Regulation Of Exocytosis Research Articles

CryoVesNet: A dedicated framework for synaptic vesicle segmentation in cryo-electron tomograms.

Cryo-electron tomography (cryo-ET) has the potential to reveal cell structure down to atomic resolution. Nevertheless, cellular cryo-ET data is highly complex, requiring image segmentation for visualization and quantification of subcellular structures. Due to noise and anisotropic resolution in cryo-ET data, automatic segmentation based on classical computer vision approaches usually does not perform satisfactorily. Communication between neurons relies on neurotransmitter-filled synaptic vesicle (SV) exocytosis. Cryo-ET study of the spatial organization of SVs and their interconnections allows a better understanding of the mechanisms of exocytosis regulation. Accurate SV segmentation is a prerequisite to obtaining a faithful connectivity representation. Hundreds of SVs are present in a synapse, and their manual segmentation is a bottleneck. We addressed this by designing a workflow consisting of a convolutional network followed by post-processing steps. Alongside, we provide an interactive tool for accurately segmenting spherical vesicles. Our pipeline can in principle segment spherical vesicles in any cell type as well as extracellular and in vitro spherical vesicles.

Rabphilin-3A negatively regulates neuropeptide release, through its SNAP25 interaction

Neuropeptides and neurotrophins are stored in and released from dense core vesicles (DCVs). While DCVs and synaptic vesicles (SVs) share fundamental SNARE/SM proteins for exocytosis, a detailed understanding of DCV exocytosis remains elusive. We recently identified the RAB3-RIM1 pathway to be essential for DCV, but not SV exocytosis, highlighting a significant distinction between the SV and DCV secretory pathways. Whether RIM1 is the only RAB3 effector that is essential for DCV exocytosis is currently unknown. In this study, we show that rabphilin-3A (RPH3A), a known downstream effector of RAB3A, is a negative regulator of DCV exocytosis. Using live-cell imaging at single-vesicle resolution with RPH3A deficient hippocampal mouse neurons, we show that DCV exocytosis increased threefold in the absence of RPH3A. RAB3A-binding deficient RPH3A lost its punctate distribution, but still restored DCV exocytosis to WT levels when re-expressed. SNAP25-binding deficient RPH3A did not rescue DCV exocytosis. In addition, we show that RPH3A did not travel with DCVs, but remained stationary at presynapses. RPH3A null neurons also had longer neurites, which was partly restored when ablating all regulated secretion with tetanus neurotoxin. Taken together, these results show that RPH3A negatively regulates DCV exocytosis, potentially also affecting neuron size. Furthermore, RAB3A interaction is required for the synaptic enrichment of RPH3A, but not for limiting DCV exocytosis. Instead, the interaction of RPH3A with SNAP25 is relevant for inhibiting DCV exocytosis.

Putative retina metal/metalloid-binding proteins: molecular functions, biological processes and retina disease associations.

The mammalian retina contains high amounts of metals/metalloid-selenium. Their dyshomeostases are associated with certain retinal diseases. We carried out this bioinformatics study to identify the relationships between putative retinal metal/selenium binding proteins, their molecular functions, and biological processes. Identification of putative mouse metal/selenium binding proteins was based on known binding motifs, domains, patterns, and profiles. Annotations were obtained from Uniprot keywords 'metal binding', 'metal ion co-factors', 'selenium proteins'. Protein functions were estimated by associative frequency with key words in UniProt annotations. The raw data of five mouse proteomics PRIDE datasets (available to date) were downloaded and processed with Mascot against the mouse taxa of Uniprot (SwissProt/Trembl) and MaxQuant (version 1.6.10.43) for qualitative and quantitative datasets, respectively. Clinically relevant variants were evaluated using archives and aggregated information in ClinVar. The 438 proteins common to all the retina proteomics datasets were used to identify over-represented Gene Ontology categories. The putative mouse retinal metal/metalloid binding proteins identified are mainly involved in: (1) metabolic processes (enzymes), (2) homeostasis, (3) transport (vesicle mediated, transmembrane, along microtubules), (4) cellular localization, (5) regulation of signalling and exocytosis, (6) organelle organization, (7) (de)phosphorylation, and (8) complex assembly. Twenty-one proteins were identified as involved in response to light stimulus and/or visual system development. An association of metal ion binding proteins rhodopsin, photoreceptor specific nuclear receptor, calcium binding protein 4 with disease-related mutations in inherited retinal conditions was identified, where the mutations affected an area within or in close proximity to the metal binding site or domain. These findings suggest a functional role for the putative metal/metalloid binding site in retinal proteins in certain retinal disorders.

