Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales

Guo, Yuting; Di, Li; Zhang, Siwei; Yang, Yanrui; Li, Jiajia; Wang, Xinyu; Liu, Chong; Milkie, Daniel E.; Moore, Regan P.; Tulu, U. Serdar; Kiehart, Daniel P.; Hu, Junjie; Lippincott‐Schwartz, Jennifer; Betzig, Eric; Dong, Li

doi:10.1016/j.cell.2018.09.057

Cited by 503 publications

(536 citation statements)

References 53 publications

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“…Alternating RTN4a membrane constriction and CLIMP-63 spacer functions along ER tubules could explain the lumenal ER periodicity that we report here. Indeed, a recent study of lumenal mEmerald-KDEL dynamics in peripheral ER tubules attributed the periodic distribution of this reporter to constrictions and bulges along the tubule length (19), as observed by EM (13,(35)(36)(37). However, ER tubule width is below the 70-80 nm STED resolution obtained in this study and our data cannot therefore report on changes in ER tubule width.…”

Section: Discussioncontrasting

confidence: 70%

“…STED imaging of lumenal (ERmoxGFP) or membrane (Sec61bGFP) ER reporters, of CRISPR/Cas9 knock-in calreticulin-GFP expressed at endogenous levels, and of various antibody labeled ER resident proteins, shows that ER proteins are localized to discrete puncta interspersed with lumenal domains along ER tubules. Periodic distribution of ER reporters along ER tubules can be observed in various publications studying ER using various super-resolution microscope approaches and EM (13,(27)(28)(29)(30)(31)(32) and has more recently been reported by live cell GI-SIM (19). However, the nanodomain organization of ER tubules, and the mechanisms that underlie it have yet to be characterized.…”

Section: Discussionmentioning

confidence: 96%

“…A more recent STED analysis identified the presence of dynamic nanoholes in peripheral ER sheets (17). Single molecule super-resolution particle tracking of an ER lumenal reporter demonstrated the existence of active ER lumenal flow in peripheral ER tubules (18) while high-speed, super resolution GI-SIM microscopy identified lumenal bulges and constrictions along ER tubules (19).…”

Section: Introductionmentioning

confidence: 99%

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Reticulon and CLIMP-63 control nanodomain organization of peripheral ER tubules

Gao

Zhu

Liu

et al. 2019

Preprint

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The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon and CLIMP-63, respectively. Here, STimulated Emission Depletion (STED) super-resolution microscopy shows that reticulon4a (RTN4a) and CLIMP-63 also regulate the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ERmoxGFP, membrane Sec61bGFP, knock-in calreticulin-GFP and antibody-labeled ER resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. RTN4a segregates away from and restricts lumenal blob length while CLIMP-63 associates with and increases lumenal blob length. RTN4a and CLIMP-63 also regulate the nanodomain distribution of ER resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that RTN4a and CLIMP-63 regulate dynamic nanoscale lumenal compartmentalization along peripheral ER tubules. RTN4a enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER shaping proteins reticulon and CLIMP-63 therefore regulate lumenal ER nanodomain heterogeneity, interaction with ER resident proteins and dynamics in peripheral ER tubules.

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Section: Discussioncontrasting

confidence: 70%

Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

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Reticulon and CLIMP-63 control nanodomain organization of peripheral ER tubules

Gao

Zhu

Liu

et al. 2019

Preprint

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“…In addition, we can observe the detachment of a filament at a knot within the network and, direct following, its attachment to another point, which is a typical behavior for ER ( fig. 12d, first row) [38]. Directly at this point the fiber then elongates over a long distance ( fig.…”

Section: Live-cell Imagingmentioning

confidence: 90%

DMD-based super-resolution structured illumination microscopy visualizes live cell dynamics at high speed and low cost

Sandmeyer

Lachetta

Sandmeyer

et al. 2019

Preprint

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“…For optimal SNR, we use the laser illumination in the critical angle fluorescence microscopy mode (Nakata and Hirokawa, 2003;Sinkó et al, 2014), also called highly inclined and laminated optical sheet (HILO) (Tokunaga et al, 2008) or grazing incidence (Guo et al, 2018). This consists in the inclination of the laser angle until reaching the critical angle, a point where the sample is illuminated by a ~1-2 µm thick horizontal light sheet above the coverslip, avoiding fluorescence from the upper part of the cell and imaging medium.…”

Section: Hardware Setupmentioning

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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Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales

Cited by 503 publications

References 53 publications

Reticulon and CLIMP-63 control nanodomain organization of peripheral ER tubules

Reticulon and CLIMP-63 control nanodomain organization of peripheral ER tubules

DMD-based super-resolution structured illumination microscopy visualizes live cell dynamics at high speed and low cost

About samples, giving examples: Optimized Single Molecule Localization Microscopy

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