Gelsolin-mediated actin filament severing in crowded environments

Heidings, James B.; Demosthene, Bryan; Merlino, Theresa R; Castaneda, Nicholas; Kang, Hyeran

doi:10.1016/j.bbrc.2020.08.041

Cited by 15 publications

(14 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Pyrene actin (>99% purity) was purchased from Cytoskeleton Inc. (Denver, CO, USA) and mixed with unlabeled actin monomers to make 20% labeled pyrene actin. Ca 2+ -bound pyrene labeled G-actin was exchanged to Mg 2+ as previously described in [ 68 ]. Graphene flakes (dissolved in ddH 2 O, dilution factor ×100) were then added to Mg 2+ -G-actin.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Graphene Enhances Actin Filament Assembly Kinetics and Modulates NIH-3T3 Fibroblast Cell Spreading

Park

Kravchuk

Krishnaprasad

et al. 2022

IJMS

Self Cite

View full text Add to dashboard Cite

Actin plays critical roles in various cellular functions, including cell morphogenesis, differentiation, and movement. The assembly of actin monomers into double-helical filaments is regulated in surrounding microenvironments. Graphene is an attractive nanomaterial that has been used in various biomaterial applications, such as drug delivery cargo and scaffold for cells, due to its unique physical and chemical properties. Although several studies have shown the potential effects of graphene on actin at the cellular level, the direct influence of graphene on actin filament dynamics has not been studied. Here, we investigate the effects of graphene on actin assembly kinetics using spectroscopy and total internal reflection fluorescence microscopy. We demonstrate that graphene enhances the rates of actin filament growth in a concentration-dependent manner. Furthermore, cell morphology and spreading are modulated in mouse embryo fibroblast NIH-3T3 cultured on a graphene surface without significantly affecting cell viability. Taken together, these results suggest that graphene may have a direct impact on actin cytoskeleton remodeling.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Functionalized coverslips were prepared using a modified protocol based on Winterhoff et al [ 68 , 69 ]. Briefly, coverslips were sonicated at 60 °C for 45 min in 1 M KOH, 1 M HCl, and 70% ethanol.…”

Section: Methodsmentioning

confidence: 99%

Graphene Enhances Actin Filament Assembly Kinetics and Modulates NIH-3T3 Fibroblast Cell Spreading

Park

Kravchuk

Krishnaprasad

et al. 2022

IJMS

Self Cite

View full text Add to dashboard Cite

show abstract

“…In a living cell, actin bundles induced by ABPs are formed in a crowded cytoplasm; therefore, it is important to understand how crowding modulates ABP-induced bundling. Changes in filament bending stiffness and conformations in crowded environments [ 39 ] can influence interactions between filaments and ABPs, including actin-crosslinking proteins (e.g., fascin and α-actinin) [ 9 ] and severing proteins (e.g., gelsolin) [ 65 ]. Crowders with different sizes and concentrations [PEG and methylcellulose (MC)] have been shown to affect the organization patterns and potentially nucleation/growth of microtubule bundles crosslinked with microtubule-associated protein (MAP65) [ 66 ].…”

Section: The Influence Of Crowding and Cation Interactions On The Organization And Mechanics Of Actin Bundles Crosslinked By Actin-bindinmentioning

confidence: 99%

Regulation of Actin Bundle Mechanics and Structure by Intracellular Environmental Factors

2021

Self Cite

View full text Add to dashboard Cite

The mechanical and structural properties of actin cytoskeleton drive various cellular processes, including structural support of the plasma membrane and cellular motility. Actin monomers assemble into double-stranded helical filaments as well as higher-ordered structures such as bundles and networks. Cells incorporate macromolecular crowding, cation interactions, and actin-crosslinking proteins to regulate the organization of actin bundles. Although the roles of each of these factors in actin bundling have been well-known individually, how combined factors contribute to actin bundle assembly, organization, and mechanics is not fully understood. Here, we describe recent studies that have investigated the mechanisms of how intracellular environmental factors influence actin bundling. This review highlights the effects of macromolecular crowding, cation interactions, and actin-crosslinking proteins on actin bundle organization, structure, and mechanics. Understanding these mechanisms is important in determining in vivo actin biophysics and providing insights into cell physiology.

show abstract

“…Purification of actin monomers (G-actin) from rabbit skeletal muscle acetone powder (PelFreeze Biologicals Inc., Rogers, AR, USA) was performed through gel filtering G-actin over Sephacryl S300 size exclusion column equilibrated in buffer A (0.2 mM CaCl 2 , 1 mM NaN 3 , 2 mM Tris-HCl pH 8.0, 0.2 mM ATP, and 0.5 mM DTT) as previously described (Kang et al, 2012;Castaneda et al, 2018;Castaneda et al, 2019;Heidings et al, 2020). G-actin bound with Ca 2+ was subjected to cation exchange by ethylene glycol-bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) to Mg 2+ with the addition of 0.2 mM EGTA and MgCl 2 concentration equal to the initial G-actin concentration plus 10 μM.…”

Section: Sample Preparationmentioning

confidence: 99%

“…G-actin bound with Ca 2+ was subjected to cation exchange by ethylene glycol-bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) to Mg 2+ with the addition of 0.2 mM EGTA and MgCl 2 concentration equal to the initial G-actin concentration plus 10 μM. Following the cation exchange, polymerization of G-to actin filaments (F-actin) was performed (Kang et al, 2012;Castaneda et al, 2018;Castaneda et al, 2019;Heidings et al, 2020).…”

Section: Sample Preparationmentioning

confidence: 99%

Actin Bundle Nanomechanics and Organization Are Modulated by Macromolecular Crowding and Electrostatic Interactions

Castaneda

Feuillie

Molinari

et al. 2021

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

The structural and mechanical properties of actin bundles are essential to eukaryotic cells, aiding in cell motility and mechanical support of the plasma membrane. Bundle formation occurs in crowded intracellular environments composed of various ions and macromolecules. Although the roles of cations and macromolecular crowding in the mechanics and organization of actin bundles have been independently established, how changing both intracellular environmental conditions influence bundle mechanics at the nanoscale has yet to be established. Here we investigate how electrostatics and depletion interactions modulate the relative Young’s modulus and height of actin bundles using atomic force microscopy. Our results demonstrate that cation- and depletion-induced bundles display an overall reduction of relative Young’s modulus depending on either cation or crowding concentrations. Furthermore, we directly measure changes to cation- and depletion-induced bundle height, indicating that bundles experience alterations to filament packing supporting the reduction to relative Young’s modulus. Taken together, our work suggests that electrostatic and depletion interactions may act counteractively, impacting actin bundle nanomechanics and organization.

show abstract

Gelsolin-mediated actin filament severing in crowded environments

Cited by 15 publications

References 33 publications

Graphene Enhances Actin Filament Assembly Kinetics and Modulates NIH-3T3 Fibroblast Cell Spreading

Graphene Enhances Actin Filament Assembly Kinetics and Modulates NIH-3T3 Fibroblast Cell Spreading

Regulation of Actin Bundle Mechanics and Structure by Intracellular Environmental Factors

Actin Bundle Nanomechanics and Organization Are Modulated by Macromolecular Crowding and Electrostatic Interactions

Contact Info

Product

Resources

About