The liver has a key role in inter-organ communication by secreting most circulating plasma proteins. However, the mechanisms governing hepatic protein secretion remain unclear. Here we show that hepatic protein secretion follows a diurnal rhythm regulated by food intake in humans and mice. Using liver microsomal proteomics, we find that proteins implicated in the early secretory pathway, such as protein glycosylation and folding in the endoplasmic reticulum (ER) and Golgi apparatus, exhibit a rhythmic expression profile, which is abolished in Bmal1-knockout mice. Mechanistically, we show that hepatic glycogenolysis provides substrates for protein N-glycosylation. In mice, perturbing hepatic glycogenolysis with pharmacological or nutritional interventions leads to ER stress and attenuates diurnal protein secretion. We confirm these results in humans, as genetic variants associated with glycogen storage disease and congenital disorders of glycosylation also alter hepatic protein secretion. Overall, our work uncovers hepatic glycogen metabolism as a circadian regulator of protein secretion.

Although several open-source, easy-to-assemble light-sheet microscope platforms already exist-such as mesoSPIM, OpenSPIM, and OpenSpin-they are optimized for imaging large specimens and lack the resolution required to visualize subcellular features, such as organelles or cytoskeletal architectures. In contrast, lattice light-sheet microscopy (LLSM) achieves the resolution necessary to resolve such fine structures but, in its open-source implementation, can be alignment- and maintenance-intensive, often requiring specialist expertise. To address this gap, we developed Altair light-sheet fluorescence microscopy (LSFM), a high-resolution, open-source, sample-scanning light-sheet microscope specifically designed for subcellular imaging. By optimizing the optical pathway in silico, we created a custom baseplate that greatly simplifies alignment and assembly. The system integrates streamlined optoelectronics and optomechanics with seamless operation through our open-source software, navigate. Altair-LSFM achieves lateral and axial resolutions of approximately 235 and 350 nm, respectively, across a 266 µm field of view after deconvolution. We validate the system's capabilities by imaging sub-diffraction fluorescent nanospheres and visualizing fine structural details in mammalian cells, including microtubules, actin filaments, nuclei, and Golgi apparatus. We further demonstrate its live-cell imaging capabilities by visualizing microtubules and vimentin intermediate filaments in actively migrating cells.

Abstract The Golgi apparatus orchestrates post-translational modification and vesicular trafficking, with guanosine diphosphatases (GDPases) facilitating nucleotide-sugar transport by hydrolyzing guanosine diphosphate (GDP) to guanosine monophosphate (GMP). Although GDPase functions are well known in yeast and human-pathogenic fungi, their roles in filamentous fungi, including plant pathogens, remain largely unexplored. Here, we investigated the biological functions of the Golgi GDPases Gda1 and Ynd1 in the wheat head blight pathogen Fusarium graminearum . While loss of FgYND1 only resulted in minor growth problems, deletion of FgGDA1 caused pleiotropic deficiencies in mycelial development, conidiation, sexual reproduction, and virulence. Notably, FgYND1 overexpression partially rescued the defects of the FgGDA1 deletion mutant in the developmental and invasive processes, indicating overlapping yet non-equivalent functions. Glycoproteomics analysis revealed global remodeling of glycoprotein profiles in the FgGDA1 deletion mutant, with prominent changes among enzymes in carbon metabolism, consistent with disrupted Golgi-dependent glycosylation. According to our results, FgGda1 is the primary driver of protein glycosylation-dependent fungal development and virulence in F. graminearum , with FgYnd1 providing only partial redundancy.

Development and characterization of a membrane-permeant GRASP65-mimetic peptide that inhibits Golgi unlinking and cell cycle progression.

