The pathway of collagen secretion.

COPII vesicles mediate export of secretory cargo from the endoplasmic reticulum (ER). However, a standard COPII vesicle with a diameter of 60-90 nm is too small to export collagens that are composed of rigid triple helices of up to 400 nm in length. How do cells pack and secrete such bulky molecules? This issue is fundamentally important, as collagens constitute approximately 25% of our dry body weight and are essential for almost all cell-cell interactions. Recently, a potential mechanism for the biogenesis of mega-transport carriers was identified, involving packing collagens and increasing the size of COPII coats. Packing is mediated by TANGO1, which binds procollagen VII in the lumen and interacts with the COPII proteins Sec23/Sec24 on the cytoplasmic side of the ER. Cullin3, an E3 ligase, and its specific adaptor protein, KLHL12, ubiquitinate Sec31, which could increase the size of COPII coats. Recruitment of these proteins and their specific interactors into COPII-mediated vesicle biogenesis may be all that is needed for the export of bulky collagens from the ER. Nonetheless, we present an alternative pathway in which TANGO1 and COPII cooperate to export collagens without generating a mega-transport carrier.

Membrane fission: the biogenesis of transport carriers.

Membrane-bound transport carriers are used to transfer cargo between membranes of the secretory and the endocytic pathways. The generation of these carriers can be classified into three steps: segregation of cargo away from the residents of a donor compartment (cargo sorting), generation of membrane curvature commensurate with the size of the cargo (membrane budding or tubulation), and finally separation of the nascent carrier from the donor membrane by a scission or membrane fission event. This review summarizes advances in our understanding of some of the best-characterized proteins required for the membrane fission that separates a transport carrier from its progenitor compartment: the large GTPase dynamin, the small guanine nucleotide-binding (G) proteins of the Arf family, BAR (Bin-amphiphysin-Rvs) domain proteins, and protein kinase D. These proteins share their ability to insert into membranes and oligomerize to create the large curvatures; however, the overall process of fission that involves these proteins appears to be quite different.

COPII vesicles get supersized by ubiquitin.

Some proteins are too big to fit into conventional COPII-coated vesicles, which raises the question of how large cargo, such as procollagen fibrils, are exported from the endoplasmic reticulum. Jin et al. (2012) in Nature now report that the creation of oversized vesicles is facilitated by the ubiquitination of the COPII component Sec31p.

A model for collagen secretion by intercompartmental continuities.

Newly synthesized secretory proteins are exported from the endoplasmic reticulum (ER) at specialized subcompartments called exit sites (ERES). Cargoes like procollagen are too large for export by the standard COPII-coated vesicle of 60 nm average diameter. We have previously suggested that procollagen is transported from the ER to the next secretory organelle, the ER-Golgi intermediate compartment (ERGIC), in TANGO1-dependent interorganelle tunnels. In the theoretical model presented here, we suggest that intrinsically disordered domains of TANGO1 in the ER lumen induce an entropic contraction, which exerts a force that draws procollagen toward the ERES. Within this framework, molecular gradients of pH and/or HSP47 between the ER and ERGIC create a force in the order of tens of femto-Newtons. This force is substantial enough to propel procollagen from the ER at a speed of approximately 1 nm · s-1. This calculated speed and the quantities of collagen secreted are similar to its observed physiological secretion rate in fibroblasts, consistent with the proposal that ER export is the rate-limiting step for procollagen secretion. Hence, the mechanism we propose is theoretically adequate to explain how cells can utilize molecular gradients and export procollagens at a rate commensurate with physiological needs.

The ulcerative colitis-associated gene FUT8 regulates the quantity and quality of secreted mucins.

Mucins are the main macrocomponents of the mucus layer that protects the digestive tract from pathogens. Fucosylation of mucins increases mucus viscoelasticity and its resistance to shear stress. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here, we show that levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients. Recapitulating this FUT8 overexpression in mucin-producing HT29-18N2 colonic cell line increases delivery of MUC1 to the plasma membrane and extracellular release of MUC2 and MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to removal from the cell surface than mucins secreted by FUT8-depleted cells (FUT8 KD). FUT8 KD causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. These data fit well with the Fut8-/- mice phenotype, which are protected from UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins. Together, our data reveal that FUT8 modifies the biophysical properties of mucus by controlling levels of cell surface MUC1 and quantity and quality of secreted MUC2 and MUC5AC. We suggest that these changes in mucus viscoelasticity likely facilitate bacterial-epithelial interactions leading to inflammation and UC progression.

TANGO1 facilitates cargo loading at endoplasmic reticulum exit sites.

