scholarly journals Intrinsic Dynamic Behavior of Fascin in Filopodia

2007 ◽  
Vol 18 (10) ◽  
pp. 3928-3940 ◽  
Author(s):  
Yvonne S. Aratyn ◽  
Thomas E. Schaus ◽  
Edwin W. Taylor ◽  
Gary G. Borisy
Keyword(s):  
Dynamic Behavior ◽  
Fluorescence Recovery After Photobleaching ◽  
Reaction Diffusion ◽  
Computational Approach ◽  
Cross Linking ◽  
Rapid Cycling ◽  
Reaction Diffusion Model ◽  
Cross Linker ◽  
Intrinsic Dynamic

Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s−1 and that it undergoes diffusion at moderate rates with a coefficient of 6 μm2s−1. This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction–diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25–60 actin monomers.

scholarly journals Dynamic partitioning of mitotic kinesin-5 cross-linkers between microtubule-bound and freely diffusing states

2008 ◽  
Vol 182 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Dhanya K. Cheerambathur ◽  
Ingrid Brust-Mascher ◽  
Gul Civelekoglu-Scholey ◽  
Jonathan M. Scholey
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Reaction Diffusion ◽  
Cross Linking ◽  
Spindle Matrix ◽  
Dynamic Partitioning ◽  
Spindle Poles ◽  
Reaction Diffusion Model ◽  
Dynamic Mobility ◽  
Spindle Microtubules ◽  
Transgenic Drosophila

The dynamic behavior of homotetrameric kinesin-5 during mitosis is poorly understood. Kinesin-5 may function only by binding, cross-linking, and sliding adjacent spindle microtubules (MTs), or, alternatively, it may bind to a stable “spindle matrix” to generate mitotic movements. We created transgenic Drosophila melanogaster expressing fluorescent kinesin-5, KLP61F-GFP, in a klp61f mutant background, where it rescues mitosis and viability. KLP61F-GFP localizes to interpolar MT bundles, half spindles, and asters, and is enriched around spindle poles. In fluorescence recovery after photobleaching experiments, KLP61F-GFP displays dynamic mobility similar to tubulin, which is inconsistent with a substantial static pool of kinesin-5. The data conform to a reaction–diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely. KLP61F appears to transiently bind MTs, moving short distances along them before detaching. Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.

scholarly journals Role of fascin in filopodial protrusion

2006 ◽  
Vol 174 (6) ◽  
pp. 863-875 ◽  
Author(s):  
Danijela Vignjevic ◽  
Shin-ichiro Kojima ◽  
Yvonne Aratyn ◽  
Oana Danciu ◽  
Tatyana Svitkina ◽  
...  
Keyword(s):  
Rna Interference ◽  
Dynamic Behavior ◽  
Actin Filaments ◽  
Cellular Localization ◽  
Fluorescence Recovery After Photobleaching ◽  
Cross Linking ◽  
Wild Type ◽  
Actin Organization ◽  
Cross Linker

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t1/2 < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.

scholarly journals An in vitro–in silico interface platform for spatiotemporal analysis of pattern formation in collective epithelial cells

2016 ◽  
Vol 8 (8) ◽  
pp. 861-868 ◽  
Author(s):  
M. Hagiwara
Keyword(s):  
Cell Culture ◽  
Pattern Formation ◽  
Epithelial Cells ◽  
In Silico ◽  
Reaction Diffusion ◽  
Spatiotemporal Analysis ◽  
Bronchial Epithelial ◽  
Reaction Diffusion Model ◽  
2D Pattern

The mechanisms of 2D pattern formation in bronchial epithelial cells were dynamically analyzed by controlled cell culture and a reaction-diffusion model.

Experimental characterization and computational modeling of hydrogel cross-linking for bioprinting applications

2019 ◽  
Vol 42 (10) ◽  
pp. 548-557 ◽  
Author(s):  
Aidin Hajikhani ◽  
Franca Scocozza ◽  
Michele Conti ◽  
Michele Marino ◽  
Ferdinando Auricchio ◽  
...  
Keyword(s):  
Calcium Chloride ◽  
Reaction Diffusion ◽  
Chloride Diffusion ◽  
Chemical System ◽  
Cross Linking ◽  
Modeling Framework ◽  
Reaction Diffusion Model ◽  
Constant Diffusivity ◽  
Low Toxicity ◽  
Element Discretizations

Alginate-based hydrogels are extensively used to create bioinks for bioprinting, due to their biocompatibility, low toxicity, low costs, and slight gelling. Modeling of bioprinting process can boost experimental design reducing trial-and-error tests. To this aim, the cross-linking kinetics for the chemical gelation of sodium alginate hydrogels via calcium chloride diffusion is analyzed. Experimental measurements on the absorbed volume of calcium chloride in the hydrogel are obtained at different times. Moreover, a reaction-diffusion model is developed, accounting for the dependence of diffusive properties on the gelation degree. The coupled chemical system is solved using finite element discretizations which include the inhomogeneous evolution of hydrogel state in time and space. Experimental results are fitted within the proposed modeling framework, which is thereby calibrated and validated. Moreover, the importance of accounting for cross-linking-dependent diffusive properties is highlighted, showing that, if a constant diffusivity property is employed, the model does not properly capture the experimental evidence. Since the analyzed mechanisms highly affect the evolution of the front of the solidified gel in the final bioprinted structure, the present study is a step towards the development of reliable computational tools for the in silico optimization of protocols and post-printing treatments for bioprinting applications.

