Visualizing and discovering cellular structures with super-resolution microscopy

Sigal, Yaron M.; Zhou, Ruobo; Zhuang, Xiaowei

doi:10.1126/science.aau1044

Cited by 570 publications

(420 citation statements)

References 101 publications

Supporting

Mentioning

418

Contrasting

Order By: Relevance

“…To investigate cytoskeletal changes at the ultrastructural level, we fixed the cells for immunofluorescence labeling, and performed 3D-STORM SRM to achieve ≈20 nm spatial resolution optically. [23][24][25][26][27] Notably, 3D-STORM revealed substantial ultrastructural changes in the vimentin intermediate filaments ( Figure 1A,B and Figure S2A, Supporting Information), whereas conventional, diffraction-limited microscopy did not well resolve the filaments ( Figure 1B). For the milder treatment of 50% HBSS buffer, where changes in cell morphology were moderate ( Figure S1A, Supporting Information), 3D-STORM showed that the initially long and continuous vimentin filaments, which often ran through the entire cell lengths, all broke down into short segments a few micrometers in length ( Figure 1A,B).…”

Section: Resultsmentioning

confidence: 99%

Hypotonic Stress Induces Fast, Reversible Degradation of the Vimentin Cytoskeleton via Intracellular Calcium Release

Pan

Ping

et al. 2019

Advanced Science

View full text Add to dashboard Cite

The dynamic response of the cell to osmotic changes is critical to its physiology and is widely exploited for cell manipulation. Here, using three‐dimensional stochastic optical reconstruction microscopy (3D‐STORM), a super‐resolution technique, the hypotonic stress‐induced ultrastructural changes of the cytoskeleton of a common fibroblast cell type are examined. Unexpectedly, these efforts lead to the discovery of a fast, yet reversible dissolution of the vimentin intermediate filament system that precedes ultrastructural changes of the supposedly more dynamic actin and tubulin cytoskeletal systems as well as changes in cell morphology. In combination with calcium imaging and biochemical analysis, it is shown that the vimentin‐specific fast cytoskeletal degradation under hypotonic stress is due to proteolysis by the calcium‐dependent protease calpain. The process is found to be activated by the hypotonic stress‐induced calcium release from intracellular stores, and is therefore efficiently suppressed by inhibiting any part of the IP3‐Ca2+‐calpain pathway established in this study. Together, these findings highlight an unexpected, fast degradation mechanism for the vimentin cytoskeleton in response to external stimuli, and point to the significant, yet previously overlooked physiological impacts of hypotonic stress‐induced intracellular calcium release on cell ultrastructure and function.

show abstract

Section: Resultsmentioning

confidence: 99%

Hypotonic Stress Induces Fast, Reversible Degradation of the Vimentin Cytoskeleton via Intracellular Calcium Release

Pan

Ping

et al. 2019

Advanced Science

View full text Add to dashboard Cite

show abstract

“…Pioneering electron microscopy (EM) studies have revealed parallel arrays of microtubules within axons that support vesicular transport over long distances (Peters et al, 1991). Only recently has optical super-resolution microscopy been able to unveil striking actin assemblies within axons (Papandréou and Leterrier, 2018;Sigal et al, 2018): the axon is lined by a membrane-associated periodic scaffold (MPS) composed of circumferential actin rings spaced every ~185 nm by tetramers of spectrins (D'Este et al, 2015;Leterrier et al, 2015;Xu et al, 2013;Zhong et al, 2014). A molecular and mechanistic understanding of actin rings and the MPS is still lacking, as EM has not yet visualized this assembly despite its presence in virtually all neurons (D'Este et al, 2016;Dubey et al, 2018;He et al, 2016;Unsain et al, 2018b).…”

Section: Introductionmentioning

confidence: 99%

Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings

Vassilopoulos

Gibaud

Jimenez

et al. 2019

Preprint

View full text Add to dashboard Cite

Recent super-resolution microscopy studies have unveiled a periodic scaffold of actin rings regularly spaced by spectrins under the plasma membrane of axons. However, ultrastructural details are unknown, limiting a molecular and mechanistic understanding of these enigmatic structures. Here, we combine platinum-replica electron and optical super-resolution microscopy to investigate the cortical cytoskeleton of axons at the ultrastructural level. Immunogold labeling and correlative super-resolution/ electron microscopy allow us to unambiguously resolve actin rings as braids made of two long, intertwined actin filaments connected by a dense mesh of aligned spectrins. This molecular arrangement contrasts with the currently assumed model of actin rings made of short, capped actin filaments. Along the proximal axon, we resolved the presence of phospho-myosin light chain and the scaffold connection with microtubules via ankyrin G. We propose that braided rings explain the observed stability of the actin-spectrin scaffold and ultimately participate in preserving the axon integrity.

show abstract

“…This study does not determine the precise subcellular protein localization in human endoneurial microvascular endothelial cells. Immunogold electron microscopy and super resolution microscopy provide avenues to more precisely determine the in situ subcellular localization of specific molecules restricted to the human BNB, particularly components of the restrictive intercellular junctional complex such as CLDN4, or determine the luminal or abluminal localization of transporters such as SLC1A1 prior to functional studies. This is an important future direction.…”

Section: Discussionmentioning

confidence: 99%

“…Immunogold electron microscopy and super resolution microscopy [46][47][48][49] ORCID Eroboghene E. Ubogu https://orcid.org/0000-0002-8307-9994…”

Section: Discussionmentioning

confidence: 99%

In situ molecular characterization of endoneurial microvessels that form the blood‐nerve barrier in normal human adult peripheral nerves

Ouyang

Dong

Ubogu

2019

J Peripheral Nervous Sys

View full text Add to dashboard Cite

The blood‐nerve barrier (BNB) formed by tight junction‐forming endoneurial microvessels located in the innermost compartment of peripheral nerves, and the perineurium serve to maintain the internal microenvironment required for normal signal transduction. The specific molecular components that define the normal adult human BNB are not fully known. Guided by data derived from the adult human BNB transcriptome, we evaluated the in situ expression of 25 junctional complex, transporter, cell membrane, and cytoskeletal proteins in four histologically normal adult sural nerves by indirect fluorescent immunohistochemistry to determine proteins specifically expressed by restrictive endoneurial microvascular endothelium. Using Ulex Europaeus Agglutinin‐1 expression to detect endothelial cells, we ascertained that the selected proteins were uniformly expressed in ≥90% of endoneurial microvessels. P‐glycoprotein (also known as adenosine triphosphate‐binding cassette subfamily B member 1) and solute carrier family 1 member 1 demonstrated restricted expression by endoneurial endothelium only, with classic tight junction protein claudin‐5 also expressed on fenestrated epineurial macrovessels, and vascular‐specific adherens junction protein cadherin‐5 also expressed by the perineurium. The expression profiles of the selected proteins provide significant insight into the molecular composition of normal adult peripheral nerves. Further work is required to elucidate the human adult BNB molecular signature in order to better understand its development and devise strategies to restore function in peripheral neuropathies.

show abstract

Visualizing and discovering cellular structures with super-resolution microscopy

Cited by 570 publications

References 101 publications

Hypotonic Stress Induces Fast, Reversible Degradation of the Vimentin Cytoskeleton via Intracellular Calcium Release

Hypotonic Stress Induces Fast, Reversible Degradation of the Vimentin Cytoskeleton via Intracellular Calcium Release

Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings

In situ molecular characterization of endoneurial microvessels that form the blood‐nerve barrier in normal human adult peripheral nerves

Contact Info

Product

Resources

About