A genome-wide screen revealed previously unidentified components required for transport and Golgi organization (TANGO). We now provide evidence that one of these proteins, TANGO1, is an integral membrane protein localized to endoplasmic reticulum (ER) exit sites, with a luminal SH3 domain and a cytoplasmic proline-rich domain (PRD). Knockdown of TANGO1 inhibits export of bulky collagen VII from the ER. The SH3 domain of TANGO1 binds to collagen VII; the PRD binds to the COPII coat subunits, Sec23/24. In this scenario, PRD binding to Sec23/24 subunits could stall COPII carrier biogenesis to permit the luminal domain of TANGO1 to guide SH3-bound cargo into a growing carrier. All cells except those of hematopoietic origin express TANGO1. We propose that TANGO1 exports other cargoes in cells that do not secrete collagen VII. However, TANGO1 does not enter the budding carrier, which represents a unique mechanism to load cargo into COPII carriers.

Novel Dent disease 1 cellular models reveal biological processes underlying ClC-5 loss-of-function.

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressing to chronic kidney disease. Although mutations in the electrogenic Cl-/H+ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype-phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. To further discover the mechanisms linking ClC-5 loss-of-function to proximal tubule dysfunction, we have generated novel DD1 cellular models depleted of ClC-5 and carrying ClC-5 mutants p.(Val523del), p.(Glu527Asp) and p.(Ile524Lys) using the human proximal tubule-derived RPTEC/TERT1 cell line. Our DD1 cellular models exhibit impaired albumin endocytosis, increased substrate adhesion and decreased collective migration, correlating with a less differentiated epithelial phenotype. Despite sharing functional features, these DD1 cell models exhibit different gene expression profiles, being p.(Val523del) ClC-5 the mutation showing the largest differences. Gene set enrichment analysis pointed to kidney development, anion homeostasis, organic acid transport, extracellular matrix organization and cell-migration biological processes as the most likely involved in DD1 pathophysiology. In conclusion, our results revealed the pathways linking ClC-5 mutations with tubular dysfunction and, importantly, provide new cellular models to further study DD1 pathophysiology.

Patient outcomes following implementation of an enhanced recovery after surgery pathway for patients with metastatic spine tumors.

BACKGROUND: Metastatic spine tumor surgery consists of palliative operations performed on frail patients with multiple medical comorbidities. Enhanced recovery after surgery (ERAS) programs involve an evidence-based, multidisciplinary approach to improve perioperative outcomes. This study presents clinical outcomes of a metastatic spine tumor ERAS pathway implemented at a tertiary cancer center. METHODS: The metastatic spine tumor ERAS program launched in April 2019, and data from January 2018 to May 2020 were reviewed. Measured outcomes included the following: hospital length of stay (LOS), time to ambulation, urinary catheter duration, time to resumption of diet, intraoperative fluid intake, estimated blood loss (EBL), and intraoperative and postoperative day 0-5 cumulative opioid use (morphine milligram equivalent [MME]). RESULTS: A total of 390 patients were included in the final analysis: 177 consecutive patients undergoing metastatic spine tumor surgery enrolled in the ERAS program and 213 consecutive pre-ERAS patients. Although the mean case durations were similar in the ERAS and pre-ERAS cohorts (265 vs. 274 min; p = .22), the ERAS cohort had decreased EBL (157 vs. 215 ml; p = .003), decreased postoperative day 0-5 cumulative mean opioid use (178 vs. 396 MME; p < .0001), earlier ambulation (mean, 34 vs. 57 h; p = .0001), earlier discontinuation of urinary catheters (mean, 36 vs. 56 h; p < .001), and shorter LOS (5.4 vs. 7.5 days; p < .0001). CONCLUSIONS: The implementation of a multidisciplinary ERAS program designed for metastatic spine tumor surgery led to improved clinical quality metrics, including shorter hospitalizations and significant reductions in opioid consumption.

Sphingolipid metabolic flow controls phosphoinositide turnover at the trans-Golgi network.

Sphingolipids are membrane lipids globally required for eukaryotic life. The sphingolipid content varies among endomembranes with pre- and post-Golgi compartments being poor and rich in sphingolipids, respectively. Due to this different sphingolipid content, pre- and post-Golgi membranes serve different cellular functions. The basis for maintaining distinct subcellular sphingolipid levels in the presence of membrane trafficking and metabolic fluxes is only partially understood. Here, we describe a homeostatic regulatory circuit that controls sphingolipid levels at the trans-Golgi network (TGN). Specifically, we show that sphingomyelin production at the TGN triggers a signalling pathway leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol and sphingolipid transport to the trans-Golgi network, PtdIns(4)P consumption interrupts this transport in response to excessive sphingomyelin production. Based on this evidence, we envisage a model where this homeostatic circuit maintains a constant lipid composition in the trans-Golgi network and post-Golgi compartments, thus counteracting fluctuations in the sphingolipid biosynthetic flow.

Unconventional protein secretion triggered by nutrient starvation.

