scholarly journals Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine.

1984 ◽  
Vol 99 (3) ◽  
pp. 1066-1075 ◽  
Author(s):  
E D Salmon ◽  
M McKeel ◽  
T Hays
Keyword(s):  
Rate Constant ◽  
Electron Micrographs ◽  
Average Length ◽  
Intrinsic Rate ◽  
Microtubule Assembly ◽  
Spindle Microtubules ◽  
Intracellular Concentrations ◽  
High Concentrations

At metaphase, the amount of tubulin assembled into spindle microtubules is relatively constant; the rate of tubulin association equals the rate of dissociation. To measure the intrinsic rate of dissociation, we microinjected high concentrations of colchicine, or its derivative colcemid, into sea urchin embryos at metaphase to bind the free tubulin, thereby rapidly blocking polymerization. The rate of microtubule disassembly was measured from a calibrated video signal by the change in birefringent retardation (BR). After an initial delay after injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM, BR decreased rapidly and simultaneously throughout the central spindle and aster. Measured BR in the central half-spindle decreased exponentially to 10% of its initial value within a characteristic period of approximately 20 s; the rate constant, k = 0.11 +/- 0.023 s-1, and the corresponding half-time, t 1/2, of BR decay was approximately 6.5 +/- 1.1 s in this concentration range. Below 0.1 mM colchicine or colcemid, the rate at which BR decreased was concentration dependent. Electron micrographs showed that the rapid decrease in BR corresponded to the disappearance of nonkinetochore microtubules; kinetochore fiber microtubules were differentially stable. As a control, lumicolchicine, which does not bind to tubulin with high affinity, was shown to have no effect on spindle BR at intracellular concentrations of 0.5 mM. If colchicine and colcemid block only polymerization, then the initial rate of tubulin dissociation from nonkinetochore spindle microtubules is in the range of 180-992 dimers per second. This range of rates is based on k = 11% of the initial polymer per second and an estimate from electron micrographs that the average length of a half-spindle microtubule is 1-5.5 micron. Much slower rates of tubulin association are predicted from the characteristics of end-dependent microtubule assembly measured previously in vitro when the association rate constant is corrected for the lower rate of tubulin diffusion in the embryo cytoplasm. Various possibilities for this discrepancy are discussed.

scholarly journals A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end.

1987 ◽  
Vol 105 (5) ◽  
pp. 2203-2215 ◽  
Author(s):  
D L Gard ◽  
M W Kirschner
Keyword(s):  
Rate Constant ◽  
Fold Increase ◽  
Microtubule Assembly ◽  
Molar Ratio ◽  
Mammalian Brain ◽  
Protein Factor ◽  
Neuronal Tissues ◽  
Assembly Rate

We have isolated a protein factor from Xenopus eggs that promotes microtubule assembly in vitro. Assembly promotion was associated with a 215-kD protein after a 1,000-3,000-fold enrichment of activity. The 215-kD protein, termed Xenopus microtubule assembly protein (XMAP), binds to microtubules with a stoichiometry of 0.06 mol/mol tubulin dimer. XMAP is immunologically distinct from the Xenopus homologues to mammalian brain microtubule-associated proteins; however, protein species immunologically related to XMAP with different molecular masses are found in Xenopus neuronal tissues and testis. XMAP is unusual in that it specifically promotes microtubule assembly at the plus-end. At a molar ratio of 0.01 mol XMAP/mol tubulin the assembly rate of the microtubule plus-end is accelerated 8-fold while the assembly rate of the minus-end is increased only 1.8-fold. Under these conditions XMAP promotes a 10-fold increase in the on-rate constant (from 1.4 s-1.microM-1 for microtubules assembled from pure tubulin to 15 s-1.microM-1), and a 10-fold decrease in off-rate constant (from 340 to 34 s-1). Given its stoichiometry in vivo, XMAP must be the major microtubule assembly factor in the Xenopus egg. XMAP is phosphorylated during M-phase of both meiotic and mitotic cycles, suggesting that its activity may be regulated during the cell cycle.

scholarly journals Pressure-induced depolymerization of spindle microtubules. II. Thermodynamics of in vivo spindle assembly.

1975 ◽  
Vol 66 (1) ◽  
pp. 114-127 ◽  
Author(s):  
E D Salmon
Keyword(s):  
Hydrostatic Pressure ◽  
Equilibrium Constant ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Condensation Polymerization ◽  
A Value ◽  
Spindle Microtubules ◽  
Brain Microtubules

