Lipid and protein content profiling of isolated native autophagic vesicles

Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin-proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses

Unprecedented staining of polar lipids by a luminescent rhenium complex revealed by FTIR microspectroscopy in adipocytes.

Fourier transform infrared (FTIR) microspectroscopy and confocal imaging have been used to demonstrate that the neutral rhenium(i) tricarbonyl 1,10-phenanthroline complex bound to 4-cyanophenyltetrazolate as the ancillary ligand is able to localise in regions with high concentrations of polar lipids such as phosphatidylethanolamine (PE), sphingomyelin, sphingosphine and lysophosphatidic acid (LPA) in mammalian adipocytes

Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains

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 storag

Autophagy ENDing unproductive phase-separated endocytic protein deposits

Selective disposal of a wide range of cellular entities by macroautophagy/autophagy is achieved through a special class of proteins called autophagy receptors, which link corresponding cargo to the membrane-bound autophagosomal protein Atg8/LC3. In pursuit of novel autophagy receptors and their cargo, we uncovered a previously undescribed autophagy pathway for removal of aberrant clathrin-mediated endocytosis (CME) protein condensates in S. cerevisiae. Of these CME proteins, Ede1 functions as an autophagy receptor, harboring distinct Atg8-binding domains and driving phase separation into condensates. The aberrant CME condensates at the plasma membrane (PM) exhibit a drop-like structure surrounded by a fenestrated ER, which are engulfed in pieces in an Ede1-dependent manner by autophagy. Thus, our work suggests that aberrant CME is a target for autophagic degradation, with the scaffold protein Ede1 serving as a built-in autophagy receptor that monitors the assembly status of the CME machinery

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozenhydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM)