scholarly journals The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

2019 ◽  
Author(s):  
Johanna Roostalu ◽  
Claire Thomas ◽  
Nicholas I. Cade ◽  
Simone Kunzelmann ◽  
Ian A. Taylor ◽  
...  
Keyword(s):  
Single Molecule ◽  
Critical Importance ◽  
Gtp Hydrolysis ◽  
Diverse Range ◽  
Slowing Down ◽  
High Affinity ◽  
Microtubule Stability ◽  
Kinetics Of ◽  
Microtubule Function

ABSTRACTMicrotubules are bistable cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage 1. Growing microtubules are thought to be stabilized by a GTP cap at their ends 2-5. The nature of this cap, however, is still poorly understood. How GTP hydrolysis determines the properties of the GTP cap and hence microtubule stability is unclear. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends 6. Whether these regulatory platforms at growing microtubule ends are identical to the GTP cap is not known. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate in microscopy-based in vitro reconstitutions that EB proteins bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps and consequently hyper-stable microtubules. Single molecule experiments reveal that the microtubule conformation gradually changes in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability; and establish that the GTP cap coincides with the EB-binding regulatory hub that modulates microtubule cytoskeleton function in cells.

scholarly journals The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Johanna Roostalu ◽  
Claire Thomas ◽  
Nicholas Ian Cade ◽  
Simone Kunzelmann ◽  
Ian A Taylor ◽  
...  
Keyword(s):  
Critical Importance ◽  
Binding Region ◽  
Gtp Hydrolysis ◽  
Diverse Range ◽  
Slowing Down ◽  
High Affinity ◽  
Microtubule Stability ◽  
Conformation Changes ◽  
Kinetics Of ◽  
Microtubule Function

Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length determines microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

scholarly journals Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

2019 ◽  
Vol 30 (12) ◽  
pp. 1369-1376 ◽  
Author(s):  
Tim N. Baldering ◽  
Marina S. Dietz ◽  
Karl Gatterdam ◽  
Christos Karathanasis ◽  
Ralph Wieneke ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Superresolution Microscopy ◽  
Superresolution Imaging ◽  
Molecular Information ◽  
Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

scholarly journals Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

2006 ◽  
Vol 175 (6) ◽  
pp. 947-955 ◽  
Author(s):  
Takushi Miyoshi ◽  
Takahiro Tsuji ◽  
Chiharu Higashida ◽  
Maud Hertzog ◽  
Akiko Fujita ◽  
...  
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Dissociation Rate ◽  
Actin Network ◽  
Lim Kinase ◽  
Capping Protein ◽  
Filament Dynamics ◽  
Actin Turnover ◽  
Kinetics Of

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s−1, respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.

scholarly journals Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.

1991 ◽  
Vol 114 (1) ◽  
pp. 73-81 ◽  
Author(s):  
R A Walker ◽  
N K Pryer ◽  
E D Salmon
Keyword(s):  
Dynamic Instability ◽  
Elongation Rate ◽  
Gtp Hydrolysis ◽  
Microtubule Stability ◽  
Assembly Mechanism ◽  
High Elongation ◽  
Microtubule Growth ◽  
Hydrolysis Of ◽  
Brain Tubulin

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build-up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.

scholarly journals Phagocytosis of Advanced Glycation End Products (AGEs) in Macrophages Induces Cell Apoptosis

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Yuan Gao ◽  
Hidenori Wake ◽  
Yuta Morioka ◽  
Keyue Liu ◽  
Kiyoshi Teshigawara ◽  
...  
Keyword(s):  
Advanced Glycation End Products ◽  
Reducing Sugars ◽  
Vascular Lesions ◽  
Advanced Glycation ◽  
Diverse Range ◽  
Intracellular Ros ◽  
Glycation End Products ◽  
End Products ◽  
Kinetics Of

Advanced glycation end products (AGEs) are the products of a series of nonenzymatic modifications of proteins by reducing sugars. AGEs play a pivotal role in development of diabetic complications and atherosclerosis. Accumulation of AGEs in a vessel wall may contribute to the development of vascular lesions. Although AGEs have a diverse range of bioactivities, the clearance process of AGEs from the extracellular space, including the incorporation of AGEs into specific cells, subcellular localization, and the fate of AGEs, remains unclear. In the present study, we examined the kinetics of the uptake of AGEs by mouse macrophage J774.1 cells in vitro and characterized the process. We demonstrated that AGEs bound to the surface of the cells and were also incorporated into the cytoplasm. The temperature- and time-dependent uptake of AGEs was saturable with AGE concentration and was inhibited by cytochalasin D but not chlorpromazine. We also observed the granule-like appearance of AGE immunoreactivity in subcellular localizations in macrophages. Higher concentrations of AGEs induced intracellular ROS and 4-HNE, which were associated with activation of the NF-κB pathway and caspase-3. These results suggest that incorporation of AGEs occurred actively by endocytosis in macrophages, leading to apoptosis of these cells through NF-κB activation.

