Real‐Time pH‐Dependent Self‐Assembly of Ionisable Lipids from COVID‐19 Vaccines and In Situ Nucleic Acid Complexation
Haitao YuAngelina AngelovaBorislav AngelovBrendan DyettLauren MatthewsYiran ZhangMohamad El MohamadXudong CaiSepideh ValimehrCalum J. DrummondJiali Zhai
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Abstract Ionisable amino‐lipid is a key component in lipid nanoparticles (LNPs), which plays a crucial role in the encapsulation of RNA molecules, allowing efficient cellular uptake and then releasing RNA from acidic endosomes. Herein, we present direct evidence for the remarkable structural transitions, with decreasing membrane curvature, including from inverse micellar, to inverse hexagonal, to two distinct inverse bicontinuous cubic, and finally to a lamellar phase for the two mainstream COVID‐19 vaccine ionisable ALC‐0315 and SM‐102 lipids, occurring upon gradual acidification as encountered in endosomes. The millisecond kinetic growth of the inverse cubic and hexagonal structures and the evolution of the ordered structural formation upon ionisable lipid‐RNA/DNA complexation are quantitatively revealed by in situ synchrotron radiation time‐resolved small angle X‐ray scattering coupled with rapid flow mixing. We found that the final self‐assembled structural identity, and the formation kinetics, were controlled by the ionisable lipid molecular structure, acidic bulk environment, lipid compositions, and nucleic acid molecular structure/size. The implicated link between the inverse membrane curvature of LNP and LNP endosomal escape helps future optimisation of ionisable lipids and LNP engineering for RNA and gene delivery.Recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane. To determine how recycling endosomes perform their functions, searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers. However, it remains unclear whether all the conventional markers have identical localizations. Here we report finding that three well-known recycling endosome markers, i.e., Arf6, Rab11 and transferrin receptor (TfR), have different intracellular localizations in PC12 cells. The results of immunofluorescence analyses showed that the signals of endogenous Arf6, Rab11 and TfR in nerve growth factor-stimulated PC12 cells generally differed, although there was some overlapping. Our findings provide new information about recycling endosome markers, and they highlight the heterogeneity of recycling endosomes.
Transferrin receptor
Colocalization
Retromer
Intracellular transport
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Abstract The separation of functional early and late endosomes from other cellular compartments by free‐flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells [1, 2]. Here, using 125 I‐labeled anti‐secretory component antibodies ([ 125 I]SC Ab) and FITC‐labeled asialoorosomucoid (FITC‐ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16°C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20°C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37°C, transcytotic endosomes carrying [ 125 I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC‐ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common ( i.e. early endosomes) and two distinct endosome subpopulations ( i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.
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We have previously characterized the trafficking of transferrin (Tf) through HEp2 human carcinoma cells (Ghosh, R. N., D. L. Gelman, and F. R. Maxfield, 1994. J. Cell Sci. 107:2177-2189). Early endosomes in these cells are comprised of both sorting endosomes and recycling compartments, which are distinct separate compartments. Endocytosed Tf initially appears in punctate sorting endosomes that also contain recently endocytosed LDL. After short loading pulses, Tf rapidly sorts from LDL with first-order kinetics (t1/2 approximately 2.5 min), and it enters the recycling compartment before leaving the cell (t1/2 approximately 7 min). Here, we report a second, slower rate for Tf to leave sorting endosomes after HEp2 cells were labeled to steady state with fluorescein Tf instead of the brief pulse used previously. We determined this rate using digital image analysis to measure the Tf content of sorting endosomes that also contained LDL. With an 11-min chase, the Tf in sorting endosomes was 24% of steady-state value. This was in excess of the amount expected (5% of steady state) from the rate of Tf exit after short filling pulses. The excess could not be accounted for by reinternalization of recycled cell surface Tf, implying that either some Tf was retained in sorting endosomes, or that Tf was delivered back to the sorting endosomes from the recycling compartment. The former is unlikely since nearly all sorting endosomes contain detectable Tf after an 11-min chase, even though more than one third of the sorting endosomes were formed during the chase time. Furthermore, while observing living cells by confocal microscopy, we saw vesicle movements that appeared to be fluorescent Tf returning from recycling compartments to sorting endosomes. The slow rate of exit after steady-state labeling was similar to the Tf exit rate from the cell, suggesting an equilibration of Tf throughout the early endosomal system by this retrograde pathway. This retrograde traffic may be important for delivering molecules from the recycling compartment, which is a long-lived organelle, to sorting endosomes, which are transient.
