Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites.

The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca2+ transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca2+ transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca2+ transients. Multi-modal SIM analysis revealed that Ca2+ transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca2+ transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca2+ transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.

TMCO1 Is an ER Ca(2+) Load-Activated Ca(2+) Channel.

Maintaining homeostasis of Ca(2+) stores in the endoplasmic reticulum (ER) is crucial for proper Ca(2+) signaling and key cellular functions. The Ca(2+)-release-activated Ca(2+) (CRAC) channel is responsible for Ca(2+) influx and refilling after store depletion, but how cells cope with excess Ca(2+) when ER stores are overloaded is unclear. We show that TMCO1 is an ER transmembrane protein that actively prevents Ca(2+) stores from overfilling, acting as what we term a "Ca(2+) load-activated Ca(2+) channel" or "CLAC" channel. TMCO1 undergoes reversible homotetramerization in response to ER Ca(2+) overloading and disassembly upon Ca(2+) depletion and forms a Ca(2+)-selective ion channel on giant liposomes. TMCO1 knockout mice reproduce the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, a developmental disorder linked to TMCO1 dysfunction, and exhibit severe mishandling of ER Ca(2+) in cells. Our findings indicate that TMCO1 provides a protective mechanism to prevent overfilling of ER stores with Ca(2+) ions.

Imaging elemental events of store-operated Ca2+ entry in invading cancer cells with plasmalemmal targeted sensors.

STIM1- and Orai1-mediated store-operated Ca2+ entry (SOCE) constitutes the major Ca2+ influx in almost all electrically non-excitable cells. However, little is known about the spatiotemporal organization at the elementary level. Here, we developed Orai1-tethered or palmitoylated biosensor GCaMP6f to report subplasmalemmal Ca2+ signals. We visualized spontaneous discrete and long-lasting transients ('Ca2+ glows') arising from STIM1-Orai1 in invading melanoma cells. Ca2+ glows occurred preferentially in single invadopodia and at sites near the cell periphery under resting conditions. Re-addition of external Ca2+ after store depletion elicited spatially synchronous Ca2+ glows, followed by high-rate discharge of asynchronous local events. Knockout of STIM1 or expression of the dominant-negative Orai1-E106A mutant markedly decreased Ca2+ glow frequency, diminished global SOCE and attenuated invadopodial formation. Functionally, invadopodial Ca2+ glows provided high Ca2+ microdomains to locally activate Ca2+/calmodulin-dependent Pyk2 (also known as PTK2B), which initiates the SOCE-Pyk2-Src signaling cascade required for invasion. Overall, the discovery of elemental Ca2+ signals of SOCE not only unveils a previously unappreciated gating mode of STIM1-Orai1 channels in situ, but also underscores a critical role of the spatiotemporal dynamics of SOCE in orchestrating complex cell behaviors such as invasion. This article has an associated First Person interview with the first author of the paper.

ORF3a of SARS-CoV-2 promotes lysosomal exocytosis-mediated viral egress.

Viral entry and egress are important determinants of virus infectivity and pathogenicity. ß-coronaviruses, including the COVID-19 virus SARS-CoV-2 and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. Here, we show that SARS-CoV-2 ORF3a, but not SARS-CoV ORF3a, promotes lysosomal exocytosis. SARS-CoV-2 ORF3a facilitates lysosomal targeting of the BORC-ARL8b complex, which mediates trafficking of lysosomes to the vicinity of the plasma membrane, and exocytosis-related SNARE proteins. The Ca2+ channel TRPML3 is required for SARS-CoV-2 ORF3a-mediated lysosomal exocytosis. Expression of SARS-CoV-2 ORF3a greatly elevates extracellular viral release in cells infected with the coronavirus MHV-A59, which itself lacks ORF3a. In SARS-CoV-2 ORF3a, Ser171 and Trp193 are critical for promoting lysosomal exocytosis and blocking autophagy. When these residues are introduced into SARS-CoV ORF3a, it acquires the ability to promote lysosomal exocytosis and inhibit autophagy. Our results reveal a mechanism by which SARS-CoV-2 interacts with host factors to promote its extracellular egress.

TMCO1-mediated Ca2+ leak underlies osteoblast functions via CaMKII signaling.

