Summary Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.

This protocol describes sample preparation strategies for correlative 3D cryo-SIM and cryo soft X-ray tomography. In addition, the authors provide a direct comparison and recommendations regarding the selection and use of fiducials for 3D correlation. TWEET A new protocol for sample preparation and fiducial selection for correlative cryo-SIM and cryo-soft X-ray tomography. #CLXT #cryoimaging #cellstructure #correlativeimaging COVER TEASER Correlative cryo-SIM and cryo-soft X-ray tomographyAbstract 3D correlative microscopy methods have revolutionised biomedical research allowing the acquisition of multi-dimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometre resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample resulting in the immediate requirement for protocols that facilitate hassle-free unambiguous cross correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialisation regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 hours and 3.5 days to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).

Autoantibodies to a polymerase III transcription factor, La (SS-B), are frequently detected in the serum of patients with Sjogren's syndrome and systemic lupus erythematosus. To define the humoral immune response to this protein, we analyzed the patterns of antibody recognition toward 13 recombinant La peptides by immunoblotting and determined the heterogeneity of antibodies reactive with the immunodominant epitopes. The smallest epitopes that were strongly antigenic and recognized by > 70% of sera tested (immunodominant) were encoded by the subclones BgX and XA located in the 5' and 3' halves of the La cDNA, respectively. Conformation of the immunodominant La peptides played a major role in antibody recognition. Although greater diversity in antibody binding to carboxyl-terminal La peptides was observed, the overall pattern of peptide recognition by anti-La antibodies was similar in different diseases. The antibody responses to the immunodominant peptides were strongly correlated (r = 0.68, P < 0.001). One-and two-dimensional isoelectric focusing of affinity purified IgG anti-La peptide antibodies revealed restricted heterogeneity and oligoclonal bands (K light chains). These observations suggest that anti-La antibodies are induced and/or maintained by the self antigen and that their diversity is constrained either by mechanisms related to tolerance or by affinity maturation of the humoral immune response. (J. Clin. Invest. 1990. 85:325-333.) autoantibody -epitope mapping * La (SS-B) * systemic lupus erythematosus

O‐GlcNAcylation is a ubiquitous post‐translational modification that is extremely labile and plays a significant role in physiology, including the heart. Sustained activation of cardiac O‐GlcNAcylation is frequently associated with alterations in cellular metabolism, leading to detrimental effects on cardiovascular function. This is particularly true during conditions such as diabetes, hypertension, cardiac remodelling, heart failure and arrhythmogenesis. Paradoxically, transient elevation of cardiac protein O‐GlcNAcylation can also exert beneficial effects in the heart. There is compelling evidence to suggest that a complex interaction between O‐GlcNAcylation and phosphorylation also exists in the heart. Beyond direct functional consequences on cardiomyocytes, O‐GlcNAcylation also acts indirectly by altering the function of transcription factors that affect downstream signalling. This review focuses on the potential cardioprotective role of protein O‐GlcNAcylation during ischaemia‐reperfusion injury, the deleterious consequences of chronically elevated O‐GlcNAc levels, the interplay between O‐GlcNAcylation and phosphorylation in the cardiomyocytes and the effects of O‐GlcNAcylation on other major non‐myocyte cell types in the heart.

FK506-binding proteins 12.6 (FKBP12.6) and 12 (FKBP12) tightly associate with the cardiac ryanodine receptor (RyR2). Studies suggest that dissociation of FKBP12.6 from mutant forms of RyR2 contributes to store overload-induced Ca(2+) release (SOICR) and Ca(2+)-triggered arrhythmias. However, these findings are controversial. Previous studies focused on the effect of FKBP12.6 on the initiation of SOICR and did not explore changes in the termination of Ca(2+) release. Less is known about FKBP12. We aimed to determine the effect of FKBP12.6 and FKBP12 on the termination of SOICR. Using single-cell imaging, in cells expressing wild-type RyR2, we found that FKBP12.6 and FKBP12 significantly increase the termination threshold of SOICR without changing the activation threshold of SOICR. This effect, dependent on the association of each FKBP with RyR2, reduced the magnitude of Ca(2+) release but had no effect on the propensity for SOICR. In contrast, neither FKBP12.6 nor FKBP12 was able to regulate an arrhythmogenic variant of RyR2, despite a conserved protein interaction. Our results suggest that both FKBP12.6 and FKBP12 play critical roles in regulating RyR2 function by facilitating the termination of SOICR. The inability of FKBPs to mediate a similar effect on the mutant RyR2 represents a novel mechanism by which mutations within RyR2 lead to arrhythmia.