Correlative multicolor 3D SIM and STORM microscopy

Hamel, Virginie; Guichard, Paul; Fournier, Mathias; Guiet, Romain; Flückiger, Isabelle; Seitz, Arne; Gönczy, Pierre

doi:10.1364/boe.5.003326

Cited by 39 publications

(28 citation statements)

References 17 publications

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“…S2A). SIM axial resolution is ∼150 nm, which is twice that of confocal microscopy and enables a more detailed view of the primary cilium, which is less than 400 nm in diameter (30). Here, PKD2-GFP trafficked to the PKD2 Null cell primary cilia, as assessed by GFP colocalization with antibodies raised against epitopes ADP ribosylation factor-like GTPase 13B (ARL13B), adenylate cyclase 3 (AC3), and acetylated tubulin (AT) (SI Appendix, Table S2).…”

Section: Resultsmentioning

confidence: 99%

Molecular dysregulation of ciliary polycystin-2 channels caused by variants in the TOP domain

Vien

Wang

et al. 2020

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

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Genetic variants in PKD2 which encodes for the polycystin-2 ion channel are responsible for many clinical cases of autosomal dominant polycystic kidney disease (ADPKD). Despite our strong understanding of the genetic basis of ADPKD, we do not know how most variants impact channel function. Polycystin-2 is found in organelle membranes, including the primary cilium—an antennae-like structure on the luminal side of the collecting duct. In this study, we focus on the structural and mechanistic regulation of polycystin-2 by its TOP domain—a site with unknown function that is commonly altered by missense variants. We use direct cilia electrophysiology, cryogenic electron microscopy, and superresolution imaging to determine that variants of the TOP domain finger 1 motif destabilizes the channel structure and impairs channel opening without altering cilia localization and channel assembly. Our findings support the channelopathy classification of PKD2 variants associated with ADPKD, where polycystin-2 channel dysregulation in the primary cilia may contribute to cystogenesis.

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Section: Resultsmentioning

confidence: 99%

Molecular dysregulation of ciliary polycystin-2 channels caused by variants in the TOP domain

Vien

Wang

et al. 2020

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

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“…Primary antibodies were 1:300 rabbit CrSAS-6 (ref. 30), 1:200 rabbit Bld10p16 (gift from Masafumi Hirono) and 1:1,000 mouse glutamylated tubulin (GT335, Adipogen) for Fig. 2d–f.…”

Section: Methodsmentioning

confidence: 99%

“…Secondary antibodies were 1:1,000 goat anti-rabbit coupled to Alexa 568 and 1:1,000 goat anti-mouse coupled to Alexa 488. Imaging was carried out on a 3D NSIM Nikon microscope as described30.…”

Section: Methodsmentioning

confidence: 99%

Cell-free reconstitution reveals centriole cartwheel assembly mechanisms

et al. 2017

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How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS-6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.

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“…Most recently, Hamel et al . used 3D-SIM to obtain multi-color SIM data and compare them with the higher resolution obtained by single molecule localization microscopy 19 .…”

mentioning

confidence: 99%

Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations

Mönkemöller

Øie

Hübner

et al. 2015

Sci Rep

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Liver sinusoidal endothelial cells (LSECs) act as a filter between blood and the hepatocytes. LSECs are highly fenestrated cells; they contain transcellular pores with diameters between 50 to 200 nm. The small sizes of the fenestrae have so far prohibited any functional analysis with standard and advanced light microscopy techniques. Only the advent of super-resolution optical fluorescence microscopy now permits the recording of such small cellular structures. Here, we demonstrate the complementary use of two different super-resolution optical microscopy modalities, 3D structured illumination microscopy (3D-SIM) and single molecule localization microscopy in a common optical platform to obtain new insights into the association between the cytoskeleton and the plasma membrane that supports the formation of fenestrations. We applied 3D-SIM to multi-color stained LSECs to acquire highly resolved overviews of large sample areas. We then further increased the spatial resolution for imaging fenestrations by single molecule localization microscopy applied to select small locations of interest in the same sample on the same microscope setup. We optimized the use of fluorescent membrane stains for these imaging conditions. The combination of these techniques offers a unique opportunity to significantly improve studies of subcellular ultrastructures such as LSEC fenestrations.

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Correlative multicolor 3D SIM and STORM microscopy

Cited by 39 publications

References 17 publications

Molecular dysregulation of ciliary polycystin-2 channels caused by variants in the TOP domain

Molecular dysregulation of ciliary polycystin-2 channels caused by variants in the TOP domain

Cell-free reconstitution reveals centriole cartwheel assembly mechanisms

Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations

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