Clathrin coats partially preassemble and subsequently bend during endocytosis

Mund, Markus; Tschanz, Aline; Wu, Yule; Frey, Felix J.; Mehl, Johanna L.; Kaksonen, Marko; Avinoam, Ori; Schwarz, Ulrich S.; Ries, Jonas

doi:10.1083/jcb.202206038

Cited by 16 publications

(16 citation statements)

References 57 publications

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“…Although 3DSIM can interpret the 3D hollow structure of CCPs, sparse deconvolution can make the hollow structure clearer. This is consistent with the physiological structure of CCPs, 32 and similar results were reported with 3D single-molecule localization microscopy 33 . Then, we use MRA deconvolution 34 for the processing of tubulin structure of the low signal-to-noise ratio in Fig.…”

Section: Resultssupporting

confidence: 86%

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High-speed autopolarization synchronization modulation three-dimensional structured illumination microscopy

Li,

Cao,

Ren

et al. 2023

Adv. Photon. Nexus

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In recent years, notable progress has been achieved in both the hardware and algorithms of structured illumination microscopy (SIM). Nevertheless, the advancement of three-dimensional structured illumination microscopy (3DSIM) has been impeded by challenges arising from the speed and intricacy of polarization modulation. We introduce a high-speed modulation 3DSIM system, leveraging the polarizationmaintaining and modulation capabilities of a digital micromirror device (DMD) in conjunction with an electrooptic modulator. The DMD-3DSIM system yields a twofold enhancement in both lateral (133 nm) and axial (300 nm) resolution compared to wide-field imaging and can acquire a data set comprising 29 sections of 1024 pixels × 1024 pixels, with 15 ms exposure time and 6.75 s per volume. The versatility of the DMD-3DSIM approach was exemplified through the imaging of various specimens, including fluorescent beads, nuclear pores, microtubules, actin filaments, and mitochondria within cells, as well as plant and animal tissues. Notably, polarized 3DSIM elucidated the orientation of actin filaments. Furthermore, the implementation of diverse deconvolution algorithms further enhances 3D resolution. The DMD-based 3DSIM system presents a rapid and reliable methodology for investigating biomedical phenomena, boasting capabilities encompassing 3D superresolution, fast temporal resolution, and polarization imaging.

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Section: Resultssupporting

confidence: 86%

“…This is consistent with the physiological structure of CCPs, 32 and similar results were reported with 3D single-molecule localization microscopy. 33 Then, we use MRA deconvolution 34 for the processing of tubulin structure of the low signal-to-noise ratio in Fig. 6(d).…”

Section: Computational 3d Deconvolutionmentioning

confidence: 99%

High-speed autopolarization synchronization modulation three-dimensional structured illumination microscopy

Li,

Cao,

Ren

et al. 2023

Adv. Photon. Nexus

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“…Class I oligomers have a diameter of ~75 nm that is smaller than the 100-200 nm diameter of clathrin pits and vesicles [23,24]. The Class II pits and vesicles have a blob area of 56,581 nm 2 that corresponds closely to the previously reported 54,000 nm 2 median surface area of clathrin pits imaged by SMLM [25]. Consistently, most hollow blobs identified by SMLM in Figure 1 are classified as Class II blobs.…”

Section: Discussionsupporting

confidence: 81%

“…Fig. 1) [25,26]. Euclidean distance analysis for 27 features (excluding volume and area), showed that Class I and Class II are highly similar between HeLa and Cos7 cells (Supp.…”

Section: Superresnet Smlm Network Analysis Detects Three Classes Of C...mentioning

confidence: 94%

Super-resolution single molecule network analysis (SuperResNET) detects changes to clathrin structure by small molecule inhibitors

Wong,

Khater,

Hallgrimson

et al. 2024

Preprint

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Specificity of small molecules for their target molecule in the cell is critical to determine their effective use as biologics and therapeutics. Small molecule inhibitors of clathrin endocytosis, Pitstop 2, and the dynamin inhibitor Dynasore, have off-target effects and their specificity has been challenged. Here, we used SuperResNET to apply network analysis to 20 nm resolution dSTORM single-molecule localization microscopy (SMLM) to test whether Pitstop 2 and Dynasore alter the morphology of clathrin coated pits in intact cells. SuperResNET analysis of dSTORM data from HeLa and Cos7 cells identifies three classes of clathrin structures: small oligomers (Class I); pits and vesicles (Class II); and larger clusters corresponding to fused clathrin pits and clathrin plaques (Class III). SuperResNET analysis of high resolution MinFlux imaging identifies Class 1 oligomers as well as Class 2 structures including morphologically identifiable clathrin pits and vesicles. SuperResNET feature analysis of dSTORM data shows that Pitstop 2 and Dynasore induce the formation of distinct homogenous populations of clathrin structures in HeLa cells. Pitstop 2 blobs are smaller and more elongated than those induced by Dynasore, indicating that these two clathrin inhibitors arrest clathrin endocytosis at distinct stages. Pitstop 2 and Dynasore are not impacting clathrin structure via actin depolymerization as the actin depolymerizing agent latrunculin A (LatA) induced larger heterogeneous clathrin structures. Ternary analysis of SuperResNET shape features presents a distinct profile for Pitstop 2 Class II structures. The most representative Pitstop blobs align with and resemble MinFlux clathrin pits while control structures resemble Minflux clathrin vesicles. SuperResNET analysis of SMLM data is therefore a highly sensitive approach to detect the effect of small molecules on target molecule structure in situ in the cell.

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“…By significantly extending the imaging throughput of fluorescence nanoscopy, PRIME-PAINT and the other recent, large-FOV modalities − bring exciting new opportunities. Similar to cryo-EM single-particle analysis, which revolutionized structural biology, recent work has used nanoscopy data to infer dynamic protein complexes. , Large-scale nanoscopic image data sets will synergize with machine-learning-based image analysis tools such as WEKA and LocMoFit to significantly accelerate biomedical discovery. , Additionally, the ability to image FFPE sections at nanometer resolution across mm 2 FOVs (Figure and Figure S14) is an important step toward clinical applications, for which PRIME-PAINT and the likes represent a leap forward compared with prior attempts using electron microscopy (EM) − or fluorescence nanoscopy with a small FOV. , …”

Section: Conclusion and Discussionmentioning

confidence: 99%

Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and Microfluidics-Enhanced DNA-PAINT

Rames,

Kenison,

Heineck

et al. 2023

Chemical & Biomedical Imaging

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Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-ofview (FOV) of around 50 μm × 50 μm at once and more recently up to ∼200 μm × 200 μm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints to the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust singlemolecule localization with up to ∼520 μm × 520 μm single FOVs and 25−40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm 2 sample areas can be completed in as little as 40 min per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality, particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ∼10 6 caveolae structures in ∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.

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Clathrin coats partially preassemble and subsequently bend during endocytosis

Cited by 16 publications

References 57 publications

High-speed autopolarization synchronization modulation three-dimensional structured illumination microscopy

High-speed autopolarization synchronization modulation three-dimensional structured illumination microscopy

Super-resolution single molecule network analysis (SuperResNET) detects changes to clathrin structure by small molecule inhibitors

Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and Microfluidics-Enhanced DNA-PAINT

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