Messenger RNA transcription levels of neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex components in the olfactory nerve system of the anadromous Pacific salmon, masu salmon Oncorhynchus masou.

Anadromous Pacific salmon (genus Oncorhynchus) are known for homing behavior to their natal rivers based on olfactory imprinted memories during seaward migration. The SNARE complex is a regulator of vesicle exocytosis from the presynaptic membrane. Our previous study suggested that its component genes (Snap25, Stx1, and Vamp2) are more highly expressed in the olfactory nervous system (ONS) during the migration stages associated with olfactory imprinting in the evolutionary species of Pacific salmon, such as chum (O. keta) and pink (O. gorbuscha) salmon. Masu salmon (O. masou) has a significantly different life history from these species, living longer in rivers and being a more primitive Pacific salmon species. In this study, the transcription of snare mRNAs in the ONS was analyzed using mainly male wild masu salmon. Five cDNAs encoding masu salmon SNAREs, which are well conserved among vertebrates, were isolated and sequenced. Each snare mRNA was highly expressed in age 1+ (yearling) parr prior to smoltification, particularly in the olfactory bulb. Their transcription status was significantly different from that of chum and pink salmon, which showed high expression in earlier under-yearling juveniles. The present results and our previous studies indicate that snare mRNAs are highly transcripted until the seaward migration, reflecting neural development and neuroplasticity of the ONS for olfactory imprinting.

Vitamin C Drives Reentrant Actin Phase Transition: Biphasic Exocytosis Regulation Revealed by Single-Vesicle Electrochemistry.

Unveiling molecular mechanisms that dominate protein phase dynamics has been a pressing need for deciphering the intricate intracellular modulation machinery. While ions and biomacromolecules have been widely recognized for modulating protein phase separations, effects of small molecules that essentially constitute the cytosolic chemical atmosphere on the protein phase behaviors are rarely understood. Herein, we report that vitamin C (VC), a key small molecule for maintaining a reductive intracellular atmosphere, drives reentrant phase transitions of myosin II/F-actin (actomyosin) cytoskeletons. The actomyosin bundle condensates dissemble in the low-VC regime and assemble in the high-VC regime in vitro or inside neuronal cells, through a concurrent myosin II protein aggregation-dissociation process with monotonic VC concentration increase. Based on this finding, we employ in situ single-cell and single-vesicle electrochemistry to demonstrate the quantitative modulation of catecholamine transmitter vesicle exocytosis by intracellular VC atmosphere, i.e., exocytotic release amount increases in the low-VC regime and decreases in the high-VC regime. Furthermore, we show how VC regulates cytomembrane-vesicle fusion pore dynamics through counteractive or synergistic effects of actomyosin phase transitions and the intracellular free calcium level on membrane tensions. Our work uncovers the small molecule-based reversive protein phase regulatory mechanism, paving a new way to chemical neuromodulation and therapeutic repertoire expansion.

A chemical genetic screen with the EXO70 inhibitor Endosidin2 uncovers potential modulators of exocytosis in Arabidopsis.

Exocytosis plays an essential role in delivering proteins, lipids, and cell wall polysaccharides to the plasma membrane and extracellular spaces. Accurate secretion through exocytosis is key to normal plant development as well as responses to biotic and abiotic stresses. During exocytosis, an octameric protein complex named the exocyst facilitates the tethering of secretory vesicles to the plasma membrane. Despite some understanding of molecular and cellular aspects of exocyst function obtained through reverse genetics and direct interaction assays, knowledge about upstream modulators and genetic interactors remains limited. Traditional genetic screens encounter practical issues in exocyst subunit mutant backgrounds, such as lethality of certain knockout mutants and/or potential redundancy of EXO70 homologs. To address these challenges, this study leverages the tunable and reversible nature of chemical genetics, employing Endosidin2 (ES2)-a synthetic inhibitor of EXO70-for a large-scale chemical genetic mutant screen in Arabidopsis. This approach led to the identification of 70 ES2-hypersensitive mutants, named es2s. Through a whole-genome sequencing-based mapping strategy, 14 nonallelic es2s mutants were mapped and the candidate mutations reported here. In addition, T-DNA insertion lines were tested as alternative alleles to identify causal mutations. We found that T-DNA insertion alleles for DCP5, VAS1/ISS1, ArgJ, and MEF11 were hypersensitive to ES2 for root growth inhibition. This research not only offers new genetic resources for systematically identifying molecular players interacting with the exocyst in Arabidopsis but also enhances understanding of the regulation of exocytosis.

