Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular ...carriers from endosomal membranes
. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders
. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.
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.
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.
•Multi- physical properties-based label-free particle sorting.•Tunable acoustofluidic system integrated with elasto-inertial particle focusing.•Particle separation at submicron size ...difference.•Separation of particles with the same size but different material properties.
Acoustophoretic manipulation has been drawing great attention in the area of microfluidics-based cell/particle isolation and sorting due to its advantages of high biocompatibility, contactless manipulation and fabrication simplicity. Most label-free microfluidic particle sorting methodologies are solely based on a single physical property for example particle size, density or material composition, which hinder their broad usage in various applications. In this work, we demonstrate a tunable acoustofluidic device for continuous particle separation based on multi-physical properties such as size, density, compressibility and speed of sound. The tunable acoustofluidic system utilizes an elasto-inertial particle focusing technique to align particles into a single line and integrates a slanted interdigitated transducer (SFIT) that generates a tunable travelling surface acoustic wave to separate the particles with improved sorting accuracy and efficiency. The presented acoustofluidic sorting device allows the generation of a variable frequency from 99 MHz to 247 MHz corresponding to the wavelength 40 μm and 16 μm using the SFIT. We have successfully sorted 5.26 μm from 5 μm polystyrene particles at 90 % accuracy with a relative size difference of 5.2 %. Furthermore, a theoretical analysis of acoustic radiation force (ARF) exerted on rigid particles of different materials (i.e., polystyrene, PLGA, PMMA) is presented. Based on the optimal frequencies identified in the theoretical analysis, we have also experimentally demonstrated effective sorting of the three above-mentioned materials of particles with the same particle size.
Sorting a set of items is a task that can be useful by itself or as a building block for more complex operations. That is why a lot of effort has been put into finding sorting algorithms that sort ...large sets as efficiently as possible. But the more sophisticated and complex the algorithms become, the less efficient they are for small sets of items due to large constant factors. A relatively simple sorting algorithm that is often used as a base case sorter is insertion sort, because it has small code size and small constant factors influencing its execution time. We aim to determine if there is a faster way to sort small sets of items to provide an efficient base case sorter. We looked at sorting networks, at how they can improve the speed of sorting few elements, and how to implement them in an efficient manner using conditional moves. Since sorting networks need to be implemented explicitly for each set size, providing networks for larger sizes becomes less efficient due to increased code sizes. To also enable the sorting of slightly larger base cases, we adapted sample sort to Register Sample Sort, to break down those larger sets into sizes that can in turn be sorted by sorting networks. From our experiments we found that when sorting only small sets of integers, the sorting networks outperform insertion sort by a factor of at least 1.76 for any array size between six and 16, and by a factor of 2.72 on average across all machines and array sizes. When integrating sorting networks as a base case sorter into Quicksort, we achieved far less performance improvements over using insertion sort, which is probably due to the networks having a larger code size and cluttering the L1 instruction cache. The same effect occurs when including Register Sample Sort as a base case sorter for IPS4o. But for x86 machines that have a larger L1 instruction cache of 64 KiB or more, we obtained speedups of 12.7% when using sorting networks as a base case sorter in std::sort, and of 5%–6% when integrating Register Sample Sort as a base case sorter into IPS4o, each in comparison to using insertion sort as the base case sorter. In conclusion, the desired improvement in speed could only be achieved under special circumstances, but the results clearly show the potential of using conditional moves in the field of sorting algorithms.
Recognising the signals for endosomal trafficking Weeratunga, Saroja; Paul, Blessy; Collins, Brett M.
Current opinion in cell biology,
August 2020, 2020-08-00, 20200801, Letnik:
65
Journal Article
Recenzirano
The endosomal compartment is a major sorting station controlling the balance between endocytic recycling and lysosomal degradation, and its homeostasis is emerging as a central factor in various ...neurodegenerative diseases such as Alzheimer's and Parkinson's. Membrane trafficking is generally coordinated by the recognition of specific signals in transmembrane protein cargos by different transport machineries. A number of different protein trafficking complexes are essential for sequence-specific recognition and retrieval of endosomal cargos, recycling them to other compartments and acting to counter-balance the default endosomal sorting complex required for transport–mediated degradation pathway. In this review, we provide a summary of the key endosomal transport machineries, and the molecular mechanisms by which different cargo sequences are specifically recognised.
