Calcium flow at ER-TGN contact sites facilitates secretory cargo export.

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains mysterious, as the cytosol has a nanomolar steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), allowing protein-mediated exchange of molecular species such as lipids. Here, we show that the TGN export of secretory proteins requires the integrity of ER-TGN MCS and inositol 3 phosphate receptor (IP3R)-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real time. These data show that ER-TGN MCS facilitates the Ca2+ transfer required for Ca2+-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology.

Cab45 deficiency leads to the mistargeting of progranulin and prosaposin and aberrant lysosomal positioning.

The trans-Golgi Network (TGN) sorts molecular "addresses" and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 oligomerizes upon transient Ca2+ influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, we used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, we observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function.

Liquid-liquid phase separation facilitates the biogenesis of secretory storage granules.

Insulin is synthesized by pancreatic ß-cells and stored into secretory granules (SGs). SGs fuse with the plasma membrane in response to a stimulus and deliver insulin to the bloodstream. The mechanism of how proinsulin and its processing enzymes are sorted and targeted from the trans-Golgi network (TGN) to SGs remains mysterious. No cargo receptor for proinsulin has been identified. Here, we show that chromogranin (CG) proteins undergo liquid-liquid phase separation (LLPS) at a mildly acidic pH in the lumen of the TGN, and recruit clients like proinsulin to the condensates. Client selectivity is sequence-independent but based on the concentration of the client molecules in the TGN. We propose that the TGN provides the milieu for converting CGs into a "cargo sponge" leading to partitioning of client molecules, thus facilitating receptor-independent client sorting. These findings provide a new receptor-independent sorting model in ß-cells and many other cell types and therefore represent an innovation in the field of membrane trafficking.

The Discovery of the Buffer Capacity of Various Types of Polyelectrolyte Microcapsules.

Polyelectrolyte microcapsules, which are obtained by the method of alternate adsorption of oppositely charged polyelectrolytes onto colloidal particles of micron size, are widely used in science and industry. Nevertheless, the properties of microcapsules are still poorly understood. In particular, there is no information in the literature on the buffer capacity. However, information on the presence of a buffer capacity and an understanding of its mechanisms can both simplify the use of microcapsules and expand the scope of their application. In this regard, the buffer capacity of various types of microcapsules was studied. It was found that polyelectrolyte microcapsules consisting of polyallylamine, and polystyrene sulfonate have a buffer capacity. In addition, in an acidic medium, the buffer capacity of microcapsules containing BSA is significantly greater than that of microcapsules without protein. This is due to the fact that BSA contributes to the buffering of microcapsules. Differences in the behaviour of the buffer capacity of microcapsules with the composition (PAH/PSS)3 and (PSS/PAH)3 were found. In addition, a hypothesis has been proposed that regions of unbound polyallylamine are responsible for the buffering properties of polyelectrolyte microcapsules. This hypothesis is confirmed by the fact that incubation of microcapsules in 0.5 M NaCl increases the amount of unbound polyallylamine, which leads to an increase in the buffer capacity of microcapsules at alkaline pH values higher than the buffer capacity of capsules in an aqueous solution.

1-deoxysphingolipids bind to COUP-TF to modulate lymphatic and cardiac cell development.

Identification of physiological modulators of nuclear hormone receptor (NHR) activity is paramount for understanding the link between metabolism and transcriptional networks that orchestrate development and cellular physiology. Using libraries of metabolic enzymes alongside their substrates and products, we identify 1-deoxysphingosines as modulators of the activity of NR2F1 and 2 (COUP-TFs), which are orphan NHRs that are critical for development of the nervous system, heart, veins, and lymphatic vessels. We show that these non-canonical alanine-based sphingolipids bind to the NR2F1/2 ligand-binding domains (LBDs) and modulate their transcriptional activity in cell-based assays at physiological concentrations. Furthermore, inhibition of sphingolipid biosynthesis phenocopies NR2F1/2 deficiency in endothelium and cardiomyocytes, and increases in 1-deoxysphingosine levels activate NR2F1/2-dependent differentiation programs. Our findings suggest that 1-deoxysphingosines are physiological regulators of NR2F1/2-mediated transcription.

Sending out molecules from the TGN.

The sorting of secreted cargo proteins and their export from the trans-Golgi network (TGN) remains an enigma in the field of membrane trafficking; although the sorting mechanisms of many transmembrane proteins have been well described. The sorting of secreted proteins at the TGN is crucial for the release of signaling factors, as well as extracellular matrix proteins. These proteins are required for cell-cell communication and integrity of an organism. Missecretion of these factors can cause diseases such as neurological disorders, autoimmune disease, or cancer. The major open question is how soluble proteins that are not associated with the membrane are packed into TGN derived transport carriers to facilitate their transport to the plasma membrane. Recent investigations have identified novel types of protein and lipid machinery that facilitate the packing of these molecules into a TGN derived vesicle. In addition, novel research has uncovered an exciting link between cargo sorting and export in which TGN structure and dynamics, as well as TGN/endoplasmic reticulum contact sites, play a significant role. Here, we have reviewed the progress made in our understanding of these processes.

Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3.

Seizure protein 6 (SEZ6) is required for the development and maintenance of the nervous system, is a major substrate of the protease BACE1 and is linked to Alzheimer's disease (AD) and psychiatric disorders, but its molecular functions are not well understood. Here, we demonstrate that SEZ6 controls glycosylation and cell surface localization of kainate receptors composed of GluK2/3 subunits. Loss of SEZ6 reduced surface levels of GluK2/3 in primary neurons and reduced kainate-evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 in vitro and in vivo prevented modification of GluK2/3 with the human natural killer-1 (HNK-1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post-endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases.

Fam20C regulates protein secretion by Cab45 phosphorylation.

The TGN is a key compartment for the sorting and secretion of newly synthesized proteins. At the TGN, soluble proteins are sorted based on the instructions carried in their oligosaccharide backbones or by a Ca2+-mediated process that involves the cargo-sorting protein Cab45. Here, we show that Cab45 is phosphorylated by the Golgi-specific protein kinase Fam20C. Mimicking of phosphorylation translocates Cab45 into TGN-derived vesicles, which goes along with an increased export of LyzC, a Cab45 client. Our findings demonstrate that Fam20C plays a key role in the export of Cab45 clients by fine-tuning Cab45 oligomerization and thus impacts Cab45 retention in the TGN.

Dppa2/4 Facilitate Epigenetic Remodeling during Reprogramming to Pluripotency.

As somatic cells are converted into induced pluripotent stem cells (iPSCs), their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratios, DNA methylation patterns, and enhancer and promoter status. The molecular machinery underlying this process is largely unknown. Here, we show that embryonic stem cell (ESC)-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer, and are required for efficient reprogramming of mouse and human cells. When co-expressed with Oct4, Klf4, Sox2, and Myc (OKSM) factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in 2 to 4 days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway and contribute to downregulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.

A Non-canonical BCOR-PRC1.1 Complex Represses Differentiation Programs in Human ESCs.

Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems.

Ethyl-2-amino-pyrrole-3-carboxylates are novel potent anticancer agents that affect tubulin polymerization, induce G2/M cell-cycle arrest, and effectively inhibit soft tissue cancer cell growth in vitro.

Microtubules are known to be one of the most attractive and validated targets in cancer therapy. However, the clinical use of drugs that affect the dynamic state of microtubules has been hindered by chemoresistance and toxicity issues. Accordingly, the development of novel agents that target microtubules is needed. Here, we report the identification of novel compounds with pirrole and carboxylate structures: ethyl-2-amino-pyrrole-3-carboxylates (EAPCs) that provide potent cytotoxic activities against multiple soft tissue cancer cell lines in vitro. Using the MTS cell proliferation assay, we assessed the activity of EAPCs on various cancer cell lines including leiomyosarcoma SK-LMS-1, rhabdomyosarcoma RD, gastrointestinal stromal tumor GIST-T1, A-673 Ewing's sarcoma, and U-2 OS osteosarcoma. We found that in the majority of cases, two EAPC compounds (EAPC-20 and EAPC-24) considerably inhibited cancer cell proliferation in vitro. The growth-inhibitory effects of EAPC-20 and EAPC-24 were time and dose dependent. The molecular mechanisms of action of these compounds were because of the inhibition of tubulin polymerization and induction of a robust G2/M cell-cycle arrest, leading to considerable accumulation of tumor cells in the M-phase. Finally, EAPCs induced tumor cell death by apoptotic pathways. The above-mentioned effects were also observed in most soft tissue tumor cell lines and the gastrointestinal stromal tumor cell line investigated. Taken together, our data identify potent antitumor activity of EAPCs in vitro, thus providing a novel scaffold with which to develop potent chemotherapeutic agents for cancer therapy.

Gut-Associated Plasmacytoid Dendritic Cells Display an Immature Phenotype and Upregulated Granzyme B in Subjects with HIV/AIDS.

Plasmacytoid dendritic cells (pDCs) in the periphery of subjects with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) decrease over time, and the fate of these cells has been the subject of ongoing investigation. Previous studies using animal models as well as studies with humans suggest that these cells may redistribute to the gut. Other studies using animal models propose that the periphery pDCs are depleted and gut is repopulated with naive pDCs from the bone marrow. In the present study, we utilized immunohistochemistry to survey duodenum biopsies of subjects with HIV/AIDS and controls. We observed that subjects with HIV/AIDS had increased infiltration of Ki-67(+)/CD303(+) pDCs, a phenotype consistent with bone marrow-derived pre-pDCs. In contrast, Ki-67(+)/CD303(+) pDCs were not observed in control biopsies. We additionally observed that gut-associated pDCs in HIV/AIDS cases upregulate the proapoptotic enzyme granzyme B; however, no granzyme B was observed in the pDCs of control biopsies. Our data are consistent with reports in animal models that suggest periphery pDCs are depleted by exhaustion and that naive pDCs egress from the bone marrow and ultimately infiltrate the gut mucosa. Additionally, our observation of granzyme B upregulation in naive pDCs may identify a contributing factor to the gut pathology associated with HIV infection.