Trafficking Of Complexes Research Articles

The subcellular distribution of miRNA isoforms, tRNA-derived fragments, and rRNA-derived fragments depends on nucleotide sequence and cell type

BackgroundMicroRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent most of the small non-coding RNAs (sncRNAs) found in cells. Members of these three classes modulate messenger RNA (mRNA) and protein abundance and are dysregulated in diseases. Experimental studies to date have assumed that the subcellular distribution of these molecules is well-understood, independent of cell type, and the same for all isoforms of a sncRNA.ResultsWe tested these assumptions by investigating the subcellular distribution of isomiRs, tRFs, and rRFs in biological replicates from three cell lines from the same tissue and same-sex donors that model the same cancer subtype. In each cell line, we profiled the isomiRs, tRFs, and rRFs in the nucleus, cytoplasm, whole mitochondrion (MT), mitoplast (MP), and whole cell. Using a rigorous mathematical model we developed, we accounted for cross-fraction contamination and technical errors and adjusted the measured abundances accordingly. Analyses of the adjusted abundances show that isomiRs, tRFs, and rRFs exhibit complex patterns of subcellular distributions. These patterns depend on each sncRNA’s exact sequence and the cell type. Even in the same cell line, isoforms of the same sncRNA whose sequences differ by a few nucleotides (nts) can have different subcellular distributions.ConclusionsSncRNAs with similar sequences have different subcellular distributions within and across cell lines, suggesting that each isoform could have a different function. Future computational and experimental studies of isomiRs, tRFs, and rRFs will need to distinguish among each molecule’s various isoforms and account for differences in each isoform’s subcellular distribution in the cell line at hand. While the findings add to a growing body of evidence that isomiRs, tRFs, rRFs, tRNAs, and rRNAs follow complex intracellular trafficking rules, further investigation is needed to exclude alternative explanations for the observed subcellular distribution of sncRNAs.

Proximity interactome of lymphatic VE-cadherin reveals mechanisms of junctional remodeling and reelin secretion

The adhesion receptor vascular endothelial (VE)-cadherin transduces an array of signals that modulate crucial lymphatic cell behaviors including permeability and cytoskeletal remodeling. Consequently, VE-cadherin must interact with a multitude of intracellular proteins to exert these functions. Yet, the full protein interactome of VE-cadherin in endothelial cells remains a mystery. Here, we use proximity proteomics to illuminate how the VE-cadherin interactome changes during junctional reorganization from dis-continuous to continuous junctions, triggered by the lymphangiogenic factor adrenomedullin. These analyses identified interactors that reveal roles for ADP ribosylation factor 6 (ARF6) and the exocyst complex in VE-cadherin trafficking and recycling. We also identify a requisite role for VE-cadherin in the in vitro and in vivo control of secretion of reelin—a lymphangiocrine glycoprotein with recently appreciated roles in governing heart development and injury repair. This VE-cadherin protein interactome shines light on mechanisms that control adherens junction remodeling and secretion from lymphatic endothelial cells.

Autosomal Recessive Spastic Paraplegia Type 51 Caused by Homozygous Mutation of the AP4E1 Gene: A Case Report of a 17-Years-Old Boy

Autosomal recessive spastic paraplegia-51 (SPG51) is a rare neurodevelopmental disorder caused by a homozygous mutation in the AP4E1 gene, located on chromosome 15q21. Spastic paraplegia-51 (SPG51) is an extremely rare autosomal recessive neurodevelopmental disorder characterized by neonatal hypotonia that progresses to hypertonia and spasticity. Mutations in the AP-4E1 gene on chromosome 15q21.2, which is part of the AP-4 complex, can lead to autosomal recessive spastic paraplegia 51 (SPG51). Different mutations in this gene have been identified in affected individuals, leading to disruption of the AP4 complex and vesicular trafficking processes. The specific characteristics of SPG51 are, due to only few cases, not well understood due to limited reports of affected families. Affected individuals also have global developmental delay with impaired intellectual development and poor or absent speech. Studies linked to an additive symptom of persistent stuttering. Affected individuals typically present with hypotonia in the neonatal period, which progresses to muscular hypertonia, especially in the lower limbs. They may also exhibit contractures, talipes equinovarus, decreased muscle mass, short stature, and microcephaly. Severe mental retardation, absent speech, and dysmorphic facial features are common. Some patients may experience seizures. Neurological examination often reveals spastic paraplegia of the lower limbs, and brain imaging may show atrophy of the cerebellar vermis and cortical atrophy. We present an extremely rare case of a 17 years-old boy with autosomal recessive spastic paraplegia type 51.