Large, multinucleated cells, like syncytiotrophoblasts (STB), are not readily analyzed by standard methods used for single cells, such as single-cell RNA-sequencing and fluorescence-activated ...cellular sorting (FACS). Here we have demonstrated that fluorescence-activated nuclear sorting (FANS) is suitable to analyze nuclei from STB. Human pluripotent stem cells (PSCs) can be differentiated into a mixed trophoblast populations comprising approximately 20 % STB by treatment with BMP4 (Bone Morphogenetic Protein-4), plus A83-01 and PD173074, inhibitors of activin and FGF2 signaling, respectively (the BAP model) in about a week. Here we demonstrate that FANS can be used to separate two types of STB nuclei from the nine different clusters of trophoblast nuclei previously identified in the BAP model by single nucleus RNA sequencing (snRNAseq). Rather than using cell surface markers, as in FACS, transcription factors in various combinations were employed to target specific nuclear types. Nuclei were isolated at d 8 of BAP differentiation of H1 human embryonic stem cells and fixed in 4 % paraformaldehyde. After permeabilization in 0.1 % triton X-100, nuclei were incubated for 3 and 1 h at 4 °C with primary and secondary antibodies respectively and nuclear samples were then subjected to FANS. By using markers identified by snRNA and immunohistochemistry, nuclei were first sorted into a Topoisomerase-1, or TOP1, bright population and then into the two STB subpopulations by using antibodies to JUNB (Jun B Proto-Oncogene) and TFCP2L1 (Transcription Factor CP2 Like 1). The protocol established here is simple, straightforward, and efficient and can be used on a relatively large scale to sort individual subtypes of nuclei from mixed populations of trophoblasts for further analysis.
•Syncytiotrophoblast are too large and fragile for traditional cell sorting methods.•Fluorescence activated nuclear sorting is suited to analysis of syncytiotrophoblast.•A protocol for flow cytometric analysis of syncytiotrophoblast nuclei is presented.
Differential evolution (DE) is one of the best evolutionary algorithms (EAs). The effort of improving its performance has received great research attentions, such as adaptive DE (JADE). Based on the ...analysis on the aspects that may improve the performance of JADE, we introduce a modified JADE version with sorting crossover rate (CR). In JADE, CR values are generated based on mean value and Gaussian distribution. In the proposed algorithm, a smaller CR value is assigned to individual with better fitness value. Therefore, the components of the individuals, which have better fitness values, can appear in the offspring with higher possibility. In addition, the better offspring generated from last iteration are supposed to have better schemes, hence these schemes are preserved in next offspring generation procedure. This modified version is called as JADE algorithm with sorting CR (JADE_sort). The experiments results with several excellent algorithms show the effectiveness of JADE_sort.
Sorting machines use computer vision (CV) to separate food items based on various attributes. For instance, sorting based on size and colour are commonly used in commercial machines. However, ...detecting external defects using CV remains an open problem. This paper presents an experimental contribution to external defect detection using deep learning. An uncensored dataset with 43,843 images including external defects was built during this study. The dataset is heavily imbalanced towards the healthy class, and it is available online. Deep residual neural network (ResNet) classifiers were trained that are capable of detecting external defects using feature extraction and fine-tuning. The results show that fine-tuning outperformed feature extraction, revealing the benefit of training additional layers when sufficient data samples are available. The best model was a ResNet50 with all its layers fine-tuned. This model achieved an average precision of 94.6% on the test set. The optimal classifier had a recall of 86.6% while maintaining a precision of 91.7%. The posterior class-conditional distributions of the classifier scores showed that the key to classifier success lies in its almost ideal healthy class distribution. The results also explain why the model does not confuse stems/calyxes with external defects. The best model constitutes a milestone for detecting external defects using CV. Because deep learning does not require feature engineering or prior knowledge about the dataset content, the methodology may also work well with other foods.
•Deep learning detects different types of external defects without feature engineering.•Fine tuning outperformed feature extraction in external defect detection.•The best model has Average Precision = 94.6 %.•The best model has 86.6 % Recall and 91.7 % Precision at the optimal threshold.•The best model does not confuse external defects with stems/calyxes.
Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal sorting complex required for transport III ...(ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a microtubule-interacting and trafficking domain-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction, in concert with a more open conformation of the Ist1 core, resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.
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