The small GTPase Ran defines nuclear pore complex asymmetry.

Nuclear pore complexes (NPCs) bridge across the nuclear envelope and mediate nucleocytoplasmic exchange. They consist of hundreds of nucleoporin building blocks and exemplify the structural complexity of macromolecular assemblies. To ensure transport directionality, different nucleoporin complexes are attached to the cytoplasmic and nuclear face of the NPC.

In situ cryo-ET visualization of mitochondrial depolarization and mitophagic engulfment.

Defective mitochondrial quality control in response to loss of mitochondrial membrane polarization is implicated in Parkinson's disease by mutations in and . Parkin-expressing U2 osteosarcoma (U2OS) cells were treated with the depolarizing agents oligomycin and antimycin A (OA) and subjected to cryo-focused ion beam milling and in situ cryo-electron tomography. Mitochondria were fragmented and devoid of matrix calcium phosphate crystals.

cryo-ET visualization of mitochondrial depolarization and mitophagic engulfment.

Defective mitochondrial quality control in response to loss of mitochondrial membrane polarization is implicated in Parkinson's disease by mutations in and . Application of cryo-electron tomography (cryo-ET) made it possible to visualize the consequences of mitochondrial depolarization at higher resolution than heretofore attainable. Parkin-expressing U2OS cells were treated with the depolarizing agents oligomycin and antimycin A (OA), subjected to cryo-FIB milling, and mitochondrial structure was characterized by cryo-ET.

To degrade or not to degrade: how phase separation modulates selective autophagy.

Selective macroautophagy/autophagy relies on newly formed double-membrane compartments, known as phagophores, to sequester and recycle diverse cellular components, including organelles, biomolecular condensates and protein aggregates, maturing into autophagosomes that fuse with the vacuole/lysosome. Autophagosomes originate at the cargo-vacuole/ER interface, where autophagy factors assemble into the phagophore assembly site (PAS). However, how autophagy proteins organize on the surface of structurally and biophysically different cargoes, and achieve spatial confinement at the PAS to support autophagosome formation remains unclear.

Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST.

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multiomic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in and mutants, where lysosomes accumulate cholesterol.

Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy.

Autophagy is a key cellular quality control mechanism. Nutrient stress triggers bulk autophagy, which nonselectively degrades cytoplasmic material upon formation and liquid-liquid phase separation of the autophagy-related gene 1 (Atg1) complex. In contrast, selective autophagy eliminates protein aggregates, damaged organelles and other cargoes that are targeted by an autophagy receptor.

Osmotic stress induces formation of both liquid condensates and amyloids by a yeast prion domain.

Liquid protein condensates produced by phase separation are involved in the spatiotemporal control of cellular functions, while solid fibrous aggregates (amyloids) are associated with diseases and/or manifest as infectious or heritable elements (prions). Relationships between these assemblies are poorly understood. The Saccharomyces cerevisiae release factor Sup35 can produce both fluid liquid-like condensates (e.

Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited.

Selective Autophagy of Macromolecular Complexes: What Does It Take to be Taken?

Proteins are known to perform an astonishing array of functions thanks to their ability to cooperate and modulate each other's properties. Inside cells, proteins can assemble into large multi-subunit complexes to carry out complex cellular functions. The correct assembly and maintenance of the functional state of macromolecular protein complexes is crucial for human health.

Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST.

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multi-omic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in and mutants, where lysosomes accumulate cholesterol.

Combinatorial selective ER-phagy remodels the ER during neurogenesis.

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy.

Protein condensates as flexible platforms for membrane traffic.

With an essential role in nearly every physiological process and disease state, trafficking vesicles are fundamental to cell biology. Canonical understanding of membrane traffic has been driven by key achievements in structural biology. Nonetheless, discoveries over the past few years progressively point to the critical role of intrinsically disordered domains and proteins, which lack a well-defined secondary structure.

Autophagy as a caretaker of nuclear integrity.

Due to their essential functions, dysregulation of nuclear pore complexes (NPCs) is strongly associated with numerous human diseases, including neurodegeneration and cancer. On a cellular level, longevity of scaffold nucleoporins in postmitotic cells of both C. elegans and mammals renders them vulnerable to age-related damage, which is associated with an increase in pore leakiness and accumulation of intranuclear aggregates in rat brain cells.

Combinatorial selective ER-phagy remodels the ER during neurogenesis.

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis: a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodeling, as autophagy-deficient neurons display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy.

How Membrane Contact Sites Shape the Phagophore.

During macroautophagy, phagophores establish multiple membrane contact sites (MCSs) with other organelles that are pivotal for proper phagophore assembly and growth. In , phagophore contacts have been observed with the vacuole, the ER, and lipid droplets. In situ imaging studies have greatly advanced our understanding of the structure and function of these sites.

In situ snapshots along a mammalian selective autophagy pathway.

Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are carriers responsible for delivering their content to a lytic compartment for destruction. The cargo can be of diverse origin, ranging from macromolecular complexes to protein aggregates, organelles, and even invading pathogens.

FACS-mediated isolation of native autophagic vesicles.

Autophagosome isolation enables the thorough investigation of structural components and engulfed materials. Recently, we introduced a novel antibody-based FACS-mediated method for isolation of native macroautophagic/autophagic vesicles and confirmed the quality of the preparations. We performed phospholipidomic and proteomic analyses to characterize autophagic vesicle-associated phospholipids and protein cargoes under different autophagy conditions.

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.

In situ structural analysis reveals membrane shape transitions during autophagosome formation.

Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells.

Cryo-EM structures of Gid12-bound GID E3 reveal steric blockade as a mechanism inhibiting substrate ubiquitylation.

Protein degradation, a major eukaryotic response to cellular signals, is subject to numerous layers of regulation. In yeast, the evolutionarily conserved GID E3 ligase mediates glucose-induced degradation of fructose-1,6-bisphosphatase (Fbp1), malate dehydrogenase (Mdh2), and other gluconeogenic enzymes. "GID" is a collection of E3 ligase complexes; a core scaffold, RING-type catalytic core, and a supramolecular assembly module together with interchangeable substrate receptors select targets for ubiquitylation.

Intracellular localization of the proteasome in response to stress conditions.

The ubiquitin-proteasome system fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation.

Bypassing the nuclear gate: A non-canonical entry pathway for the mitochondrial pyruvate dehydrogenase complex.

Zervopoulos et al. (2022) propose a non-canonical nuclear import pathway for the functional mitochondrial pyruvate dehydrogenase complex (PDC), facilitated by dynamic MFN2-mediated tethering of mitochondria to the nuclear envelope upon exposure to proliferative stimuli.

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, frozen-hydrated 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.