Identifying the Molecular Cause of Extreme Endoplasmic Reticulum Dilation in Pediatric Osteosarcoma and Its Relationship to the Disease

Pediatric osteosarcoma tumors are characterized by an unusual abundance of grossly dilated endoplasmic reticulum and an immense genomic instability that has complicated identifying new effective molecular therapeutic targets. Here we report a novel molecular signature that encompasses the majority of 108 patient tumor samples, PDXs and osteosarcoma cell lines. These tumors exhibit reduced expression of four critical COPII vesicle proteins that has resulted in the accumulation of procollagen-I protein within ‘hallmark’ dilated ER. Using CRISPR activation technology, increased expression of only SAR1A and SEC24D to physiologically normal levels was sufficient to restore both collagen-I secretion and resolve dilated ER morphology to normal.

miR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles

Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV.

Sequences in the cytoplasmic tail of SARS-CoV-2 Spike facilitate expression at the cell surface and syncytia formation

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain, and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. Here we report a proteomic screen for cellular factors that interact with the cytoplasmic tail of S. We confirm interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding site promotes exit from the endoplasmic reticulum, and although binding to COPI should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

Mutant Cyclin F Impedes COPII Vesicle-Mediated ER-Golgi Trafficking and ER-Associated Degradation, Inducing ER Stress and Golgi Fragmentation in ALS/FTD

BackgroundMutations in the CCNF gene encoding cyclin F are associated with sporadic and familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying pathophysiological mechanisms are unknown. Proper functioning of the endoplasmic reticulum (ER) is essential for physiological cellular function. MethodsWe used human neuroblastoma SH-SY5Y and human embryonic kidney HEK293T cell lines and mouse primary neurons-overexpressing two familial ALS cyclin F mutants to examine whether mutant ALS/FTD-associated cyclin F perturbs key functions of the ER and Golgi compartments. Specific cellular assays were used to examine ER-Golgi transport (VSVGts045), the budding of vesicles from ER membranes and ER-associated degradation (ERAD). Immunocytochemistry was used to examine the morphology of the Golgi and ER-exit sites, and to detect ER stress and apoptosis. Western blotting was used to examine the content of vesicles budding from ER membranes and the interaction between Sec 31 and cyclin F. Flow cytometry was used to examine cell death.Results We demonstrated that mutant cyclin F inhibited protein transport from the ER to Golgi apparatus by a mechanism involving aberrant vesicle sorting from the ER. It also impeded ER-associated degradation, whereby misfolded ER proteins are ubiquitinated and degraded by the proteasome. This was associated with induction of ER stress and Golgi fragmentation, leading to apoptosis. Conclusion Together, these results demonstrate that ER dysfunction is a pathogenic pathway associated with ALS/FTD-variant cyclin F.

A new type of ERGIC-ERES membrane contact mediated by TMED9 and SEC12 is required for autophagosome biogenesis

Under stress, the endomembrane system undergoes reorganization to support autophagosome biogenesis, which is a central step in autophagy. How the endomembrane system remodels has been poorly understood. Here we identify a new type of membrane contact formed between the ER-Golgi intermediate compartment (ERGIC) and the ER-exit site (ERES) in the ER-Golgi system, which is essential for promoting autophagosome biogenesis induced by different stress stimuli. The ERGIC-ERES contact is established by the interaction between TMED9 and SEC12 which generates a short distance opposition (as close as 2-5 nm) between the two compartments. The tight membrane contact allows the ERES-located SEC12 to transactivate COPII assembly on the ERGIC. In addition, a portion of SEC12 also relocates to the ERGIC. Through both mechanisms, the ERGIC-ERES contact promotes formation of the ERGIC-derived COPII vesicle, a membrane precursor of the autophagosome. The ERGIC-ERES contact is physically and functionally different from the TFG-mediated ERGIC-ERES adjunction involved in secretory protein transport, and therefore defines a unique endomembrane structure generated upon stress conditions for autophagic membrane formation.

MIA3/TANGO1 enables efficient secretion by constraining COPII vesicle budding

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum. In vertebrates, this includes transport and Golgi organization protein 1 (TANGO1), encoded by the Mia3 gene. Here, using genome engineering of human cells light microscopy, secretion assays, and proteomics, we show loss of Mia3/TANGO1 results in formation of numerous vesicles and a loss of early secretory pathway integrity. This restricts secretion not only of large proteins like procollagens but of all types of secretory cargo. Our data shows that Mia3/TANGO1 constrains the propensity of COPII to form vesicles promoting instead the formation of the ER-Golgi intermediate compartment. Thus, Mia3/TANGO1 facilities the secretion of complex and high volume cargoes from vertebrate cells.

Collagen has a unique SEC24 preference for efficient export from the endoplasmic reticulum

ABSTRACTProcollagen requires COPII coat proteins for export from the endoplasmic reticulum (ER). SEC24 is the major component of the COPII proteins that selects cargo during COPII vesicle assembly. There are four paralogs (A to D) of SEC24 in mammals, which are classified into two subgroups. Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans and sec24d mutant fish also recapitulate this phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. To explore this possibility, we generated Sec24d knockout (KO) mice. Homozygous KO mice died prior to bone development. When we analyzed embryonic and extraembryonic tissues of mutant animals, we observed tissue-dependent defects of procollagen processing and ER export. The spacial patterns of these defects mirrored with SEC24B deficiency. By systematically knocking down the expression of Sec24 paralogs, we determined that, in addition to SEC24C and SEC24D, SEC24A and SEC24B also contribute to collagen secretion. In contrast, fibronectin 1 preferred either SEC24C or SEC24D. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.

Export Control: Post-transcriptional Regulation of the COPII Trafficking Pathway

The coat protein complex II (COPII) mediates forward trafficking of protein and lipid cargoes from the endoplasmic reticulum. COPII is an ancient and essential pathway in all eukaryotes and COPII dysfunction underlies a range of human diseases. Despite this broad significance, major aspects of COPII trafficking remain incompletely understood. For example, while the biochemical features of COPII vesicle formation are relatively well characterized, much less is known about how the COPII system dynamically adjusts its activity to changing physiologic cues or stresses. Recently, post-transcriptional mechanisms have emerged as a major mode of COPII regulation. Here, we review the current literature on how post-transcriptional events, and especially post-translational modifications, govern the COPII pathway.