The mouse Balbiani body regulates primary oocyte quiescence via RNA storage.

In mammalian females, the transition from dormancy in primordial follicles to follicular development is critical for maintaining ovarian function and reproductive longevity. In mice, the quiescent primary oocyte of the primordial follicle contains a Balbiani body (B-body), an organelle aggregate comprised of a spherical structure of Golgi complexes. Here we show that the structure of the B-body is maintained by microtubules and actin. The B-body stores mRNA-capping enzyme and 597 mRNAs associated with mRNA-decapping enzyme 1 A (DCP1A). Gene ontology analysis results indicate that proteins encoded by these mRNAs function in enzyme binding, cellular component organization and packing of telomere ends. Pharmacological depolymerization of microtubules or actin led to B-body disassociation and nascent protein synthesis around the dissociated B-bodies within three hours. An increased number of activated developing follicles were observed in ovaries with prolonged culture and the in vivo mouse model. Our results indicate that the mouse B-body is involved in the activation of dormant primordial follicles likely via translation of the B-body-associated RNAs in primary oocytes.

Sec23IP recruits VPS13B/COH1 to ER exit site-Golgi interface for tubular ERGIC formation.

VPS13B/COH1 is the only known causative factor for Cohen syndrome, an early-onset autosomal recessive developmental disorder with intellectual inability, developmental delay, joint hypermobility, myopia, and facial dysmorphism as common features, but the molecular basis of VPS13B/COH1 in pathogenesis remains largely unclear. Here, we identify Sec23 interacting protein (Sec23IP) at the ER exit site (ERES) as a VPS13B adaptor that recruits VPS13B to ERES-Golgi interfaces. VPS13B interacts directly with Sec23IP via the VPS13 adaptor binding domain (VAB), and the interaction promotes the association between ERES and the Golgi. Disease-associated missense mutations of VPS13B-VAB impair the interaction with Sec23IP. Knockout of VPS13B or Sec23IP blocks the formation of tubular ERGIC, an unconventional cargo carrier that expedites ER-to-Golgi transport. In addition, depletion of VPS13B or Sec23IP delays ER export of procollagen, suggesting a link between procollagen secretion and joint laxity in patients with Cohen disease. Together, our study reveals a crucial role of VPS13B-Sec23IP interaction at the ERES-Golgi interface in the pathogenesis of Cohen syndrome.

S-acylation of NLRP3 provides a nigericin sensitive gating mechanism that controls access to the Golgi.

NLRP3 is an inflammasome seeding pattern recognition receptor activated in response to multiple danger signals which perturb intracellular homeostasis. Electrostatic interactions between the NLRP3 polybasic (PB) region and negatively charged lipids on the trans-Golgi network (TGN) have been proposed to recruit NLRP3 to the TGN. In this study, we demonstrate that membrane association of NLRP3 is critically dependant on S-acylation of a highly conserved cysteine residue (Cys-130), which traps NLRP3 in a dynamic S-acylation cycle at the Golgi, and a series of hydrophobic residues preceding Cys-130 which act in conjunction with the PB region to facilitate Cys-130 dependent Golgi enrichment. Due to segregation from Golgi localised thioesterase enzymes caused by a nigericin induced breakdown in Golgi organisation and function, NLRP3 becomes immobilised on the Golgi through reduced de-acylation of its Cys-130 lipid anchor, suggesting that disruptions in Golgi homeostasis are conveyed to NLRP3 through its acylation state. Thus, our work defines a nigericin sensitive S-acylation cycle that gates access of NLRP3 to the Golgi.

A nanoscale visual exploration of the pathogenic effects of bacterial extracellular vesicles on host cells.

