Stages Of Vesicle Formation Research Articles

Counting AP2 using quantitative correlative light and electron microscopy (quant-CLEM).

Clathrin-mediated-endocytosis (CME) constitutes a ubiquitous and essential mechanism for the internalization of membrane segments or extracellular cargo in eukaryotic cells. Together with the pathway's namesake protein clathrin, dozens of other proteins contribute to the binding of specific cargo, coating the plasma membrane, inducing membrane curvature and eventual budding of vesicles. Facilitated by their characteristic honeycomb structure, clathrin-coated membranes have proven an ideal subject for correlative light and electron microscopy (CLEM) imaging. Combination of this approach with super-resolution light microscopy allowed mapping the precise locations of numerous key proteins during different stages of vesicle formation.

From Otic Induction to Hair Cell Production: Pax2EGFP Cell Line Illuminates Key Stages of Development in Mouse Inner Ear Organoid Model.

Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.

Clathrin polymerization exhibits high mechano-geometric sensitivity.

How tension modulates cellular transport has become a topic of interest in the recent past. However, the effect of tension on clathrin assembly and vesicle growth remains less understood. Here, we use the classical Helfrich theory to predict the energetic cost that clathrin is required to pay to remodel the membrane at different stages of vesicle formation. Our study reveals that this energetic cost is highly sensitive to not only the tension in the membrane but also to the instantaneous geometry of the membrane during shape evolution. Our study predicts a sharp reduction in clathrin coat size in the intermediate tension regime (0.01-0.1 mN m-1). Remarkably, the natural propensity of the membrane to undergo bending beyond the Ω shape causes a significant decrease in the energy needed from clathrin to drive vesicle growth. Our studies in mammalian cells confirm a reduction in clathrin coat size in an increased tension environment. In addition, our findings suggest that the two apparently distinct clathrin assembly modes, namely coated pits and coated plaques, observed in experimental investigations might be a consequence of varied tensions in the plasma membrane. Overall, the mechano-geometric sensitivity revealed in this study might also be at play during the polymerization of other membrane remodeling proteins.

Accelerated evolution of 3'avian FOXE1 genes, and thyroid and feather specific expression of chicken FoxE1

BackgroundThe forkhead transcription factor gene E1 (FOXE1) plays an important role in regulation of thyroid development, palate formation and hair morphogenesis in mammals. However, avian FOXE1 genes have not been characterized and as such, codon evolution of FOXE1 orthologs in a broader evolutionary context of mammals and birds is not known.ResultsIn this study we identified the avian FOXE1 gene in chicken, turkey and zebra finch, all of which consist of a single exon. Chicken and zebra finch FOXE1 are uniquely located on the sex-determining Z chromosome. In situ hybridization shows that chicken FOXE1 is specifically expressed in the developing thyroid. Its expression is initiated at the placode stage and is maintained during the stages of vesicle formation and follicle primordia. Based on this expression pattern, we propose that avian FOXE1 may be involved in regulating the evagination and morphogenesis of thyroid. Chicken FOXE1 is also expressed in growing feathers. Sequence analysis identified two microdeletions in the avian FOXE1 genes, corresponding to the loss of a transferable repression domain and an engrailed homology motif 1 (Eh1) C-terminal to the forkhead domain. The avian FOXE1 proteins exhibit a significant sequence divergence of the C-terminus compared to those of amphibian and mammalian FOXE1. The codon evolution analysis (dN/dS) of FOXE1 shows a significantly increased dN/dS ratio in the avian lineages, consistent with either a relaxed purifying selection or positive selection on a few residues in avian FOXE1 evolution. Further site specific analysis indicates that while relaxed purifying selection is likely to be a predominant cause of accelerated evolution at the 3'-region of avian FOXE1, a few residues might have evolved under positive selection.ConclusionsWe have identified three avian FOXE1 genes based on synteny and sequence similarity as well as characterized the expression pattern of the chicken FOXE1 gene during development. Our evolutionary analyses suggest that while a relaxed purifying selection is likely to be the dominant force driving accelerated evolution of avian FOXE1 genes, a few residues may have evolved adaptively. This study provides a basis for future genetic and comparative biochemical studies of FOXE1.

