Inhibition of the mitochondria-shaping protein Opa1 restores sensitivity to Gefitinib in a lung adenocarcinomaresistant cell line.

Drug resistance limits the efficacy of chemotherapy and targeted cancer treatments, calling for the identification of druggable targets to overcome it. Here we show that the mitochondria-shaping protein Opa1 participates in resistance against the tyrosine kinase inhibitor gefitinib in a lung adenocarcinoma cell line. Respiratory profiling revealed that oxidative metabolism was increased in this gefitinib-resistant lung cancer cell line. Accordingly, resistant cells depended on mitochondrial ATP generation, and their mitochondria were elongated with narrower cristae. In the resistant cells, levels of Opa1 were increased and its genetic or pharmacological inhibition reverted the mitochondrial morphology changes and sensitized them to gefitinib-induced cytochrome c release and apoptosis. In vivo, the size of gefitinib-resistant lung orthotopic tumors was reduced when gefitinib was combined with the specific Opa1 inhibitor MYLS22. The combo gefitinib-MYLS22 treatment increased tumor apoptosis and reduced its proliferation. Thus, the mitochondrial protein Opa1 participates in gefitinib resistance and can be targeted to overcome it.

Vacuolar protein Tag1 and Atg1-Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae.

Under starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10-12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

Pulmonary neuroendocrine cells: physiology, tissue homeostasis and disease.

Mammalian lungs have the ability to recognize external environments by sensing different compounds in inhaled air. Pulmonary neuroendocrine cells (PNECs) are rare, multi-functional epithelial cells currently garnering attention as intrapulmonary sensors; PNECs can detect hypoxic conditions through chemoreception. Because PNEC overactivation has been reported in patients suffering from respiratory diseases - such as asthma, chronic obstructive pulmonary disease, bronchopulmonary dysplasia and other congenital diseases - an improved understanding of the fundamental characteristics of PNECs is becoming crucial in pulmonary biology and pathology. During the past decade, murine genetics and disease models revealed the involvement of PNECs in lung ventilation dynamics, mechanosensing and the type 2 immune responses. Single-cell RNA sequencing further unveiled heterogeneous gene expression profiles in the PNEC population and revealed that a small number of PNECs undergo reprogramming during regeneration. Aberrant large clusters of PNECs have been observed in neuroendocrine tumors, including small-cell lung cancer (SCLC). Modern innovation of imaging analyses has enabled the discovery of dynamic migratory behaviors of PNECs during airway development, perhaps relating to SCLC malignancy. This Review summarizes the findings from research on PNECs, along with novel knowledge about their function. In addition, it thoroughly addresses the relevant questions concerning the molecular pathology of pulmonary diseases and related therapeutic approaches.

P73 C-terminus is dispensable for multiciliogenesis.

The p53 family transcriptional factor p73 plays a pivotal role in development. Ablation of p73 results in severe neurodevelopmental defects, chronic infections, inflammation and infertility. In addition to this, Trp73-\- mice display severe alteration in the ciliated epithelial lining and the full-length N-terminal isoform TAp73 has been implicated in the control of multiciliogenesis transcriptional program. With our recently generated Trp73Δ13/Δ13 mouse model, we interrogate the physiological role of p73 C-terminal isoforms in vivo. Trp73Δ13/Δ13 mice lack exon 13 in Trp73 gene, producing an ectopic switch from the C-terminal isoforms p73α to p73ß. Trp73Δ13/Δ13 mice show a pattern of expression of TAp73 comparable to the wild-type littermates, indicating that the α to ß switch does not significantly alter the expression of the gene in this cell type. Moreover, Trp73Δ13/Δ13 do not display any significant alteration in the airway ciliated epithelium, suggesting that in this context p73ß can fully substitute the function of the longer isoform p73α. Similarly, Trp73Δ13/Δ13 ciliated epithelium of the brain ependyma also does appear defective. In this district however expression of TAp73 is not detectable, indicating that expression of the gene might be compensated by alternative mechanisms. Overall our work indicates that C-terminus p73 is dispensable for the multiciliogenesis program and suggests a possible tissue-specific effect of p73 alternative splicing.

The formation of solar-neighbourhood stars in two generations separated by 5 billion years.

