Vascular smooth muscle cell-derived nerve growth factor regulates sympathetic collateral branching to innervate blood vessels in embryonic skin.

Blood vessels serve as intermediate conduits for the extension of sympathetic axons towards target tissues, while also acting as crucial targets for their homeostatic processes encompassing the regulation of temperature, blood pressure, and oxygen availability. How sympathetic axons innervate not only blood vessels but also a wide array of target tissues is not clear. Here we show that in embryonic skin, after the establishment of co-branching between sensory nerves and blood vessels, sympathetic axons invade the skin alongside these sensory nerves and extend their branches towards these blood vessels covered by vascular smooth muscle cells (VSMCs). Our mosaic labeling technique for sympathetic axons shows that collateral branching predominantly mediates the innervation of VSMC-covered blood vessels by sympathetic axons. The expression of nerve growth factor (NGF), previously known to induce collateral axon branching in culture, can be detected in the vascular smooth muscle cell (VSMC)-covered blood vessels, as well as sensory nerves. Indeed, VSMC-specific Ngf knockout leads to a significant decrease of collateral branching of sympathetic axons innervating VSMC-covered blood vessels. These data suggest that VSMC-derived NGF serves as an inductive signal for collateral branching of sympathetic axons innervating blood vessels in the embryonic skin.

Impact of capillary and sarcolemmal proximity on mitochondrial structure and energetic function in skeletal muscle.

Mitochondria within skeletal muscle cells are located either between the muscle contractile apparatus (interfibrillar mitochondria, IFM) or beneath the cell membrane (subsarcolemmal mitochondria, SSM), with several structural and functional differences reported between IFM and SSM. However, recent 3D imaging studies demonstrate that mitochondria are particularly concentrated in the proximity of capillaries embedded in sarcolemmal grooves rather than in proximity to the sarcolemma itself (paravascular mitochondria, PVM). To evaluate the impact of capillary vs. sarcolemmal proximity, we compared the structure and function of skeletal muscle mitochondria located either lateral to embedded capillaries (PVM), adjacent to the sarcolemma but not in PVM pools (SSM) or interspersed between sarcomeres (IFM). Mitochondrial morphology and interactions were assessed by 3D electron microscopy coupled with machine learning segmentation, whereas mitochondrial energy conversion was assessed by two-photon microscopy of mitochondrial membrane potential, content, calcium, NADH redox and flux in live, intact cells. Structurally, although PVM and SSM were similarly larger than IFM, PVM were larger, rounder and had more physical connections to neighbouring mitochondria compared to both IFM and SSM. Functionally, PVM had similar or greater basal NADH flux compared to SSM and IFM, respectively, despite a more oxidized NADH pool and a greater membrane potential, signifying a greater activation of the electron transport chain in PVM. Together, these data indicate that proximity to capillaries has a greater impact on resting mitochondrial energy conversion and distribution in skeletal muscle than the sarcolemma alone. KEY POINTS: Capillaries have a greater impact on mitochondrial energy conversion in skeletal muscle than the sarcolemma. Paravascular mitochondria are larger, and the outer mitochondrial membrane is more connected with neighbouring mitochondria. Interfibrillar mitochondria are longer and have greater contact sites with other organelles (i.e. sarcoplasmic reticulum and lipid droplets). Paravascular mitochondria have greater activation of oxidative phosphorylation than interfibrillar mitochondria at rest, although this is not regulated by calcium.

Reorganization of mitochondria-organelle interactions during postnatal development in skeletal muscle.

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.

A protocol for measuring plasma membrane tension in the Drosophila ovary using fluorescence lifetime imaging microscopy.

Mechanosensation of plasma membrane tension by various mechanoresponsive machineries is crucial for regulating stem cell fate, cell adhesion, and tissue morphogenesis. Here, we present a protocol for evaluating plasma membrane stretching during the differentiation of Drosophila ovarian cyst using a fluorescent lipid tension reporter (Flipper-TR). We describe the steps for microphone setup, ovary dissection, Flipper-TR staining, fluorescence lifetime imaging microscopy imaging, and image processing and analysis. This protocol demonstrates the utility of Flipper-TR for investigating the impact of mechanical forces in living tissue. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

Deep learning-based aberration compensation improves contrast and resolution in fluorescence microscopy.