An AAGAB-to-CCDC32 handover mechanism controls the assembly of the AP2 adaptor complex

Vesicular transport relies on multimeric trafficking complexes to capture cargo and drive vesicle budding and fusion. Faithful assembly of the trafficking complexes is essential to their functions but remains largely unexplored. Assembly of AP2 adaptor, a heterotetrameric protein complex regulating clathrin-mediated endocytosis, is assisted by the chaperone AAGAB. Here, we found that AAGAB initiates AP2 assembly by stabilizing its α and σ2 subunits, but the AAGAB:α:σ2 complex cannot recruit additional AP2 subunits. We identified CCDC32 as another chaperone regulating AP2 assembly. CCDC32 recognizes the AAGAB:α:σ2 complex, and its binding leads to the formation of an α:σ2:CCDC32 ternary complex. The α:σ2:CCDC32 complex serves as a template that sequentially recruits the µ2 and β2 subunits of AP2 to complete AP2 assembly, accompanied by CCDC32 release. The AP2-regulating function of CCDC32 is disrupted by a disease-causing mutation. These findings demonstrate that AP2 is assembled by a handover mechanism switching from AAGAB-based initiation complexes to CCDC32-based template complexes. A similar mechanism may govern the assembly of other trafficking complexes exhibiting the same configuration as AP2.

Abstract Tu119: The exocyst trafficking complex mediates fuel transporter membrane delivery in cardiomyocytes.

Introduction: To maintain homeostasis, cardiomyocytes (CMs) primarily rely on fatty acid β-oxidation for ATP production facilitated by substrate uptake via fuel transporters. The glucose transporter, GLUT4, and fatty acid translocase, CD36, are directed to the cardiomyocyte sarcolemma in response to insulin signaling and AMP-activated protein kinase (AMPK) signaling. In adverse conditions like acute hypoxia and ischemia, these cells undergo a metabolic shift to favor glycolysis. Previous reports showed that under these conditions, CD36 moved away from the sarcolemma as GLUT4 was delivered to the cell membrane, contributing to a shift from fatty acid oxidation to glycolysis. Investigating CD36 and GLUT4 membrane delivery in cardiomyocytes is key to better understanding cardiac energy metabolism under normal and disease conditions. Prior work in adipocytes, and our own studies in skeletal muscle showed that the exocyst trafficking complex is critical for membrane delivery of GLUT4 and CD36 in response to insulin and AMPK. Our objective was to determine if in cardiomyocytes GLUT4 and CD36 trafficking, and subsequent fuel uptake is dependent on the exocyst. Methods and Results: We demonstrate that the exocyst is recruited to GLUT4 and CD36-vesicles in cultured H9C2 cells in response to stimuli with proximity ligation assays. Using on-cell Western analysis and fuel uptake assays we also show that the exocyst is essential for induced GLUT4 and CD36 cell surface delivery and subsequent energy substrate uptake, as inhibition of the exocyst attenuates stimulated surface translocation of both fuel transporters and subsequent fluorescent fuel analog uptake. Our earlier work revealed that protein levels of exocyst subunits are increased in murine heart tissue following myocardial infarction. To examine the role of the exocyst in fuel uptake under ischemic conditions, we used an in vitro model of simulated ischemia, and found similar alterations in exocyst subunit expression in this model at both mRNA and protein levels. This suggests an increased demand for exocyst activity. Conclusion: Further research will explore how ischemia impacts exocyst-mediated trafficking and if the exocyst contributes to the glycolytic shift under ischemia in cardiomyocytes.

In Vivo Proximity Cross-Linking and Immunoprecipitation of Cell Wall Epitopes Identify Proteins Associated with the Biosynthesis of Matrix Polysaccharides.