Pseudorabies virus hijacks the Rab6 protein to promote viral assembly and egress

Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky’s disease, or pseudorabies, in swine populations. Rab6, a member of the small GTPase family, is implicated in various membrane trafficking processes, particularly exocytosis regulation. Its involvement in PRV infection, however, has not been documented previously. In our study, we observed a significant increase in the Rab6 mRNA and protein levels in both PK-15 porcine kidney epithelial cells and porcine alveolar macrophages, as well as in the lungs and spleens of mice infected with PRV. The overexpression of wild-type Rab6 and its GTP-bound mutant facilitated PRV proliferation, whereas the GDP-bound mutant form of Rab6 had no effect on viral propagation. These findings indicated that the GTPase activity of Rab6 was crucial for the successful spread of PRV. Further investigations revealed that the reduction in Rab6 levels through knockdown significantly hampered PRV proliferation and disrupted virus assembly and egress. At the molecular level, Rab6 was found to interact with the PRV glycoproteins gB and gE, both of which are essential for viral assembly and egress. Our results collectively suggest that PRV exploits Rab6 to expedite its assembly and egress and identify Rab6 as a promising novel target for therapeutic treatment for PRV infection.

Abstract 156: Pyruvate Kinase M2 Regulates SNAP23-mediated Platelet Granule Exocytosis, Neutrophil Extracellular Traps Formation And Experimental Venous Thrombosis

Introduction: Several factors released from activated platelets via exocytosis contribute to venous thrombosis (VT) by potentiating neutrophil extracellular traps formation (NETosis) at the sites of inflammation. The metabolic enzyme pyruvate kinase M2 (PKM2) contributes to platelet activation and arterial thrombosis. PKM2 possess protein kinase activity. The regulatory role of PKM2 in platelet granule exocytosis, NETosis, and VT has not been investigated yet. Aim: To test the hypothesis that dimeric PKM2 regulates SNAP23-mediated platelet granule exocytosis, and thereby contributes to NETosis, and VT. Methods: We utilized platelet-specific PKM2 –/– (PKM2 fl/fl PF4Cre + ), littermate (PKM2 fl/fl ) mice, and wild-type (WT) mice treated with small molecule ML265 (limits PKM2 dimerization) or vehicle to test the susceptibility to VT in inferior vena cava (IVC)-stenosis model. In vitro NETs formation was evaluated by co-incubating WT neutrophils with agonist (thrombin or convulxin)-stimulated platelets. The SNAP23 phosphorylation and PF4 release were measured using Western blot. Results: PKM2 fl/fl PF4Cre + or WT mice pre-treated with ML265 exhibited reduced susceptibility to VT in the 48 hours IVC-stenosis model as evaluated by less thrombus burden (% incidence, length, and weight, n=11-12/group, P<0.05 vs. controls). Myography revealed that limiting PKM2 dimerization improves the venous wall function post-VT. Activated PKM2 –/– or ML265 pre-treated WT platelets exhibited decreased SNAP23 phosphorylation and PF4 secretion suggesting a regulatory role for PKM2 in SNAP23 exocytosis and PF4 secretion. Furthermore, neutrophils stimulated with activated platelets from either PKM2 fl/fl PF4Cre + or WT mice pre-treated with ML265 showed reduced NETosis. Finally, we demonstrate that the % of the area covered by the thrombus is profoundly reduced in the ML265-treated human whole blood perfused over a tissue factor-coated surface at the venous shear. Conclusions: Dimeric PKM2 is a novel regulator of SNAP23-mediated platelet granule exocytosis. Targeting dimeric PKM2 in platelets inhibits NETosis in neutrophils stimulated with activated platelets and reduces susceptibility to experimental VT.