The present experiments were designed to test whether the simple equilibrium assembly model proposed by Inoué could predict variations in spindle microtubule assembly in response to changes in hydrostatic pressure as it does for changes in temperature. The results were also analyzed according to a model based on nucleated condensation polymerization since this recently appears to be the mechanism by which purified brain microtubules are assembled in vitro. Equilibrium birefringence (BR) of the meiotic metaphase-arrested spindle was measured in vivo as a function of hydrostatic pressure and temperature in Chaetopterus oocytes using a miniature microscope pressure chamber. Increasing pressure in steps to 3,000 psi at temperatures below 22 degrees C did produce decreases in spindle equilibrium BR predictable directly from the simple equilibrium model of spindle assembly. Thermodynamic analysis of the pressure data yielded a value of delta V congruent to 400 ml/mol of polymerizing unit. Theoretical curves based on the nucleated condensation model can also be made to fit the data, but semilog plots of the dependence of the equilibrium constant versus pressure and versus reciprocal temperature are biphasic, suggesting that either the size of the polymerizing unit changes or more than one equilibrium constant governs the assembly reaction. That the same value of delta V, 90 ml/mol, was estimated from both the majority of the spindle BR data and data for the assembly of neural microtubules in vitro supports the possibility that spindle microtubules are assembled by a nucleated condensation mechanism.

scholarly journals Microtubule assembly and disassembly at alkaline pH

1981 ◽  
Vol 89 (1) ◽  
pp. 45-53 ◽  
Author(s):  
CS Regula ◽  
Pfeiffer JR ◽  
RD Berlin
Keyword(s):  
Steady State ◽  
Critical Concentration ◽  
Ph Dependence ◽  
Intrinsic Rate ◽  
Bovine Brain ◽  
Microtubule Assembly ◽  
Alkaline Ph ◽  
Direct Assembly

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of "treadmilling." The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

scholarly journals Enterococcus faecalis exploits the human fibrinolytic system to drive excess collagenolysis: implications in gut healing and identification of druggable targets

2020 ◽  
Vol 318 (1) ◽  
pp. G1-G9 ◽  
Author(s):  
Richard A. Jacobson ◽  
Kiedo Wienholts ◽  
Ashley J. Williamson ◽  
Sara Gaines ◽  
Sanjiv Hyoju ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Healing Process ◽  
Abdominal Sepsis ◽  
Infectious Complications ◽  
Fibrinolytic System ◽  
Clinical Safety ◽  
High Concentrations ◽  
Clinically Significant

Perforations, anastomotic leak, and subsequent intra-abdominal sepsis are among the most common and feared complications of invasive interventions in the colon and remaining intestinal tract. During physiological healing, tissue protease activity is finely orchestrated to maintain the strength and integrity of the submucosa collagen layer in the wound. We (Shogan, BD et al. Sci Trans Med 7: 286ra68, 2015.) have previously demonstrated in both mice and humans that the commensal microbe Enterococcus faecalis selectively colonizes wounded colonic tissues and disrupts the healing process by amplifying collagenolytic matrix-metalloprotease activity toward excessive degradation. Here, we demonstrate for the first time, to our knowledge, a novel collagenolytic virulence mechanism by which E. faecalis is able to bind and locally activate the human fibrinolytic protease plasminogen (PLG), a protein present in high concentrations in healing colonic tissue. E. faecalis-mediated PLG activation leads to supraphysiological collagen degradation; in this study, we demonstrate this concept both in vitro and in vivo. This pathoadaptive response can be mitigated with the PLG inhibitor tranexamic acid (TXA) in a fashion that prevents clinically significant complications in validated murine models of both E. faecalis- and Pseudomonas aeruginosa-mediated colonic perforation. TXA has a proven clinical safety record and is Food and Drug Administration approved for topical application in invasive procedures, albeit for the prevention of bleeding rather than infection. As such, the novel pharmacological effect described in this study may be translatable to clinical trials for the prevention of infectious complications in colonic healing. NEW & NOTEWORTHY This paper presents a novel mechanism for virulence in a commensal gut microbe that exploits the human fibrinolytic system and its principle protease, plasminogen. This mechanism is targetable by safe and effective nonantibiotic small molecules for the prevention of infectious complications in the healing gut.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

Application of the EYTEX™ System to the Evaluation of Cosmetic Products and their Ingredients

1992 ◽  
Vol 20 (1) ◽  
pp. 146-163
Author(s):  
Francis H. Kruszewski ◽  
Laura H. Hearn ◽  
Kyle T. Smith ◽  
Janice J. Teal ◽  
Virginia C. Gordon ◽  
...  
Keyword(s):  
Double Blind ◽  
Eye Irritation ◽  
Ocular Irritation ◽  
Rabbit Eye ◽  
Wide Range ◽  
High Concentrations ◽  
Alkaline Membrane ◽  
Positive Agreement

