The unfolded protein response regulates ER exit sites via SNRPB-dependent RNA splicing and contributes to bone development.

Splicing and endoplasmic reticulum (ER)-proteostasis are two key processes that ultimately regulate the functional proteins that are produced by a cell. However, the extent to which these processes interact remains poorly understood. Here, we identify SNRPB and other components of the Sm-ring, as targets of the unfolded protein response and novel regulators of export from the ER. Mechanistically, The Sm-ring regulates the splicing of components of the ER export machinery, including Sec16A, a component of ER exit sites. Loss of function of SNRPB is causally linked to cerebro-costo-mandibular syndrome (CCMS), a genetic disease characterized by bone defects. We show that heterozygous deletion of SNRPB in mice resulted in bone defects reminiscent of CCMS and that knockdown of SNRPB delays the trafficking of type-I collagen. Silencing SNRPB inhibited osteogenesis in vitro, which could be rescued by overexpression of Sec16A. This rescue indicates that the role of SNRPB in osteogenesis is linked to its effects on ER-export. Finally, we show that SNRPB is a target for the unfolded protein response, which supports a mechanistic link between the spliceosome and ER-proteostasis. Our work highlights components of the Sm-ring as a novel node in the proteostasis network, shedding light on CCMS pathophysiology.

Truncated variants of MAGEL2 are involved in the etiologies of the Schaaf-Yang and Prader-Willi syndromes.

The neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11-q13. A deletion of the SNORD116 cluster, encoding small nucleolar RNAs, or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS, respectively. By investigation of their subcellular localization, we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2, a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases, we identified protein interaction partners for WT or mutant MAGEL2, in particular the survival motor neuron protein (SMN), involved in spinal muscular atrophy, and the fragile-X-messenger ribonucleoprotein (FMRP), involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism, while regulation of transcription was mainly observed for WT MAGEL2. Hence, we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus, including the SNORD116 cluster. Thereby, we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116, SNORD115, and SNORD109A, as well as protein-coding genes MKRN3 and SNRPN, thus bridging the gap between PWS and SYS.

Inhibition of Autophagy Alters Intracellular Transport of APP Resulting in Increased APP Processing.

Alzheimer's disease (AD) pathology is characterized by amyloid beta (Aß) plaques and dysfunctional autophagy. Aß is generated by sequential proteolytic cleavage of amyloid precursor protein (APP), and the site of intracellular APP processing is highly debated, which may include autophagosomes. Here, we investigated the involvement of autophagy, including the role of ATG9 in APP intracellular trafficking and processing by applying the RUSH system, which allows studying the transport of fluorescently labeled mCherry-APP-EGFP in a systematic way, starting from the endoplasmic reticulum. HeLa cells, expressing the RUSH mCherry-APP-EGFP system, were investigated by live cell imaging, immunofluorescence, and Western blot. We found that mCherry-APP-EGFP passed through the Golgi faster in ATG9 knockout cells. Furthermore, ATG9 deletion shifted mCherry-APP-EGFP from early endosomes and lysosomes toward the plasma membrane concomitant with reduced endocytosis. Importantly, this alteration in mCherry-APP-EGFP transport resulted in increased secreted mCherry-soluble APP and C-terminal fragment-EGFP. These effects were also phenocopied by pharmacological inhibition of ULK1, indicating that autophagy is regulating the intracellular trafficking and processing of APP. These findings contribute to the understanding of the role of autophagy in APP metabolism and could potentially have implications for new therapeutic approaches for AD.

Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1.

Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.

Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy.

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

The PIDDosome activates p53 in response to supernumerary centrosomes.

Centrosomes, the main microtubule-organizing centers in animal cells, are replicated exactly once during the cell division cycle to form the poles of the mitotic spindle. Supernumerary centrosomes can lead to aberrant cell division and have been causally linked to chromosomal instability and cancer. Here, we report that an increase in the number of mature centrosomes, generated by disrupting cytokinesis or forcing centrosome overduplication, triggers the activation of the PIDDosome multiprotein complex, leading to Caspase-2-mediated MDM2 cleavage, p53 stabilization, and p21-dependent cell cycle arrest. This pathway also restrains the extent of developmentally scheduled polyploidization by regulating p53 levels in hepatocytes during liver organogenesis. Taken together, the PIDDosome acts as a first barrier, engaging p53 to halt the proliferation of cells carrying more than one mature centrosome to maintain genome integrity.

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins.

Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT-dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin-5/EG5 and kinesin-12/KIF15, and our data suggest that the MT-flux driving force is transmitted from non-KT-MTs to KT-MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT-flux rate correlates with spindle length, and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we find that MT-flux is required to regulate spindle length by counteracting kinesin 13/MCAK-dependent MT-depolymerization. Thus, our study unveils the long-sought mechanism of MT-flux in human cells as relying on the coordinated action of four kinesins to compensate for MT-depolymerization and regulate spindle length.

Feedback control of the Gpr161-Gαs-PKA axis contributes to basal Hedgehog repression in zebrafish.

Hedgehog (Hh) ligands act as morphogens to direct patterning and proliferation during embryonic development. Protein kinase A (PKA) is a central negative regulator of Hh signalling, and in the absence of Hh ligands, PKA activity prevents inappropriate expression of Hh target genes. The orphan G-protein-coupled receptor Gpr161 contributes to the basal Hh repression machinery by activating PKA. Gpr161 acts as an A-kinase-anchoring protein, and is itself phosphorylated by PKA, but the functional significance of PKA phosphorylation of Gpr161 in the context of Hh signalling remains unknown. Here, we show that loss of Gpr161 in zebrafish leads to constitutive activation of medium and low, but not maximal, levels of Hh target gene expression. Furthermore, we find that PKA phosphorylation-deficient forms of Gpr161, which we show directly couple to Gαs, display an increased sensitivity to Shh, resulting in excess high-level Hh signalling. Our results suggest that PKA feedback-mediated phosphorylation of Gpr161 may provide a mechanism for fine-tuning Gpr161 ciliary localisation and PKA activity.

Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown.

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

The lipid environment modulates cardiolipin and phospholipid constitution in wild type and tafazzin-deficient cells.

Deficiency of the transacylase tafazzin due to loss of function variants in the X-chromosomal TAFAZZIN gene causes Barth syndrome (BTHS) with severe neonatal or infantile cardiomyopathy, neutropenia, myopathy, and short stature. The condition is characterized by drastic changes in the composition of cardiolipins, a mitochondria-specific class of phospholipids. Studies examining the impact of tafazzin deficiency on the metabolism of other phospholipids have so far generated inhomogeneous and partly conflicting results. Recent studies showed that the cardiolipin composition in cells and different murine tissues is highly dependent on the surrounding lipid environment. In order to study the relevance of different lipid states and tafazzin function for cardiolipin and phospholipid homeostasis we conducted systematic modulation experiments in a CRISPR/Cas9 knock-out model for BTHS. We found that-irrespective of tafazzin function-the composition of cardiolipins strongly depends on the nutritionally available lipid pool. Tafazzin deficiency causes a consistent shift towards cardiolipin species with more saturated and shorter acyl chains. Interestingly, the typical biochemical BTHS phenotype in phospholipid profiles of HEK 293T TAZ knock-out cells strongly depends on the cellular lipid context. In response to altered nutritional lipid compositions, we measured more pronounced changes on phospholipids that were largely masked under standard cell culturing conditions, therewith giving a possible explanation for the conflicting results reported so far on BTHS lipid phenotypes.