Eukaryotic cells consist of various organelles, each responsible for specific biological processes, making the understanding of protein subcellular localization essential for determining their potential functions. However, the rapid polar growth of pollen tubes requires careful consideration of organelle trafficking when analyzing subcellular localization in related studies. Fluorescence-tagged organelle markers have shown limited utility for studying pollen during the reproductive stage. In this study, we developed pollen-specific fluorescent marker sets for organelles and other cell structures using the promoter of the OsTAPE gene, which is highly expressed in mature pollen and pollen tubes of rice (Oryza sativa L.). These marker sets enable the visualization of cell membranes, nuclei, endoplasmic reticulum, Golgi apparatus, prevacuolar compartments, and filamentous actin by tagging fluorescent proteins (FP) at the amino N-terminal end. Specifically designed to accommodate the rapid tube elongation of rice pollen, this system offers a valuable resource for gene function research and colocalization analysis, helping to elucidate the pollen tube elongation process. This study expands the potential for using fluorescent labeling in monocotyledonous plants like rice during reproductive stages, facilitating gene function studies under varying environmental conditions through subcellular localization analysis in growing pollen tubes.

Organelle-targeted and stimuli-responsive theranostic system for combinational phototherapies of cancer through synergistic cell apoptosis and pyroptosis.

ERLIN1: A central regulator of protein quality control, lipid homeostasis, and cellular signaling at the endoplasmic reticulum.

The COG3 subunit interacts with SNX1 to enhance salt tolerance in Arabidopsis.

Ghrelin-producing cells (ghrelin cells) in rat fundic glands were analyzed three-dimensionally by combining serial section scanning electron microscopy with immunogold labeling to elucidate their ultrastructural characteristics. This approach enabled unambiguous identification of ghrelin cells and the three-dimensional (3D) reconstruction of their organelles with special reference to primary cilium. Rat ghrelin cells were morphologically classified into two types: ciliated ghrelin cells possessing primary cilia and non-ciliated ghrelin cells. In ciliated cells, primary cilia protruded from basal bodies located near the Golgi apparatus and were largely or entirely enclosed within ciliary pockets. Ghrelin-positive secretory granules were electron-dense and spherical. Ciliated and non-ciliated cells contained both large (250-350 nm) and small (100-200 nm) ghrelin-positive granules. In ciliated cells, the granules were densely accumulated in a localized region of the cytoplasm, being opposite the Golgi apparatus across the nucleus; in non-ciliated cells they were densely distributed throughout the cytoplasm. The ultrastructure of the basement membrane and overall 3D configuration of the Golgi apparatus also differed between the two cell types. Our results provide novel insights into the morphological organization of ghrelin cells, whose 3D ultrastructural features have been unclear in conventional two-dimensional transmission electron microscopy using single ultrathin sections.

Serial section scanning electron microscopy (SEM) is useful for revealing the three-dimensional (3D) architecture of organelles by acquiring backscattered electron images of ultrathin serial sections of resin-embedded tissues on solid substrates. However, comprehensive analyses of organelle function require a combination of ultrastructural and molecular localization data. In the present study, we developed a novel 3D immuno-electron microscopy (immuno-EM) approach that combines Tokuyasu cryosectioning with serial section SEM to elucidate the spatial distribution of organelle-associated proteins. Thick cryosections of tissues were immunolabeled with primary antibodies and FluoroNanogold-conjugated secondary antibodies, followed by gold enhancement, resin embedding, and serial sectioning and SEM. Serial tomographic images of organelles were aligned and segmented to generate 3D reconstructions. To demonstrate the effectiveness of the method, we visualized the localization of GM130, a representative cis-Golgi matrix protein, in a 3D model of the Golgi apparatus in rat pituitary gonadotropes. The 3D model revealed a spherical Golgi apparatus composed of five cisternae arranged in cis-trans order, with GM130 localized on the outer cisternae, consistent with previous findings. Our 3D immuno-EM technique enables the detailed 3D visualization of the Golgi apparatus and other organelles as well as analyses of the spatial distribution of target proteins in their 3D reconstructions.

