Dynamic configurations of meiotic DNA-break hotspot determinant proteins

Appropriate DNA double-strand-break (DSB) and crossover distributions are required for proper meiotic chromosome segregation. Schizosaccharomyces pombe linear element proteins (LinEs) determine DSB hotspots; LinE-bound hotspots form 3D clusters over ∼200 kb chromosomal regions. Here, we investigated LinE configurations and distributions in live cells using super-resolution fluorescence microscopy. We found LinEs form two chromosomal structures, dot-like and linear structures, in both zygotic and azygotic meiosis. Dot-like LinE structures appeared around the time of meiotic DNA replication, underwent dotty-to-linear-to-dotty configurational transitions, and disassembled before the first meiotic division. DSB formation and repair did not detectably influence LinE structure formation, but failure of DSB formation delayed disassembly. Recombination-deficient LinE missense mutants formed dot-like but not linear LinE structures. Our quantitative study reveals a transient form of LinE structures and suggests a novel role for LinE proteins in regulating meiotic events, such as DSB repair. We discuss the relation of LinEs and the synaptonemal complex in other species.

Label2label: Training a neural network to selectively restore cellular structures in fluorescence microscopy

Immunofluorescence (IF) microscopy is routinely used to visualise the spatial distribution of proteins that dictates their cellular function. However, unspecific antibody binding often results in high cytosolic background signals, decreasing the image contrast of a target structure. Recently, convolutional neural networks (CNNs) were successfully employed for image restoration in IF microscopy, but current methods cannot correct for those background signals. We report a new method that trains a CNN to reduce unspecific signals in IF images; we name this method label2label (L2L). In L2L, a CNN is trained with image pairs of two non-identical labels that target the same cellular structure. We show that after L2L training a network predicts images with significantly increased contrast of a target structure, which is further improved after implementing a multi-scale structural similarity loss function. Here, our results suggest that sample differences in the training data decrease hallucination effects that are observed with other methods. We further assess the performance of a cycle generative adversarial network, and show that a CNN can be trained to separate structures in superposed IF images of two targets.

Diverse mechanisms regulate contractile ring assembly for cytokinesis in the two-cell C. elegans embryo

Cytokinesis occurs at the end of mitosis due to the ingression of a contractile ring that cleaves the daughter cells. The core machinery regulating this crucial process is conserved among metazoans. Multiple pathways control ring assembly, but their contribution in different cell types is not known. We found that in the C. elegans embryo, AB and P1 cells fated to be somatic tissue and germline, respectively, have different cytokinesis kinetics supported by distinct myosin levels and organization. Through perturbation of RhoA or polarity regulators and the generation of tetraploid strains, we found that ring assembly is controlled by multiple fate-dependent factors that include myosin-levels, and mechanisms that respond to cell size. Active Ran coordinates ring position with the segregating chromatids in HeLa cells by forming an inverse gradient with importins that control the cortical recruitment of anillin. We found that the Ran pathway regulates anillin in AB cells, but functions differently in P1 cells. We propose that ring assembly delays in P1 cells caused by low myosin and Ran signaling coordinate the timing of ring closure with their somatic neighbours.

Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies

Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. While the formation and functions of EVs have been investigated extensively in cultured cells, studies of EVs in vivo have remained scarce. We report here that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. We defined two distinct EV subpopulations, one of which contains the Munc13-4 homologue Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion (anastomosis) events. The formation of Stac-positive luminal EVs depends on the tracheal tip-cell-specific GTPase Arl3, which is also required for the formation of Stac-positive multivesicular bodies, suggesting that Stac-EVs derive from fusion of Stac-MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-MVB formation and tube fusion. We propose that Stac-MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35, and Stac/Munc13-4.