Abstract 193: Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9

Abstract Cell shape change and motility are essential in metastasis and involve remodeling of the actin cytoskeleton, cell adhesions, and plasma membrane. How these cytoskeletal and membrane remodeling are regulated and coordinated remains largely unknown. We previously identified TRIM9 as a regulator of actin dynamics and exocytosis in developing neurons. Deletion of murine Trim9 impairs neuronal migration, netrin-induced axon turning, and axonal and dendritic branching, and increased exocytosis and filopodial stability. In addition, we have shown TRIM9 alters dynamics of the actin polymerase VASP at filopodia tips via non-degrative mono-ubiquitination. TRIM9 is expressed in other motile cells, but the non-neuronal role of TRIM9 remains unknown. TRIM9 was identified as a possible prognostic biomarker in melanoma and high expression correlates with low patient survival. Melanomas undergo phenotype switching, where three distinct phenotypes exist that are associated with differences in gene expression. The three phenotypes consist of a melanocytic proliferative state, a neural crest-like invasive state, an intermediate state. Single cell RNAseq (scRNASeq) data from patient-derived melanoma indicate TRIM9 is highly expressed in the proliferative melanocytic state, which correlates with poor prognosis. We hypothesize that TRIM9 coordinates actin dynamics, adhesion, and exocytosis in melanoma. We show that TRIM9 protein is enriched in several human melanoma lines. Here we examine the role of TRIM9 in regulation of focal adhesions, exocytosis, migration, and invasion in two of these lines. Genetic loss of TRIM9 increased random migration velocity but reduced directional persistence. Interestingly, we find that TRIM9 plays a role in promoting bleb-like morphology and inhibits the ability to durotax on shallow gradients on soft substrate. Fluorescence recovery after photobleaching (FRAP), Total internal reflection fluorescence microscopy (TIRF), and widefield microscopy revealed that loss of TRIM9 results in increased focal adhesions, cell size, and altered dynamics of focal adhesion proteins VASP, zyxin, and paxillin dynamics. In addition, TRIM9 knockout cells exhibit reduced filopodial length and density and altered filopodial localization of VASP. In-gel zymography indicates that TRIM9 knockout cells also display an increased degradative capacity. Current studies are investigating how loss of TRIM9 alters parameters of cell contractility, lamellipodia, invadopodia, and exocytosis to define the role of TRIM9 in melanoma motility. Together these findings suggest TRIM9 reduces adhesion and migration, increases proliferation and blebbing and may be an important regulator of phenotype switching in melanoma. Citation Format: Kimberly Lukasik, Jordan Brooks, Sam Redinbo, James Bear, Stephanie Gupton. Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 193.

Induction of Exocytosis Rescues Lysosomal GM2 Accumulation in Tay-Sachs Disease

Introduction. The Tay-Sachs disease is a progressive neurodegenerative disorder that is caused by a genetic mutation in the HEXA gene coding the lysosomal α-subunit of β-hexosaminidase A. Currently, there is no effective treatment for Tay-Sachs. Induction of exocytosis as a potential treatment approach is suggested to restore lysosomal enlargement in several lysosomal storage diseases. Here, we aimed to test the therapeutic potential of two small molecules, δ-tocopherol and hydroxypropyl-β-cyclodextrin, in fibroblast and neuroglia cells derived from Hexa-/-Neu3-/- mice and Tay-Sachs patients. Method. The effect of two small molecules on lysosomal enlargement and GM2 accumulation in lysosomes was examined by LysoTracker staining and immunocytochemical colocalization analysis for GM2 and LAMP1. qRT-PCR and fluorometric enzyme assay were also used to investigate the effect of combined treatment on the level of neuraminidase 1, a negative regulator of exocytosis. Results. Single treatment with δ-tocopherol (5-40 μM) and hydroxypropyl-β-cyclodextrin (10-50 μM) for 48 hours led to significant induction of lysosomal exocytosis. We demonstrated that the combined treatment with δ-tocopherol (10 μM) and hydroxypropyl-β-cyclodextrin (25 μM) resulted in a significant reduction of lysosomal GM2 and downregulation of lysosomal Neu1 expression. Conclusion. In this study, we demonstrated that inducing exocytosis by δ-tocopherol and hydroxypropyl-β-cyclodextrin might have therapeutic potential to reduce GM2 storage and pathology in Tay-Sachs cells.