Identification of proteins involved in cell wall matrix polysaccharide biosynthesis is crucial to understand plant cell wall biology. We utilized in vivo cross-linking and immunoprecipitation with cell wall antibodies that recognized xyloglucan, xylan, mannan, and homogalacturonan to capture proteins associated with matrix polysaccharides in Arabidopsis protoplasts. The use of cross-linkers allowed us to capture proteins actively associated with cell wall polymers, including those directly interacting with glycans via glycan-protein (GP) cross-linkers and those associated with proteins linked to glycans via a protein-protein (PP) cross-linker. Immunoprecipitations led to the identification of 65 Arabidopsis protein IDs localized in the Golgi, ER, plasma membrane, and others without subcellular localization data. Among these, we found several glycosyltransferases directly involved in polysaccharide synthesis, along with proteins related to cell wall modification and vesicle trafficking. Protein interaction networks from DeepAraPPI and AtMAD databases showed interactions between various IDs, including those related to cell-wall-associated proteins and membrane/vesicle trafficking proteins. Gene expression and coexpression analyses supported the presence and relevance of the proteins to the cell wall processes. Reverse genetic studies using T-DNA insertion mutants of selected proteins revealed changes in cell wall composition and saccharification, further supporting their potential roles in cell wall biosynthesis. Overall, our approach represents a novel approach for studying cell wall polysaccharide biosynthesis and associated proteins, providing advantages over traditional immunoprecipitation techniques. This study provides a list of putative proteins associated with different matrix polysaccharides for further investigation and highlights the complexity of cell wall biosynthesis and trafficking within plant cells.

Flp/FRT-mediated disruption of ptex150 and exp2 in Plasmodium falciparum sporozoites inhibits liver-stage development

Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.

Strengthening National Regulations in Combating Cross-Border Trafficking: Empirical Approach and the Way Forward

At practical level, both nationally and internationally, various efforts have been made to deal with increasingly acute human trafficking. However, as it turns out in practice, all existing legal instruments have not had a deterrent effect and have broken the chain of human trafficking to this day. Even more so in Indonesia. This article focuses on examining the answer to the problem, namely the national policy to prevent human trafficking and the efforts that can be made to eradicate increasingly complex human trafficking. This qualitative study is related to the policy and implementation of international and national policies to address human trafficking in cross-country border legal research. This paper provides information on the latest trends in research. The results show that the current moment of globalization is witnessing an extraordinary movement of people, legitimate and illegitimate, across national and international borders. This global movement of people has created panic across borders. It manifests itself in strengthening border controls and tightening immigration laws as a threat to the nation-state's security

Atypical chemokine receptors in the immune system.

Leukocyte migration is a fundamental component of innate and adaptive immune responses as it governs the recruitment and localization of these motile cells, which is crucial for immune cell priming, effector functions, memory responses and immune regulation. This complex cellular trafficking system is controlled to a large extent via highly regulated production of secreted chemokines and the restricted expression of their membrane-tethered G-protein-coupled receptors. The activity of chemokines and their receptors is also regulated by a subfamily of molecules known as atypical chemokine receptors (ACKRs), which are chemokine receptor-like molecules that do not couple to the classical signalling pathways that promote cell migration in response to chemokine ligation. There has been a great deal of progress in understanding the biology of these receptors and their functions in the immune system in the past decade. Here, we describe the contribution of the various ACKRs to innate and adaptive immune responses, focussing specifically on recent progress. This includes recent findings that have defined the role for ACKRs in sculpting extracellular chemokine gradients, findings that broaden the spectrum of chemokine ligands recognized by these receptors, candidate new additions to ACKR family, and our increasing understanding of the role of these receptors in shaping the migration of innate and adaptive immune cells.

Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells

BackgroundThe C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with “cancer hallmarks” and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT).Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions.Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities.MethodsStructured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model.ResultsWe identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. ConclusionsThis study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Interferon-induced MXB protein restricts vimentin-dependent viral infection

Type I interferon (IFN) inhibits a wide spectrum of viruses through stimulating the expression of antiviral proteins. As an IFN-induced protein, myxovirus resistance B (MXB) protein was reported to inhibit multiple highly pathogenic human viruses. It remains to be determined whether MXB employs a common mechanism to restrict different viruses. Here, we find that IFN alters the subcellular localization of hundreds of host proteins, and this IFN effect is partially lost upon MXB depletion. The results of our mechanistic study reveal that MXB recognizes vimentin (VIM) and recruits protein kinase B (AKT) to phosphorylate VIM at amino acid S38, which leads to reorganization of the VIM network and impairment of intracellular trafficking of virus protein complexes, hence causing a restriction of virus infection. These results highlight a new function of MXB in modulating VIM-mediated trafficking, which may lead towards a novel broad-spectrum antiviral strategy to control a large group of viruses that depend on VIM for successful replication.