The Golgi apparatus is a central hub for protein trafficking and signaling, yet its rapid imaging and cell-selective disruption remain challenging. Here, we report cycling molecular assemblies (CyMA) for fast Golgi imaging and cell-selective interference. CyMA precursors are acetylated amphiphilic thiopeptides that traverse plasma membrane and are deacetylated by intracellular thioesterases. This exposes thiols that undergo palmitoylation by Golgi-resident palmitoyl acyltransferases utilizing palmitoyl-CoA. The resulting palmitoylated peptides self-assemble into dynamic nanostructures (i.e., CyMA) localized at theGolgi. Their continuous, reversible S-acylation enables near-instantaneous Golgi imaging. Replacing fluorophore with a biphenyl motif promotes CyMA accumulation and disrupts functions such as protein modifications, trafficking, and secretion, leading to cell death. This study establishes dynamic supramolecular assembly as an active and selective strategy for Golgi-targeting, pleiotropically interfering with Golgi functions, which may be applicable to targeting other organelles by utilizing alternative enzyme switches to enable kinetic trapping.

Introduction Papillary thyroid cancer (PTC) is the most common type of endocrine malignancy caused by genetic mutations, hormonal imbalances, and environmental factors. However, recurrent infections, and metastasis in PTC patients remain challenged due to complexity of traditional methods. Baicalein (BA) is a kind of natural flavonoid that exhibits the anti-cancer, anti-inflammatory, anti-tumor, and anti-viral activities. The molecular mechanism of baicalein in pathogenesis of PTC remains unclear. This study was designed to explore the inhibitory effects of BA against PTC by mediating the Golgi apparatus reprogramming via PLAU and suppressing the TPL2/MEK2/ERK2 pathway. Methods Transcriptomic analysis was performed to explore the gene expression profiles. Molecular docking was employed to identify the potential targets to elucidate the molecular mechanism of action of BA. Results PLAU, an up-regulated DEG, is implicated in tumor development, lymph node metastasis, and infiltration levels of neutrophils and dendritic cells in thyroid cancer patients. Molecular docking analysis revealed that serum levels of uPA protein encoded by PLAU and Plau mRNA were elevated in PTC patients with metastasis and BRAF mutation. BA treatment upregulates PLAU gene expression, but this increased PLAU protein subsequently interacts with and inhibited by BA, leading to downstream pathway suppression. Conclusion It was concluded it could be served as a promising therapeutic strategy for the treatment of PTC.

Bone metastases dramatically worsen breast cancer (BC) prognosis reducing overall survival. Natural killer (NK) cells recognize and eliminate malignant cells through the interaction with NKG2D receptor ligands (NKG2DLs) on cancer cells. Tumors often evade NK surveillance by downregulating the NKG2DLs expression and avoid recognition, but whether this occurs in bone metastases remains unclear. This study investigates mechanisms of NKG2DLs downregulation in primary BC and bone metastases (BoMet). Expression and localization of the NKG2D/NKG2DL axis components were investigated in BC tissues (with and without metastases), paired bone metastatic ductal carcinoma (bmDC), BoMet, and in BC cells lines of varying invasiveness. In bmDC and BoMet, major histocompatibility complex class I chain-related protein A and B (MICA/B) and UL16-binding protein 2 (ULBP2) localized in perinuclear area, contrasting with predominantly cytosolic distribution in non-metastatic BC. Similarly, invasive MDA-MB-231 and MDA-BoM-1833 showed NKG2DLs perinuclear localization and co-localization with the Golgi apparatus, while less invasive MCF7 showed a prominent cytosolic distribution. Accumulation of NKG2DLs in membrane and cytoskeletal fractions further supports this pattern. Additionally, when N-glycosilation is impaired, NKG2DLs fail to reach the cell surface in metastatic cell lines, while are still transported through the Golgi apparatus and delivered to the plasma membrane, resulting in increased surface expression irrespective of correct glycosylation. Our findings suggest that invasive and bone-metastatic breast cancer cells are more dependent on correct glycosylation and intracellular trafficking for NKG2DL surface expression than non-metastatic breast cancer cells. This difference may have important implications for potential immune evasion mechanisms and for the development of therapeutic strategies targeting bone metastases in breast cancer.