Serotonergic regulation of catecholamine exocytosis from adrenal chromaffin cells involves two mechanistically and temporally distinct pathways

Serotonergic regulation of catecholamine exocytosis from adrenal chromaffin cells involves two mechanistically and temporally distinct pathways

THU277 Deletion Or inhibition Of Ornithine Decarboxylase Protects Islet β-cell Health And Is Associated With Preserved Residual C-peptide In Persons With Recent Onset Type 1 Diabetes

Abstract Disclosure: B. Hammoud: None. L.A. DiMeglio: None. A. Kulkarni: None. A. Hull: None. S. Woerner: None. S. Cabrera: None. L.D. Mastrandrea: None. F. Ouyang: None. S.N. Perkins: None. C. Evans-Molina: None. T.L. Mastracci: None. S.A. Tersey: None. S. Sarkar: None. E. Nakayasu: None. B. Webb-Robertson: None. E. Gerner: None. R.G. Mirmira: None. E.K. Sims: None. Inhibition of polyamine biosynthesis using α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, reduces β cell stress and type 1 diabetes (T1D) incidence in preclinical animal models. However, the cellular and molecular mechanisms by which polyamines promote β-cell stress and the tolerability and effectiveness of their depletion by DFMO in humans with T1D remain unknown. We hypothesized that β-cell protection from stress occurs via cell-autonomous effects of polyamine depletion. To test this hypothesis, we generated β-cell specific knockout of ornithine decarboxylase in mice, performed comprehensive proteomics and transcriptomics on human islets treated with DFMO under inflammatory conditions in vitro, and performed a clinical study of DFMO tolerability, safety, and efficacy in persons with new-onset T1D. β-cell-specific deletion of the gene encoding ornithine decarboxylase in mice demonstrated protection of these animals against toxin (streptozotocin)-induced β-cell loss and hyperglycemia, implicating a cell-autonomous role for ornithine decarboxylase in β-cell stress. RNA sequencing and proteomics analyses of human islets exposed to a cytokine mix (1000 U/mL IFNγ + 50 U/mL IL-1β) +/- 5 mM DFMO identified 130 differentially regulated transcripts and 701 differentially regulated proteins. Pathway analysis implicated endosomal transport, vesicle organization and regulation of exocytosis, mRNA processing, and antigen cross presentation as mechanisms underlying DFMO effects on the islet response. To test DFMO in humans, we performed a 3-site randomized, placebo-controlled, double masked dose-ranging study of 3 months daily oral DFMO in persons with recent-onset T1D (<100 days). Dosing at 125, 250, 500, 750 or 1000 mg/m2/day was tested in 41 persons (12-34 y/o, 59% male, mean HbA1c 7.3%), with 6-10 participants/dosing group. Only mild and moderate adverse effects (AEs) were noted. Possible drug-related AEs included mild-moderate gastrointestinal symptoms, headache, upper respiratory infections, a pump site infection, and anemia. Urinary levels of the ODC product putrescine were decreased (p=0.05) in the 1000 mg/m2 dosing group. Compared to placebo, individuals receiving 750 and 1000 mg/m2 doses had significantly higher mixed-meal C-peptide area under the curve values at the 6-month post-randomization visit (p<0.05 vs. placebo). In conclusion, β-cell ODC inhibition protects β-cell health and function in the context of the proinflammatory T1D milieu. A 3-month course of oral DFMO was well tolerated with a favorable AE profile in persons with recent-onset T1D. Higher doses were associated with greater β-function compared to placebo. DFMO may have utility as a β-cell preservation therapy in recent-onset T1D. Presentation: Thursday, June 15, 2023

Gβγ-SNAP25 exocytotic brake removal enhances insulin action, promotes adipocyte browning, and protects against diet-induced obesity.

Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o GPCRs by Gβγ subunit inhibition of 2 mechanisms: decreased calcium entry and direct interaction of Gβγ with soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) that decreased Gβγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact and removing GPCR-Gβγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. Snap25Δ3/Δ3 mice exhibited enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in Snap25Δ3/Δ3 mice challenged with a high-fat diet. Glucose homeostasis, whole-body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in Snap25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting that basal autonomic activity was required. Direct electrode stimulation of sympathetic neuron exocytosis from Snap25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gβγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combating metabolic disease.

The post-translational regulation of transcription factor EB (TFEB) in health and disease.