Two roads diverge for cellulose synthase complex trafficking

Cellulose, an abundant and essential component of plant cell walls, is made by cellulose synthase complexes at the plasma membrane (PM). Recently, Liu et al. uncovered molecular mechanisms that suggest the existence of two distinct pathways for cellulose synthase trafficking from the Golgi apparatus to the PM.

The TRAPPIII subunit, Trs85, has a dual role in the trafficking of cellulose synthase complexes in Arabidopsis.

Plant cell walls are essential for defining plant growth and development, providing structural support to the main body and responding to abiotic and biotic cues. Cellulose, the main structural polymer of plant cell walls, is synthesized at the plasma membrane by cellulose synthase complexes (CSCs). The construction and transport of CSCs to and from the plasma membrane is poorly understood but is known to rely on the coordinated activity of cellulose synthase-interactive protein 1 (CSI1), a key regulator of CSC trafficking. In this study, we found that Trs85, a TRAPPIII complex subunit, interacted with CSI1 in vitro. Using functional genetics and live-cell imaging, we have shown that trs85-1 mutants have reduced cellulose content, stimulated CSC delivery, an increased population of static CSCs and deficient clathrin-mediated endocytosis in the primary cell wall. Overall, our findings suggest that Trs85 has a dual role in the trafficking of CSCs, by negatively regulating the exocytosis and clathrin-mediated endocytosis of CSCs.

Endolysosomal trafficking controls yolk granule biogenesis in vitellogenic Drosophila oocytes.

Endocytosis and endolysosomal trafficking are essential for almost all aspects of physiological functions of eukaryotic cells. As our understanding on these membrane trafficking events are mostly from studies in yeast and cultured mammalian cells, one challenge is to systematically evaluate the findings from these cell-based studies in multicellular organisms under physiological settings. One potentially valuable in vivo system to address this challenge is the vitellogenic oocyte in Drosophila, which undergoes extensive endocytosis by Yolkless (Yl), a low-density lipoprotein receptor (LDLR), to uptake extracellular lipoproteins into oocytes and package them into a specialized lysosome, the yolk granule, for storage and usage during later development. However, by now there is still a lack of sufficient understanding on the molecular and cellular processes that control yolk granule biogenesis. Here, by creating genome-tagging lines for Yl receptor and analyzing its distribution in vitellogenic oocytes, we observed a close association of different endosomal structures with distinct phosphoinositides and actin cytoskeleton dynamics. We further showed that Rab5 and Rab11, but surprisingly not Rab4 and Rab7, are essential for yolk granules biogenesis. Instead, we uncovered evidence for a potential role of Rab7 in actin regulation and observed a notable overlap of Rab4 and Rab7, two Rab GTPases that have long been proposed to have distinct spatial distribution and functional roles during endolysosomal trafficking. Through a small-scale RNA interference (RNAi) screen on a set of reported Rab5 effectors, we showed that yolk granule biogenesis largely follows the canonical endolysosomal trafficking and maturation processes. Further, the data suggest that the RAVE/V-ATPase complexes function upstream of or in parallel with Rab7, and are involved in earlier stages of endosomal trafficking events. Together, our study provides s novel insights into endolysosomal pathways and establishes vitellogenic oocyte in Drosophila as an excellent in vivo model for dissecting the highly complex membrane trafficking events in metazoan.

Quantifying mitochondrial protein import by mePRODmt proteomics.

Mitochondrial protein import is crucial for maintaining cellular health and homeostasis. Disruptions in this process have been linked to various diseases. Traditional methods for studying mitochondrial protein import predominantly focus on individual proteins and lack the dynamic resolution needed to fully appreciate the complexity of mitochondrial proteostasis and protein trafficking. To address these limitations, we developed a technique called mitochondria-specific multiplexed enhanced protein dynamics (mePRODmt). This method is a novel application of the mePROD methodology and utilizes pulsed stable isotope labeling with amino acids in cell culture (pSILAC)-based proteomics approach to study transient mitochondrial protein import. This chapter outlines the mePRODmt protocol, which includes the preparation of heavy SILAC-labeled peptides for boosting overall mitochondrial peptide signals (booster), SILAC labeling of cultured cells under experimental conditions, mitochondria isolation, sample preparation for multiplex proteomics using tandem mass tags (TMT) for isobaric labeling, recommended liquid chromatography-mass spectrometry (LC-MS) settings for reporter ion quantitation and a data analysis pipeline to analyze pSILAC-TMT data.

