Kinetics of actin networks formation measured by time resolved particle-tracking microrheology

ATP-assisted actin network self assembly in vitro is acompanied by an overshoot of the viscoelastic moduli followed by a relaxation to steady-state values.

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Quantitative regulation of the dynamic steady state of actin networks

eLife ◽

10.7554/elife.42413 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 6

Author(s):

Angelika Manhart ◽

Téa Aleksandra Icheva ◽

Christophe Guerin ◽

Tobbias Klar ◽

Rajaa Boujemaa-Paterski ◽

...

Keyword(s):

Mathematical Model ◽

Steady State ◽

Heterogeneous Networks ◽

In Silico ◽

Actin Network ◽

Network Nodes ◽

Actin Networks ◽

Cell Functions ◽

Selective Disassembly

Principles of regulation of actin network dimensions are fundamentally important for cell functions, yet remain unclear. Using both in vitro and in silico approaches, we studied the effect of key parameters, such as actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks reached equilibrium and became treadmilling. At the trailing edge, the network disintegrated into large fragments. A mathematical model predicts the network length as a function of width, actin and ADF/Cofilin concentrations. Local depletion of ADF/Cofilin by binding to actin is significant, leading to wider networks growing longer. A single rate of breaking network nodes, proportional to ADF/Cofilin density and inversely proportional to the square of the actin density, can account for the disassembly dynamics. Selective disassembly of heterogeneous networks by ADF/Cofilin controls steering during motility. Our results establish general principles on how the dynamic steady state of actin network emerges from biochemical and structural feedbacks.

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In situ Time-Resolved Small-Angle X-ray Scattering Reveals the Formation Kinetics of Polyelectrolyte Complex Micelles

10.26434/chemrxiv.9876395.v1 ◽

2019 ◽

Author(s):

Hao Wu ◽

Jeffrey Ting ◽

Siqi Meng ◽

Matthew Tirrell

Keyword(s):

Small Angle ◽

Self Assembly ◽

Electrostatic Interactions ◽

Polyelectrolyte Complex ◽

Time Resolved ◽

X Ray ◽

X Ray Scattering ◽

Kinetics Of ◽

Ray Scattering

We have directly observed the <i>in situ</i> self-assembly kinetics of polyelectrolyte complex (PEC) micelles by synchrotron time-resolved small-angle X-ray scattering, equipped with a stopped-flow device that provides millisecond temporal resolution. This work has elucidated one general kinetic pathway for the process of PEC micelle formation, which provides useful physical insights for increasing our fundamental understanding of complexation and self-assembly dynamics driven by electrostatic interactions that occur on ultrafast timescales.

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The assembly of microtubule protein in vitro. The kinetic role in microtubule elongation of oligomeric fragments containing microtubule-associated proteins

Biochemical Journal ◽

10.1042/bj2270439 ◽

1985 ◽

Vol 227 (2) ◽

pp. 439-455 ◽

Cited By ~ 15

Author(s):

P M Bayley ◽

F M M Butler ◽

D C Clark ◽

E J Manser ◽

S R Martin

Keyword(s):

Self Assembly ◽

Protein Concentration ◽

Wavelength Dependence ◽

Electrophoretic Analysis ◽

Slow Phase ◽

Condensation Polymerization ◽

Fast Phase ◽

Microtubule Protein ◽

Kinetics Of

The kinetics of assembly were studied for bovine and pig microtubule protein in vitro over a range of conditions of pH, temperature, nucleotide and protein concentration. The kinetics are in general biphasic with two major processes of similar amplitude but separated in rate by one order of magnitude. Rates and amplitudes are complex functions of solution conditions. The rates of the fast phase and the slow phase attain limiting values as a function of increasing protein concentration, and are more stringently limited at pH 6.5 than pH 6.95. Such behaviour indicates that mechanisms other than the condensation polymerization of tubulin dimer become rate-limiting at higher protein concentration. The constancy of the wavelength-dependence of light-scattering and ultrastructural criteria indicate that microtubules of normal morphology are formed in both phases of the assembly process. Electrophoretic analysis of assembling microtubule protein shows that MAP- (microtubule-associated-protein-)rich microtubules are formed during the fast phase. The rate of dissociation of oligomeric species on dilution of microtubule protein closely parallels the fast-phase rate in magnitude and temperature-dependence. We propose that the rate of this process constitutes an upper limit to the rate of the fast phase of assembly. The kinetics of redistribution of MAPs from MAP-rich microtubules may be a factor limiting the slow-phase rate. A working model is derived for the self-assembly of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles are assigned to MAP-containing fragments in both phases of microtubule elongation. Variations in kinetic behaviour with solution conditions are inferred to derive from the nature and properties of fragments formed from oligomeric species after the rapid temperature jump. The model accounts for the limiting rate behaviour and indicates experimental criteria to be applied in evaluating the relative contributions of alternative pathways.