It is usually assumed that eukaryotic cells secrete only proteins that contain a signal sequence for Sec61 mediated translocation into the lumen of endoplasmic reticulum (ER). Surprisingly however, many proteins, such as superoxide dismutase (SOD)1, acyl-CoA binding protein (Acb1), interleukin 1ß, fibroblast growth factor 2 and the adipokine Unpaired2, to name a few, are secreted even though they lack a signal sequence. The discovery that these proteins are secreted has presented a new challenge and we describe here a common pathway by which SOD1 and Acb1 are specifically secreted upon nutrient starvation. Their secretion follows a type III unconventional pathway, requiring the exposure of a di-acidic motif, which we propose promotes their capture into a membrane compartment called CUPS (compartment for unconventional protein secretion). We suggest that CUPS, composed of membranes derived from the Golgi apparatus and endosomes, serves as a major sorting station prior to release of SOD1 and Acb1 into the extracellular space. The trafficking of these signal sequence lacking proteins therefore has functional similarities to conventional protein secretion in that they rely on membrane bounded compartments for their sorting and transport, but bypass the need of Sec61 for translocating into the ER and COPII and COPI for their intracellular transfers. This review is part of a Special Issue of SCDB on "unconventional protein secretion" edited by Walter Nickel and Catherine Rabouille.

Sodium channel TRPM4 and sodium/calcium exchangers (NCX) cooperate in the control of Ca2+-induced mucin secretion from goblet cells.

Regulated mucin secretion is essential for the formation of the mucus layer that protects the underlying epithelial cells from foreign particles. Alterations in the quantity or quality of secreted mucins are therefore detrimental to airway and colon physiology. Based on various biochemical assays in several human cell lines, we report here that Na+/Ca2+ exchanger 2 (NCX2) works in conjunction with transient receptor potential cation channel subfamily M member 4 (TRPM4), and perhaps TRPM5, Na+ channels to control Ca2+-mediated secretion of both mucin 2 (MUC2) and MUC5AC from HT29-18N2 colonic cancer cells. Differentiated normal bronchial epithelial (NHBE) cells and tracheal cells from patients with cystic fibrosis (CFT1-LC3) expressed only TRPM4 and all three isoforms of NCXs. Blocking the activity of TRPM4 or NCX proteins abrogated MUC5AC secretion from NHBE and CFT1-LC3 cells. Altogether, our findings reveal that NCX and TRPM4/TRPM5 are both required for mucin secretion. We therefore propose that these two proteins could be potential pharmacological targets to control mucus-related pathologies such as cystic fibrosis.

Protein kinase D regulates metabolism and growth by controlling secretion of insulin like peptide.

Mechanisms coupling growth and metabolism are conserved in Drosophila and mammals. In metazoans, such coupling is achieved across tissue scales through the regulated secretion of chemical messengers such as insulin that control the metabolism and growth of cells. Although the regulated secretion of Insulin like peptide (dILP) is key to normal growth and metabolism in Drosophila, the sub-cellular mechanisms that regulate dILP release remain poorly understood. We find that reduced function of the only protein kinase D in Drosophila (dPKDH) results in delayed larval growth and development associated with abnormal sugar and lipid metabolism, reduced insulin signalling and accumulation of dILP2 in the neurosecretory IPCs of the larval brain. These phenotypes are rescued by tissue-selective reconstitution of dPKD in the neurosecretory cells of dPKDH. Selective downregulation of dPKD activity in the neurosecretory IPCs phenocopies the growth defects, metabolic abnormalities and dILP2 accumulation seen in dPKDH. Thus, dPKD mediated secretion of dILP2 from neurosecretory cells during development is necessary for normal larval growth.

Unconventional protein secretion: an evolving mechanism.

The process by which proteins are secreted without entering the classical endoplasmic reticulum (ER)-Golgi complex pathway, in eukaryotic cells, is conveniently called unconventional protein secretion. Recent studies on one such protein called Acb1 have revealed a number of components involved in its secretion. Interestingly, conditions that promote the secretion of Acb1 trigger the biogenesis of a new compartment called CUPS (Compartment for Unconventional Protein Secretion). CUPS form near the ER exit site but lack ER-specific proteins. Other proteins that share some of the features common with the secretion of Acb1 are interleukin-1ß and tissue transglutaminase. Here I will review recent advances made in the field and propose a new model for unconventional protein secretion.

MEK1 inactivates Myt1 to regulate Golgi membrane fragmentation and mitotic entry in mammalian cells.

