High-Throughput 3D Imaging Flow Cytometry of Suspended Adherent 3D Cell Cultures.

3D cell cultures are indispensable in recapitulating in vivo environments. Among the many 3D culture methods, culturing adherent cells on hydrogel beads to form spheroid-like structures is a powerful strategy for maintaining high cell viability and functions in the adherent states. However, high-throughput, scalable technologies for 3D imaging of individual cells cultured on the hydrogel scaffolds are lacking. This study reports the development of a high throughput, scalable 3D imaging flow cytometry platform for analyzing spheroid models. This platform is realized by integrating a single objective fluorescence light-sheet microscopy with a microfluidic device that combines hydrodynamic and acoustofluidic focusing techniques. This integration enabled unprecedentedly high-throughput and scalable optofluidic 3D imaging, processing 1310 spheroids consisting of 28 117 cells min-1 . The large dataset obtained enables precise quantification and comparison of the nuclear morphology of adhering and suspended cells, revealing that the adhering cells have smaller nuclei with less rounded surfaces. This platform's high throughput, robustness, and precision for analyzing the morphology of subcellular structures in 3D culture models hold promising potential for various biomedical analyses, including image-based phenotypic screening of drugs with spheroids or organoids.

A mechanism that ensures non-selective cytoplasm degradation by autophagy.

In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting double-membrane vesicle autophagosome for lysosomal/vacuolar degradation. Here, we discover a mechanism that allows the isolation membrane to expand with a large opening to ensure non-selective cytoplasm sequestration within the autophagosome. A sorting nexin complex that localizes to the opening edge of the isolation membrane plays a critical role in this process. Without the complex, the isolation membrane expands with a small opening that prevents the entry of particles larger than about 25 nm, including ribosomes and proteasomes, although autophagosomes of nearly normal size eventually form. This study sheds light on membrane morphogenesis during autophagosome formation and selectivity in autophagic degradation.

Phase 1 Clinical Trial of PPMX-T003, a Novel Human Monoclonal Antibody Specific for Transferrin Receptor 1, to Evaluate Its Safety, Pharmacokinetics, and Pharmacodynamics.

This study aimed to evaluate the safety, pharmacokinetics, and pharmacodynamics of PPMX-T003, a novel human monoclonal antibody for transferrin receptor 1 (TFR1), in healthy individuals. Forty participants were enrolled and randomized to PPMX-T003 dose groups (n = 6/group) and the placebo group (n = 10). The safety and pharmacokinetics profiles were assessed according to the sequential, ascending single-dose intravenous infusions of PPMX-T003 from 0.008 mg/kg to 0.25 mg/kg. Adverse events (AEs) after PPMX-T003 administration occurred in 16 of 30 participants. Any severe AE and AE incidence were not reported, but they tended to increase depending on the dose. Laboratory tests, vital signs, and standard 12-lead electrocardiogram showed no clinically relevant changes. Five participants experienced an infusion-related reaction but recovered on days 5-10. Regarding pharmacokinetics, PPMX-T003 has a nonlinear elimination pattern. PPMX-T003 in the 0.25 mg/kg group showed apparent (>50%) decreased serum levels of reticulocytes from day 3 and sustained moderate (<10%) fall of hematocrit and hemoglobin counts from day 7. In conclusion, the antibody-mediated blockade of TFR1 elicited the expected fall in blood cell levels and showed an acceptable safety profile, supporting the continuing development of PPMX-T003 as a new candidate for polycythemia vera treatment.

Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus.

In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to the forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane via its transmembrane domain and also associated with the inner nuclear membrane via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that autophagosome formation-coupled Atg39 crowding causes the NE to protrude toward the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 crowding in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.

Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis.

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.

Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation.

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36-Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36-Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.

Super-assembly of ER-phagy receptor Atg40 induces local ER remodeling at contacts with forming autophagosomal membranes.

The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles.

