Live-Cell Super-resolution Reveals F-Actin and Plasma Membrane Dynamics at the T Cell Synapse

Ashdown, George W.; Burn, Garth L.; Williamson, David J.; Pandžić, Elvis; Peters, Ruby; Holden, Michael R.; Ewers, Helge; Shao, Lin; Wiseman, Paul W.; Owen, Dylan M.

doi:10.1016/j.bpj.2017.01.038

Cited by 55 publications

(51 citation statements)

References 51 publications

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“…By contrast, in α-actinin depleted cells, the average flow vector deviated from the symmetry axis ( Figure 4B, C). This observation was consistent with a recent observation of a bias in the actin flow at the immunological synapse in response to a-actinin depletion (Ashdown et al, 2017) The retrograde flow of contractile-actin bundles is involved in nucleus positioning (Gomes et al, 2005) with which they interact. Asymmetric actin flow could therefore affect the proper positioning of the nucleus.…”

Section: Alpha-actinin Knock-down Deviates Actin Flow and Nucleus Possupporting

confidence: 92%

Spatial integration of mechanical forces by alpha-actinin establishes actin network symmetry

Senger

Pitaval

Ennomani

et al. 2019

Preprint

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Cell and tissue morphogenesis depend on the production and spatial organization of tensional forces in the actin cytoskeleton. Actin network architecture is complex because it is made of distinct modules in which filaments adopt a variety of organizations. The assembly and dynamics of these modules is well described but the self-organisation rules directing the global network architecture are much less understood. Here we investigated the mechanism regulating the interplay between network architecture and the geometry of cell's extracellular environment. We found that α-actinin, a filament crosslinker, is essential for network symmetry to be consistent with extracellular microenvironment symmetry. It appeared to be required for the interconnection of transverse arcs with radial fibres to ensure an appropriate balance between forces at cell adhesions and across the entire actin network. Furthermore, the connectivity of the actin network appeared necessary for the cell ability to integrate and adapt to complex patterns of extracellular cues as they migrate. Altogether, our study has unveiled a role of actin-filament crosslinking in the physical integration of mechanical forces throughout the entire cell, and the role of this integration in the establishment and adaptation of intracellular symmetry axes in accordance with the geometry of extracellular cues.

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Section: Alpha-actinin Knock-down Deviates Actin Flow and Nucleus Possupporting

confidence: 92%

Spatial integration of mechanical forces by alpha-actinin establishes actin network symmetry

Senger

Pitaval

Ennomani

et al. 2019

Preprint

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“…The earliest evidence on actin-membrane interactions was reported several from several biochemical studies (Branton et al, 1981;Luna and Hitt, 1992;Hitt and Luna, 1994;Cowin and Burke, 1996). In general, actin interacts with the membrane mainly through indirect interactions, although direct interactions have also been reported (Sasaki et al, 2014;Ashdown and Burn, 2017). Indirect interactions occur through cytoskeletal elements linked to transmembrane proteins or actin binding proteins (ABPs) that interact with the inner leaflet of the plasma membrane or organelle membrane (Senju and Lappalainen, 2019;Lemiere et al, 2016;Fritzsche et al, 2017;Papadopulos, 2017).…”

Section: Interactions Of the Actin Cytoskeleton With Cell Membranesmentioning

confidence: 99%

Actin Cytoskeleton and Membrane Interactions: Role in GPCR Function and Organization

Prakash

Sengupta²,

Krishna³

2019

PINSA

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Cell signaling networks are generally initiated at the cell membrane and mediated by receptors such as the G proteincoupled receptors (GPCRs). Adjacent to the cell membrane lies a complex network of proteins-the actin cytoskeleton to which several structural and physiological roles have been attributed. An emerging role of the cytoskeleton is to modulate GPCR function and organization, either directly or indirectly. The GPCR-cytoskeleton cross-talk is a complex hierarchical process where each step has its own set of rules and combinations. Due to the inherent complexity involved at each step and the multiple spatio-temporal levels, a complete picture is yet to emerge. In this review article, we provide an overview of actin-membrane interactions and how they modulate GPCR function and organization. In this context, we briefly discuss the structural characterization of actin monomers and filaments together with their interactions with partner proteins, such as the actin binding proteins. The effect of actin interactions on membrane domains is examined that assumes significance in light of membrane modulated effects in GPCR function. We aim to bring together concepts in GPCR signaling and cytoskeletal dynamics towards addressing emerging concepts in GPCR function.

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“…As an alternative, PAINT of actin has been performed using phalloidin coupled to a DNA docking strand or actin-targeting affimers (Schlichthaerle et al, 2018a). It is also possible to use fluorescent LifeAct that transiently binds to actin in fixed samples to generate an SMLM image, a technique called Integrating exchangeable single-molecule localization (IRIS) (Ashdown et al, 2017;Kiuchi et al, 2015;Tas et al, 2018). In our hands, these alternatives are significantly slower than STORM with fluorescent phalloidin to generate a high-quality image.…”

Section: Actin Stainingmentioning

confidence: 99%

About samples, giving examples: Optimized Single Molecule Localization Microscopy

Jimenez

Friedl

Leterrier

2019

Preprint

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Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

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Live-Cell Super-resolution Reveals F-Actin and Plasma Membrane Dynamics at the T Cell Synapse

Cited by 55 publications

References 51 publications

Spatial integration of mechanical forces by alpha-actinin establishes actin network symmetry

Spatial integration of mechanical forces by alpha-actinin establishes actin network symmetry

Actin Cytoskeleton and Membrane Interactions: Role in GPCR Function and Organization

About samples, giving examples: Optimized Single Molecule Localization Microscopy

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