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.

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.

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 prerequisite for meaningful biological discovery.

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.

No abstract

In recent years, the development of analytical approaches to super-resolution microscopy has highlighted the possibility of recovering super-resolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit without specialized hardware. Here, we present eSRRF (enhanced-SRRF), a significant improvement over our initial method, enhancing image fidelity to the underlying structure and resolution. Especially, eSRRF uses automated data-driven parameter optimization, including an estimation of the number of frames necessary for optimal reconstruction. We demonstrate the improved fidelity of the images reconstructed with eSRRF, and highlight its versatility and ease of use over a wide range of microscopy techniques and biological systems. We also extend eSRRF to 3D super-resolution microscopy by combining it with multi-focus microscopy (MFM), obtaining volumetric super-resolution imaging of live cells with acquisition speed of ~1 volume/second.

The Apolipoprotein E4 (APOE4) is the major allelic risk factor for late-onset Alzheimer′s disease (AD). APOE4 associates with a pro-inflammatory phenotype increasingly considered as critical in AD initiation and progression. Yet, the mechanisms driving an APOE4-dependent neuroinflammation remain unelucidated. Leveraging patient specific human induced Pluripotent Stem Cells (iPSCs) we demonstrate inflammatory chronicity and hyperactivated responses upon cytokines in human APOE4 astrocytes via a novel mechanism. We uncovered that APOE4 represses Transgelin 3 (TAGLN3), a new interacting partner of IκBα, thus increasing the NF-κB activity. The transcriptional repression of TAGLN3 was shown to result from an APOE4-dependent histone deacetylase (HDAC) activity. The functional relevance of TAGLN3 was demonstrated by the attenuation of APOE4-driven neuroinflammation after TAGLN3 supplementation. Importantly, TAGLN3 downregulation was confirmed in the brain of AD patients. Our findings highlight the APOE4-TAGLN3 axis as a new pathogenic pathway that paves the way for the development of therapeutics to prevent maladaptive inflammatory responses in APOE4 carriers, while placing TAGLN3 downregulation as a potential biomarker of AD.

In recent years, the development of analytical approaches to super-resolution microscopy has highlighted the possibility of recovering super-resolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit without specialized hardware. Here, we present eSRRF (enhanced-SRRF), a significant improvement over our initial method, enhancing image fidelity to the underlying structure and resolution. Especially, eSRRF uses automated data-driven parameter optimization, including an estimation of the number of frames necessary for optimal reconstruction. We demonstrate the improved fidelity of the images reconstructed with eSRRF and highlight its versatility and ease of use over a wide range of microscopy techniques and biological systems. We also extend eSRRF to 3D super-resolution microscopy by combining it with multi-focus microscopy (MFM), obtaining volumetric super-resolution imaging of live cells with acquisition speed of ~1 volume/second.