TORC1 inactivation stimulates autophagy of nucleoporin and nuclear pore complexes.

The mechanisms underlying turnover of the nuclear pore complex (NPC) and the component nucleoporins (Nups) are still poorly understood. In this study, we found that the budding yeast Saccharomyces cerevisiae triggers NPC degradation by autophagy upon the inactivation of Tor kinase complex 1. This degradation largely depends on the selective autophagy-specific factor Atg11 and the autophagy receptor-binding ability of Atg8, suggesting that the NPC is degraded via receptor-dependent selective autophagy. Immunoelectron microscopy revealed that NPCs embedded in nuclear envelope-derived double-membrane vesicles are sequestered within autophagosomes. At least two pathways are involved in NPC degradation: Atg39-dependent nucleophagy (selective autophagy of the nucleus) and a pathway involving an unknown receptor. In addition, we found the interaction between Nup159 and Atg8 via the Atg8-family interacting motif is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term "NPC-phagy" and "nucleoporinophagy."

COPII vesicles contribute to autophagosomal membranes.

A hallmark of autophagy is the de novo formation of double-membrane vesicles called autophagosomes, which sequester various cellular constituents for degradation in lysosomes or vacuoles. The membrane dynamics underlying the biogenesis of autophagosomes, including the origin of the autophagosomal membrane, are still elusive. Although previous studies suggested that COPII vesicles are closely associated with autophagosome biogenesis, it remains unclear whether these vesicles serve as a source of the autophagosomal membrane. Using a recently developed COPII vesicle-labeling system in fluorescence and immunoelectron microscopy in the budding yeast Saccharomyces cerevisiae, we show that the transmembrane cargo Axl2 is loaded into COPII vesicles in the ER. Axl2 is then transferred to autophagosome intermediates, ultimately becoming part of autophagosomal membranes. This study provides a definitive answer to a long-standing, fundamental question regarding the mechanisms of autophagosome formation by implicating COPII vesicles as a membrane source for autophagosomes.

Two distinct mechanisms target the autophagy-related E3 complex to the pre-autophagosomal structure.

In autophagy, Atg proteins organize the pre-autophagosomal structure (PAS) to initiate autophagosome formation. Previous studies in yeast revealed that the autophagy-related E3 complex Atg12-Atg5-Atg16 is recruited to the PAS via Atg16 interaction with Atg21, which binds phosphatidylinositol 3-phosphate (PI3P) produced at the PAS, to stimulate conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine. Here, we discover a novel mechanism for the PAS targeting of Atg12-Atg5-Atg16, which is mediated by the interaction of Atg12 with the Atg1 kinase complex that serves as a scaffold for PAS organization. While autophagy is partially defective without one of these mechanisms, cells lacking both completely lose the PAS localization of Atg12-Atg5-Atg16 and show no autophagic activity. As with the PI3P-dependent mechanism, Atg12-Atg5-Atg16 recruited via the Atg12-dependent mechanism stimulates Atg8 lipidation, but also has the specific function of facilitating PAS scaffold assembly. Thus, this study significantly advances our understanding of the nucleation step in autophagosome formation.

The Atg2-Atg18 complex tethers pre-autophagosomal membranes to the endoplasmic reticulum for autophagosome formation.