BACKGROUND: Bacterial extracellular vesicles (EVs) are pivotal mediators of intercellular communication and influence host cell biology, thereby contributing to the pathogenesis of infections. Despite their significance, the precise effects of bacterial EVs on the host cells remain poorly understood. This study aimed to elucidate ultrastructural changes in host cells upon infection with EVs derived from a pathogenic bacterium, Staphylococcus aureus (S. aureus). RESULTS: Using super-resolution fluorescence microscopy and high-voltage electron microscopy, we investigated the nanoscale alterations in mitochondria, endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and microtubules of skin cells infected with bacterial EVs. Our results revealed significant mitochondrial fission, loss of cristae, transformation of the ER from tubular to sheet-like structures, and fragmentation of the Golgi apparatus in cells infected with S. aureus EVs, in contrast to the negligible effects observed following S. epidermidis EV infection, probably due to the pathogenic factors in S. aureus EV, including protein A and enterotoxin. These findings indicate that bacterial EVs, particularly those from pathogenic strains, induce profound ultrastructural changes of host cells that can disrupt cellular homeostasis and contribute to infection pathogenesis. CONCLUSIONS: This study advances the understanding of bacterial EV-host cell interactions and contributes to the development of new diagnostic and therapeutic strategies for bacterial infections.

TMC7 deficiency causes acrosome biogenesis defects and male infertility in mice.

Transmembrane channel-like (TMC) proteins are a highly conserved ion channel family consisting of eight members (TMC1-TMC8) in mammals. TMC1/2 are components of the mechanotransduction channel in hair cells, and mutations of TMC1/2 cause deafness in humans and mice. However, the physiological roles of other TMC proteins remain largely unknown. Here, we show that Tmc7 is specifically expressed in the testis and that it is required for acrosome biogenesis during spermatogenesis. Tmc7-/- mice exhibited abnormal sperm head, disorganized mitochondrial sheaths, and reduced number of elongating spermatids, similar to human oligo-astheno-teratozoospermia. We further demonstrate that TMC7 is colocalized with GM130 at the cis-Golgi region in round spermatids. TMC7 deficiency leads to aberrant Golgi morphology and impaired fusion of Golgi-derived vesicles to the developing acrosome. Moreover, upon loss of TMC7 intracellular ion homeostasis is impaired and ROS levels are increased, which in turn causes Golgi and endoplasmic reticulum stress. Taken together, these results suggest that TMC7 is required to maintain pH and ion homeostasis, which is needed for acrosome biogenesis. Our findings unveil a novel role for TMC7 in acrosome biogenesis during spermiogenesis.

Cytokines on the way to secretion.

The activation of immune cells by pro-inflammatory or immunosuppressive stimuli is followed by the secretion of immunoregulatory cytokines which serve as messengers to activate the immune response in target cells. Although the mechanisms that control the secretion of cytokines by immune cells are not yet fully understood, several key aspects of this process have recently emerged. This review focuses on cytokine release via exocytosis and highlights the routes of cytokine trafficking leading to constitutive and regulated secretion as well as the impact of sorting receptors on this process. We discuss the involvement of cytoskeletal rearrangements in vesicular transport, secretion, and formation of immunological synapses. Finally, we describe the non-classical pathways of cytokine release that are independent of vesicular ER-Golgi transport. Instead, these pathways are based on processing by inflammasome or autophagic mechanisms. Ultimately, understanding the molecular mechanisms behind cytokine release may help to identify potential therapeutic targets in diseases associated with altered immune responses.

Cytosolic FKBPL and ER-resident CKAP4 co-regulates ER-phagy and protein secretion.

Endoplasmic reticulum quality control is crucial for maintaining cellular homeostasis and adapting to stress conditions. Although several ER-phagy receptors have been identified, the collaboration between cytosolic and ER-resident factors in ER fragmentation and ER-phagy regulation remains unclear. Here, we perform a phenotype-based gain-of-function screen and identify a cytosolic protein, FKBPL, functioning as an ER-phagy regulator. Overexpression of FKBPL triggers ER fragmentation and ER-phagy. FKBPL has multiple protein binding domains, can self-associate and might act as a scaffold connecting CKAP4 and LC3/GABARAPs. CKAP4 serves as a bridge between FKBPL and ER-phagy cargo. ER-phagy-inducing conditions increase FKBPL-CKAP4 interaction followed by FKBPL oligomerization at the ER, leading to ER-phagy. In addition, FKBPL-CKAP4 deficiency leads to Golgi disassembly and lysosome impairment, and an increase in ER-derived secretory vesicles and enhances cytosolic protein secretion via microvesicle shedding. Taken together, FKBPL with the aid of CKAP4 induces ER fragmentation and ER-phagy, and FKBPL-CKAP4 deficiency facilitates protein secretion.