Transmembrane and coiled-coil domains 1 (TMCO1) is a recently identified Ca2+ leak channel in the endoplasmic reticulum. TMCO1 dysfunction in humans is associated with dysmorphism, mental retardation, glaucoma and the occurrence of cancer. Here we show an essential role of TMCO1 in osteogenesis mediated by local Ca2+/CaMKII signaling in osteoblasts. TMCO1 levels were significantly decreased in bone from both osteoporosis patients and bone-loss mouse models. Tmco1-/- mice exhibited loss of bone mass and altered microarchitecture characteristic of osteoporosis. In the absence of TMCO1, decreased HDAC4 phosphorylation resulted in nuclear enrichment of HADC4, which leads to deacetylation and degradation of RUNX2, the master regulator of osteogenesis. We further demonstrate that TMCO1-mediated Ca2+ leak provides local Ca2+ signals to activate the CaMKII-HDAC4-RUNX2 signaling axis. The establishment of TMCO1 as a pivotal player in osteogenesis uncovers a novel potential therapeutic target for ameliorating osteoporosis.

Programmed Cell Death 5 Provides Negative Feedback on Cardiac Hypertrophy Through the Stabilization of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a Protein.

PDCD5 (programmed cell death 5) is ubiquitously expressed in tissues, including the heart; however, the mechanism underlying the cardiac function of PDCD5 has not been understood. We investigated the mechanisms of PDCD5 in the pathogenesis of cardiac hypertrophy. Cardiac-specific PDCD5 knockout mice developed severe cardiac hypertrophy and impaired cardiac function, whereas PDCD5 protein was significantly increased in transverse aortic constriction mouse hearts and phenylephrine-stimulated cardiomyocytes. Overexpression of PDCD5 inhibited phenylephrine-induced cardiomyocyte hypertrophy, and knockdown of PDCD5 induced cardiomyocyte hypertrophy and aggravated phenylephrine-induced hypertrophy. The expression of PDCD5 protein was regulated by NFATc2 (nuclear factor of activated T cells c2) during hypertrophy. SERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase 2a) expression was decreased in PDCD5-deficient mouse hearts because of increased ubiquitination. PDCD5-deficient cardiomyocytes displayed decreased calcium uptake rate, slowed decay of Ca2+ transients, decreased calcium stores, and diastolic dysfunction. Moreover, reintroduction of PDCD5 in PDCD5-deficient mouse hearts reserved SERCA2a protein, suppressed NFATc2 protein, and rescued the hypertrophy and cardiac dysfunction. Our results revealed that PDCD5 is a novel target of NFATc2 in the hypertrophic heart and provides negative feedback to protect the heart against excessive hypertrophy via the stabilization of SERCA2a protein.

6-hydroximino-4-aza-A-homo-cholest-3-one and related analogue as a potent inducer of apoptosis in cancer cells.

Here we report that 6-hydroximino-4-aza-A-homo-cholest-3-one and 6-hydroxyl-4-aza-A-homo-cholest-3-one, new steroidal lactams were synthesized recently, displayed antiproliferative activity against some cancer cells through inducing cancer cell apoptosis by activation of the intrinsic pathway. The apoptotic function of the compounds was demonstrated by release of cytochrome C, activation of caspase 3 and annexin V labeling. Furthermore, the compound was able to inhibit tumor growth in an athymic mouse model.

Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells.

Accumulating evidence has shown that dysfunctional mitochondria can be selectively removed by mitophagy. Dysregulation of mitophagy is implicated in the development of neurodegenerative disease and metabolic disorders. How individual mitochondria are recognized for removal and how this process is regulated remain poorly understood. Here we report that FUNDC1, an integral mitochondrial outer-membrane protein, is a receptor for hypoxia-induced mitophagy. FUNDC1 interacted with LC3 through its typical LC3-binding motif Y(18)xxL(21), and mutation of the LC3-interaction region impaired its interaction with LC3 and the subsequent induction of mitophagy. Knockdown of endogenous FUNDC1 significantly prevented hypoxia-induced mitophagy, which could be reversed by the expression of wild-type FUNDC1, but not LC3-interaction-deficient FUNDC1 mutants. Mechanistic studies further revealed that hypoxia induced dephosphorylation of FUNDC1 and enhanced its interaction with LC3 for selective mitophagy. Our findings thus offer insights into mitochondrial quality control in mammalian cells.