Molecular height measurement by cell surface optical profilometry (CSOP)

Son, Sungmin; Takatori, Sho C.; Belardi, Brian; Podolski, Marija; Bakalar, Matthew; Fletcher, Daniel A.

doi:10.1101/2019.12.31.892075

Cited by 3 publications

(1 citation statement)

References 69 publications

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“…In addition to measurements of thickness and proximity to the plasma membrane, STORM imaging has been used in a similar approach to measure the distribution of actin regulatory proteins within the actin cortex. Measuring differences in the intensity profile of fluorophores targeted to different proteins or the plasma membrane presents a valuable technique for precisely measuring distances, that has been applied not only to the actin cytoskeleton but also for characterizing membrane protein height (Son et al, 2020).…”

Section: Cortical Actinmentioning

confidence: 99%

Quantifying cytoskeletal organization from optical microscopy data

Desroches,

Harris

2024

Front. Cell Dev. Biol.

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The actin cytoskeleton plays a pivotal role in a broad range of physiological processes including directing cell shape and subcellular organization, determining cell mechanical properties, and sensing and transducing mechanical forces. The versatility of the actin cytoskeleton arises from the ability of actin filaments to assemble into higher order structures through their interaction with a vast set of regulatory proteins. Actin filaments assemble into bundles, meshes, and networks, where different combinations of these structures fulfill specific functional roles. Analyzing the organization and abundance of different actin structures from optical microscopy data provides a valuable metric for assessing cell physiological function and changes associated with disease. However, quantitative measurements of the size, abundance, orientation, and distribution of different types of actin structure remains challenging both from an experimental and image analysis perspective. In this review, we summarize image analysis methods for extracting quantitative values that can be used for characterizing the organization of actin structures and provide selected examples. We summarize the potential sample types and metric reported with different approaches as a guide for selecting an image analysis strategy.

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Section: Cortical Actinmentioning

confidence: 99%

Quantifying cytoskeletal organization from optical microscopy data

Desroches,

Harris

2024

Front. Cell Dev. Biol.

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Membrane fusion studied by colloidal probes

et al. 2021

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Membrane-coated colloidal probes combine the benefits of solid-supported membranes with a more complex three-dimensional geometry. This combination makes them a powerful model system that enables the visualization of dynamic biological processes with high throughput and minimal reliance on fluorescent labels. Here, we want to review recent applications of colloidal probes for the study of membrane fusion. After discussing the advantages and disadvantages of some classical vesicle-based fusion assays, we introduce an assay using optical detection of fusion between membrane-coated glass microspheres in a quasi two-dimensional assembly. Then, we discuss free energy considerations of membrane fusion between supported bilayers, and show how colloidal probes can be combined with atomic force microscopy or optical tweezers to access the fusion process with even greater detail.

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Molecular height measurement by cell surface optical profilometry (CSOP)

Son

Takatori

Belardi

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example, by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and the role of the molecular landscape in cell function.

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Molecular height measurement by cell surface optical profilometry (CSOP)

Cited by 3 publications

References 69 publications

Quantifying cytoskeletal organization from optical microscopy data

Quantifying cytoskeletal organization from optical microscopy data

Membrane fusion studied by colloidal probes

Molecular height measurement by cell surface optical profilometry (CSOP)

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