Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale.
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ABSTRACT:
SUBMITTER: Paramasivam P
PROVIDER: S-EPMC8666849 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Through substitution mapping studies, we previously identified that a <330kb region from a rat strain with no renal pathology (the Lewis rat), which when introgressed onto the genetic background of a rat with renal disease (the Dahl Salt-sensitive (S) rat), caused an increase rather than the expected decrease in proteinuria. The purpose of this study was to prioritize a candidate gene and further delineate the mechanism underlying the observed increased in proteinuria. A higher level of proteinuria independent of dietary salt was observed in the congenic rat at a very young age (50-52 day old). The critical congenic segment was further mapped to <42.5kb containing a single candidate gene, rififylin. Rififylin was expressed 1.59 fold higher in the congenic strain compared with S. Overexpression of rififylin is known to delay recycling of endosomes. Renal transcriptome analysis indicated that Atp1a1 one of the most highly differentially expressed genes. Atp1a1 was 5.33 fold higher in the congenic strain compared with S. The protein product of Atp1a1, the alpha subunit of Na+K+ATPase, was also significantly higher in the endosomes of proximal tubules from the congenic strain compared with S. To determine whether the higher amounts of this protein in the endosomes is due to a delay in recycling of endosomes caused by the overexpression of rififylin in the congenic strain, recycling of exogenously labeled-transferrin by single cell cultures of proximal tubules was monitored by confocal microscopy. Recycling of transferrin was significantly delayed in the congenic strain compared with S. These results suggest that impaired endosomal recycling in the proximal tubules from the congenic strain caused by the overexpression of rififylin is a novel molecular mechanism linked to the observed increase in proteinuria of the congenic strain. Three male S control and 3 male congenic S.LEW(10)x12x2x3x5 rats born on the same day were selected, weaned at 30 days of age, and caged with 1 congenic and 1 S rat per cage. They were raised on a low-salt (0.3%) diet (Harlan Teklad diet TD 7034; Harlan–Sprague-Dawley) and sacrificed at 53 days of age and total RNAs were isolated from the kidney. The isolated RNA from each animal was used for the cRNA preparation. cRNA was prepared and fragmented as suggested by Affymetrix technical manual, and simultaneously hybridized (15 µg adjusted cRNA for each chip) to Rat Expression Array 230 2.0 (3' IVT Expression Analysis). Statistical analyses of the microarray data were performed with BH adjustment using R statistical package (version 2.8.1).
2011-12-17 | E-GEOD-30770 | biostudies-arrayexpress
Project description:Hermansky-Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2-deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2-deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.
| S-EPMC4442807 | biostudies-literature
Project description:Through substitution mapping studies, we previously identified that a <330kb region from a rat strain with no renal pathology (the Lewis rat), which when introgressed onto the genetic background of a rat with renal disease (the Dahl Salt-sensitive (S) rat), caused an increase rather than the expected decrease in proteinuria. The purpose of this study was to prioritize a candidate gene and further delineate the mechanism underlying the observed increased in proteinuria. A higher level of proteinuria independent of dietary salt was observed in the congenic rat at a very young age (50-52 day old). The critical congenic segment was further mapped to <42.5kb containing a single candidate gene, rififylin. Rififylin was expressed 1.59 fold higher in the congenic strain compared with S. Overexpression of rififylin is known to delay recycling of endosomes. Renal transcriptome analysis indicated that Atp1a1 one of the most highly differentially expressed genes. Atp1a1 was 5.33 fold higher in the congenic strain compared with S. The protein product of Atp1a1, the alpha subunit of Na+K+ATPase, was also significantly higher in the endosomes of proximal tubules from the congenic strain compared with S. To determine whether the higher amounts of this protein in the endosomes is due to a delay in recycling of endosomes caused by the overexpression of rififylin in the congenic strain, recycling of exogenously labeled-transferrin by single cell cultures of proximal tubules was monitored by confocal microscopy. Recycling of transferrin was significantly delayed in the congenic strain compared with S. These results suggest that impaired endosomal recycling in the proximal tubules from the congenic strain caused by the overexpression of rififylin is a novel molecular mechanism linked to the observed increase in proteinuria of the congenic strain.
2011-12-17 | GSE30770 | GEO
Project description:Endosomal escape is a key step for intracellular drug delivery of nucleic acids, but reliable and sensitive methods for its quantitation remain an unmet need. In order to rationally optimize the mRNA transfection efficiency of a library of polymeric materials, we designed a deactivated Renilla luciferase-derived molecular probe whose activity can be restored only in the cytosol. This probe can be coencapsulated with mRNA in the same delivery vehicle, thereby accurately measuring its endosomal escape efficiency. We examined a library of poly(amine-co-ester) (PACE) polymers with different end groups using this probe and observed a strong correlation between endosomal escape and transfection efficiency (R2 = 0.9334). In addition, we found that mRNA encapsulation efficiency and endosomal escape, but not uptake, were determinant factors for transfection efficiency. The polymers with high endosomal escape/transfection efficiency in vitro also showed good transfection efficiency in vivo, and mRNA expression was primarily observed in spleens after intravenous delivery. Together, our study suggests that the luciferase probe can be used as an effective tool to quantitate endosomal escape, which is essential for rational optimization of intracellular drug delivery systems.