Transferrin receptor
Cell Sorting
Sorting nexin
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Phosphatidylinositol 4-kinase IIα (PtdIns4KIIα) localizes to the trans-Golgi network and endosomal compartments and has been implicated in the regulation of endosomal traffic, but the roles of both its enzymatic activity and the site of its action have not been elucidated. This study shows that PtdIns4KIIα is required for production of endosomal phosphatidylinositol 4-phosphate (PtdIns(4)P) on early endosomes and for the sorting of transferrin and epidermal growth factor receptor into recycling and degradative pathways. Depletion of PtdIns4KIIα with small interfering RNA significantly reduced the amount of vesicular PtdIns(4)P on early endosomes but not on Golgi membranes. Cells depleted of PtdIns4KIIα had an impaired ability to sort molecules destined for recycling from early endosomes. We further identify the Eps15 homology domain–containing protein 3 (EHD3) as a possible endosomal effector of PtdIns4KIIα. Tubular endosomes containing EHD3 were shortened and became more vesicular in PtdIns4KIIα-depleted cells. Endosomal PtdIns(4,5)P 2 was also significantly reduced in PtdIns4KIIα-depleted cells. These results show that PtdIns4KIIα regulates receptor sorting at early endosomes through a PtdIns(4)P-dependent pathway and contributes substrate for the synthesis of endosomal PtdIns(4,5)P 2 .
Pleckstrin homology domain
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After endocytosis, lysosomally targeted ligands pass through a series of endosomal compartments. The endocytic apparatus that accomplishes this passage may be considered to take one of two forms: (a) a system in which lysosomally targeted ligands pass through preexisting, long-lived early sorting endosomes and are then selectively transported to long-lived late endosomes in carrier vesicles, or (b) a system in which lysosomally targeted ligands are delivered to early sorting endosomes which themselves mature into late endosomes. We have previously shown that sorting endosomes in CHO cells fuse with newly formed endocytic vesicles (Dunn, K. W., T. E. McGraw, and F. R. Maxfield. 1989. J. Cell Biol. 109:3303-3314) and that previously endocytosed ligands lose their accessibility to fusion with a half-time of approximately 8 min (Salzman, N. H., and F. R. Maxfield. 1989. J. Cell Biol. 109:2097-2104). Here we have studied the properties of individual endosomes by digital image analysis to distinguish between the two mechanisms for entry of ligands into late endosomes. We incubated TRVb-1 cells (derived from CHO cells) with diO-LDL followed, after a variable chase, by diI-LDL, and measured the diO content of diI-containing endosomes. As the chase period was lengthened, an increasing percentage of the endosomes containing diO-LDL from the initial incubation had no detectable diI-LDL from the second incubation, but those endosomes that contained both probes showed no decrease in the amount of diO-LDL per endosomes. These results indicate that (a) a pulse of fluorescent LDL is retained by individual sorting endosomes, and (b) with time sorting endosomes lose the ability to fuse with primary endocytic vesicles. These data are inconsistent with a preexisting compartment model which predicts that the concentration of ligand in sorting endosomes will decline during a chase interval, but that the ability of the stable sorting endosome to receive newly endocytosed ligands will remain high. These data are consistent with a maturation mechanism in which the sorting endosome retains and accumulates lysosomally directed ligands until it loses its ability to fuse with newly formed endocytic vesicles and matures into a late endosome. We also find that, as expected according to the maturation model, new sorting endosomes are increasingly labeled during the chase period indicating that new sorting endosomes are continuously formed to replace those that have matured into late endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)
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Most antibody-drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.
Conjugate
Linker
Antibody-drug conjugate
Bispecific antibody
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Objective To design the ribozymes which may cleave the mRNA of human TIMP-1. Establishing plasmids transcripting the ribozymes to produce abundant ribozymes. Methods The TIMP-1 mRNA was taken as the target RNA. Ribozymes were designed according to the hammerhead structure described by Symons. To establish clones of TIMP-1 ribozymes use P 1.5. Then obtain abundant ribozymes by transcripting. Results Three ribozymes targeting the nt 123, nt 299 and nt 353 on TIMP-1 mRNA were designed. The ribozyme gene has been synthesized, plasmids cloned and abundant ribozymes obtained. Conclusion Computer assisted design is indispensable in studying ribozyme. The establishment of plasmids transcripting ribozymes is the key step of studying their activity.
Ligase ribozyme
Hammerhead ribozyme
Cleave
Mammalian CPEB3 ribozyme
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Cells internalize extracellular solutes, ligands and proteins and lipids in the plasma membrane (PM) by endocytosis. The removal of membrane from the PM is counteracted by endosomal recycling pathways that return the endocytosed proteins and lipids back to the PM. Recycling to the PM can occur from early endosomes. However, many cells have a distinct subpopulation of endosomes that have a mildly acidic pH of 6.5 and are involved in the endosomal recycling. These endosomes are dubbed recycling endosomes (REs). In recent years, studies have begun to reveal that function of REs is not limited to the endosomal recycling. In this review, I summarize the nature of membrane trafficking pathways that pass through REs and the cell biological roles of these pathways.
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