Qiming Sun

The Golgi apparatus is essential for protein secretion and cellular homeostasis, yet its organization and turnover in living tissues remain poorly understood. Golgiphagy, the selective autophagic degradation of the Golgi, has emerged as a key quality control pathway, but its physiological regulation in vivo remains largely unknown. Here, we generated two reporter mouse lines for constitutive or Cre-dependent visualization of Golgi architecture and Golgiphagy, enabling quantitative single-cell analysis across tissues. These models revealed striking cell type– and tissue-specific heterogeneity in basal Golgiphagy, including higher activity in renal proximal than distal tubules and in cerebellar than cortical neurons. Starvation and lipopolysaccharide-induced inflammation also promoted Golgi remodeling and Golgiphagy in multiple organs, particularly the liver, spleen, and kidney. Together, these reporter mice provide a powerful genetic toolkit for studying Golgi dynamics in vivo and establish that Golgiphagy is a spatially heterogeneous and stress-responsive process under physiological conditions.

Cell survival requires tight coordination between growth-promoting metabolism and cellular quality-control pathways, yet how these processes are integrated remains unclear. Here we identify the conserved glycolytic enzyme PGAM1 as a metabolic-autophagy checkpoint that links glycolysis to autophagy initiation independently of its catalytic activity. Using complementary yeast and mammalian systems we show that PGAM1 functions as a molecular scaffold that recruits phosphatidylinositol 3-kinase complex I to the phagophore assembly site, thereby licensing autophagosome biogenesis. This autophagy-regulatory function is genetically essential, evolutionarily conserved and functionally separable from glycolysis. It is regulated by Atg1/ULK1-mediated phosphorylation that enhances Atg14 binding under starvation. Functionally, PGAM1 coordinates anabolic growth and stress-induced survival to maintain cellular homeostasis. In cancer, PGAM1 upregulation enhances both glycolytic flux and autophagy capacity. Disruption of either function markedly impairs tumour growth, establishing PGAM1 as a homeostatic checkpoint that is hijacked in cancer to drive both proliferation and stress tolerance.

Endoplasmic reticulum autophagy (ER-phagy) is a selective autophagy pathway in which receptor proteins target ER membranes and proteins for degradation, yet its role in Alzheimer’s disease (AD) remains unclear. Here, we identify FAM134B/RETREG1 as a specific ER-phagy receptor mediating amyloid precursor protein (APP) degradation. FAM134B directly interacts with ER-localized wild-type and familial mutant APP via their C-terminal domains and recruits LC3 through its LC3-interacting region (LIR) to promote APP delivery to phagophores for lysosomal degradation. In AD, epigenetic silencing at the FAM134B promoter suppresses its transcription by limiting TFEB/TFE3 binding despite their nuclear enrichment. This transcriptional suppression impairs ER-phagy, leading to APP accumulation and exacerbated AD pathology. AAV-mediated hippocampal expression of wild-type, but not LIR-mutant, FAM134B in 5XFAD mice restores ER-phagy, enhances APP clearance, reduces Aβ deposition, preserves synaptic and myelin integrity, and improves cognitive performance. These findings establish FAM134B downregulation as an upstream pathogenic event in AD, suggesting ER-phagy enhancement as a promising strategy to suppress Aβ generation at its source.

