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After endocytosis, transmembrane cargo reaches endosomes, where it encounters complexes dedicated to opposing functions: recycling and degradation. Microdomains containing endosomal sorting complexes required for transport (ESCRT)-0 component Hrs [hepatocyte growth factor-regulated tyrosine kinase substrate (HGRS-1) in Caenorhabditis elegans] mediate cargo degradation, concentrating ubiquitinated cargo and organizing the activities of ESCRT. At the same time, retromer associated sorting nexin one (SNX-1) and its binding partner, J-domain protein RME-8, sort cargo away from degradation, promoting cargo recycling to the Golgi. Thus, we hypothesized that there could be important regulatory interactions between retromer and ESCRT that balance degradative and recycling functions. Taking advantage of the naturally large endosomes of the C. elegans coelomocyte, we visualized complementary ESCRT-0 and RME-8/SNX-1 microdomains in vivo and assayed the ability of retromer and ESCRT microdomains to regulate one another. We found in snx-1(0) and rme-8(ts) mutants increased endosomal coverage and intensity of HGRS-1–labeled microdomains, as well as increased total levels of HGRS-1 bound to membranes. These effects are specific to SNX-1 and RME-8, as loss of other retromer components SNX-3 and vacuolar protein sorting-associated protein 35 (VPS-35) did not affect HGRS-1 microdomains. Additionally, knockdown of hgrs-1 had little to no effect on SNX-1 and RME-8microdomains, suggesting directionality to the interaction. Separation of the functionally distinct ESCRT-0 and SNX-1/RME-8 microdomains was also compromised in the absence of RME-8 and SNX-1, a phenomenonwe observed to be conserved, as depletion of Snx1 and Snx2 in HeLa cells also led to greater overlap of Rme-8 and Hrs on endosomes.

The molecular events that determine the recycling versus degradation fates of internalized membrane proteins remain poorly understood. Two of the three members of the SNX-FERM family, SNX17 and SNX31, utilize their FERM domain to mediate endocytic trafficking of cargo proteins harboring the NPxY/NxxY motif. In contrast, SNX27 does not recycle NPxY/NxxY-containing cargo but instead recycles cargo containing PDZ-binding motifs via its PDZ domain. The underlying mechanism governing this divergence in FERM domain binding is poorly understood. Here, we report that the FERM domain of SNX27 is functionally distinct from SNX17 and interacts with a novel DLF motif localized within the N terminus of SNX1/2 instead of the NPxY/NxxY motif in cargo proteins. The SNX27-FERM-SNX1 complex structure reveals that the DLF motif of SNX1 binds to a hydrophobic cave surrounded by positively charged residues on the surface of SNX27. The interaction between SNX27 and SNX1/2 is critical for efficient SNX27 recruitment to endosomes and endocytic recycling of multiple cargoes. Finally, we show that the interaction between SNX27 and SNX1/2 is critical for brain development in zebrafish. Altogether, our study solves a long-standing puzzle in the field and suggests that SNX27 and SNX17 mediate endocytic recycling through fundamentally distinct mechanisms.

Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.

Metabolic demands fluctuate rhythmically and rely on coordination between the circadian clock and nutrient-sensing signalling pathways, yet mechanisms of their interaction remain not fully understood. Surprisingly, we find that class 3 phosphatidylinositol-3-kinase (PI3K), known best for its essential role as a lipid kinase in endocytosis and lysosomal degradation by autophagy, has an overlooked nuclear function in gene transcription as a coactivator of the heterodimeric transcription factor and circadian driver Bmal1–Clock. Canonical pro-catabolic functions of class 3 PI3K in trafficking rely on the indispensable complex between the lipid kinase Vps34 and regulatory subunit Vps15. We demonstrate that although both subunits of class 3 PI3K interact with RNA polymerase II and co-localize with active transcription sites, exclusive loss of Vps15 in cells blunts the transcriptional activity of Bmal1–Clock. Thus, we establish non-redundancy between nuclear Vps34 and Vps15, reflected by the persistent nuclear pool of Vps15 in Vps34-depleted cells and the ability of Vps15 to coactivate Bmal1–Clock independently of its complex with Vps34. In physiology we find that Vps15 is required for metabolic rhythmicity in liver and, unexpectedly, it promotes pro-anabolic de novo purine nucleotide synthesis. We show that Vps15 activates the transcription of Ppat , a key enzyme for the production of inosine monophosphate, a central metabolic intermediate for purine synthesis. Finally, we demonstrate that in fasting, which represses clock transcriptional activity, Vps15 levels are decreased on the promoters of Bmal1 targets, Nr1d1 and Ppat . Our findings open avenues for establishing the complexity for nuclear class 3 PI3K signalling for temporal regulation of energy homeostasis. Alkhoury et al. show that the class 3 phosphatidylinositol-3-kinase Vps15 subunit coactivates the circadian clock transcription factor Bmal1–Clock for metabolic rhythmicity in the liver and promotes pro-anabolic de novo purine synthesis.

