Autophagy, an essential cellular process for maintaining cellular homeostasis and eliminating harmful cytoplasmic objects, involves the de novo formation of double‐membraned autophagosomes that engulf and degrade cellular debris, protein aggregates, damaged organelles, and pathogens. Central to this process is the phagophore, which forms from donor membranes rich in lipids synthesized at various cellular sites, including the endoplasmic reticulum (ER), which has emerged as a primary source. The ER‐associated omegasomes, characterized by their distinctive omega‐shaped structure and accumulation of phosphatidylinositol 3‐phosphate (PI3P), (...) play a pivotal role in autophagosome formation. Omegasomes are thought to serve as platforms for phagophore assembly by recruiting essential proteins such as DFCP1/ZFYVE1 and facilitating lipid transfer to expand the phagophore. Despite the critical importance of phagophore biogenesis, many aspects remain poorly understood, particularly the complete range of proteins involved in omegasome dynamics, and the detailed mechanisms of lipid transfer and membrane contact site formation. (shrink)

Macroautophagy is a major degradation mechanism of cell components via the lysosome. Macroautophagy greatly contributes to not only cell homeostasis but also the prevention of various diseases. Because macroautophagy proceeds through multi‐step reactions, researchers often face a persistent question of how macroautophagic activity can be measured correctly. To make a straightforward determination of macroautophagic activity, diverse monitoring assays have been developed. Direct measurement of lysosome‐dependent degradation of radioisotopically labeled cell proteins has long been applied. Meanwhile, indirect monitoring procedures have been (...) developed. In these assays, autophagosome marker proteins, microtubule‐associated proteins 1A/1B light chain 3B‐II (LC3B‐II) and gamma‐aminobutyric acid receptor‐associated protein‐II (GABARAP‐II) have been analyzed and the validity of the assays strongly depends on appropriate assessment of the fluctuation of LC3‐II and/or GABARAP‐II levels in the presence or absence of lysosomal inhibitors. This article describes these monitoring methods, paying special attention to the principles and characteristics of each procedure. (shrink)

There is a debate regarding the function of Drp1, a GTPase involved in mitochondrial fission, during the elimination of mitochondria by autophagy. A number of experiments indicate that Drp1 is needed to eliminate mitochondria during mitophagy, either by reducing the mitochondrial size or by providing a noncanonical mitophagy function. Yet, other convincing experimental results support the conclusion that Drp1 is not necessary. Here, we review the possible functions for Drp1 in mitophagy and autophagy, depending on tissues, organisms and stresses, and (...) discuss these apparent discrepancies. In this regard, it appears that the reduction of mitochondria size is often required for mitophagy but not always in a Drp1‐dependent manner. Finally, we speculate on Drp1‐independent mitochondrial fission mechanism that may take place during mitophagy and on noncanonical roles, which Drp1 may play such as modulating organelle contact sites dynamic during the autophagosome formation. (shrink)

Phosphatidylinositol 3‐phosphate (PtdIns3P) is generated on the cytosolic leaflet of cellular membranes, primarily by phosphorylation of phosphatidylinositol by class II and class III phosphatidylinositol 3‐kinases. The bulk of this lipid is found on the limiting and intraluminal membranes of endosomes, but it can also be detected in domains of phagosomes, autophagosome precursors, cytokinetic bridges, the plasma membrane and the nucleus. PtdIns3P controls cellular functions through recruitment of specific protein effectors, many of which contain FYVE or PX domains. Cellular processes (...) known to be controlled by PtdIns3P and its effectors include endosomal fusion, sorting and motility, autophagy, cytokinesis, regulated exocytosis and signal transduction. Here we discuss how Ptdins3P is generated on specific cellular membranes, how its localizations and functions can be studied, and how its effectors serve to control cellular functions.Editor's suggested further reading in BioEssays:Phosphatidylinositol 4,5‐bisphosphate: Targeted production and signalingAbstractHow does SHIP1/2 balance PtdIns(3,4)P2 and does it signal independently of its phosphatase activity?AbstractPhosphatidylinositol‐3,4,5‐trisphosphate: Tool of choice for class I PI 3‐kinasesAbstractPhosphatidylinositol‐4‐phosphate: The Golgi and beyondAbstract. (shrink)

Two key questions in the autophagy field are the mechanisms that underlie the signals for autophagy initiation and the source of membrane for expansion of the nascent membrane, the phagophore. In this review, we discuss recent findings highlighting the role of the classical endosomal pathway, from plasma membrane to lysosome, in the formation and expansion of the phagophore and subsequent degradation of the autophagosome contents. We also highlight the striking conservation of regulatory factors between the two pathways, including those (...) regulating membrane budding and fusion, and the role of the lysosome in sensing the nutrient status of the cell, regulating mTORC1 activity, and ultimately the initiation of autophagy.Editor's suggested further reading in BioEssays The evolution of dynamin to regulate clathrin‐mediated endocytosis Abstract. (shrink)