The biogenesis of double-membrane vesicles called autophagosomes, which sequester and transport intracellular material for degradation in lysosomes or vacuoles, is a central event in autophagy. This process requires a unique set of factors called autophagy-related (Atg) proteins. The Atg proteins assemble to organize the preautophagosomal structure (PAS), at which a cup-shaped membrane, the isolation membrane (or phagophore), forms and expands to become the autophagosome. The molecular mechanism of autophagosome biogenesis remains poorly understood. Previous studies have shown that Atg2 forms a complex with the phosphatidylinositol 3-phosphate (PI3P)-binding protein Atg18 and localizes to the PAS to initiate autophagosome biogenesis; however, the molecular function of Atg2 remains unknown. In this study, we show that Atg2 has two membrane-binding domains in the N- and C-terminal regions and acts as a membrane tether during autophagosome formation in the budding yeast Saccharomyces cerevisiae An amphipathic helix in the C-terminal region binds to membranes and facilitates Atg18 binding to PI3P to target the Atg2-Atg18 complex to the PAS. The N-terminal region of Atg2 is also involved in the membrane binding of this protein but is dispensable for the PAS targeting of the Atg2-Atg18 complex. Our data suggest that this region associates with the endoplasmic reticulum (ER) and is responsible for the formation of the isolation membrane at the PAS. Based on these results, we propose that the Atg2-Atg18 complex tethers the PAS to the ER to initiate membrane expansion during autophagosome formation.

The PP2A-like Protein Phosphatase Ppg1 and the Far Complex Cooperatively Counteract CK2-Mediated Phosphorylation of Atg32 to Inhibit Mitophagy.

Mitophagy plays an important role in mitochondrial quality control. In yeast, phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 (CK2) upon induction of mitophagy is a prerequisite for interaction of Atg32 with Atg11 (an adaptor protein for selective autophagy) and following delivery of mitochondria to the vacuole for degradation. Because CK2 is constitutively active, Atg32 phosphorylation must be precisely regulated to prevent unrequired mitophagy. We found that the PP2A (protein phosphatase 2A)-like protein phosphatase Ppg1 was essential for dephosphorylation of Atg32 and inhibited mitophagy. We identified the Far complex proteins, Far3, Far7, Far8, Far9, Far10, and Far11, as Ppg1-binding proteins. Deletion of Ppg1 or Far proteins accelerated mitophagy. Deletion of a cytoplasmic region (amino acid residues 151-200) of Atg32 caused the same phenotypes as in ppg1Δ cells, which suggested that dephosphorylation of Atg32 by Ppg1 required this region. Therefore, Ppg1 and the Far complex cooperatively dephosphorylate Atg32 to prevent excessive mitophagy.

Structural Basis for Receptor-Mediated Selective Autophagy of Aminopeptidase I Aggregates.

Selective autophagy mediates the degradation of various cargoes, including protein aggregates and organelles, thereby contributing to cellular homeostasis. Cargo receptors ensure selectivity by tethering specific cargo to lipidated Atg8 at the isolation membrane. However, little is known about the structural requirements underlying receptor-mediated cargo recognition. Here, we report structural, biochemical, and cell biological analysis of the major selective cargo protein in budding yeast, aminopeptidase I (Ape1), and its complex with the receptor Atg19. The Ape1 propeptide has a trimeric coiled-coil structure, which tethers dodecameric Ape1 bodies together to form large aggregates. Atg19 disassembles the propeptide trimer and forms a 2:1 heterotrimer, which not only blankets the Ape1 aggregates but also regulates their size. These receptor activities may promote elongation of the isolation membrane along the aggregate surface, enabling sequestration of the cargo with high specificity.

Phospholipid methylation controls Atg32-mediated mitophagy and Atg8 recycling.

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy-inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria-anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8-PMME, a mitophagy-incompetent lipid conjugate of the autophagy-related ubiquitin-like modifier. Amelioration of Atg32 expression and attenuation of Atg8-PMME conjugation markedly rescue mitophagy in opi3-null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32-mediated mitophagy.

Receptor-mediated selective autophagy degrades the endoplasmic reticulum and the nucleus.