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MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

10.1101/2020.09.21.306522 ◽

2020 ◽

Author(s):

Chiara Galloni ◽

Davide Carra ◽

Jasmine V. G. Abella ◽

Svend Kjær ◽

Pavithra Singaravelu ◽

...

Keyword(s):

Functional Properties ◽

Actin Filament ◽

Actin Assembly ◽

Actin Network ◽

Network Stability ◽

Actin Networks ◽

Cellular Processes ◽

Actin Turnover ◽

Filament Networks

AbstractThe Arp2/3 complex (Arp2, Arp3 and ARPC1-5) is essential to generate branched actin filament networks for many cellular processes. Human Arp3, ARPC1 and ARPC5 exist as two isoforms but the functional properties of Arp2/3 iso-complexes is largely unexplored. Here we show that Arp3B, but not Arp3 is subject to regulation by the methionine monooxygenase MICAL2, which is recruited to branched actin networks by coronin-1C. Although Arp3 and Arp3B iso-complexes promote actin assembly equally efficiently in vitro, they have different cellular properties. Arp3B turns over significantly faster than Arp3 within the network and upon its depletion actin turnover decreases. Substitution of Arp3B Met293 by Thr, the corresponding residue in Arp3 increases actin network stability, and conversely, replacing Arp3 Thr293 with Gln to mimic Met oxidation promotes network disassembly. Thus, MICAL2 regulates a subset of Arp2/3 complexes to control branched actin network disassembly.

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Temperature-dependent Self assembly of biofilaments during red blood cell sickling

10.1101/2021.03.07.433773 ◽

2021 ◽

Author(s):

Arabinda Behera ◽

Oshin Sharma ◽

Debjani Paul ◽

Anirban Sain

Keyword(s):

Self Assembly ◽

Kinetic Monte Carlo ◽

Lag Time ◽

Vital Role ◽

Temperature Dependent ◽

Opposite Case ◽

Assembly Kinetics ◽

The Mean ◽

Kinetics Of

Molecular self-assembly plays vital role in various biological functions. However, when aberrant molecules self-assemble to form large aggregates, it can give rise to various diseases. For example, the sickle cell disease and Alzheimer’s disease are caused by self-assembled hemoglobin fibers and amyloid plaques, respectively. Here we study the assembly kinetics of such fibers using kinetic Monte-Carlo simulation. We focus on the initial lag time of these highly stochastic processes, during which self-assembly is very slow. The lag time distributions turn out to be similar for two very different regimes of polymerization, namely, a) when polymerization is slow and depolymerization is fast, and b) the opposite case, when polymerization is fast and depolymerization is slow. Using temperature dependent on- and off-rates for hemoglobin fiber growth, reported in recent in-vitro experiments, we show that the mean lag time can exhibit non-monotonic behaviour with respect to change of temperature.

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Myosin 1b Flattens and Prunes Branched Actin Filaments

10.1101/2020.02.26.966663 ◽

2020 ◽

Author(s):

Julien Pernier ◽

Antoine Morchain ◽

Valentina Caorsi ◽

Aurélie Bertin ◽

Hugo Bousquet ◽

...

Keyword(s):

Total Internal Reflection ◽

Molecular Motor ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence ◽

Actin Network ◽

Tirf Microscopy ◽

Actin Networks ◽

Cellular Processes

AbstractMotile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Using in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show in this report that this molecular motor flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and in the dynamics of actin networks in different cellular regions.Short summaryUsing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show that myosin flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches

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Tropomyosin and α-actinin cooperation inhibits fimbrin association with actin filament networks in fission yeast

10.1101/169961 ◽

2017 ◽

Author(s):

Jenna R. Christensen ◽

Kaitlin E. Homa ◽

Meghan E. O’Connell ◽

David R. Kovar

Keyword(s):