Transvaginal ultrasound (TVS) is used routinely in gynecological care in Australia to manage gynecological health concerns. Typically, TVS is well tolerated by patients, with low levels of discomfort reported. Trans and gender diverse people assigned female at birth may experience gender dysphoria or testosterone-related anatomical changes, which could make such intimate examinations physically difficult or emotionally distressing. However, to date, no studies have considered the impact of gender identity on individuals' experiences of TVS. To fill this research gap, we explored the experiences of TVS among trans and gender diverse individuals assigned female at birth within Australia. We conducted semi-structured interviews with trans and gender diverse individuals assigned female at birth who have experienced TVS in Australia. We analyzed all interviews in line with Braun and Clarke's reflexive thematic analysis. Ten trans and gender diverse individuals aged between 18 and 50 years old participated in this study. From their interviews, we developed three overarching themes: (1) It's a bit like being a detective, (2) So I could properly say, "I don't want this done," and (3) I definitely felt like a novelty. Participants described a range of positive and negative experiences with TVS, with issues related to cisnormativity in documentation, staff attitudes, and inadequate informed consent consistently highlighted. Trans and gender diverse people face challenges in accessing inclusive gynecological care in Australia. Our findings highlight a need for improved informed consent guidelines, better education and training for health professionals, and more inclusive clinic documentation to promote inclusive care.

Mannans with β-1,4-linked backbones are common cell wall components of algae and land plants. Prior challenges to enhance β-mannan content in plants point to unclear metabolic bottlenecks and the potential for hidden biosynthetic players. Endo-β-MANNANASEs (MANs) are the main glycosyl hydrolases that mobilize extracellular β-mannans during seed germination. However, we found that Arabidopsis man2 man5 seeds resemble β-mannan biosynthetic mutants. CELLULOSE SYNTHASE-LIKE A (CSLA) overexpression restored β-mannan synthesis in the man double mutant and increased the distribution of crystalline polymers but impaired the release of other mucilaginous polysaccharides. Using yeast synthetic biology, we dissected the functional interplay of MAN enzymes with CSLAs. Intracellular MAN2 and MAN5 reduced the quantity of insoluble β-mannan but elevated the content of water-soluble carbohydrates. We propose that Arabidopsis MAN2/5 and orthologous crop enzymes with a transmembrane domain sustain hemicellulose production in the Golgi apparatus by cleaving insoluble β-mannan polymers into hydrophilic counterparts.

Homeostatic pathways maintain the lipid composition of organelle membranes, and mechanistic links between lipid sensing, synthesis, and trafficking are lacking. Acute depletion of cell cholesterol elicits an increase in the rate of very-long-chain (VLC) sphingomyelin synthesis in the Golgi apparatus, thereby promoting cholesterol retention in the plasma membrane. Stable isotope metabolic analyses and lipid trafficking assays showed that the increase in VLC-sphingomyelin results from an increase in the rate of coatomer II-dependent trafficking of VLC-ceramide from the endoplasmic reticulum to the Golgi apparatus. An integral membrane protein of the coatomer II network, cTAGE5, is required for endoplasmic reticulum-to-Golgi trafficking of ceramide and cTAGE5 overexpression caused herniations of the endoplasmic reticulum network that entrapped a synthetic ceramide analog to which cTAGE5 could be photochemically cross-linked. We propose that cTAGE5 is a ceramide sensor for export of VLC-ceramide from the endoplasmic reticulum exit site.

Bidirectional trafficking between the trans-Golgi network (TGN) and endolysosomal compartments lies at the intersection of biosynthetic and degradative pathways. At the center of this trafficking route is the adaptor protein complex 1 (AP1), a heterotetramer essential for cargo recognition and vesicle budding. Here, we identified Male-Enhanced Antigen 1 (MEA1), a previously uncharacterized protein, as a critical AP1 regulator. Loss of MEA1 resulted in depletion of AP1 subunits and impaired trafficking of AP1-dependent cargoes. Mechanistically, MEA1 acts as a bi-handed chaperone, simultaneously engaging and stabilizing the μ1 and β1 subunits of AP1. The MEA1-stabilized μ1 and β1 collide with the γ and σ1 subunits stabilized by Alpha- and Gamma-Adaptin Binding Protein (AAGAB), another bi-handed chaperone, leading to formation of the tetrameric AP1 adaptor and release of both chaperones. These findings identify MEA1 as a key AP1 regulator and uncover a dual chaperone collision mechanism potentially generalizable to multiprotein complex assembly.

