Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane

Stone, Matthew B.; Shelby, Sarah A.; Veatch, Sarah L.

doi:10.1021/acs.chemrev.6b00716

Cited by 150 publications

(137 citation statements)

References 251 publications

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“…The cascades for signal transduction usually begin at the cell surface, and for this reason the plasma membrane can be considered as the main hub for cellular signalling 1 . However, drawing conclusions about membrane behaviour and architecture proves challenging, not least because poorly understood or still unknown processes influence its dynamics 2,3 . Our current knowledge shows that plasma membrane is a vastly complex and intricate system 4 .…”

Section: Introductionmentioning

confidence: 99%

Impact of nanoscale hindrances on the relationship between lipid packing and diffusion in model membranes

Beckers

Urbančič

Sezgin

2020

Preprint

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Membrane models have allowed for precise study of the plasma membrane's biophysical properties, helping to unravel both structural and dynamic motifs within cell biology. Free standing and supported bilayer systems are popular models to reconstitute the membrane related processes. Although it is well-known that each have their advantages and limitations, comprehensive comparison of their biophysical properties is still lacking. Here, we compare the diffusion and lipid packing in giant unilamellar vesicles, planar and spherical supported membranes and cell-derived giant plasma membrane vesicles. We apply florescence correlation spectroscopy, spectral imaging and super-resolution STED-FCS to study the diffusivity, lipid packing and nanoscale architecture of these membrane systems, respectively.Our data show that lipid packing and diffusivity is tightly correlated in free-standing bilayers.However, nanoscale interactions in the supported bilayers cause deviation from this correlation.This data is essential to develop accurate theoretical models of the plasma membrane and will serve as a guideline for suitable model selection in future studies to reconstitute biological processes.

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Section: Introductionmentioning

confidence: 99%

Impact of nanoscale hindrances on the relationship between lipid packing and diffusion in model membranes

Beckers

Urbančič

Sezgin

2020

Preprint

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“…We believe that our approach, coupled with advances in localization of various membrane proteins [68], can bring us closer to the goal of in silico biology with realistic geometries. To accelerate this goal of routine cell modeling, experimentalists can contribute by sharing segmented datasets from their work along with biological questions of interest.…”

Section: Discussionmentioning

confidence: 99%

GAMer 2: A System for 3D Mesh Processing of Cellular Electron Micrographs

Lee

Laughlin

Beaumelle

et al. 2019

Preprint

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Recent advances in electron microscopy have, for the first time, enabled imaging of single cells in 3D at a nanometer length scale resolution. An uncharted frontier for in silico biology is the ability to simulate cellular processes using these observed geometries. Enabling such simulations will require a system for going from electron micrographs to 3D volume meshes, which can then form the basis of computer simulations of such processes using numerical techniques such as the Finite Element Method (FEM). In this paper, we develop an end-to-end pipeline for this task by adapting and extending computer graphics mesh processing and smoothing algorithms. Our workflow makes use of our recently rewritten mesh processing software, GAMer 2, which implements several mesh conditioning algorithms and serves as a platform to connect different pipeline steps. We apply this pipeline to a series of electron micrographs of neuronal dendrite morphology explored at three different length scales and demonstrate that the resultant meshes are suitable for finite element simulations. Our pipeline, which consists of free and open-source community driven tools, is a step towards routine physical simulations of biological processes in realistic geometries. We posit that a new frontier at the intersection of computational technologies and single cell biology is now open. Innovations in algorithms to reconstruct and simulate cellular length scale phenomena based on emerging structural data will enable realistic physical models and advance discovery. Author summary3D imaging of cellular components and associated reconstruction methods have made great strides in the past decade, opening windows into the complex intraceullar organization. These advances also mean that computational tools need to be developed to work with these images not just for purposes of visualization but also for biophysical simulations. Here, we describe a pipeline that takes images from electron microscopy as input and produces smooth surface and volume meshes as output. These meshes are suitable for building high-quality finite element simulations of cellular processes modeled by ordinary and partial differential equations, bringing us closer to realizing the goal of generating high-resolution simulations of such phenomena in realistic geometries. We demonstrate the utility of this pipeline by meshing 3D reconstructions of dendritic spines, calculating 1 the curvatures of the different component membranes, and conducting finite-element simulations of reaction-diffusion equations using the generated meshes. The software tools employed in our pipeline are community driven, open source, and free. We believe that technologies such as those presented will enable a new frontier in biophysical simulations in realistic geometries.

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“…These tight puncta are most likely a consequence of observing the same mEos3.2 fluorophore multiple times over the course of imaging. This over-counting effect is common to super-resolution localization measurements and does not indicate that peptides are themselves organized into clusters (Veatch et al, 2012;Stone et al, 2017b). Cells were also imaged using interference contrast microscopy (ICM; Fig 2B).…”

Section: Markers Of Ordered and Disordered Phases Sort With Respect Tmentioning

confidence: 95%

Membrane domains and phosphatase exclusion produce robust signaling responses in B cells engaged with natural ligands and artificial cross-linkers presented on lipid bilayers

Nunez

Wisser

Veatch

2019

Preprint

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B cells respond to a wide variety of antigens with varying valiancy and mode of presentation to the highly expressed B cell receptor (BCR). We previously demonstrated that clustering the IgM isotype of BCR with an artificial soluble cross-linker led to the stabilization of an ordered phase-like domain. This domain sorted minimal peptides and full-length proteins to generate local hot-spots for tyrosine phosphorylation at receptor clusters, facilitating receptor activation. BCR can also be activated through interactions with natural ligands or artificial cross-linkers presented at bilayer surfaces, where it is proposed that alternate mechanisms play important roles in receptor clustering and activation, including one mechanism involving the exclusion phosphatases due to their bulky extracellular domains.The goals of the current study are to determine if markers of membrane phases are sorted by BCR clusters formed through engagement with bilayer-presented natural ligands or cross-linkers, and to estimate the contribution that membrane phase partitioning plays in organizing regulatory proteins with respect to BCR clusters under these stimulation conditions. We use super-resolution fluorescence localization microscopy to find that BCR engagement with either a bilayer-presented natural ligand or artificial cross-linker generates ordered phase-like domains that are more robust than those observed with soluble cross-linkers. In addition, we provide evidence that interactions between regulatory proteins and BCR are partially determined through their preference for ordered membrane domains and present a minimal model of receptor activation that incorporates both ordered domains and steric exclusion mechanisms to produce a more sensitive response. Overall, this work highlights that cells are capable of integrating multiple interaction modalities to give rise to cellular functions, likely conferring flexibility and robustness to cellular responses.

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Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane

Cited by 150 publications

References 251 publications

Impact of nanoscale hindrances on the relationship between lipid packing and diffusion in model membranes

Impact of nanoscale hindrances on the relationship between lipid packing and diffusion in model membranes

GAMer 2: A System for 3D Mesh Processing of Cellular Electron Micrographs

Membrane domains and phosphatase exclusion produce robust signaling responses in B cells engaged with natural ligands and artificial cross-linkers presented on lipid bilayers

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