scholarly journals The glucocorticoid receptor DNA-binding domain recognizes RNA hairpin structures with high affinity

2019 ◽  
Vol 47 (15) ◽  
pp. 8180-8192 ◽  
Author(s):  
Nicholas V Parsonnet ◽  
Nickolaus C Lammer ◽  
Zachariah E Holmes ◽  
Robert T Batey ◽  
Deborah S Wuttke
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Diverse Range ◽  
High Affinity ◽  
Rna Hairpin

AbstractThe glucocorticoid receptor (GR) binds the noncoding RNA Gas5 via its DNA-binding domain (DBD) with functional implications in pro-apoptosis signaling. Here, we report a comprehensive in vitro binding study where we have determined that GR-DBD is a robust structure-specific RNA-binding domain. GR-DBD binds to a diverse range of RNA hairpin motifs, both synthetic and biologically derived, with apparent mid-nanomolar affinity while discriminating against uniform dsRNA. As opposed to dimeric recognition of dsDNA, GR-DBD binds to RNA as a monomer and confers high affinity primarily through electrostatic contacts. GR-DBD adopts a discrete RNA-bound state, as assessed by NMR, distinct from both free and DNA-bound. NMR and alanine mutagenesis suggest a heightened involvement of the C-terminal α-helix of the GR-DBD in RNA-binding. RNA competes for binding with dsDNA and occurs in a similar affinity range as dimer binding to the canonical DNA element. Given the prevalence of RNA hairpins within the transcriptome, our findings strongly suggest that many RNAs have potential to impact GR biology.

scholarly journals Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase

2020 ◽  
Vol 117 (21) ◽  
pp. 11257-11264 ◽  
Author(s):  
Wei-Hsuan Lan ◽  
Sheng-Yao Lin ◽  
Chih-Yuan Kao ◽  
Wen-Hsuan Chang ◽  
Hsin-Yi Yeh ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Molecule ◽  
Meiotic Recombination ◽  
Direct Evidence ◽  
Protein Protein Interaction ◽  
Filament Assembly ◽  
Tethered Particle Motion ◽  
Physical Interactions ◽  
Kinetics Of

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly ofSaccharomyces cerevisiaeDmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5′-to-3′ polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein–protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.

Kinetics Of Factor Viii Inhibitors In Three Non-Hemophiliac Patients

1981 ◽  
Author(s):  
S L McClellan ◽  
C V Hussey ◽  
J E Fobian ◽  
A V Pisciotta
Keyword(s):  
Factor Viii ◽  
Single Molecule ◽  
Gamma Globulin ◽  
Normal Activity ◽  
Direct Binding ◽  
Low Levels ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Kinetics Of

Factor VIII (VIII) inhibitors were isolated from the gamma globulin fraction of the plasmas of three non-hemophilic patients. One of these patients is thought to have acquired the inhibitor secondary to Hodgkin’s disease while the other two were apparently drug-related. These patients presented without significant prior replacement therapy and showed elevated levels of von Willebrand factor (VIIIVW-R) as measured by Ristocetin aggregation of fixed washed platelets and elevated levels of antigenic VIII (VIIIAGN) using a CELIA technique. The procoagulant VIII (VIIIAHF) activities, however, ranged from 12-20% of normal activity (one-stage method) and the inhibitors were present in levels of 6-50 Bethesda Units/ml of plasma.The kinetics of the interaction between these inhibitors and VIII were studied to further characterize the inhibitors. In vitro inactivation of VIII by the inhibitors demonstrated a two-step kinetic model similar to that proposed by Biggs. Iii vivo neutralization of transfused VIII appeared to follow the same model. The high levels of VIIIAGN and VIIIVW-R in the presence of low levels of VIIIAHF suggest binding of the inhibitor to the procoagulant moiety. Further, two-step inactivation of the procoagulant activity suggests binding of more than one molecule of inhibitor near rather than at the active procoagulant site which results in increasing stearic inhibition of the active site. This is in contrast to simple direct binding of a single molecule of inhibitor as described with bimolecular second order kinetics.Finally, the presence of high levels of VIIIAGN with markedly attenuated VIIIAHF activities implies relatively slow clearance of the inhibitor-VIII complex.

scholarly journals Polymerization of FtsZ, a Bacterial Homolog of Tubulin

2001 ◽  
Vol 276 (15) ◽  
pp. 11743-11753 ◽  
Author(s):  
Laura Romberg ◽  
Martha Simon ◽  
Harold P. Erickson
Keyword(s):  
Steady State ◽  
Critical Concentration ◽  
Gtp Hydrolysis ◽  
Nucleotide Hydrolysis ◽  
High Affinity ◽  
Hydrolysis Rates ◽  
Bacterial Homolog ◽  
Nucleation Phase ◽  
Maximum Steady State

FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis.In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers. Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or “isodesmic”) assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration. We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme. The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates. It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolarKD.

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