TMX5/TXNDC15, a natural trapping mutant of the PDI family is a client of the proteostatic factor ERp44.

The ER is the organelle of nucleated cells that produces lipids, sugars, and proteins. More than 20 ER-resident members of the protein disulfide isomerase (PDI) family regulate formation, isomerization, and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4, and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least-known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multipass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57, and ERp44. The association between TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER localization of TMX5 in pre-Golgi compartments. Thus, TMX5 belongs to the family of proteins including Ero1α, Ero1ß, Prx4, ERAP1, and SUMF1 that operate in pre-Golgi compartments but lack localization sequences required to position themselves and rely on ERp44 engagement for proper intercompartmental distribution.

Organelle Communication with the Nucleus.

Compartmentalization of cellular components is critical to the spatiotemporal and environmental regulation of biochemical activities inside a cell, ensures the proper division of cellular labor and resources, and increases the efficiency of metabolic processes. However, compartmentalization also poses a challenge as organelles often need to communicate across these compartments to complete reaction pathways. These communication signals are often critical aspects of the cellular response to changing environmental conditions. A central signaling hub in the cell, the nucleus communicates with mitochondria, lysosomes, the endoplasmic reticulum, and the Golgi body to ensure optimal organellar and cellular performance. Here we review different mechanisms by which these organelles communicate with the nucleus, focusing on anterograde and retrograde signaling of mitochondria, localization-based signaling of lysosomes, the unfolded protein response of the endoplasmic reticulum, and evidence for nucleus-Golgi signaling. We also include a brief overview of some less well-characterized mechanisms of communication between non-nuclear organelles.

Molecular basis of TMED9 oligomerization and entrapment of misfolded protein cargo in the early secretory pathway.

Intracellular accumulation of misfolded proteins causes serious human proteinopathies. The transmembrane emp24 domain 9 (TMED9) cargo receptor promotes a general mechanism of cytotoxicity by entrapping misfolded protein cargos in the early secretory pathway. However, the molecular basis for this TMED9-mediated cargo retention remains elusive. Here, we report cryo-electron microscopy structures of TMED9, which reveal its unexpected self-oligomerization into octamers, dodecamers, and, by extension, even higher-order oligomers. The TMED9 oligomerization is driven by an intrinsic symmetry mismatch between the trimeric coiled coil domain and the tetrameric transmembrane domain. Using frameshifted Mucin 1 as an example of aggregated disease-related protein cargo, we implicate a mode of direct interaction with the TMED9 luminal Golgi-dynamics domain. The structures suggest and we confirm that TMED9 oligomerization favors the recruitment of coat protein I (COPI), but not COPII coatomers, facilitating retrograde transport and explaining the observed cargo entrapment. Our work thus reveals a molecular basis for TMED9-mediated misfolded protein retention in the early secretory pathway.

Subcellular activation of ß-adrenergic receptors using a spatially restricted antagonist.

Gprotein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of Food and Drug Administration-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of tools to separate GPCR signaling at the plasma membrane from the ones initiated at intracellular compartments. We leveraged the structural and pharmacological information available for ß-adrenergic receptors (ßARs) and focused on ß1AR as exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell-impermeable ßAR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited ß1AR on the plasma membrane. In contrast, a cell-permeable ßAR antagonist containing a nonsulfonated fluorophore efficiently inhibited both the plasma membrane and Golgi pools of ß1ARs. Furthermore, the cell-impermeable antagonist selectively inhibited the phosphorylation of PKA downstream effectors near the plasma membrane, which regulate sarcoplasmic reticulum (SR) Ca2+ release in adult cardiomyocytes, while the ß1AR Golgi pool remained active. Our tools offer promising avenues for investigating compartmentalized ßAR signaling in various contexts, potentially advancing our understanding of ßAR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.