The Nucleolar δ Isoform of Adapter Protein SH2B1 Enhances Morphological Complexity and Function of Cultured Neurons

The adapter protein SH2B1 is recruited to neurotrophin receptors including TrkB, receptor for brain-derived neurotrophic factor (BDNF). Herein, we demonstrate that the four alternatively spliced isoforms of SH2B1 are important determinants of neuronal architecture and neurotrophin-induced gene expression. Primary hippocampal neurons from Sh2b1−/- (KO) mice exhibit decreased neurite complexity and length and BDNF-induced expression of synapse-related immediate early genes Egr1 and Arc. Reintroduction of each SH2B1 isoform into KO neurons increases neurite complexity; the brain-specific δ isoform also increases total neurite length. Human obesity-associated variants, when expressed in SH2B1δ, alter neurite complexity, suggesting that a decrease or increase in neurite branching may have deleterious effects that contribute to the severe childhood obesity and neurobehavioral abnormalities associated with these variants. Surprisingly, in contrast to SH2B1α, β, and γ, which localize primarily in the cytoplasm and plasma membrane, SH2B1δ localizes primarily in nucleoli. Some SH2B1δ is also present in the plasma membrane and nucleus. Nucleolar localization, driven by two highly basic regions unique to SH2B1δ, is required for SH2B1δ to maximally increase neurite complexity and BDNF-induced expression of Egr1, Arc, and FosL1.

Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells

Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including Bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain non-neuronal proteins LL5β and KANK1, which in migrating cells organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. While previous analyses in vitro and in neurons suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

First person – Hannah Black and Rachel Livingstone

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Hannah Black and Rachel Livingstone are co-first authors on ‘ Knockout of syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion’, published in JCS. Hannah conducted the research described in this article while a PhD student in Professor Nia Bryant and Professor Gwyn Gould's lab at the Henry Wellcome Laboratory for Cell Biology, University of Glasgow, UK. She is now a postdoc in the lab of Professor Nia Bryant at the Department of Biology, University of York, UK, investigating membrane trafficking of the glucose transporter protein GLUT4. Rachel is a PhD student in the lab of Professor Gwyn Gould at the Henry Wellcome Laboratory for Cell Biology, University of Glasgow, UK, where she is also investigating membrane trafficking of GLUT4.

High content Image Analysis to study phenotypic heterogeneity in endothelial cell monolayers

Endothelial cells (EC) are heterogeneous across and within tissues, reflecting distinct, specialised functions. EC heterogeneity has been proposed to underpin EC plasticity independently from vessel microenvironments. However, heterogeneity driven by contact-dependent or short-range cell-cell crosstalk cannot be evaluated with single cell transcriptomic approaches as spatial and contextual information is lost. Nonetheless, quantification of EC heterogeneity and understanding of its molecular drivers is key to developing novel therapeutics for cancer, cardiovascular diseases and for revascularisation in regenerative medicine. Here, we developed an EC profiling tool (ECPT) to examine individual cells within intact monolayers. We used ECPT to characterise different phenotypes in arterial, venous and microvascular EC populations. In line with other studies, we measured heterogeneity in terms of cell cycle, proliferation, and junction organisation. ECPT uncovered a previously under-appreciated single-cell heterogeneity in NOTCH activation. We correlated cell proliferation with different NOTCH activation states at the single cell and population levels. The positional and relational information extracted with our novel approach is key to elucidating the molecular mechanisms underpinning EC heterogeneity.

Dishevelled coordinates phosphoinositide kinases PI4KIIIα and PIP5KIγ for efficient PtdInsP2 synthesis

Phosphatidylinositol(4,5)-bisphosphate (PtdInsP2) is an important modulator of many cellular processes and its abundance in the plasma membrane is closely regulated. We examined the hypothesis that the scaffolding protein Dishevelled can bind the lipid kinases PI4K and PIP5K, facilitating synthesis of PtdInsP2 directly from PtdIns. This report used several assays for PtdInsP2 to examine the cooperative function of phosphoinositide kinases and Dishevelled in the context of two receptor signaling cascades. Simultaneous overexpression of PI4KIIIα and PIP5KIγ had a synergistic effect on PtdInsP2 synthesis that was recapitulated by overexpression of Dishevelled. Increasing the activity of Dishevelled by overexpression increased resting plasma membrane PtdInsP2. Knockdown of Dishevelled reduced resting plasma membrane PtdInsP2 and slowed PtdInsP2 resynthesis following receptor activation. We confirm that Dishevelled promotes coupling of PI4KIIIα and PIP5KIγ and show that this interaction is essential for efficient resynthesis of PtdInsP2 following receptor activation.

Isoform-dependent lysosomal degradation and internalization of Apolipoprotein E requires autophagy proteins

The human Apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found in cells stably expressing each APOE isoform that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-golgi compartment is degraded by autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that may contribute to AD pathogenesis.