The chemical compositions of stars encode those of the gas from which they formed, providing important clues regarding the formation histories of galaxies. A powerful diagnostic is the abundance of α elements (O, Mg, Si, S, Ca and Ti) relative to iron, [α/Fe]. The α elements are synthesized and injected into the interstellar medium by type II supernovae, which occur about ten million years after their originating stars form; by contrast, iron is returned to the interstellar medium by type Ia supernovae, which occur after a much longer timescale of roughly one billion years1. Periods of rapid star formation therefore tend to produce high-[α/Fe] stellar populations (because only type II supernovae have time to contribute to interstellar-medium enrichment as the stellar population forms), whereas low-[α/Fe] stars require periods of star formation that last more than a few billion years (over which timescales type Ia supernovae begin to affect the elemental composition of the interstellar medium more strongly than type II supernovae). The existence of two distinct groups of stars in the solar neighbourhood2-7, one with high [α/Fe] and the other with low [α/Fe], therefore suggests two different origins, but the mechanism by which this bimodal distribution arose remains unknown. Here we use a model of disk-galaxy evolution to show that the two episodes of star formation8 predicted by the 'cold flow' theory of galactic gas accretion9,10 also explain the observed chemical bimodality. In this scenario, the high-[α/Fe] stars form early, during an initial phase of accretion that involves infalling streams of cold primordial gas. There is then a hiatus of around two billion years until the shock-heated gas in the galactic dark-matter halo has cooled as a result of radiation and can itself commence accretion. The low-[α/Fe] stars form during this second phase. The peaks in these two star-formation episodes are separated by around five billion years. In addition, the large-scale variation in the abundance patterns of these two stellar populations that has been observed for the Milky Way5,7 is partially explained by the spatial variation in this gas-accretion history.

Mitochondrial dynamics coordinate cell differentiation.

Cells differentiate into specific and functional lineages to build up tissues. It has been shown in several tissues that mitochondrial morphology, levels of "mitochondria-shaping" proteins, and mitochondrial functions change upon differentiation. In this review, we highlight the significance of mitochondrial dynamics and functions in tissue development, cell differentiation, and reprogramming processes. Signalling cascades are critical for tissue stem cell maintenance and cell fate determination, and growing evidence demonstrates mitochondria could act as a centre of intra and extracellular signals to coordinate signalling pathways, such as Notch, Wnt, and YAP/TAZ signalling. Just an organelle, however, emerges as a master regulator of cell differentiation, and can be a target to manipulate cell fates.

Directed Migration of Pulmonary Neuroendocrine Cells toward Airway Branches Organizes the Stereotypic Location of Neuroepithelial Bodies.

The airway epithelium consists of diverse cell types, including neuroendocrine (NE) cells. These cells are thought to function as chemoreceptors and as a component of the stem cell niche as well as the cells of origin in small-cell lung cancer. NE cells often localize at bifurcation points of airway tubes, forming small clusters called neuroepithelial bodies (NEBs). To investigate NEB development, we established methods for 3D mapping and ex vivo 4D imaging of developing lungs. We found that NEBs localize at stereotypic positions in the bifurcation area irrespective of variations in size. Notch-Hes1 signaling contributes to the differentiation of solitary NE cells, regulating their number but not localization. Live imaging revealed that individual NE cells migrate distally to and cluster at bifurcation points, driving NEB formation. We propose that NEB development is a multistep process involving differentiation of individual NE cells and their directional migration to organize NEBs.

Region-specific regulation of cell proliferation by FGF receptor signaling during the Wolffian duct development.

The Wolffian duct (WD) is a primordium of the male reproductive tract and kidney collecting duct system. Fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase (RTK) family, are essential for kidney development. Although the functions of FGFR signaling in kidney morphogenesis have been analyzed, their function in WD development has not been comprehensively investigated. Here, we demonstrate that Fgfr2 is the major Fgfr gene expressed throughout the WD epithelia and that it is essential for the maintenance of the WD, specifically in the caudal part of the WD. Hoxb7-Cre mediated inactivation of Fgfr2 in the mouse WD epithelia resulted in the regression of the caudal part of the WD and abnormal male reproductive tract development. Cell proliferation and expression of the downstream target genes of RTK signaling (Etv4 and Etv5) were decreased in the caudal part of the WD epithelia in the mutant embryos. Cranial (rostral) WD formation and ureteric budding were not affected. Ret, Etv4, and Etv5 expression were sustained in the ureteric bud of the mutant embryos. Taken together, these data suggest region-specific requirements for FGFR2 signaling in the developing caudal WD epithelia.

Mild reduction of plasmalogens causes rhizomelic chondrodysplasia punctata: functional characterization of a novel mutation.

Rhizomelic chondrodysplasia punctata (RCDP) is an autosomal recessive disorder due to the deficiency in ether lipid synthesis. RCDP type 1, the most prominent type, is caused by the dysfunction of the receptor of peroxisome targeting signal type 2, Pex7 (peroxisomal biogenesis factor 7), and the rest of the patients, RCDP types 2 and 3, have defects in peroxisomal enzymes catalyzing the initial two steps of alkyl-phospholipid synthesis, glyceronephosphate O-acyltransferase and alkylglycerone phosphate synthase (Agps). We herein investigated defects of two patients with RCDP type 3. Patient 1 had a novel missense mutation, T1533G, resulting in the I511M substitution in Agps. The plasmalogen level was mildly reduced, whereas the protein level and peroxisomal localization of Agps-I511M in fibroblasts were normal as in the control fibroblasts. Structure prediction analysis suggested that the mutated residue was located in the helix α15 on the surface of V-shaped active site tunnel in Agps, likely accounting for the mild defects of plasmalogen synthesis. These results strongly suggest that an individual with mildly affected level of plasmalogen synthesis develops RCDP. In fibroblasts from patient 2, the expression of AGPS mRNA and Agps protein was severely affected, thereby giving rise to the strong reduction of plasmalogen synthesis.