Visualization of ER-to-Golgi trafficking of procollagen X.

Collagen is the most abundant protein in the extracellular matrix of animals, and 28 types of collagen have been reported in humans. We previously analyzed the endoplasmic reticulum (ER)-to-Golgi transport of fibril-forming type III collagen (Hirata et al., 2022) and network-forming type IV collagen (Matsui et al., 2020), both of which have long collagenous triple-helical regions. To understand the ER-to-Golgi trafficking of various types of collagens, we analyzed the transport of short-chain type X collagen in this study. We fused cysteine-free GFP to the N-telopeptide region of procollagen X (GFP-COL10A1), as employed in our previous analysis of procollagens III and IV, and analyzed its transport by live-cell imaging. Procollagen X was transported to the Golgi apparatus via vesicular and tubular carriers containing ERGIC53 and RAB1B, similar to those used for procollagen III. Carriers containing procollagen X probably used the same transport processes as those containing conventional cargoes such as α1-antitrypsin. SAR1, TANGO1, SLY1/SCFD1, and BET3/TRAPPC3 were required for trafficking of procollagen X, which are different from the factors required for trafficking of procollagens III (SAR1, TANGO1, and CUL3) and IV (SAR1 and SLY1/SCFD1). These findings reveal that accommodation of various types of collagens with different shapes into carriers may require fine-tuning of the ER-to-Golgi transport machinery.Key words: collagen, GFP-procollagen X, ER-to-Golgi trafficking, export from ER, TANGO1.

Two functionally interchangeable Vps9 isoforms mediate pollen tube penetration of style.

Style penetration by pollen tubes is essential for reproductive success, a process requiring canonical Rab5s in Arabidopsis. However, functional loss of Arabidopsis Vps9a, the gene encoding for guanine nucleotide exchange factor (GEF) of Rab5s, did not affect male transmission, implying the presence of a compensation program or redundancy. By combining genetic, cytological, and molecular approaches, we report that Arabidopsis Vps9b is a pollen-preferential gene, redundantly mediating pollen tube penetration of style with Vps9a. Vps9b is functionally interchangeable with Vps9a, whose functional distinction results from distinct expression profiles. Functional loss of Vps9a and Vps9b results in the mis-targeting of Rab5-dependent tonoplast proteins, defective vacuolar biogenesis, disturbed distribution of post-Golgi vesicles, increased cellular turgor, cytosolic acidification, and disrupted organization of actin microfilaments (MF) in pollen tubes, which collectively lead to the failure of pollen tubes to grow through style.

Spatial organization of adenylyl cyclase and its impact on dopamine signaling in neurons.

The cAMP cascade is increasingly recognized to transduce physiological effects locally through spatially limited cAMP gradients. However, little is known about how adenylyl cyclase enzymes that initiate cAMP gradients are localized. Here we address this question in physiologically relevant striatal neurons and investigate how AC localization impacts downstream signaling function. We show that the major striatal AC isoforms are differentially sorted between ciliary and extraciliary domains of the plasma membrane, and that one isoform, AC9, is uniquely concentrated in endosomes. We identify key sorting determinants in the N-terminal cytoplasmic domain responsible for isoform-specific localization. We further show that AC9-containing endosomes accumulate activated dopamine receptors and form an elaborately intertwined network with juxtanuclear PKA stores bound to Golgi membranes. Finally, we provide evidence that endosomal localization enables AC9 to selectively elevate PKA activity in the nucleus relative to the cytoplasm. Together, these results reveal a precise spatial landscape of the cAMP cascade in neurons and a key role of AC localization in directing downstream PKA signaling to the nucleus.

Cell-Sonar, a Novel Method for Intracellular Tracking of Secretory Pathways.