Different yeast species, including Ogataea polymorpha, are often used as hosts for recombinant protein production. One of the most important factors limiting such applications is yeast-specific modifications of glycoside chains attached to secretory proteins. This problem can potentially be solved by the identification and inactivation of genes responsible for these modifications. Previously we demonstrated that the exceptional resistance of O. polymorpha to vanadate depends on the ABV1 gene responsible for the mannosylphosphorylation of protein glycoside chain in the Golgi apparatus. Here we show that mutations altering protein glycosylation in the secretory pathway can be selected in the abv1Δ mutant by screening for vanadate resistance. For one such mutant, we identified the responsible gene, which encodes a putative α-1,2-mannosyltransferase. To ensure the absence of phosphomannosylation, both O. polymorpha genes, ABV1 and MNN4, which encode mannosylphosphate transferase homologs, were inactivated. Some vanadate resistant mutants generated in this strain showed defects in N-glycosylation of a recombinant glycoprotein. This demonstrates that the effects of N-glycosylation on vanadate resistance in O. polymorpha are not mediated by phosphomannosylation per se and that identification of certain genes responsible for N-glycosylation in this yeast can be performed via selection of vanadate resistant clones.

Polytopic copper (Cu)-ATPases are central regulators of the essential micronutrient copper in all organisms. In polarized epithelia, the vertebrate homologues ATP7A and ATP7B undergo copper-induced trafficking from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively, to mediate efflux of excess copper. To probe (1) inter-domain interactions that drive trafficking and (2) the extent of divergence between homologous domains constituting Cu-ATPases, we replaced the copper-binding N-terminal (NT), nucleotide-binding (NBD) and/or C-terminal (CT) domains of ATP7B with those of ATP7A. The functionally active chimeras exhibited distinct trafficking phenotypes. Notably, the ATP7B-NT substitution led to constitutive basolateral membrane trafficking, whereas simultaneous NT-NBD substitution led to steady-state TGN localization, suggesting that interaction between the two domains, as confirmed by in vitro NT-NBD-binding studies, might be essential for TGN localization. Interestingly, reciprocal replacement of the ATP7A-NBD and -NT with that from ATP7B did not rescue membrane localization, indicating that domain compatibility is restricted, suggesting greater evolutionary divergence of ATP7B domains. Analysing orthologous Cu-ATPase domain-sequences from diverse organisms, however, revealed similar evolutionary relationships between the NT and NBD, suggesting their co-evolution. We thus correlate the copper-responsive trafficking ability of Cu-ATPases with evolutionary stringency imparted onto Cu-ATPase domains.

Retrograde transport from endosomes to the trans-Golgi network (TGN) is essential for intracellular trafficking, yet its molecular mechanism remains poorly understood. In Fusarium graminearum, 10 Rab GTPases associated with the Golgi-associated retrograde protein (GARP) complex were identified through immunoprecipitation followed by mass spectrometry (IP-MS). Among these, only the deletion of FgRAB6 disrupted the proper localisation of the GARP complex to the TGN. FgRab6 directly interacts with the GARP subunit FgVps52 via a conserved Q73 residue, which is critical for fungal growth and pathogenicity. Notably, this Q73-dependent interaction is evolutionarily conserved across eukaryotic species. Upon GTP activation, FgRab6 recruits FgVps52 to the TGN, thereby facilitating the assembly of the GARP complex through the sequential recruitment of additional subunits, including FgVps51, FgVps53 and FgVps54. The fully assembled GARP complex subsequently recruits the retromer complex and ensures the precise localisation of the SNARE proteins FgSnc1, FgTlg1 and FgTlg2 at the endosomes and the TGN. Disruption of this pathway severely compromises fungal development and virulence. Collectively, these findings identify a FgRab6-GARP-retromer-coordinated vesicle trafficking pathway that mediates the retrograde transport of SNARE proteins, which is critical for the pathogenicity of F. graminearum. This work provides new mechanistic insights into vesicular transport and highlights potential targets for antifungal intervention.