Transcription factor EB (TFEB) is a basic helix-loop-helix leucine zipper transcription factor that acts as a master regulator of lysosomal biogenesis, lysosomal exocytosis, and macro-autophagy. TFEB contributes to a wide range of physiological functions, including mitochondrial biogenesis and innate and adaptive immunity. As such, TFEB is an essential component of cellular adaptation to stressors, ranging from nutrient deprivation to pathogenic invasion. The activity of TFEB depends on its subcellular localisation, turnover, and DNA-binding capacity, all of which are regulated at the post-translational level. Pathological states are characterised by a specific set of stressors, which elicit post-translational modifications that promote gain or loss of TFEB function in the affected tissue. In turn, the resulting increase or decrease in survival of the tissue in which TFEB is more or less active, respectively, may either benefit or harm the organism as a whole. In this way, the post-translational modifications of TFEB account for its otherwise paradoxical protective and deleterious effects on organismal fitness in diseases ranging from neurodegeneration to cancer. In this review, we describe how the intracellular environment characteristic of different diseases alters the post-translational modification profile of TFEB, enabling cellular adaptation to a particular pathological state.

Short Survey on the Protein Modifications in Plasma during SARS-CoV-2 Infection.

Although the COVID-19 pandemic has ended, it is important to understand the pathology of severe SARS-CoV-2 infection associated with respiratory failure and high mortality. The plasma proteome, including protein modification by lipid peroxidation products in COVID-19 survivors (COVID-19; n = 10) and deceased individuals (CovDeath; n = 10) was compared in samples collected upon admission to the hospital, when there was no difference in their status, with that of healthy individuals (Ctr; n = 10). The obtained results show that COVID-19 development strongly alters the expression of proteins involved in the regulation of exocytosis and platelet degranulation (top 20 altered proteins indicated by analysis of variance; p-value (False Discovery Rate) cutoff at 5%). These changes were most pronounced in the CovDeath group. In addition, the levels of 4-hydroxynonenal (4-HNE) adducts increased 2- and 3-fold, whereas malondialdehyde (MDA) adducts increased 7- and 2.5-fold, respectively, in COVID-19 and CovDeath groups. Kinases and proinflammatory proteins were particularly affected by these modifications. Protein adducts with 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) were increased 2.5-fold in COVID-19 patients, including modifications of proteins such as p53 and STAT3, whereas CovDeath showed a decrease of approximately 60% compared with Ctr. This study for the first time demonstrates the formation of lipid metabolism products-protein adducts in plasma from survived and deceased COVID-19 patients, significantly distinguishing them, which may be a predictor of the course of SARS-CoV-2 infection.

Knockdown of Secernin 1 inhibit cell invasion and migration by activating the TGF-β/Smad3 pathway in oral squamous cell carcinomas

Secernin-1 (SCRN1) is a regulator of exocytosis in mast cells. Recently, SCRN1 was reported to be correlated with the prognosis of colorectal cancer and gastric cancer, but its functional effects on oral squamous cell carcinoma (OSCC) remain unclear. Our aim was to explore the expression pattern and the migration and invasion effects of the newly identified SCRN1 in OSCC. Western blotting (WB) was performed to measure SCRN1 expression in human OSCC tissue samples and OSCC cell lines. The effects of SCRN1 on OSCC cell proliferation, invasion and migration were analyzed by cell counting kit-8 and Transwell assays. The expression levels of TGF-β, Smad3 and phosphorylated Smad3 (p-Smad3) were measured by WB. The secretion of matrix metalloproteinase (MMP)-2 and MMP-9 was determined by the enzyme-linked immunosorbent assay. The expression of SCRN1 was significantly elevated in OSCC tissues and cell lines. SCRN1 knockdown reduced the expression of TGF-β and p-Smad3 in OSCC cells. TGF-β stimulation promoted proliferation, invasion and migration and enhanced the expression of p-Smad3 and the secretion of MMP9 in SCRN1-knockdown OSCC cell lines. Our study demonstrated that SCRN1 is upregulated in OSCC. Further analyses demonstrated that SCRN1 promotes the proliferation, invasion and migration of OSCC cells via TGF-β/Smad3 signaling.

Melanin's Journey from Melanocytes to Keratinocytes: Uncovering the Molecular Mechanisms of Melanin Transfer and Processing.