Distinct beta-arrestin coupling and intracellular trafficking of metabotropic glutamate receptor homo- and heterodimers.

The metabotropic glutamate receptors (mGluRs) are family C, dimeric G protein-coupled receptors (GPCRs), which play critical roles in synaptic transmission. Despite an increasing appreciation of the molecular diversity of this family, how distinct mGluR subtypes are regulated remains poorly understood. We reveal that different group II/III mGluR subtypes show markedly different beta-arrestin (β-arr) coupling and endocytic trafficking. While mGluR2 is resistant to internalization and mGluR3 shows transient β-arr coupling, which enables endocytosis and recycling, mGluR8 and β-arr form stable complexes, which leads to efficient lysosomal targeting and degradation. Using chimeras and mutagenesis, we pinpoint carboxyl-terminal domain regions that control β-arr coupling and trafficking, including the identification of an mGluR8 splice variant with impaired internalization. We then use a battery of high-resolution fluorescence assays to find that heterodimerization further expands the diversity of mGluR regulation. Together, this work provides insight into the relationship between GPCR/β-arr complex formation and trafficking while revealing diversity and intricacy in the regulation of mGluRs.

Human microglia, microglial LRRK2, and tau pathogenesis: a complex relationship

AbstractBackgroundEmerging evidence indicates leucine‐rich repeat kinase 2 (LRRK2) may be involved in tau uptake and spread throughout the brain. Microglia, the brain‐resident phagocytes, become one of the highest LRRK2 expressing cells under inflammatory conditions. Rodent microglia were also shown capable of secreting seeding‐competent tau into the extracellular space. We set out to examine the contribution of human microglia and microglial LRRK2 to tau pathogenesis.MethodIsogenic LRRK2 wild‐type (WT) and knockout (KO) induced pluripotent stem cell (iPSC) lines, together with Parkinson’s disease (PD) patient‐isolated LRRK2 G2019S‐mutated iPSC lines were differentiated into an in‐vitro model of human microglia, and incubated with in‐house purified, endotoxin‐free, human recombinant 2N4R tau in monomeric or in vitro aggregated form to examine the cellular response.ResultMicroglia internalise recombinant monomeric and aggregated tau by overlapping but distinct mechanisms. RNA‐seq analysis was performed to reveal changes in microglial gene expression upon tau internalisation. We further identified LRP1 as one of the major microglia receptors mediating soluble tau entry, and LRRK2 bona‐fide substrates, Rab8a and Rab10, as the Rab GTPases involved in the downstream LRP1‐tau complex trafficking. The internalised soluble tau is readily degraded but intracellular clearance of tau aggregates is concentration‐ and incubation‐length dependent, and inversely associated with microglial tau secretion. Following tau fibril incubation, the intracellular non‐cleared tau and tau secreted by microglia, shows seeding competency, supporting the role for microglia in tau prion‐like spreading. Finally, we describe the influence of microglial LRRK2 on tau processing. Compared to LRRK2 WT, tau uptake and clearance is accelerated in G2019S microglia. However, tau fibrils processed by G2019S microglia, though reduced in the amount, template pathogenic conformation faster and more consistently onto naive tau substrate.ConclusionCollectively, we provide detailed characterisation of the response of human iPSC‐microglia to tau protein in its native and pathogenic form, and show that microglial LRRK2 influences tau processing in a complex way that warrants further investigation.

Adiponectin Promote TPO Response for Treating ITP By Relating Rab6A-myosin9 Trafficking Vehicle Carrying c-Mpl to Cell Surface