ER-resident Gi2protein controls sar1 translocation onto the ER during budding of transport vesicles

In our previous study, fluoride ([AlF(4) ](-) ) disturbed ER-to-Golgi transport through the activation of ER-resident heterotrimeric G protein (ER-G protein). Therefore, ER-G protein may be implicated in ER-to-Golgi transport at the early stage prior to coat protein assembly. Sar1 translocation onto the endoplasmic reticulum (ER) membrane is suppressed by non-selective protein kinase inhibitor H89, suggesting the participation of H89-sensitive kinase in this process. To investigate the involvement of ER-G protein in ER-to-Golgi transport, the effect of G(i) protein activator (mastoparan 7) was examined on Sar1 translocation onto the ER in a cell-free system consisting of microsome membrane and cytosol. Sar1 translocation onto the microsome membrane was induced by addition of GTPγS in the cell-free system. Translocation of Sar1 by GTPγS was suppressed significantly by both H89 and mastoparan 7. Mastoparan 7 suppressed the translocation of Sar1 onto the microsome membrane with dosage dependency, but mastoparan 17, the inactive analog of mastoparan 7, had no effect on Sar1 translocation. The suppressive effect of mastoparan 7 was recovered by treatment with pertussis toxin (IAP). Moreover, G(i2) protein was detected on the microsome membrane by western blotting for heterotrimeric G(i) proteins. These results indicate that ER-G(i2) protein modulated Sar1 translocation onto the ER, suggesting that ER-resident G(i2) protein is an important negative regulator of vesicular transport at the early stage of vesicle formation before coat protein assembly on the ER.

Structure of the Eps15–stonin2 complex provides a molecular explanation for EH-domain ligand specificity

Eps15 homology (EH) domain-containing proteins play a key regulatory role in intracellular membrane trafficking and cell signalling. EH domains serve as interaction platforms for short peptide motifs comprising the residues NPF within natively unstructured regions of accessory proteins. The EH-NPF interactions described thus far are of very low affinity and specificity. Here, we identify the presynaptic endocytic sorting adaptor stonin2 as a high-affinity ligand for the second EH domain (EH2) of the clathrin accessory protein Eps15. Calorimetric data indicate that both NPF motifs within stonin2 interact with EH2 simultaneously and with sub-micromolar affinity. The solution structure of this complex reveals that the first NPF motif binds to the conserved site on the EH domain, whereas the second motif inserts into a novel hydrophobic pocket. Our data show how combination of two EH-attachment sites provides a means for modulating specificity and allows discrimination from a large pool of potential binding partners containing NPF motifs.

Mosaic Organization of the Endocytic Pathway

Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Besides possible recycling routes to the plasma membrane and various organelles, previously described endocytic pathways (e.g., clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway) all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs), we identify an endocytic pathway that moves material to nonacidic Lamp1-positive late endosomes. Trafficking via this endocytic route is fully independent of Rab5 and Rab7 but requires the Rab14 protein. The pathway taken by CPPs differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already, as it is not affected by a series of compounds that inhibit macropinocytosis or clathrin-mediated endocytosis. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins.

Rotary dialysis: Its application to the preparation of large liposomes and large proteoliposomes (protein-lipid vesicles) with high encapsulation efficiency and efficient reconstitution of membrane proteins

An apparatus for rotary dialysis is introduced and described in detail. The component parts are inexpensive, widely available, and relatively easy to modify and assemble. The apparatus achieves increased mixing of the contents of dialysis bags by constant end-over-end rotation. This technique is particularly useful in systems where maximum contact is desired between substances which would tend to partition under standard dialysis conditions. We have applied rotary dialysis to two liposome production methods. These are (i) the calcium-EDTA-chelation method of Papahadjopoulos et al. (1), which produces large unilamellar liposomes from negatively charged phospholipids, and (ii) a procedure for the reconstituion of membrane proteins into liposomes with a large internal aqueous space, which we have developed using the calcium-EDTA-chelation technique as a point of departure. In both techniques, vesicle formation occurs when a calcium-phospholipid precipitate is dissolved by the addition of EDTA. Instead of adding a 150 m m EDTA solution directly, as described in the original method, we have used overnight rotary dialysis against buffer containing 10 m m EDTA at the vesicle formation stage. Materials are encapsulated within the aqueous interior of the vesicles at much higher efficiencies when rotary dialysis is used in either method, compared to efficiencies obtained with direct addition of EDTA (up to 37% of added material vs a maximum published efficiency of 10% for direct addition). Rotary dialysis also promotes the reconstitution of a higher proportion of the membrane proteins present in the dialysis mixture into the bilayer of large liposomes (79 vs 41.6%). It also affects the content of liposomes qualitatively, allowing better reconstitution of the Sendai virus F glycoprotein than does direct addition of EDTA. These effects may be due to the slow time course, the extensive mixing of components, and the low volume-to-phospholipid ratios maintained during vesicle formation.