BACKGROUND: Transverse tubules (T-tubules) are invaginations of the plasma membrane crucial for excitation-contraction coupling. Disruptions in T-tubule organization are frequently observed in heart diseases and are associated with impaired contractile function and malignant arrhythmias. In mammalian cells, the ESCRT (endosomal sorting complex required for transport) proteins mediate a fundamental mechanism for membrane deformation. This study aimed to elucidate the roles of key ESCRT proteins, including Chmp4b (charged multivesicular body protein 4b) and Tsg101 (tumor susceptibility gene 101), in the formation and maintenance of T-tubules. METHODS: Myocardial-specific gene deletion was achieved using Chmp4b ^F/F , Tsg101 ^F/F , and Rosa26 ^Cas9-GFP mouse strains in conjunction with adeno-associated virus 9–mediated gene editing. The polymerization state of Chmp4b was assessed through the introduction of point mutations combined with glycerol-gradient fractionation. Direct interaction between Chmp4b and membrane phospholipids was examined using genetically encoded biosensors, lipid strip binding, and liposome tubulation assays. An inducible Chmp4b knockout model was utilized to determine its role in T-tubule maintenance during adulthood. Chmp4b expression levels were analyzed in a heart failure mouse model and in human patients with dilated cardiomyopathy. RESULTS: Chmp4b gradually localizes to the dyad during postnatal development, with its deletion causing a complete loss of T-tubules and defects in cardiac structure and contractile function. Chmp4b polymerizes and binds to PtdIns(4,5)P2 (Phosphatidylinositol 4,5-bisphosphate) as well as other negatively charged membrane lipids, driving plasma membrane invagination in a process that depends on the ESCRT-I component Tsg101. In mature cardiomyocytes, Chmp4b remains anchored to the T-tubule membranes to maintain their structure, while Tsg101 detaches and becomes dispensable for T-tubule organization. Chmp4b expression was significantly reduced in heart samples from dilated cardiomyopathy patients and in a mouse model of heart failure. CONCLUSIONS: These results uncover an ESCRT-mediated membrane deformation machinery that is essential for shaping cardiomyocyte structure in physiological and disease conditions.

Inhibiting the platelet glycoprotein VI (GPVI) receptor is a promising strategy for reducing cerebral ischemia‐reperfusion injury (CIRI) without severe compromise of hemostasis, while targeting glycoprotein IIb/IIIa (integrin αIIbβ3) causes bleeding. The underlying cellular mechanism remains unclear. This study shows that megakaryocyte‐platelet‐specific deficiency of the autophagic protein Rubicon (Run domain protein as Beclin‐1 interacting and cysteine‐rich containing) accelerates stroke development and exacerbates cerebral hemorrhage. Rubicon interacts with Bruton's tyrosine kinase (Btk) to inhibit GPVI‐mediated thrombus formation, while it prevents αIIbβ3‐mediated selective autophagy and degradation of Btk to stabilize platelet thrombi. The expression of Rubicon in platelets is decreased in patients with acute ischemic‐reperfusion injury. A cell‐permeable peptide mimicking the Rubicon‐Btk interaction significantly reduces cerebral infarction volume in a mouse model. As Rubicon is dispensable for hemostasis but crucial in the reperfusion stage of CIRI, peptides mimicking its effects may offer a selective and safe therapeutic strategy.

Lysosome homeostasis is vital for cellular fitness due to the essential roles of this organelle in various pathways. Given their extensive workload, lysosomes are prone to damage, which can stimulate lysosomal quality control mechanisms such as biogenesis, repair, or autophagic removal - a process termed lysophagy. Despite recent advances highlighting lysophagy as a critical mechanism for lysosome maintenance, the extent of lysosome integrity perturbation and the magnitude of lysophagy in vivo remain largely unexplored. Additionally, the pathophysiological relevance of lysophagy is poorly understood. To address these gaps, it is necessary to develop quantifiable methods for evaluating lysosome damage and lysophagy flux in vivo. To this end, we created two transgenic mouse lines expressing a tandem fluorescent LGALS3/galectin 3 probe (tfGAL3), either constitutively or conditionally under Cre recombinase control, utilizing the property of LGALS3 to recognize damaged lysosomes. This tool enables spatiotemporal visualization of lysosome damage and lysophagy activity at single-cell resolution in vivo. Systemic analysis across various organs, tissues, and primary cultures from these lysophagy reporter mice revealed significant variations in basal lysophagy, both in vivo and in vitro. Additionally, this study identified substantial changes in lysosome integrity and lysophagy flux in different tissues under stress conditions such as starvation, acute kidney injury and diabetic modeling. In conclusion, these complementary lysophagy reporter models are valuable resources for both basic and translational research.Abbreviation: AAV: adeno-associated virus; ATG7: autophagy related 7; CA-tfGAL3: cre-recombinase-activated tandem fluorescent LGALS3; DAPI: 4',6-diamidino-2-phenylindole; DM: diabetes mellitus; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HFD: high-fat diet; Igs2/H11/Hipp11: intergenic site 2; IST1: IST1 factor associated with ESCRT-III; KI: knock-in; LAMP1: lysosomal-associated membrane protein 1; LGALS3: lectin, galactoside-binding, soluble, 3; LLOMe: L-leucyl-L-leucine methyl ester hydrobromide; MEFs: mouse embryonic fibroblasts; NaOx: sodium oxalate; PDCD6IP: programmed cell death 6 interacting protein; PTECs: proximal tubular epithelial cells; RFP: red fluorescent protein; STZ: streptozotocin; TAM: tamoxifen; tfGAL3: tandem fluorescent LGALS3; TMEM192: transmembrane protein 192.