Deterministic lateral displacement (DLD) pillar arrays are an efficient technology to sort, separate and enrich micrometre-scale particles, which include parasites, bacteria, blood cells and circulating tumour cells in blood. However, this technology has not been translated to the true nanoscale, where it could function on biocolloids, such as exosomes. Exosomes, a key target of 'liquid biopsies', are secreted by cells and contain nucleic acid and protein information about their originating tissue. One challenge in the study of exosome biology is to sort exosomes by size and surface markers. We use manufacturable silicon processes to produce nanoscale DLD (nano-DLD) arrays of uniform gap sizes ranging from 25 to 235nm. We show that at low Peclet (Pe) numbers, at which diffusion and deterministic displacement compete, nano-DLD arrays separate particles between 20 to 110nm based on size with sharp resolution. Further, we demonstrate the size-based displacement of exosomes, and so open up the potential for on-chip sorting and quantification of these important biocolloids.

In the last two decades, the fast and elitist non-dominated sorting genetic algorithm (NSGA-II) has attracted extensive research interests, and it is still one of the hottest research methods to deal with multi-objective optimization problems. Considering the importance and wide applications of NSGA-II method, we believe it is the right time to provide a comprehensive survey of the research work in this area, and also to discuss the potential in the future research. The purpose of this paper is to summarize and explore the literature on NSGA-II and another version called NSGA-III, a reference-point based many-objective NSGA-II approach. In this paper, we first introduce the concept of multi-objective optimization and the foundation of NSGA-II. Then we review the family of NSGA-II and their modifications, and classify their applications in engineering community. Finally, we present several interesting open research directions of NSGA-II for multi-objective optimization.

After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells by using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nanometer-diameter filaments, which narrowed the cortex of the intercellular bridge to a single stalk. The endosomal sorting complex required for transport (ESCRT)—III co-localized with constriction zones and was required for assembly of 17-nanometer-diameter filaments. Simultaneous spastin-mediated removal of underlying microtubules enabled full constriction at the abscission site. The identification of contractile filament helices at the intercellular bridge has broad implications for the understanding of cell division and of ESCRT-III—mediated fission of large membrane structures.

Multivesicular bodies are key endosomal compartments implicated in cellular quality control through their degradation of membrane-bound cargo proteins 1 – 3 . The ATP-consuming ESCRT protein machinery mediates the capture and engulfment of membrane-bound cargo proteins through invagination and scission of multivesicular-body membranes to form intraluminal vesicles 4 , 5 . Here we report that the plant ESCRT component FREE1 6 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation. We use a minimal physical model, reconstitution experiments and in silico simulations to identify the dynamics of this process and describe intermediate morphologies of nascent intraluminal vesicles. Furthermore, we find that condensate-wetting-induced line tension forces and membrane asymmetries are sufficient to mediate scission of the membrane neck without the ESCRT protein machinery or ATP consumption. Genetic manipulation of the ESCRT pathway in several eukaryotes provides additional evidence for condensate-mediated membrane scission in vivo. We find that the interplay between condensate and machinery-mediated scission mechanisms is indispensable for osmotic stress tolerance in plants. We propose that condensate-mediated scission represents a previously undescribed scission mechanism that depends on the physicomolecular properties of the condensate and is involved in a range of trafficking processes. More generally, FREE1 condensate-mediated membrane scission in multivesicular-body biogenesis highlights the fundamental role of wetting in intracellular dynamics and organization. Plant ESCRT component FREE1 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation.

The ESCRT (endosomal sorting complex required for transport) protein complex plays a role in budding into multivesicular bodies, in cytokinesis, and in HIV budding. Now, Jimenez et al. (p. 10.1126/science.1247136 , published online 30 January) propose a role for ESCRT proteins in wound repair at the plasma membrane. In vivo imaging, modeling, and electron microscopy were used to reveal how the ESCRTs participate in a rapid energy-independent, calcium-dependent, membrane-shedding process at the plasma membrane that reseals small wounds caused by toxins or laser treatment. ESCRT proteins repair small wounds in the plasma membrane by shearing off damaged portions. Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.

The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed β-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ- binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold.