Protein condensates in the the secretory pathway: Unraveling biophysical interactions and function.

The emergence of phase separation phenomena among macromolecules has identified biomolecular condensates as fundamental cellular organizers. These condensates concentrate specific components and accelerate biochemical reactions without relying on membrane boundaries. Although extensive studies have revealed a large variety of nuclear and cytosolic membraneless organelles, we are witnessing a surge in the exploration of protein condensates associated with the membranes of the secretory pathway, such as the endoplasmic reticulum and the Golgi apparatus. This review focuses on protein condensates in the secretory pathway and discusses their impact on the organization and functions of this cellular process. Moreover, we explore the modes of condensate-membrane association and the biophysical and cellular consequences of protein condensate interactions with secretory pathway membranes.

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.

Sorting secretory proteins.

A receptor protein called TGN46 has an important role in sorting secretory proteins into vesicles going to different destinations inside cells.

Rediscovering the intricacies of secretory granule biogenesis.

Regulated secretion, an essential cellular process, relies on secretory granules (SGs) for the controlled release of a diverse range of cargo molecules, including proteins, peptides, hormones, enzymes, and neurotransmitters. SG biogenesis encompasses cargo selection, sorting, packaging, and trafficking, with the trans-Golgi Network (TGN) playing a central role. Research in the last three decades has revealed significant components required for SG biogenesis; however, no cargo receptor transferring granule cargo from the TGN to immature SGs (ISGs) has yet been identified. Consequently, recent research has devoted significant attention to studying receptor-independent cargo sorting mechanisms, shedding new light on the complexities of regulated secretion. Understanding the underlying molecular and biophysical mechanisms behind cargo sorting into ISGs holds great promise for advancing our knowledge of cellular communication and disease mechanisms.

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.

Quantification of Protein Exit at the Trans-Golgi Network.

With one-third of all newly synthesized proteins entering the secretory pathway, correct protein sorting is essential for cellular homeostasis. In the last three decades, researchers have developed numerous biochemical, genetic, and cell biological approaches to study protein export and sorting from the trans-Golgi network (TGN). However, accurately quantifying protein transport from one compartment to the next in the secretory pathway has been challenging. The Retention Using Selective Hooks (RUSH) system is a method that allows monitoring trafficking of a protein of interest in real time, similar to a pulse-chase experiment but without the need of radiolabeling. Accurate calculations, however, are necessary and currently lacking. Here, we combine the RUSH system with live cell imaging to quantify and calculate half lives. We exemplify our approach using a soluble secreted protein (LyzC). This system will benefit membrane trafficking researchers by adding numbers to protein export and comparing the export kinetics of different cargoes and variating conditions.

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.

Cargo sorting at the trans-Golgi network at a glance.

The Golgi functions principally in the biogenesis and trafficking of glycoproteins and lipids. It is compartmentalized into multiple flattened adherent membrane sacs termed cisternae, which each contain a distinct repertoire of resident proteins, principally enzymes that modify newly synthesized proteins and lipids sequentially as they traffic through the stack of Golgi cisternae. Upon reaching the final compartments of the Golgi, the trans cisterna and trans-Golgi network (TGN), processed glycoproteins and lipids are packaged into coated and non-coated transport carriers derived from the trans Golgi and TGN. The cargoes of clathrin-coated vesicles are chiefly residents of endo-lysosomal organelles, while uncoated carriers ferry cargo to the cell surface. There are outstanding questions regarding the mechanisms of protein and lipid sorting within the Golgi for export to different organelles. Nonetheless, conceptual advances have begun to define the key molecular features of cargo clients and the mechanisms underlying their sorting into distinct export pathways, which we have collated in this Cell Science at a Glance article and the accompanying poster.

Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Live-cell Vesicle Tracking in the Secretory Pathway Using HeLa Cells.

More than 30% of the total amount of proteins synthesized in mammalian cells follow the secretory pathway in order to mature and be properly sorted to their final destinations. Among several methodologies that describe live-cell monitoring of vesicles, the Retention Using Selective Hooks (RUSH) system is a powerful one that allows to visualize cargo trafficking under physiological conditions. The present protocol describes a method to use the RUSH system in live-cell microscopy and a subsequent quantitative analysis of cargo vesicles to dissect protein trafficking. In brief, HeLa cells are transiently transfected with an MMP2-RUSH construct and vesicle trafficking is evaluated by wide-field microscopy, recording videos in 1-min time frames for 45 min. We also present a quantitative approach that can be used to identify kinetics of uncharacterized protein cargo, as well as to evaluate with more detail processes such as ER-to-Golgi vesicle trafficking. Graphic abstract: Live-cell RUSH: a tool to monitor real-time protein trafficking in the secretory pathway.

Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells.

Monitoring vesicle trafficking is an excellent tool for the evaluation of protein dynamics in living cells. Such study is key for the understanding of protein sorting and secretion. Recent developments in microscopy, as well as new methodologies developed to study synchronized trafficking of proteins, allowed a better understanding of signaling, regulation and trafficking dynamics at the secretory pathway. One of the most helpful tools so far developed is the Retention Using Selective Hooks (RUSH) system, a methodology that facilitates the evaluation of synchronized cargo trafficking by monitoring fluorescent vesicles in cells upon biotin addition. Here we present a protocol that allows the quantitative evaluation of protein cargo trafficking at different fixed time points and an analytic approach that enables a better examination of specific cargo trafficking dynamics at the secretory pathway. Graphic abstract: Schematic representation of RUSH sorting assay in mammalian cells.

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.

Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs.

Matrix metalloproteinases (MMPs) degrade several ECM components and are crucial modulators of cell invasion and tissue organization. Although much has been reported about their function in remodeling ECM in health and disease, their trafficking across the Golgi apparatus remains poorly understood. Here we report that the cis-Golgi protein nucleobindin-1 (NUCB1) is critical for MMP2 and MT1-MMP trafficking along the Golgi apparatus. This process is Ca2+-dependent and is required for invasive MDA-MB-231 cell migration as well as for gelatin degradation in primary human macrophages. Our findings emphasize the importance of NUCB1 as an essential component of MMP transport and its overall impact on ECM remodeling.

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.

Meeting Report from the 2019 "Organelle Zone" Symposium in Osaka, Japan.

On May 29 at the Osaka University Hospital, Japan, the "Organelle Zones" research grant group (see http://organellezone.org/english/) organized a one day symposium for its own members and four guest speakers, with about 60 attendees. The research group studies three different ways in which regions within organelles carry out functions distinct from other parts of the organelle. Work at this sub-organellar level is increasingly recognised as an important aspect of cell biology. The group's projects are divided into these themes with 9 Principal Investigators and 18 Co-Investigators over 5 years. The symposium, followed a similar meeting in 2018, and had 4 external speakers and 4 internal members of the consortium. The talks were divided into three sessions, each show-casing one way of sub-compartmentalising organelles into zones.

Lipid-dependent coupling of secretory cargo sorting and trafficking at the trans-Golgi network.

In eukaryotic cells, the trans-Golgi network (TGN) serves as a platform for secretory cargo sorting and trafficking. In recent years, it has become evident that a complex network of lipid-lipid and lipid-protein interactions contributes to these key functions. This review addresses the role of lipids at the TGN with a particular emphasis on sphingolipids and diacylglycerol. We further highlight how these lipids couple secretory cargo sorting and trafficking for spatiotemporal coordination of protein transport to the plasma membrane.

Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death.

The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.