Impaired megakaryocyte maturation is one of the pathogenic mechanisms of primary immune thrombocytopenia (ITP). Exploring the regulatory mechanisms of megakaryocyte maturation is important for the clinical treatment of ITP. The thrombopoietin (TPO) receptor c-Mpl on the membrane surface of megakaryocytes plays a critical role in the cellular reception of TPO signals and megakaryocyte maturation. But how the distribution of c-Mpl on the membrane surface of megakaryocytes influences the pathogenesis of ITP and the specific regulatory mechanisms are still unclear. In this project, we found that the distribution of c-Mpl on the membrane surface of megakaryocytes of ITP patients was significantly reduced, confirming that the abnormal distribution of c-Mpl is associated with the pathogenesis of ITP. Further, we applied immunoprecipitation and protein profiling to find the intracellular c-Mpl transport-related protein molecules. Motor protein myosin 9 and G protein-associated protein Rab6A were screened out. Next, we down-regulated the expression of myosin 9 and Rab6A, proved that aberrant expression of both leads to reduced transportation of the c-Mpl to membranes and impaired megakaryocyte maturation, identifying the regulatory complex for c-Mpl transport to membranes was composed of myosin 9 and Rab6A. Moreover, we confirmed that the formation of the above complex is dependent on GTP activation of Rab6A by transient overexpression of wild-type Rab6A (wt), dominant-negative (T27N) and GTPase-deficient (Q72L) Rab6A, clarified the working mechanism of how the Rab6A-myosin9 trafficking vehicle regulate c-Mpl transport to membranes. At the same time, we demonstrated that the abnormally low expression of Adiponectin in the plasma of ITP patients affects the transportation of the c-Mpl to membranes by inhibiting the interaction between Rab6A and myosin 9, thereby reducing the responsiveness of megakaryocytes to TPO and inhibiting the activation of its corresponding intracellular signaling pathways. Finally, using a mouse model of ITP disease, we demonstrated that Adiponectin could mediate the transportation of the c-Mpl to membranes by regulating the Rab6A-myosin9 complex and can improve the efficacy of TPO in the treatment of ITP. In conclusion, the present study identified a novel trafficking complex mediating the transportation of the c-Mpl to membranes, clarified the abnormal mechanism of this complex in ITP, and confirms that Adiponectin can regulate this complex to improve the efficacy of TPO, which provides a new idea for the clinical treatment of ITP.

The role of membrane trafficking and retromer complex in Parkinson's and Alzheimer's disease.

Membrane trafficking is a physiological process encompassing different pathways involved in transporting cellular products across cell membranes to specific cell locations via encapsulated vesicles. This process is required for cells to mature and function properly, allowing them to adapt to their surroundings. The retromer complex is a complex composed of nexin proteins and peptides that play a vital role in the endosomal pathway of membrane trafficking. In humans, any interference in normal membrane trafficking or retromer complex can cause profound changes such as those seen in neurodegenerative disorders such as Alzheimer's and Parkinson's. Several studies have explored the potential causative mechanisms in developing both disease processes; however, the role of retromer trafficking in their pathogenesis is becoming increasingly significant with promising therapeutic applications. This manuscript describes the processes involved in membrane transport and the roles of the retromer in the onset and progression of Alzheimer's and Parkinson's. Moreover, we will also explore how these aberrant mechanisms may serve as possible avenues for treatment development in both diseases and the prospect of its future application.

Role of the Glycine Receptor β Subunit in Synaptic Localization and Pathogenicity in Severe Startle Disease.

Startle disease is due to the disruption of recurrent inhibition in the spinal cord. Most common causes are genetic variants in genes (GLRA1, GLRB) encoding inhibitory glycine receptor (GlyR) subunits. The adult GlyR is a heteropentameric complex composed of α1 and β subunits that localizes at postsynaptic sites and replaces embryonically expressed GlyRα2 homomers. The human GlyR variants of GLRA1 and GLRB, dominant and recessive, have been intensively studied in vitro. However, the role of unaffected GlyRβ, essential for synaptic GlyR localization, in the presence of mutated GlyRα1 in vivo is not fully understood. Here, we used knock-in mice expressing endogenous mEos4b-tagged GlyRβ that were crossed with mouse Glra1 startle disease mutants. We explored the role of GlyRβ under disease conditions in mice carrying a missense mutation (shaky) or resulting from the loss of GlyRα1 (oscillator). Interestingly, synaptic targeting of GlyRβ was largely unaffected in both mouse mutants. While synaptic morphology appears unaltered in shaky animals, synapses were notably smaller in homozygous oscillator animals. Hence, GlyRβ enables transport of functionally impaired GlyRα1 missense variants to synaptic sites in shaky animals, which has an impact on the efficacy of possible compensatory mechanisms. The observed enhanced GlyRα2 expression in oscillator animals points to a compensation by other GlyRα subunits. However, trafficking of GlyRα2β complexes to synaptic sites remains functionally insufficient, and homozygous oscillator mice still die at 3 weeks after birth. Thus, both functional and structural deficits can affect glycinergic neurotransmission in severe startle disease, eliciting different compensatory mechanisms in vivo.