A mathematical model of actin filament turnover for fitting FRAP data

Halavatyi, Aliaksandr; Nazarov, Petr V.; Tanoury, Ziad Al; Apanasovich, V. V.; Yatskou, Mikalai M.; Friederich, Evelyne

doi:10.1007/s00249-009-0558-2

Cited by 13 publications

(7 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, using this method to compare two curves does not directly lead to a p -value but the significance is estimated by other means. More elaborate means of analysis are also found where fitting is performed to a stochastic model of actin treadmilling (Halavatyi et al, 2010). These models work well in in silico simulations but fitting measured data to six different parameters without fixed values is not feasible.…”

Section: Discussionmentioning

confidence: 99%

Measuring F-actin properties in dendritic spines

Koskinen

Hotulainen

2014

Front. Neuroanat.

View full text Add to dashboard Cite

During the last decade, numerous studies have demonstrated that the actin cytoskeleton plays a pivotal role in the control of dendritic spine shape. Synaptic stimulation rapidly changes the actin dynamics and many actin regulators have been shown to play roles in neuron functionality. Accordingly, defects in the regulation of the actin cytoskeleton in neurons have been implicated in memory disorders. Due to the small size of spines, it is difficult to detect changes in the actin structures in dendritic spines by conventional light microscopy imaging. Instead, to know how tightly actin filaments are bundled together, and how fast the filaments turnover, we need to use advanced microscopy techniques, such as fluorescence recovery after photobleaching (FRAP), photoactivatable green fluorescent protein (PAGFP) fluorescence decay and fluorescence anisotropy. Fluorescence anisotropy, which measures the Förster resonance energy transfer (FRET) between two GFP fluorophores, has been proposed as a method to measure the level of actin polymerization. Here, we propose a novel idea that fluorescence anisotropy could be more suitable to study the level of actin filament bundling instead of actin polymerization. We validate the method in U2OS cell line where the actin structures can be clearly distinguished and apply to analyze how actin filament organization in dendritic spines changes during neuronal maturation. In addition to fluorescence anisotropy validation, we take a critical look at the properties and limitations of FRAP and PAGFP fluorescence decay methods and offer our proposals for the analysis methods for these approaches. These three methods complement each other, each providing additional information about actin dynamics and organization in dendritic spines.

show abstract

Section: Discussionmentioning

confidence: 99%

Measuring F-actin properties in dendritic spines

Koskinen

Hotulainen

2014

Front. Neuroanat.

View full text Add to dashboard Cite

show abstract

“…This recovery can occur through the diffusion of monomeric EGFP-Actin or through nucleation of EGFP-Actin at the junctions. As diffusive recovery occurs within 50 ms (Fritzsche et al, 2013), we assumed that monomeric EGFP-Actin distribution was homogeneous (Halavatyi et al, 2010) and that the recovery of the mobile EGFP-Actin fraction was a result of F-actin polymerization. Thus, our FRAP study revealed a mobile pool of dynamic F-actin with high turnover at EC junctions.…”

Section: Formin Activity Maintains a Pool Of Dynamic F-actin At Endotmentioning

confidence: 99%

Formin-Mediated Actin Polymerization at Endothelial Junctions Is Required for Vessel Lumen Formation and Stabilization

et al. 2015

View full text Add to dashboard Cite

During blood vessel formation, endothelial cells (ECs) establish cell-cell junctions and rearrange to form multicellular tubes. Here, we show that during lumen formation, the actin nucleator and elongation factor, formin-like 3 (fmnl3), localizes to EC junctions, where filamentous actin (F-actin) cables assemble. Fluorescent actin reporters and fluorescence recovery after photobleaching experiments in zebrafish embryos identified a pool of dynamic F-actin with high turnover at EC junctions in vessels. Knockdown of fmnl3 expression, chemical inhibition of formin function, and expression of dominant-negative fmnl3 revealed that formin activity maintains a stable F-actin content at EC junctions by continual polymerization of F-actin cables. Reduced actin polymerization leads to destabilized endothelial junctions and consequently to failure in blood vessel lumenization and lumen instability. Our findings highlight the importance of formin activity in blood vessel morphogenesis.

show abstract

“…For example, one method tailored specifically to actin called sequential fluorescence decay after photoactivation (sFDAP) uses sequential photoactivation in a single region of the cell in order to obtain information about the local G-actin concentration and how it changes with respect to extracellular stimuli (Higashida et al, 2013; Kiuchi et al, 2011). However, one problem with deriving accurate information from photoactivation or photobleaching experiments is that the analysis often fails to account for all of the complex real-world details of an experiment (Carrero et al, 2003; Halavatyi et al, 2010; Sprague et al, 2004; Tardy et al, 1995). These include, but are not limited to: non-isotropic diffusion due to specific cell morphology, a substantial loss of information due to the delay between photoactivation and imaging recording, and the fact that photoactivation is non-instantaneous.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous quantification of actin monomer and filament dynamics with modeling-assisted analysis of photoactivation

Kapustina

Read

Vitriol

2016

Journal of Cell Science

View full text Add to dashboard Cite

Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments.

show abstract

A mathematical model of actin filament turnover for fitting FRAP data

Cited by 13 publications

References 30 publications

Measuring F-actin properties in dendritic spines

Measuring F-actin properties in dendritic spines

Formin-Mediated Actin Polymerization at Endothelial Junctions Is Required for Vessel Lumen Formation and Stabilization

Simultaneous quantification of actin monomer and filament dynamics with modeling-assisted analysis of photoactivation

Contact Info

Product

Resources

About