Autophagy functions in both selective and non‐selective ways to maintain cellular homeostasis. Endoplasmic reticulum autophagy (ER‐phagy) is a subclass of autophagy responsible for the degradation of the endoplasmic reticulum through selective encapsulation into autophagosomes. ER‐phagy occurs both under physiological conditions and in response to stress cues, and plays a crucial role in maintaining the homeostatic control of the organelle. Although specific receptors that target parts of the ER membrane, as well as, internal proteins for lysosomal degradation have been identified, the (...) molecular regulation of ER‐phagy has been elusive. Recent work has uncovered novel regulators of ER‐phagy that involve post‐translational modifications of ER‐resident proteins and functional cross‐talk with other cellular processes. Herein, we discuss how morphology affects the function of the peripheral ER, and how ER‐phagy modulates the turnover of this organelle. We also address how ER‐phagy is regulated at the molecular level, considering implications relevant to human diseases. (shrink)

Abnormalities in the ability of cells to properly degrade proteins have been identified in many neurodegenerative diseases. Recent work has implicated synaptojanin 1 (SynJ1) in Alzheimer's disease and Parkinson's disease, although the role of this polyphosphoinositide phosphatase in protein degradation has not been thoroughly described. Here, we dissected in vivo the role of SynJ1 in endolysosomal trafficking in zebrafish cone photoreceptors using a SynJ1‐deficient zebrafish mutant, nrca14. We found that loss of SynJ1 leads to specific accumulation of late endosomes and (...) autophagosomes early in photoreceptor development. An analysis of autophagic flux revealed that autophagosomes accumulate because of a defect in maturation. In addition we found an increase in vesicles that are highly enriched for PI(3)P, but negative for an early endosome marker in nrca14 cones. A mutational analysis of SynJ1 enzymatic domains found that activity of the 5'phosphatase, but not the Sac1 domain, is required to rescue both aberrant late endosomes and autophagosomes. Finally, modulating activity of the PI(4,5)P2 regulator, Arf6, rescued the disrupted trafficking pathways in nrca14 cones. Our study describes a specific role for SynJ1 in autophagosomal and endosomal trafficking and provides evidence that PI(4,5)P2 participates in autophagy in a neuronal cell type. -/- . (shrink)

Activity-dependent neuroprotective protein, discovered in our laboratory in 1999, has been characterized as a master gene vital for mammalian brain formation. ADNP de novo mutations in humans result in a syndromic form of autism-like spectrum disorder, including cognitive and motor deficits, the ADNP syndrome. One of the most important cellular processes associated with ADNP is the autophagy pathway, recently discovered by us as a key player in the pathophysiology of schizophrenia. In this regard, given the link between the microtubule and (...) autophagy systems, the ADNP microtubule end binding protein motif, namely, the neuroprotective NAP, was found to enhance autophagy while protecting microtubules and augmenting ADNP's association with both systems. Thus, linking autophagy and ADNP is proposed as a major target for intervention in brain diseases from autism to Alzheimer's disease and our findings introduce autophagy as a possible novel target for treating schizophrenia. Activity-dependent neuroprotective protein binds to microtubule-associated protein light chain 3, which forms the membrane of the autophagosome, a key organelle in the autophagy process and regulates beclin1 formation, an inducer of autophagy. Autophagy plays a major role in Alzheimer's disease and as recently discovered in schizophrenia and autism. (shrink)

Selective protein degradation maintains cellular homeostasis, but this process is disrupted in many diseases. Targeted protein degradation (TPD) approaches, built upon existing cellular mechanisms, are promising methods for therapeutically regulating protein levels. Here, we review the diverse palette of tools that are now available for doing so throughout the gene expression pathway and in specific cellular compartments. These include methods for directly removing targeted proteins via the ubiquitin proteasome system with proteolysis targeting chimeras (PROTACs) or dephosphorylation targeting chimeras (DEPTACs). Similar (...) effects can also be achieved through the lysosomal system with autophagy‐targeting chimeras (AUTACs), autophagosome tethering compounds (ATTECs), and lysosome targeting chimeras (LYTACs). Other methods act upstream to degrade RNAs (ribonuclease targeting chimeras; RIBOTACs) or transcription factors (transcription factor targeting chimeras; TRAFTACs), offering control throughout the gene expression process. We highlight the evolution and function of these methods and discuss their clinical implications in diverse disease contexts. (shrink)