Simple models of complex aggregation: Vesicle formation by soft repulsive spheres with dipolelike interactions

Structural and thermodynamic properties of spherical particles carrying classical spins are investigated by Monte Carlo simulations. The potential energy is the sum of short range, purely repulsive pair contributions, and spin-spin interactions. These last are of the dipole-dipole form, with however, a crucial change of sign. At low density and high temperature the system is a homogeneous fluid of weakly interacting particles and short range spin correlations. With decreasing temperature particles condense into an equilibrium population of free floating vesicles. The comparison with the electrostatic case, giving rise to predominantly one-dimensional aggregates under similar conditions, is discussed. In both cases condensation is a continuous transformation, provided the isotropic part of the interatomic potential is purely repulsive. At low temperature the model allows us to investigate thermal and mechanical properties of membranes. At intermediate temperatures it provides a simple model to investigate equilibrium polymerization in a system giving rise to predominantly two-dimensional aggregates.

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

XBP1 mRNA splicing triggers an autophagic response in endothelial cells through BECLIN-1 transcriptional activation

Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3ß (LC3-ßII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1a (IRE1a)-dependent manner. Knockdown of XBP1 or IRE1a by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3ß expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt -537 to -755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1.

The interplay between clathrin-coated vesicles and cell signalling

Internalization of cargo proteins and lipids at the cell surface occurs in both a constitutive and signal-regulated manner through clathrin-mediated and other endocytic pathways. Clathrin-coated vesicle formation is a principal uptake route in response to signalling events. Protein-lipid and protein-protein interactions control both the targeting of signalling molecules and their binding partners to membrane compartments and the assembly of clathrin coats. An emerging aspect of membrane trafficking research is now addressing how signalling cascades and vesicle coat assembly and subsequently disassembly are integrated.

Low-dose salinomycin induces anti-leukemic responses in AML and MLL

Development of anti-cancer drugs towards clinical application is costly and inefficient. Large screens of drugs, efficacious for non-cancer disease, are currently being used to identify candidates for repurposing based on their anti-cancer properties. Here, we show that low-dose salinomycin, a coccidiostat ionophore previously identified in a breast cancer screen, has anti-leukemic efficacy. AML and MLLr cell lines, primary cells and patient samples were sensitive to submicromolar salinomycin. Most strikingly, colony formation of normal hematopoietic cells was unaffected by salinomycin, demonstrating a lack of hemotoxicity at the effective concentrations. Furthermore, salinomycin treatment of primary cells resulted in loss of leukemia repopulation ability following transplantation, as demonstrated by extended recipient survival compared to controls. Bioinformatic analysis of a 17-gene signature identified and validated in primary MLLr cells, uncovered immunomodulatory pathways, hubs and protein interactions as potential transducers of low dose salinomycin treatment. Additionally, increased protein expression of p62/Sqstm1, encoded for by one of the 17 signature genes, demonstrates a role for salinomycin in aggresome/vesicle formation indicative of an autophagic response.
Together, the data support the efficacy of salinomycin as an anti-leukemic at non-hemotoxic concentrations. Further investigation alone or in combination with other therapies is warranted for future clinical trial.

Do vesicle cells of the red alga Asparagopsis (Falkenbergia stage) play a role in bromocarbon production?

The Rhodophyceae (red algae) are an established source of volatile halocarbons in the marine environment. Some species in the Bonnemaisoniaceae have been reported to contain large amounts of halogens in structures referred to as vesicle cells, suggesting involvement of these specialised cells in the production of halocarbons. We have investigated the role of vesicle cells in the accumulation and metabolism of bromide in an isolate of the red macroalga Asparagopsis (Falkenbergia stage), a species known to release bromocarbons. Studies of laboratory-cultivated alga, using light microscopy, revealed a requirement of bromide for both the maintenance and formation of vesicle cells. Incubation of the alga in culture media with bromide concentrations below 64 mg l-1 (the concentration of Br- in seawater) resulted in a decrease in the proportion of vesicle cells to pericentral cells. The abundance of vesicle cells was correlated with bromide concentration below this level. Induction of vesicle cell formation in cultures of Falkenbergia occurred at concentrations as low as 8 mg l-1, with the abundance of vesicle cells increasing with bromide concentration up to around 100 mg l-1. Further studies revealed a positive correlation between the abundance of vesicle cells and dibromomethane and bromoform production. Interestingly, however, whilst dibromomethane production was stimulated by the presence of bromide in the culture media, bromoform release remained unaffected suggesting that the two compounds are formed by different mechanisms.

An endophilin-dynamin complex promotes budding of clathrin-coated vesicles during synaptic vesicle recycling

Clathrin-mediated vesicle recycling in synapses is maintained by a unique set of endocytic proteins and interactions. We show that endophilin localizes in the vesicle pool at rest and in spirals at the necks of clathrin-coated pits (CCPs) during activity in lamprey synapses. Endophilin and dynamin colocalize at the base of the clathrin coat. Protein spirals composed of these proteins on lipid tubes in vitro have a pitch similar to the one observed at necks of CCPs in living synapses, and lipid tubules are thinner than those formed by dynamin alone. Tubulation efficiency and the amount of dynamin recruited to lipid tubes are dramatically increased in the presence of endophilin. Blocking the interactions of the endophilin SH3 domain in situ reduces dynamin accumulation at the neck and prevents the formation of elongated necks observed in the presence of GTPγS. Therefore, endophilin recruits dynamin to a restricted part of the CCP neck, forming a complex, which promotes budding of new synaptic vesicles.