| S-EPMC7195212 | biostudies-literature
Project description:Despite the increasing number of regulatory proteins identified in clathrin-independent endocytic (CIE) pathways, our understanding of the exact functions of these proteins and the sequential manner in which they function remains limited. In this study, using the Caenorhabditis elegans intestine as a model, we observed a unique structure of interconnected endosomal tubules, which is required for the basolateral recycling of several CIE cargoes including hTAC, GLUT1, and DAF-4. SEC-10 is a subunit of the octameric protein complex exocyst. Depleting SEC-10 and several other exocyst components disrupted the endosomal tubules into various ring-like structures. An epistasis analysis further suggested that SEC-10 operates at the intermediate step between early endosomes and recycling endosomes. The endosomal tubules were also sensitive to inactivation of the Rab GTPase RAB-10 and disruption of microtubules. Taken together, our data suggest that SEC-10 coordinates with RAB-10 and microtubules to form the endosomal tubular network for efficient recycling of particular CIE cargoes.
| S-EPMC4217428 | biostudies-literature
Project description:Cell surface proteins are internalized into the cell through endocytosis and either degraded within lysosomes or recycled back to the plasma membrane. While perturbations in endosomal internalization are known to modulate renal function, it is not known whether similar alterations in recycling affect renal function. Rififylin is a known regulator of endocytic recycling with E3 ubiquitin protein ligase activity. In this study, using two genetically similar strains, the Dahl Salt-sensitive rat and an S.LEW congenic strain, which had allelic variants within a?<?330?kb segment containing rififylin, we tested the hypothesis that alterations in endosomal recycling affect renal function. The congenic strain had 1.59-fold higher renal expression of rififylin. Transcriptome analysis indicated that components of both endocytosis and recycling were upregulated in the congenic strain. Transcription of Atp1a1 and cell surface content of the protein product of Atp1a1, the alpha subunit of Na(+)K(+)ATPase were increased in the proximal tubules from the congenic strain. Because rififylin does not directly regulate endocytosis and it is also a differentially expressed gene within the congenic segment, we reasoned that the observed alterations in the transcriptome of the congenic strain constitute a feedback response to the primary functional alteration of recycling caused by rififylin. To test this, recycling of transferrin was studied in isolated proximal tubules. Recycling was significantly delayed within isolated proximal tubules of the congenic strain, which also had a higher level of polyubiquitinated proteins and proteinuria compared with S. These data provide evidence to suggest that delayed endosomal recycling caused by excess of rififylin indirectly affects endocytosis, enhances intracellular protein polyubiquitination and contributes to proteinuria.
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Project description:Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.
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Project description:RNA-based therapeutics hold great promise for treating diseases and lipid nanoparticles (LNPs) represent the most advanced platform for RNA delivery. However, the fate of the LNP-mRNA after endosome-engulfing and escape from the autophagy-lysosomal pathway remains unclear. To investigate this, mRNA (encoding human erythropoietin) was delivered to cells using LNPs, which shows, for the first time, a link between LNP-mRNA endocytosis and its packaging into extracellular vesicles (endo-EVs: secreted after the endocytosis of LNP-mRNA). Endosomal escape of LNP-mRNA is dependent on the molar ratio between ionizable lipids and mRNA nucleotides. Our results show that fractions of ionizable lipids and mRNA (1:1 molar ratio of hEPO mRNA nucleotides:ionizable lipids) of endocytosed LNPs were detected in endo-EVs. Importantly, these EVs can protect the exogenous mRNA during in vivo delivery to produce human protein in mice, detected in plasma and organs. Compared to LNPs, endo-EVs cause lower expression of inflammatory cytokines.
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Project description:The inefficient translocation of proteins across biological membranes limits their application as therapeutic compounds and research tools. In most cases, translocation involves two steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules promote protein uptake but few enable efficient endosomal escape. One exception is ZF5.3, a mini-protein that exploits natural endosomal maturation machinery to translocate across endosomal membranes. Although certain ZF5.3-protein conjugates are delivered efficiently into the cytosol or nucleus, overall delivery efficiency varies widely with no obvious design rules. Here we evaluate the role of protein size and thermal stability in the ability to efficiently escape endosomes when attached to ZF5.3. Using fluorescence correlation spectroscopy, a single-molecule technique that provides a precise measure of intra-cytosolic protein concentration, we demonstrate that delivery efficiency depends on both size and the ease with which a protein unfolds. Regardless of size and pI, low-Tm cargos of ZF5.3 (including intrinsically disordered domains) bias its endosomal escape route toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal even if the Tm is high. These findings imply a novel protein- and/or lipid-dependent pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo.
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Project description:The MHC-Ib molecule MR1 presents microbial metabolites to MR1-restricted T cells (MR1Ts). Given the ubiquitous expression of MR1 and the high prevalence of human MR1Ts, it is important to understand the mechanisms of MR1-dependent antigen presentation. Here, we show that MR1-dependent antigen presentation can be distinguished between intracellular Mycobacterium tuberculosis (Mtb) infection and exogenously added antigens. Although both Mtb infection and exogenously added antigens are presented by preformed MR1, only exogenously added antigens are capable of reusing MR1 that had been bound to the folic acid metabolite 6-formylpterin (6-FP). In addition, we identify an endosomal trafficking protein, Syntaxin 4, which is specifically involved in the presentation of exogenously delivered antigens but not Mtb-dependent antigen presentation. These data reveal there are multiple ways that MR1 can sample antigens and that MR1-mediated sampling of intracellular Mtb infection is distinguishable from the sampling of exogenously added antigens.
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