The pericentriolar stacks of Golgi cisternae are separated from each other in G2 and fragmented extensively during mitosis. MEK1 is required for Golgi fragmentation in G2 and for the entry of cells into mitosis. We now report that Myt1 mediates MEK1's effects on the Golgi complex. Knockdown of Myt1 by siRNA increased the efficiency of Golgi complex fragmentation by mitotic cytosol in permeabilized and intact HeLa cells. Myt1 knockdown eliminated the requirement of MEK1 in Golgi fragmentation and alleviated the delay in mitotic entry due to MEK1 inhibition. The phosphorylation of Myt1 by MEK1 requires another kinase but is independent of RSK, Plk, and CDK1. Altogether our findings reveal that Myt1 is inactivated by MEK1 mediated phosphorylation to fragment the Golgi complex in G2 and for the entry of cells into mitosis. It is known that Myt1 inactivation is required for CDK1 activation. Myt1 therefore is an important link by which MEK1 dependent fragmentation of the Golgi complex in G2 is connected to the CDK1 mediated breakdown of Golgi into tubules and vesicles in mitosis.

Recruitment of arfaptins to the trans-Golgi network by PI(4)P and their involvement in cargo export.

The BAR (Bin/Amphiphysin/Rvs) domain proteins arfaptin1 and arfaptin2 are localized to the trans-Golgi network (TGN) and, by virtue of their ability to sense and/or generate membrane curvature, could play an important role in the biogenesis of transport carriers. We report that arfaptins contain an amphipathic helix (AH) preceding the BAR domain, which is essential for their binding to phosphatidylinositol 4-phosphate (PI(4)P)-containing liposomes and the TGN of mammalian cells. The binding of arfaptin1, but not arfaptin2, to PI(4)P is regulated by protein kinase D (PKD) mediated phosphorylation at Ser100 within the AH. We also found that only arfaptin1 is required for the PKD-dependent trafficking of chromogranin A by the regulated secretory pathway. Altogether, these findings reveal the importance of PI(4)P and PKD in the recruitment of arfaptins at the TGN and their requirement in the events leading to the biogenesis of secretory storage granules.

The Tri-Institutional Pain Registry-Analysis of Outpatient Pain Management at a Specialized Cancer Center.

Objectives: The Outpatient Pain Clinics at Memorial Sloan Kettering Cancer Center participated in developing a pain registry to gain insight on the referral and management of cancer pain as related to demographic information, cancer history, prescription records, and interventional pain procedures stored in the institutional database. Methods: Five cohorts (subsets of one another) were defined and compared to describe demographics and differences in management and outcomes by age, race, sex, and cancer type. Clinic patients were compared with the entire institution to determine factors associated with better pain relief and reduced side effects. Results: A small percentage were referred to a pain specialist. A total of 1,043 patients completed 3,544 surveys. Compared with the institution, there were higher proportions of patients age 51 to 60 years, nonwhites, and patients with thoracic, abdominal, and head and neck cancers. Medical management controlled pain with three drug categories in 40% of visits. Short-acting opioids were the only category that statistically provided good pain relief with fewer side effects. Pain scores were improved with increasing opioid dose. Management differed by sex, age, and race; women consistently had lower doses of opioids, poorer pain control, more side effects, and were prescribed a greater variety of medications. Conclusions: A limited set of medications was required to manage most patients in the clinic, supporting the continued place of opioids and the World Health Organization analgesic ladder in managing cancer pain. Women may need a more nuanced approach for obtaining the best balance of pain relief and side effects.

A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface.

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane-specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CARriers of the TGN to the cell Surface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)-G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.

Sphingomyelin organization is required for vesicle biogenesis at the Golgi complex.

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D-ceramide-C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D-ceramide-C6-treated HeLa cells revealed an increase in the levels of C6-sphingomyelin, C6-glucosylceramide, and diacylglycerol. D-ceramide-C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D-ceramide-C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6-sphingomyelin prevented liquid-ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.

Protein export at the ER: loading big collagens into COPII carriers.

COPII vesicles mediate the export of secretory cargo from endoplasmic reticulum (ER) exit sites. However, of 60-90 nm diameter COPII vesicles are too small to accommodate secreted molecules such as the collagens. The ER exit site-located proteins TANGO1 and cTAGE5 are required for the transport of collagens and therefore provide a means to understand the export of big cargo and the mechanism of COPII carrier size regulation commensurate with cargo dimensions.

Procollagen export from the endoplasmic reticulum.

Collagens are secreted into the extracellular space where they assemble into a large complex protein network to form basement membrane and extracellular matrix. Collagens are therefore essential for cell attachment, tissue organization and the overall survival of all multicellular organisms. Collagens are synthesized in the endoplasmic reticulum (ER) but they are too big to fit into a conventional coat protein complex II (COPII) transport carrier of 60-90 nm average diameter. How are these molecules exported from the ER and then transported along the secretory pathway? We describe here the involvement of special packing machinery composed of hetero oligomers of transport and Golgi organization 1 (TANGO1) and cutaneous T-cell lymphoma-associated antigen 5 (cTAGE5) in the export of procollagen VII from the ER.