Skin pigmentation ensures efficient photoprotection and relies on the pigment melanin, which is produced by epidermal melanocytes and transferred to surrounding keratinocytes. While the molecular mechanisms of melanin synthesis and transport in melanocytes are now well characterized, much less is known about melanin transfer and processing within keratinocytes. Over the past few decades, distinct models have been proposed to explain how melanin transfer occurs at the cellular and molecular levels. However, this remains a debated topic, as up to four different models have been proposed, with evidence presented supporting each. Here, we review the current knowledge on the regulation of melanin exocytosis, internalization, processing, and polarization. Regarding the different transfer models, we discuss how these might co-exist to regulate skin pigmentation under different conditions, i.e., constitutive and facultative skin pigmentation or physiological and pathological conditions. Moreover, we discuss recent evidence that sheds light on the regulation of melanin exocytosis by melanocytes and internalization by keratinocytes, as well as how melanin is stored within these cells in a compartment that we propose be named the melanokerasome. Finally, we review the state of the art on the molecular mechanisms that lead to melanokerasome positioning above the nuclei of keratinocytes, forming supranuclear caps that shield the nuclear DNA from UV radiation. Thus, we provide a comprehensive overview of the current knowledge on the molecular mechanisms regulating skin pigmentation, from melanin exocytosis by melanocytes and internalization by keratinocytes to processing and polarization within keratinocytes. A better knowledge of these molecular mechanisms will clarify long-lasting questions in the field that are crucial for the understanding of skin pigmentation and can shed light on fundamental aspects of organelle biology. Ultimately, this knowledge can lead to novel therapeutic strategies to treat hypo- or hyper-pigmentation disorders, which have a high socio-economic burden on patients and healthcare systems worldwide, as well as cosmetic applications.

Yolk granule fusion and microtubule aster formation regulate cortical granule translocation and exocytosis in zebrafish oocytes.

Dynamic reorganization of the cytoplasm is key to many core cellular processes, such as cell division, cell migration, and cell polarization. Cytoskeletal rearrangements are thought to constitute the main drivers of cytoplasmic flows and reorganization. In contrast, remarkably little is known about how dynamic changes in size and shape of cell organelles affect cytoplasmic organization. Here, we show that within the maturing zebrafish oocyte, the surface localization of exocytosis-competent cortical granules (Cgs) upon germinal vesicle breakdown (GVBD) is achieved by the combined activities of yolk granule (Yg) fusion and microtubule aster formation and translocation. We find that Cgs are moved towards the oocyte surface through radially outward cytoplasmic flows induced by Ygs fusing and compacting towards the oocyte center in response to GVBD. We further show that vesicles decorated with the small Rab GTPase Rab11, a master regulator of vesicular trafficking and exocytosis, accumulate together with Cgs at the oocyte surface. This accumulation is achieved by Rab11-positive vesicles being transported by acentrosomal microtubule asters, the formation of which is induced by the release of CyclinB/Cdk1 upon GVBD, and which display a net movement towards the oocyte surface by preferentially binding to the oocyte actin cortex. We finally demonstrate that the decoration of Cgs by Rab11 at the oocyte surface is needed for Cg exocytosis and subsequent chorion elevation, a process central in egg activation. Collectively, these findings unravel a yet unrecognized role of organelle fusion, functioning together with cytoskeletal rearrangements, in orchestrating cytoplasmic organization during oocyte maturation.

Loss of ZNF148 enhances insulin secretion in human pancreatic β cells.

Insulin secretion from pancreatic β cells is essential to the maintenance of glucose homeostasis. Defects in this process result in diabetes. Identifying genetic regulators that impair insulin secretion is crucial for the identification of novel therapeutic targets. Here, we show that reduction of ZNF148 in human islets, and its deletion in stem cell-derived β cells (SC-β cells), enhances insulin secretion. Transcriptomics of ZNF148-deficient SC-β cells identifies increased expression of annexin and S100 genes whose proteins form tetrameric complexes involved in regulation of insulin vesicle trafficking and exocytosis. ZNF148 in SC-β cells prevents translocation of annexin A2 from the nucleus to its functional place at the cell membrane via direct repression of S100A16 expression. These findings point to ZNF148 as a regulator of annexin-S100 complexes in human β cells and suggest that suppression of ZNF148 may provide a novel therapeutic strategy to enhance insulin secretion.

Rac2 regulates immune complex-mediated granule polarization and exocytosis in neutrophils.

A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase (GTPase). Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin-1A (Coro1A), a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes (IC) and relate this to intracellular Coro1A localization. We used bone marrow neutrophils (BMN) from WT and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase, MPO), secondary (lactoferrin, LTF) and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of Coro1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in MPO, MMP-2, and MMP-9, but not LTF, release, along with diminished filopodia formation, CD63 polarization, and colocalization of Coro1A with CD63 in IC-stimulated Rac2-/- BMNs. Rac2 and Coro1A were found associated with granules in CB/fMLF-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on Coro1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.