Macroautophagy (hereafter referred to as autophagy) degrades various intracellular constituents to regulate a wide range of cellular functions, and is also closely linked to several human diseases. In selective autophagy, receptor proteins recognize degradation targets and direct their sequestration by double-membrane vesicles called autophagosomes, which transport them into lysosomes or vacuoles. Although recent studies have shown that selective autophagy is involved in quality/quantity control of some organelles, including mitochondria and peroxisomes, it remains unclear how extensively it contributes to cellular organelle homeostasis. Here we describe selective autophagy of the endoplasmic reticulum (ER) and nucleus in the yeast Saccharomyces cerevisiae. We identify two novel proteins, Atg39 and Atg40, as receptors specific to these pathways. Atg39 localizes to the perinuclear ER (or the nuclear envelope) and induces autophagic sequestration of part of the nucleus. Atg40 is enriched in the cortical and cytoplasmic ER, and loads these ER subdomains into autophagosomes. Atg39-dependent autophagy of the perinuclear ER/nucleus is required for cell survival under nitrogen-deprivation conditions. Atg40 is probably the functional counterpart of FAM134B, an autophagy receptor for the ER in mammals that has been implicated in sensory neuropathy. Our results provide fundamental insight into the pathophysiological roles and mechanisms of 'ER-phagy' and 'nucleophagy' in other organisms.

Localization of Atg3 to autophagy-related membranes and its enhancement by the Atg8-family interacting motif to promote expansion of the membranes.

The E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE) to drive autophagosome formation in Saccharomyces cerevisiae. In this study, we show that Atg3 localizes to the pre-autophagosomal structure (PAS) and the isolation membrane (IM), providing crucial evidence that Atg8-PE conjugates are produced on these structures. We also find that mutations in the Atg8-family interacting motif (AIM) of Atg3 significantly impairs the PAS/IM localization of Atg3, resulting in inefficient IM expansion. It is suggested that the AIM-mediated PAS/IM localization of Atg3 facilitates membrane expansion in these structures probably by ensuring active production of Atg8-PE on the membranes.

Hrr25 triggers selective autophagy-related pathways by phosphorylating receptor proteins.

In selective autophagy, degradation targets are specifically recognized, sequestered by the autophagosome, and transported into the lysosome or vacuole. Previous studies delineated the molecular basis by which the autophagy machinery recognizes those targets, but the regulation of this process is still poorly understood. In this paper, we find that the highly conserved multifunctional kinase Hrr25 regulates two distinct selective autophagy-related pathways in Saccharomyces cerevisiae. Hrr25 is responsible for the phosphorylation of two receptor proteins: Atg19, which recognizes the assembly of vacuolar enzymes in the cytoplasm-to-vacuole targeting pathway, and Atg36, which recognizes superfluous peroxisomes in pexophagy. Hrr25-mediated phosphorylation enhances the interactions of these receptors with the common adaptor Atg11, which recruits the core autophagy-related proteins that mediate the formation of the autophagosomal membrane. Thus, this study introduces regulation of selective autophagy as a new role of Hrr25 and, together with other recent studies, reveals that different selective autophagy-related pathways are regulated by a uniform mechanism: phosphoregulation of the receptor-adaptor interaction.

Atg12-Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site.

Two autophagy-related ubiquitin-like systems have unique features: the E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to the lipid phosphatidylethanolamine, and the other ubiquitin-like protein conjugate Atg12-Atg5 promotes that conjugase activity of Atg3. Here, we elucidate the mode of this action of Atg12-Atg5 as a new E3 enzyme by using Saccharomyces cerevisiae proteins. Biochemical analyses based on structural information suggest that Atg3 requires a threonine residue to catalyze the conjugation reaction instead of the typical asparagine residue used by other E2 enzymes. Moreover, the catalytic cysteine residue of Atg3 is arranged in the catalytic center such that the conjugase activity is suppressed; Atg12-Atg5 induces a reorientation of the cysteine residue toward the threonine residue, which enhances the conjugase activity of Atg3. Thus, this study reveals the mechanism of the key reaction that drives membrane biogenesis during autophagy.

The autophagy-related protein kinase Atg1 interacts with the ubiquitin-like protein Atg8 via the Atg8 family interacting motif to facilitate autophagosome formation.

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.