most notably among children who had never worn a helmet before the ordinance. Seventy percent of children who had never worn a helmet wore one most or all of the time since the ordinance. The ordinance also appears to have prompted parents to make helmet use rules for their children. Before the ordinance, families with rules reported helmet use by 79% of youths at least most of the time. This figure increased to 86% in families with rules after the ordinance was implemented. Even among the families without rules, helmet use increased from 7% before the ordinance to 60% after the law was enacted, indicating that both the ordinance and family rules are influential.

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.

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.

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.

How well do internal medicine faculty members evaluate the clinical skills of residents?

OBJECTIVE: To determine the accuracy of faculty evaluations of residents' clinical skills and whether a structured form and instructional videotape improve accuracy. DESIGN: Randomized, controlled trial. SETTING: Twelve university and community teaching hospitals. PARTICIPANTS: A total of 203 faculty internists. INTERVENTIONS: Participants watched a videotape of one of two residents performing new patient workups. Participants were assigned to one of three groups: They used either an open-ended evaluation form or a structured form that prompted detailed observations; some participants used the structured form after seeing a videotape showing good evaluation techniques. MAIN OUTCOME MEASURES: Faculty observations of strengths and weaknesses in the residents' performance were scored. An accuracy score consisting of clinical skills of critical importance for a competent history and physical examination was calculated for each participant by raters blinded to the participants' hospital, training, subspecialty, and experience as observers. RESULTS: When observations were not prompted, participants recorded only 30% of the residents' strengths and weaknesses; accuracy among participants using structured forms increased to 60% or greater. Faculty in university hospitals were more accurate than those in community hospitals, and general internists were more accurate than subspecialists; the structured form improved performance in all groups. However, participants disagreed markedly about the residents' overall clinical competence: Thirty-one percent assessed one resident's clinical skills as unsatisfactory or marginal, whereas 69% assessed them as satisfactory or superior; 48% assessed the other resident's clinical skills as unsatisfactory or marginal, whereas 52% assessed them as satisfactory or superior. Participants also disagreed about the residents' humanistic qualities. The instructional videotape did not improve accuracy. CONCLUSIONS: A structured form improved the accuracy of observations of clinical skills, but faculty still disagreed in their assessments of clinical competence. If program directors are to certify residents' clinical competence, better and more standardized evaluation is needed.

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.

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.

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.

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)

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.

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.

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.

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.

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.

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.