In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume ultrastructural imaging. We present a toolset for adherent cells that enables tracking and finding cells, previously identified in light microscopy (LM), in the FIB-SEM, along with the automatic acquisition of high-resolution volume datasets. We detect the underlying grid pattern in both modalities (LM and EM), to identify common reference points. A combination of computer vision techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope's field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 µm accuracy while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing volume acquisition in multiple locations autonomously is possible in EM.

Dysfunction of caveolae is involved in human muscle disease, although the underlying molecular mechanisms remain unclear. In this paper, we have functionally characterized mouse and zebrafish models of caveolae-associated muscle disease. Using electron tomography, we quantitatively defined the unique three-dimensional membrane architecture of the mature muscle surface. Caveolae occupied around 50% of the sarcolemmal area predominantly assembled into multilobed rosettes. These rosettes were preferentially disassembled in response to increased membrane tension. Caveola-deficient cavin-1(-/-) muscle fibers showed a striking loss of sarcolemmal organization, aberrant T-tubule structures, and increased sensitivity to membrane tension, which was rescued by muscle-specific Cavin-1 reexpression. In vivo imaging of live zebrafish embryos revealed that loss of muscle-specific Cavin-1 or expression of a dystrophy-associated Caveolin-3 mutant both led to sarcolemmal damage but only in response to vigorous muscle activity. Our findings define a conserved and critical role in mechanoprotection for the unique membrane architecture generated by the caveolin-cavin system.

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

Phosphatidylserine (PS) plays a central role in cell signaling and in the biosynthesis of other lipids. To date, however, the subcellular distribution and transmembrane topology of this crucial phospholipid remain ill-defined. We transfected cells with a GFP-tagged C2 domain of lactadherin to detect by light and electron microscopy PS exposed on the cytosolic leaflet of the plasmalemma and organellar membranes. Cytoplasmically exposed PS was found to be clustered on the plasma membrane, and to be associated with caveolae, the trans-Golgi network, and endocytic organelles including intraluminal vesicles of multivesicular endosomes. This labeling pattern was compared with the total cellular distribution of PS as visualized using a novel on-section technique. These complementary methods revealed PS in the interior of the ER, Golgi complex, and mitochondria. These results indicate that PS in the lumenal monolayer of the ER and Golgi complex becomes exposed cytosolically at the trans-Golgi network. Transmembrane flipping of PS may contribute to the exit of cargo from the Golgi complex.

Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of "emerging" LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage.