Ectopic A-lattice seams destabilize microtubules

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe

Universities and Pricing on Higher Education Markets

Markets and prices in higher education. When can we speak of markets, and when markets exist, how are prices set

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

On becoming (un)committed: A taxonomy and test of newcomer on-boarding scenarios

How does the bond between the newcomer and the organization develop over time? Process research on temporal patterns of newcomer's early commitment formation has been very scarce because theory and appropriate longitudinal research designs in this area are lacking. From extant research we extract three process-theoretical accounts regarding how the newcomer adjustment process evolves over time: (1) Learning to Love; (2) Honeymoon Hangover; and (3) High Match, Moderate Match, or Low Match. From these scenarios we develop a taxonomy of newcomer adjustment scenarios. Further, we empirically verify these different scenarios by examining naturally occurring "trajectory classes," which are found to display strengthening, weakening, or stabilizing of the employee-organization linkage. For this, we use a sample of 72 Ph. D. graduates whose organizational commitment history was recorded in their first 25 consecutive weeks of new employment. In closing, we discuss the theoretical and practical implications of the scenario-based approach

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

A deterministic oscillatory model of microtubule growth and shrinkage for differential actions of short chain fatty acids.

Short chain fatty acids (SCFA), principally acetate, propionate, butyrate and valerate, are produced in pharmacologically relevant concentrations by the gut microbiome. Investigations indicate that they exert beneficial effects on colon epithelia. There is increasing interest in whether different SCFAs have distinct functions which may be exploited for prevention or treatment of colonic diseases including colorectal cancer (CRC), inflammatory bowel disease and obesity. Based on experimental evidence, we hypothesised that odd-chain SCFAs may possess anti-mitotic capabilities in colon cancer cells by disrupting microtubule (MT) structural integrity via dysregulation of β-tubulin isotypes. MT dynamic instability is an essential characteristic of MT cellular activity. We report a minimal deterministic model that takes a novel approach to explore the hypothesised pathway by triggering spontaneous oscillations to represent MT dynamic behaviour. The dynamicity parameters in silico were compared to those reported in vitro. Simulations of untreated and butyrate (even-chain length) treated cells reflected MT behaviour in interphase or untreated control cells. The propionate and valerate (odd-chain length) simulations displayed increased catastrophe frequencies and longer periods of MT-fibre shrinkage. Their enhanced dynamicity was dissimilar to that observed in mitotic cells, but parallel to that induced by MT-destabilisation treatments. Antimicrotubule drugs act through upward or downward modulation of MT dynamic instability. Our computational modelling suggests that metabolic engineering of the microbiome may facilitate managing CRC risk by predicting outcomes of SCFA treatments in combination with AMDs

Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell

Islands Containing Slowly Hydrolyzable GTP Analogs Promote Microtubule Rescues

Microtubules are dynamic polymers of GTP- and GDP-tubulin that undergo stochastic transitions between growing and shrinking phases. Rescues, the conversion from shrinking to growing, have recently been proposed to be to the result of regrowth at GTP-tubulin islands within the lattice of growing microtubules. By introducing mixed GTP/GDP/GMPCPP (GXP) regions within the lattice of dynamic microtubules, we reconstituted GXP islands in vitro (GMPCPP is the slowly hydrolyzable GTP analog guanosine-5′-[(α,β)-methyleno]triphosphate). We found that such islands could reproducibly induce rescues and that the probability of rescue correlated with both the size of the island and the percentage of GMPCPP-tubulin within the island. The islands slowed the depolymerization rate of shortening microtubules and promoted regrowth more readily than GMPCPP seeds. Together, these findings provide new mechanistic insights supporting the possibility that rescues could be triggered by enriched GTP-tubulin regions and present a new tool for studying such rescue events in vitro