BACKGROUND: Intracellular tracking is commonly used in trafficking research. Until today, the respective techniques have remained complex, and complicated, mostly transgenic target protein changes are necessary, often requiring expensive equipment and expert knowledge. METHODS: We present a novel method, which we term "cell-sonar", that enables the user to track expression changes of specific protein markers that serve as points of interaction. Our study provides comparable analyses of expression changes of these marker proteins by in-cell Western analyses in two otherwise isogenic cell lines that only differ in the overexpression of the tracked target protein. Using the overexpressed human adult muscle-type nicotinic acetylcholine receptor as an example, we demonstrate that cell-sonar can cover multiple intracellular compartments such as the endoplasmic reticulum, the pathway between it and the Golgi apparatus, and the endocytic pathway. RESULTS: We provide evidence for receptor maturation in the Golgi and storage in recycling endosomes, rather than the fate of increased insertion into the plasma membrane. Additionally, we demonstrate with the implementation of nicotine that the receptor's destiny is exasperated up to secondary degradation. CONCLUSIONS: Cell-sonar is an affordable, easy-to-implement, and cheap method that can be adapted to a broad variety of proteins and cellular pathways of interest to researchers.

VPS13B is localized at the interface between Golgi cisternae and is a functional partner of FAM177A1.

Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here, we show that VPS13B is localized at the interface between proximal and distal Golgi subcompartments and that Golgi complex reformation after Brefeldin A (BFA)-induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by the loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b ortholog, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in the dynamics of Golgi membranes and that VPS13B may be assisted in this function by FAM177A1.

The ORP9-ORP11 dimer promotes sphingomyelin synthesis.

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.

Anaplasma phagocytophilum effector EgeA facilitates infection by hijacking TANGO1 and SCFD1 from ER-Golgi exit sites to pathogen-occupied inclusions.

The obligatory intracellular bacterium Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging zoonosis. Anaplasma has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, ER-Golgi exit site protein of Anaplasma (EgeA). In cells infected by Anaplasma, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to Anaplasma-containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the Anaplasma inclusion surface. Furthermore, knockdown of Anaplasma EgeA or either host TANGO1 or SCFD1 significantly reduced Anaplasma infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into Anaplasma inclusions, and several ER stress marker genes were not up-regulated in Anaplasma-infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates Anaplasma nutrient acquisition and reduces ER stress for Anaplasma survival and proliferation.

Intestinal Nogo-B reduces GLP1 levels by binding to proglucagon on the endoplasmic reticulum to inhibit PCSK1 cleavage.

Glucagon-like peptide 1 (GLP1), which is mainly processed and cleaved from proglucagon in enteroendocrine cells (EECs) of the intestinal tract, acts on the GLP1 receptor in pancreatic cells to stimulate insulin secretion and to inhibit glucagon secretion. However, GLP1 processing is not fully understood. Here, we show that reticulon 4B (Nogo-B), an endoplasmic reticulum (ER)-resident protein, interacts with the major proglucagon fragment of proglucagon to retain proglucagon on the ER, thereby inhibiting PCSK1-mediated cleavage of proglucagon in the Golgi. Intestinal Nogo-B knockout in male type 2 diabetes mellitus (T2DM) mice increases GLP1 and insulin levels and decreases glucagon levels, thereby alleviating pancreatic injury and insulin resistance. Finally, we identify aberrantly elevated Nogo-B expression and inhibited proglucagon cleavage in EECs from diabetic patients. Our study reveals the subcellular regulatory processes involving Nogo-B during GLP1 production and suggests intestinal Nogo-B as a potential therapeutic target for T2DM.

The FAM114A proteins are adaptors for the recycling of Golgi enzymes.

Golgi-resident enzymes remain in place while their substrates flow through from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the Golgi to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and one COPI adaptor protein, GOLPH3, acts to recruit enzymes into vesicles in part of the stack. Here, we used proximity biotinylation to identify further components of intra-Golgi vesicles and found FAM114A2, a cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1, showed that they bind to Golgi-resident membrane proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not cause substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Drosophila mutants lacking FAM114A have defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins bind Golgi enzymes and are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.