scholarly journals Modulation of Entrapment Efficiency and In Vitro Release Properties of BSA-Loaded Chitosan Microparticles Cross-Linked with Citric Acid as a Potential Protein–Drug Delivery System

Materials ◽  
2020 ◽  
Vol 13 (8) ◽  
pp. 1989 ◽  
Author(s):  
Natalia Sedyakina ◽  
Andrey Kuskov ◽  
Kelly Velonia ◽  
Nataliya Feldman ◽  
Sergey Lutsenko ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Citric Acid ◽  
In Vitro Release ◽  
Entrapment Efficiency ◽  
Cross Linking ◽  
Release Mechanism ◽  
Model Protein ◽  
Cross Linker ◽  
The Cross

Microparticles, aimed for oral protein and peptide drug delivery, were prepared via emulsion cross-linking using citric acid as cross-linker and polyglycerol polyricinoleate as surfactant. A comparative study of the interaction between chitosan and citric acid and its effect on the resulting microparticle properties was performed using different chitosan-to-cross-linker mass ratios and pH-values during fabrication of the microparticles. Non-cross-linked and cross-linked microparticles were studied in terms of size (4–12 μm), zeta potential (−15.7 to 12.8 mV), erosion (39.7–75.6%), a model protein encapsulation efficiency (bovine serum albumin) (6.8–27.6%), and loading capacity (10.4–40%). Fourier transform infrared spectroscopy and X-ray diffraction confirmed the ionic interaction between the protonated amine groups of chitosan and the carboxylate ions of the cross-linking agent. Scanning electron microscopy revealed that the non-cross-linked microparticles had an uneven shape with wrinkled surfaces, while the cross-linked formulations were spherical in shape with smooth surfaces. On the basis of these data, the role of the surfactant and microparticle structure on the release mechanism was proposed. Control of the microparticle shape and release mechanisms is expected to be crucial in developing carriers for the controlled delivery of proteins and peptides.

scholarly journals Neuronal Calcium Wave Propagation Varies with Changes in Endoplasmic Reticulum Parameters: A Computer Model

2015 ◽  
Vol 27 (4) ◽  
pp. 898-924 ◽  
Author(s):  
Samuel A. Neymotin ◽  
Robert A. McDougal ◽  
Mohamed A. Sherif ◽  
Christopher P. Fall ◽  
Michael L. Hines ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Wave Propagation ◽  
Reaction Diffusion ◽  
High Sensitivity ◽  
Apical Dendrite ◽  
Reaction Diffusion Model ◽  
Apparent Diffusion ◽  
Text Density ◽  
Electrochemical Model

Calcium ([Formula: see text]) waves provide a complement to neuronal electrical signaling, forming a key part of a neuron’s second messenger system. We developed a reaction-diffusion model of an apical dendrite with diffusible inositol triphosphate ([Formula: see text]), diffusible [Formula: see text], [Formula: see text] receptors ([Formula: see text]s), endoplasmic reticulum (ER) [Formula: see text] leak, and ER pump (SERCA) on ER. [Formula: see text] is released from ER stores via [Formula: see text]s upon binding of [Formula: see text] and [Formula: see text]. This results in [Formula: see text]-induced-[Formula: see text]-release (CICR) and increases [Formula: see text] spread. At least two modes of [Formula: see text] wave spread have been suggested: a continuous mode based on presumed relative homogeneity of ER within the cell and a pseudo-saltatory model where [Formula: see text] regeneration occurs at discrete points with diffusion between them. We compared the effects of three patterns of hypothesized [Formula: see text] distribution: (1) continuous homogeneous ER, (2) hotspots with increased [Formula: see text] density ([Formula: see text] hotspots), and (3) areas of increased ER density (ER stacks). All three modes produced [Formula: see text] waves with velocities similar to those measured in vitro (approximately 50–90 [Formula: see text]m /sec). Continuous ER showed high sensitivity to [Formula: see text] density increases, with time to onset reduced and speed increased. Increases in SERCA density resulted in opposite effects. The measures were sensitive to changes in density and spacing of [Formula: see text] hotspots and stacks. Increasing the apparent diffusion coefficient of [Formula: see text]  substantially increased wave speed. An extended electrochemical model, including voltage-gated calcium channels and AMPA synapses, demonstrated that membrane priming via AMPA stimulation enhances subsequent [Formula: see text] wave amplitude and duration. Our modeling suggests that pharmacological targeting of [Formula: see text]s and SERCA could allow modulation of [Formula: see text] wave propagation in diseases where [Formula: see text] dysregulation has been implicated.

Tridegin, a Novel Peptidic Inhibitor of Factor XIIIa from the Leech, Haementeria ghilianii, Enhances Fibrinolysis In Vitro

1997 ◽  
Vol 77 (05) ◽  
pp. 0959-0963 ◽  
Author(s):  
Lisa Seale ◽  
Sarah Finney ◽  
Roy T Sawyer ◽  
Robert B Wallis
Keyword(s):  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Fibrinolytic Enzymes ◽  
Small Polypeptide ◽  
Tissue Plasminogen ◽  
Protein Cross Linking

SummaryTridegin is a potent inhibitor of factor Xllla from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet- free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

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