Fission Yeast ◽

Fluorescent Imaging ◽

Actin Binding ◽

Yeast Genetics ◽

Actin Network ◽

Contractile Ring ◽

Actin Networks ◽

Filament Networks ◽

Actin Patch

ABSTRACTWe previously discovered that competition between fission yeast actin binding proteins (ABPs) for association with F-actin helps facilitate their sorting to different F-actin networks. Specifically, competition between actin patch ABPs fimbrin Fim1 and cofilin Adf1 enhances each other’s activities, and rapidly displaces tropomyosin Cdc8 from the F-actin network. However, these interactions don’t explain how Fim1, a robust competitor, is prevented from associating equally well with other F-actin networks. Here, with a combination of fission yeast genetics, live cell fluorescent imaging, and in vitro TIRF microscopy, we identified the contractile ring ABP α-actinin Ain1 as a key sorting factor. Fim1 competes with Ain1 for association with F-actin, which is dependent upon their residence time on F-actin. Remarkably, although Fim1 outcompetes both contractile ring ABPs Ain1 and Cdc8 individually, Cdc8 enhances the bundling activity of Ain1 10-fold, allowing the combination of Ain1 and Cdc8 to inhibit Fim1 association with contractile ring F-actin.

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Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude

Nature Communications ◽

10.1038/s41467-019-13268-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 15

Author(s):

Shashank Shekhar ◽

Johnson Chung ◽

Jane Kondev ◽

Jeff Gelles ◽

Bruce L. Goode

Keyword(s):

Actin Filament ◽

Single Molecule ◽

Actin Filaments ◽

Binding Protein ◽

Actin Dynamics ◽

Actin Network ◽

Cellular Mechanisms ◽

Actin Networks ◽

Binding Event

AbstractCellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

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A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks

Nature Communications ◽

10.1038/s41467-021-25682-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Johanna Funk ◽

Felipe Merino ◽

Matthias Schaks ◽

Klemens Rottner ◽

Stefan Raunser ◽

...

Keyword(s):

Structural Biology ◽

Actin Filaments ◽

Actin Network ◽

Essential Factor ◽

Actin Networks ◽

Capping Protein ◽

Cellular Processes ◽

In Vitro Reconstitution ◽

Interference Mechanism

AbstractHeterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP’s C-terminal “tentacle” extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes—capping and nucleation—in branched actin network assembly.

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Time course and kinetics of proximal tubular processing of insulin

AJP Renal Physiology ◽

10.1152/ajprenal.1992.262.5.f813 ◽

1992 ◽

Vol 262 (5) ◽

pp. F813-F822 ◽

Cited By ~ 1

Author(s):

S. Nielsen

Keyword(s):

Steady State ◽

Time Course ◽

Accumulation Rate ◽

Compartmental Analysis ◽

Proximal Tubules ◽

Hydrolysis Rate ◽

Time Courses ◽

High Concentrations ◽

Kinetics Of

The present study was undertaken to determine the time courses and kinetics of the subcellular processing of 125I-insulin in isolated and in vitro perfused proximal tubules. Morphometric analysis demonstrated well-preserved ultrastructure after 90 min of perfusion. After luminal perfusion for 90 min the absorption was constant with time and reached steady state within 5 min (177 +/- 7 fg.min-1.mm-1). Also the hydrolysis rate and tubular accumulation rate were constant and averaged 84 +/- 8 and 93 +/- 10 fg.min-1.mm-1, respectively. Free 125I appeared already within 5 min of perfusion and reached steady state within 10 min. From proximal tubules perfused with 125I-insulin for 30 min and chased for 60 min, a compartmental analysis revealed two compartments; half time (t1/2) for delivery of insulin to the lysosomes was determined to be 8.5 min, and t1/2 for lysosomal degradation was 72 min. The results demonstrated that internalization by endocytic invaginations, incorporation in endocytic vacuoles, fusion with lysosomes, and hydrolysis were rapid processes and reached maximum rates within few minutes. A significant transtubular transport of insulin to the peritubular compartment was determined to be a constant rate of 11.2 +/- 0.7 fg.min-1.mm-1. Perfusion of tubules with insulin at high concentrations in the perfusate revealed that the transport was dependent on the absorbed amount and not on the perfused load, compatible with transport through the cells and not via a paracellular mechanism. The intactness of the tight junctions was supported by the following: 1) [14C]inulin leak did not increase with time and 2) enzyme-free intercellular spaces were evident after perfusion for only 5 min with microperoxidase (mol wt of 1,700). The transported 125I-insulin was trichloroacetic acid precipitable and immunoprecipitable.

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