ER-phagy, a selective autophagy process crucial for maintaining cellular homeostasis by targeting the endoplasmic reticulum (ER), has been challenging to study in vivo due to the lack of suitable spatiotemporal quantification tools. Existing methods like electron microscopy, biochemical assays, and in vitro reporters lack resolution, scalability, or physiological relevance. Here, we present a detailed protocol for generating two transgenic mouse models: ER-TRG (constitutively expressing an ER lumen-targeting tandem RFP-GFP tag) and CA-ER-TRG (Cre-recombinase-activated ER-TRG). Additionally, we outline procedures for quantitative imaging of ER-phagy in vivo, covering tissue preparation, confocal microscopy, and signal analysis. This protocol offers a robust and reproducible tool for investigating ER-phagy dynamics across various tissues, developmental stages, and pathophysiological conditions, facilitating both fundamental and translational research.

Maintaining peroxisome homeostasis is crucial for cellular function and its disruption links to metabolic and neurodegenerative disorders. We developed PO-TRG mice ubiquitously expressing RFP-GFP-SKL to enable in vivo pexophagy monitoring. The probe was validated through cellular assays, immunostaining, autophagy perturbation, and age-dependent stability assessments. The model revealed tissue-specific basal pexophagy and dynamic changes during development. High-fat diet-induced obesity significantly reduced hepatic pexophagy, demonstrating metabolic sensitivity. Comparative analysis with mitophagy reporters showed both coordinated and distinct spatiotemporal patterns. We also created an inducible model (CA-PO-TRG) that eliminated cardiac artifacts and enabled neuronal analysis. These models provide robust tools for investigating pexophagy in physiological and pathological contexts.

FAM134/RETREG family members are ER-phagy receptors that maintain cellular homeostasis by regulating endoplasmic reticulum turnover. However, possible non-ER-phagy functions of FAM134 proteins remain elusive. Here, we show that RETREG3/FAM134C functions as a selective autophagy receptor for the type I BMP receptor (BMPRIA/ALK3) and recruits BMPRIA into LC3-containing autophagosomes for subsequent degradation. FAM134C-induced degradation diminishes the availability of BMP receptors and thus the strength of BMP signaling. Inhibition of autophagy through chemical means or knockdown of key autophagy regulators, ATG5 or Beclin-1, prevents BMPR1A degradation. Additionally, disruption of the putative LC3-interacting region (LIR) motif in FAM134C completely abolishes its interaction with LC3, thereby impeding its ability to degrade BMPR1A. Moreover, FAM134C-deficient mice exhibit enhanced BMP responses in the intestines, which affects intestinal crypt regeneration. Our findings suggest that FAM134C acts as a specific receptor that controls BMP signaling through the autophagic degradation of the type I BMP receptor, independent of its canonical role in ER-phagy.