Synergistic phase separation of two pathways promotes integrin clustering and nascent adhesion formation

Case, Lindsay B.; Pasquale, Milagros De; Henry, Lisa; Rosen, Michael K.

doi:10.7554/elife.72588

Cited by 60 publications

(61 citation statements)

References 112 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Interactions between the adaptor proteins and the receptor can promote phase separation to form micron-sized condensates on the membrane that can be visualized with total internal reflection fluorescence (TIRF) microscopy. In vitro reconstitution has been used to study the role of phase separation in many plasma membrane-associated condensates including nephrin clusters ( Banjade and Rosen, 2014 ; Case et al, 2019b ), LAT clusters ( Su et al, 2016 ; Huang et al, 2019 ), integrin clusters ( Case et al, 2022 ), post synaptic density condensates ( Zeng et al, 2018 ), and growth factor receptor clusters ( Lin et al, 2022 ). Reconstitution has also been used to study intracellular membrane-associated condensates ( Banjade et al, 2022 ; Snead et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Reconstitution of Phase-Separated Signaling Clusters and Actin Polymerization on Supported Lipid Bilayers

Cheng

Ullo

Case

2022

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

Liquid–liquid phase separation driven by weak interactions between multivalent molecules contributes to the cellular organization by promoting the formation of biomolecular condensates. At membranes, phase separation can promote the assembly of transmembrane proteins with their cytoplasmic binding partners into micron-sized membrane-associated condensates. For example, phase separation promotes clustering of nephrin, a transmembrane adhesion molecule, resulting in increased Arp2/3 complex-dependent actin polymerization. In vitro reconstitution is a powerful approach to understand phase separation in biological systems. With a bottom-up approach, we can determine the molecules necessary and sufficient for phase separation, map the phase diagram by quantifying de-mixing over a range of molecular concentrations, assess the material properties of the condensed phase using fluorescence recovery after photobleaching (FRAP), and even determine how phase separation impacts downstream biochemical activity. Here, we describe a detailed protocol to reconstitute nephrin clusters on supported lipid bilayers with purified recombinant protein. We also describe how to measure Arp2/3 complex-dependent actin polymerization on bilayers using fluorescence microscopy. These different protocols can be performed independently or combined as needed. These general techniques can be applied to reconstitute and study phase-separated signaling clusters of many different receptors or to generally understand how actin polymerization is regulated at membranes.

show abstract

Section: Introductionmentioning

confidence: 99%

Reconstitution of Phase-Separated Signaling Clusters and Actin Polymerization on Supported Lipid Bilayers

Cheng

Ullo

Case

2022

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

show abstract

“…FAK acts as the molecular hub connecting integrin and downstream signaling molecules of the integrin signaling pathway [ 39 ]. Cytoskeletal proteins such as integrin, FAK and talin are co-localized and assembled together to form focal adhesion [ 40 ]. Paxillin is a cytoskeletal protein involved in actin-membrane attachment at the extracellular matrix site, which plays an important role in the integrin signaling transduction [ 41 ].…”

Section: Resultsmentioning

confidence: 99%

Electrical charge on ferroelectric nanocomposite membranes enhances SHED neural differentiation

Heng

Bai

et al. 2023

Bioactive Materials

View full text Add to dashboard Cite

“…VASP associates with aligned actin filaments in multiple cellular structures including filopodia and focal adhesions 46,47 . Interestingly, multiple proteins involved in assembly of focal adhesions have recently been found to undergo liquid-liquid phase separation [48][49][50] . However, the role of this network in aligning actin filaments is presently unknown.…”

Section: Discussionmentioning

confidence: 99%

Liquid-like VASP condensates drive actin polymerization and dynamic bundling

Graham

Chandrasekaran

Wang

et al. 2022

Preprint

View full text Add to dashboard Cite

The organization of actin filaments into bundles is required for cellular processes such as motility, morphogenesis, and cell division. Filament bundling is controlled by a network of actin binding proteins. Recently, several proteins that comprise this network have been found to undergo liquid-liquid phase separation. How might liquid-like condensates contribute to filament bundling? Here we show that the processive actin polymerase, VASP, forms liquid-like droplets under physiological conditions. As actin polymerizes within VASP droplets, elongating filaments partition to the edges of the droplet to minimize filament curvature, forming an actin-rich ring within the droplet. The rigidity of this ring is balanced by the droplet’s surface tension, as predicted by a continuum-scale computational model. However, as the ring grows thicker, its rigidity increases and eventually overcomes the surface tension of the droplet, deforming into a linear bundle. The resulting bundles contain parallel actin filaments that grow from their tips. Growing bundles zipper together upon contact with one another, an effect which is mediated by the surface tension of the liquid-like VASP droplets that encapsulate them. Once the parallel arrangement of filaments is created within a VASP droplet, it propagates through the addition of new actin monomers to achieve a length that is many times greater than the initial droplet. This droplet-based mechanism of bundling may be relevant to the assembly of cellular architectures rich in parallel actin filaments, such as filopodia, stress fibers, and focal adhesions.

show abstract

Synergistic phase separation of two pathways promotes integrin clustering and nascent adhesion formation

Cited by 60 publications

References 112 publications

Reconstitution of Phase-Separated Signaling Clusters and Actin Polymerization on Supported Lipid Bilayers

Reconstitution of Phase-Separated Signaling Clusters and Actin Polymerization on Supported Lipid Bilayers

Electrical charge on ferroelectric nanocomposite membranes enhances SHED neural differentiation

Liquid-like VASP condensates drive actin polymerization and dynamic bundling

Contact Info

Product

Resources

About