System to quantify the import of peroxisomal matrix proteins by fluorescence intensity.

Fourteen distinct peroxins are essential for peroxisome biogenesis in mammals, of which ten are involved in the import of matrix proteins into peroxisomes. Peroxisomal matrix protein import is regulated by various cellular factors; however, the mechanisms underlying this regulation are poorly understood. This is primarily because no quantitative detection method with high resolution is available to study the import of peroxisomal matrix proteins. Here, we developed a monitoring system that uses a fluorescent reporter that is stabilized in peroxisomes but is degraded in the cytosol. An FK506 binding protein 12 variant, termed destabilization domain (DD), is rapidly and constitutively degraded by proteasomes when expressed in mammalian cells. DD is reversibly protected by the addition of a specific synthetic ligand. In the absence of the ligand, a reporter molecule, enhanced GFP (EGFP) fused with DD and peroxisomal targeting signal 1 (DD-EGFP-PTS1), is largely degraded in the cytosol. By contrast, in the presence of the ligand, the reporter is stabilized and translocates into peroxisomes. Upon withdrawal of the ligand, the reporter in peroxisomes remains intact, whereas that in the cytosol is rapidly degraded. Thus, peroxisomal protein import can be readily quantified by measuring the fluorescence intensity of whole cells.

AWP1/ZFAND6 functions in Pex5 export by interacting with cys-monoubiquitinated Pex5 and Pex6 AAA ATPase.

During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.

An integrated diagnosis and therapeutic system using intra-operative 5-aminolevulinic-acid-induced fluorescence guided robotic laser ablation for precision neurosurgery.

We have developed an integrated diagnosis and therapeutic system for precision malignant gliomas resection during neurosurgery. A combination of three-dimensional (3-D) magnetic resonance imaging (MRI) navigation and 5-aminolevulinic acid (5-ALA)-induced fluorescence based intra-operative tumor diagnosis technique has been incorporated into a robotic laser ablation neurosurgery system with an automatic focusing and robotic scanning mechanism. 5-ALA is a non-fluorescent prodrug that leads to intracellular accumulation of fluorescent protoporphyrins IX (PpIX) in malignant glioma. The PpIX tends to accumulate in pathological lesions, and emits red fluorescence when excited by blue light. This fluorescence is illuminated with laser excitation, enables intra-operative identification of the position of a tumor and provides guidance for resection with laser photocoagulation. The information provided by the MRI is enhanced by the intra-operative 5-ALA fluorescence data, and this enhanced information is integrated into a robotic laser ablation system. The accuracy of the fluorescent measurement of the tumor is improved using high-precision spectral analysis. The fluorescence assists in the detection of malignant brain tumors intra-operatively and improves their removal rate.

Combination of intraoperative 5-aminolevulinic acid-induced fluorescence and 3-D MR imaging for guidance of robotic laser ablation for precision neurosurgery.

A combination of 5-aminolevulinic acid (5-ALA)-induced fluorescence and three-dimensional (3-D) magnetic resonance imaging (MRI) of a brain tumor has been incorporated into a robotic laser ablation neurosurgery system. 5-ALA is a non-fluorescent prodrug that leads to intracellular accumulation of fluorescent protoporphyrins IX (PpIX) in malignant glioma. The PpIX tends to accumulate in pathological lesions, and emits red fluorescence when excited by blue light. This fluorescence is illuminated with laser excitation, enables intra-operative identification of the position of a tumor and provides guidance for resection with laser photocoagulation. The information provided by the MRI is enhanced by the 5-ALA fluorescence data, and this enhanced information is integrated into a robotic laser ablation system. The accuracy of the fluorescent measurement of the tumor is improved using high-precision spectral analysis. The fluorescence assists in the detection of malignant brain tumors intraoperatively and improves their removal rate.

A novel robotic laser ablation system for precision neurosurgery with intraoperative 5-ALA-induced PpIX fluorescence detection.

We developed a combined system of tumor detection by 5-ALA-induced PpIX fluorescence and precise ablation by micro laser for the first time, with an automatic focusing and robotic scanning mechanism for the brain surface. 5-ALA accumulates on tumors to be metabolized to become PpIX that is a fluorescent. Intra-operative detection of 5-ALA induced PpIX fluorescence provides useful information for tumor detection. The wavelength of the micro laser is 2.8 microm close to the absorption band of water. This laser is effective only on the surface of brain tissue, enabling precise ablation at the boundary between tumor and normal tissue identified by intra-operative 5-ALA induced fluorescence. Combination tests of the fluorescence measurement and the laser ablation were performed, and it was possible to extract the area with fluorescence appropriately from the measurement data, and the micro laser with automatically scanning selectively ablated the extracted area.