465 cosmetic product formulations and raw ingredients were evaluated with the EYTEX™ system to determine the potential of this in vitro alternative for identifying eye irritation potential. The EYTEX™ system is a non-animal, biochemical procedure developed by Ropak Laboratories, Irvine, CA, that was designed to approximate the Draize rabbit eye irritation assay for the evaluation of ocular irritation. Avon Products Inc. provided all the test samples, which included over 30 different product types and represented a wide range of eye irritancy. All the EYTEX™ protocols available at the time of this study were used. Samples were evaluated double-blind with both the membrane partition assay (MPA) and the rapid membrane assay (RMA). When appropriate, the standard assay (STD) and the alkaline membrane assay (AMA) were used, as well as specific, documented protocol modifications. EYTEX™ results were correlated with rabbit eye irritation data which was obtained from the historical records of Avon Products Inc. A positive agreement of EYTEX™ results with the in vivo assay was demonstrated by an overall concordance of 80%. The assay error was 20%, of which 18% was due to an overestimation of sample irritancy (false positives) and 2% was attributed to underestimation (false negatives). Overestimation error in this study was due in part to the inability of the protocols to accurately classify test samples with very low irritation potential. Underestimation of sample irritancy was generally associated with ethoxylated materials and high concentrations of specific types of surfactants. 100% sensitivity and 85% predictability were described by the data, indicating the efficiency of EYTEX™ in identifying known irritants. A specificity rate of 39% showed the EYTEX™ assay to be weak in discerning non-irritants. However, the EYTEX™ protocols used in this study were not designed to identify non-irritants. A compatibility rate of 99% proved the effectiveness of the EYTEX™ assay in accommodating a diversity of product types. The EYTEX™ system protocols, when used appropriately, can provide a conservative means of assessing the irritant potential of most cosmetic formulations and their ingredients.

scholarly journals A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jie Zheng ◽  
Na Tian ◽  
Fei Liu ◽  
Yidian Zhang ◽  
Jingfen Su ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Protein Phosphatase ◽  
High Efficiency ◽  
Microtubule Assembly ◽  
Hyperphosphorylated Tau ◽  
Phosphatase 2A ◽  
Dependent Learning ◽  
The Brain

AbstractIntraneuronal accumulation of hyperphosphorylated tau is a hallmark pathology shown in over twenty neurodegenerative disorders, collectively termed as tauopathies, including the most common Alzheimer’s disease (AD). Therefore, selectively removing or reducing hyperphosphorylated tau is promising for therapies of AD and other tauopathies. Here, we designed and synthesized a novel DEPhosphorylation TArgeting Chimera (DEPTAC) to specifically facilitate the binding of tau to Bα-subunit-containing protein phosphatase 2A (PP2A-Bα), the most active tau phosphatase in the brain. The DEPTAC exhibited high efficiency in dephosphorylating tau at multiple AD-associated sites and preventing tau accumulation both in vitro and in vivo. Further studies revealed that DEPTAC significantly improved microtubule assembly, neurite plasticity, and hippocampus-dependent learning and memory in transgenic mice with inducible overexpression of truncated and neurotoxic human tau N368. Our data provide a strategy for selective removal of the hyperphosphorylated tau, which sheds new light for the targeted therapy of AD and related-tauopathies.

scholarly journals Drosophila Stathmin: A Microtubule-destabilizing Factor Involved in Nervous System Formation

2002 ◽  
Vol 13 (2) ◽  
pp. 698-710 ◽  
Author(s):  
Sylvie Ozon ◽  
Antoine Guichet ◽  
Olivier Gavet ◽  
Siegfried Roth ◽  
André Sobel
Keyword(s):  
Nervous System ◽  
System Development ◽  
Nervous System Development ◽  
Microtubule Assembly ◽  
Nervous Systems ◽  
Germ Cell Migration ◽  
Numerous Cell ◽  
First Time

Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins inDrosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin andstathmin family genes. They interfere with microtubule assembly in vitro, and in vivo when expressed in HeLa cells. Drosophila stathmin expression is regulated during embryogenesis: it is high in the migrating germ cells and in the central and peripheral nervous systems, a pattern resembling that of mammalian stathmin. Furthermore, RNA interference inactivation ofDrosophila stathmin expression resulted in germ cell migration arrest at stage 14. It also induced important anomalies in nervous system development, such as loss of commissures and longitudinal connectives in the ventral cord, or abnormal chordotonal neuron organization. In conclusion, a single Drosophilagene encodes phosphoproteins homologous to the entire vertebrate stathmin family. We demonstrate for the first time their direct involvement in major biological processes such as development of the reproductive and nervous systems.

Directionality of microtubule assembly: an in vivo study with the ciliate Tetrahymena

1978 ◽  
Vol 33 (1) ◽  
pp. 227-234
Author(s):  
S.F. Ng
Keyword(s):  
Tetrahymena Thermophila ◽  
Recessive Gene ◽  
Microtubule Assembly ◽  
In Vivo Study ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Posterior Portion ◽  
Temperature Sensitive Mutant

A temperature-sensitive mutant homozygous for the recessive gene molb in Tetrahymena thermophila offers opportunity for studying the direction of microtubule assembly in vivo. At 39 degrees C the mutant fails to divide properly; the 2 daughter animals remain attached and bend over each other. As revealed by protargol staining, the bending results in acute turning and breaking of some of the longitudinal microtubular bands close and parallel to the surface. Hence, 2 broken microtubular ends are available for study of the problem of directionality of microtubule assembly, by assessing which of the 2 ends regenerates. In most cases the posterior portion of the longitudinal microtubular band regenerates. The present study hence supports the conclusion based on in vitro observation in other systems that microtubule assembly is predominantly unidirectional.

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