Optical aberrations hinder fluorescence microscopy of thick samples, reducing image signal, contrast, and resolution. Here we introduce a deep learning-based strategy for aberration compensation, improving image quality without slowing image acquisition, applying additional dose, or introducing more optics into the imaging path. Our method (i) introduces synthetic aberrations to images acquired on the shallow side of image stacks, making them resemble those acquired deeper into the volume and (ii) trains neural networks to reverse the effect of these aberrations. We use simulations to show that applying the trained 'de-aberration' networks outperforms alternative methods, and subsequently apply the networks to diverse datasets captured with confocal, light-sheet, multi-photon, and super-resolution microscopy. In all cases, the improved quality of the restored data facilitates qualitative image inspection and improves downstream image quantitation, including orientational analysis of blood vessels in mouse tissue and improved membrane and nuclear segmentation in C. elegans embryos.

Mechanical stimulation from the surrounding tissue activates mitochondrial energy metabolism in Drosophila differentiating germ cells.

In multicellular lives, the differentiation of stem cells and progenitor cells is often accompanied by a transition from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). However, the underlying mechanism of this metabolic transition remains largely unknown. In this study, we investigate the role of mechanical stress in activating OXPHOS during differentiation of the female germline cyst in Drosophila. We demonstrate that the surrounding somatic cells flatten the 16-cell differentiating cyst, resulting in an increase of the membrane tension of germ cells inside the cyst. This mechanical stress is necessary to maintain cytosolic Ca2+ concentration in germ cells through a mechanically activated channel, transmembrane channel-like. The sustained cytosolic Ca2+ triggers a CaMKI-Fray-JNK signaling relay, leading to the transcriptional activation of OXPHOS in differentiating cysts. Our findings demonstrate a molecular link between cell mechanics and mitochondrial energy metabolism, with implications for other developmentally orchestrated metabolic transitions in mammals.

Post-Transplant Administration of G-CSF Impedes Engraftment of Gene Edited Human Hematopoietic Stem Cells by Exacerbating the p53-Mediated DNA Damage Response.

Granulocyte colony stimulating factor (G-CSF) is commonly used as adjunct treatment to hasten recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, the utility of G-CSF administration after ex vivo gene therapy procedures targeting human HSPCs has not been thoroughly evaluated. Here, we provide evidence that post-transplant administration of G-CSF impedes engraftment of CRISPR-Cas9 gene edited human HSPCs in xenograft models. G-CSF acts by exacerbating the p53-mediated DNA damage response triggered by Cas9- mediated DNA double-stranded breaks. Transient p53 inhibition in culture attenuates the negative impact of G-CSF on gene edited HSPC function. In contrast, post-transplant administration of G-CSF does not impair the repopulating properties of unmanipulated human HSPCs or HSPCs genetically engineered by transduction with lentiviral vectors. The potential for post-transplant G-CSF administration to aggravate HSPC toxicity associated with CRISPR-Cas9 gene editing should be considered in the design of ex vivo autologous HSPC gene editing clinical trials.

Pericentrin interacts with Kinesin-1 to drive centriole motility.

Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosomal microtubules (MTs) with motor proteins anchored at the cortex or the nuclear surface. However, it remains unknown how centrioles migrate in cellular contexts in which they do not nucleate MTs. Here, we demonstrate that during interphase, inactive centrioles move directly along the interphase MT network as Kinesin-1 cargo. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP. Finally, our studies of neural stem cell asymmetric divisions in the Drosophila brain show that the PLP-Kinesin-1 interaction is essential for the timely separation of centrioles, the asymmetry of centrosome activity, and the age-dependent centrosome inheritance.

Mitapivat increases ATP and decreases oxidative stress and erythrocyte mitochondria retention in a SCD mouse model.

Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels.

Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen.

A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.

Multiview confocal super-resolution microscopy.

Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.

Monitoring mitochondrial calcium and metabolism in the beating MCU-KO heart.

Optical methods for measuring intracellular ions including Ca2+ revolutionized our understanding of signal transduction. However, these methods are not extensively applied to intact organs due to issues including inner filter effects, motion, and available probes. Mitochondrial Ca2+ is postulated to regulate cell energetics and death pathways that are best studied in an intact organ. Here, we develop a method to optically measure mitochondrial Ca2+ and demonstrate its validity for mitochondrial Ca2+ and metabolism using hearts from wild-type mice and mice with germline knockout of the mitochondria calcium uniporter (MCU-KO). We previously reported that germline MCU-KO hearts do not show an impaired response to adrenergic stimulation. We find that these MCU-KO hearts do not take up Ca2+, consistent with no alternative Ca2+ uptake mechanisms in the absence of MCU. This approach can address the role of mitochondrial Ca2+ to the myriad of functions attributed to alterations in mitochondrial Ca2+.

Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes.

We demonstrate residual channel attention networks (RCAN) for the restoration and enhancement of volumetric time-lapse (four-dimensional) fluorescence microscopy data. First we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy four-dimensional super-resolution data, enabling image capture of over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables resolution enhancement equivalent to, or better than, other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy data as ground truth, achieving improvements of ~1.9-fold laterally and ~3.6-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluation and further enhancement of network performance.

A Step-by-Step Guide to Instant Structured Illumination Microscopy (iSIM).

Instant structured illumination microscopy (iSIM) allows for rapid multicolor three-dimensional fluorescence imaging at levels of resolution approaching twice the diffraction limit. Here we briefly describe the theory of iSIM and outline a typical hardware setup. We also provide step-by-step guides for generating a cellular-based fluorescent standard, obtaining a multicolor image with iSIM, and the post-processing steps of de-striping and deconvolution using freely distributed software to minimize time and expense. A "Notes" section is also given to inform the reader of the limitations and considerations for the methods shown. Also discussed are alternative methods, quality control checks, and considerations for two-camera alignment.

NLRP3 inflammasome and bruton tyrosine kinase inhibition interferes with upregulated platelet aggregation and in vitro thrombus formation in sickle cell mice.

The nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3) inflammasome is a critical inflammatory mechanism identified in platelets, which controls platelet activation and aggregation. We have recently shown that the platelet NLRP3 inflammasome is upregulated in sickle cell disease (SCD), which is mediated by Bruton tyrosine kinase (BTK). Here, we investigated the effect of pharmacological inhibition of NLRP3 and BTK on platelet aggregation and the formation of in vitro thrombi in Townes SCD mice. Mice were injected for 4 weeks with the NLRP3 inhibitor MCC950, the BTK inhibitor ibrutinib or vehicle control. NLRP3 activity, as monitored by caspase-1 activation, was upregulated in platelets from SCD mice, which was dependent on BTK. Large areas of platelet aggregates detected in the liver of SCD mice were decreased when mice were treated with MCC950 or ibrutinib. Moreover, platelet aggregation and in vitro thrombus formation were upregulated in SCD mice and were inhibited when mice were subjected to pharmacological inhibition of NLRP3 and BTK. Targeting the NLRP3 inflammasome might be a novel approach for antiplatelet therapy in SCD.

Circulating mitochondrial DNA is a proinflammatory DAMP in sickle cell disease.

The pathophysiology of sickle cell disease (SCD) is driven by chronic inflammation fueled by damage associated molecular patterns (DAMPs). We show that elevated cell-free DNA (cfDNA) in patients with SCD is not just a prognostic biomarker, it also contributes to the pathological inflammation. Within the elevated cfDNA, patients with SCD had a significantly higher ratio of cell-free mitochondrial DNA (cf-mtDNA)/cell-free nuclear DNA compared with healthy controls. Additionally, mitochondrial DNA in patient samples showed significantly disproportionately increased hypomethylation compared with healthy controls, and it was increased further in crises compared with steady-state. Using flow cytometry, structured illumination microscopy, and electron microscopy, we showed that circulating SCD red blood cells abnormally retained their mitochondria and, thus, are likely to be the source of the elevated cf-mtDNA in patients with SCD. Patient plasma containing high levels of cf-mtDNA triggered the formation of neutrophil extracellular traps (NETs) that was substantially reduced by inhibition of TANK-binding kinase 1, implicating activation of the cGAS-STING pathway. cf-mtDNA is an erythrocytic DAMP, highlighting an underappreciated role for mitochondria in sickle pathology. These trials were registered at www.clinicaltrials.gov as #NCT00081523, #NCT03049475, and #NCT00047996.

Mitochondrial DNA segregation and replication restrict the transmission of detrimental mutation.

Although mitochondrial DNA (mtDNA) is prone to accumulate mutations and lacks conventional DNA repair mechanisms, deleterious mutations are exceedingly rare. How the transmission of detrimental mtDNA mutations is restricted through the maternal lineage is debated. Here, we demonstrate that mitochondrial fission, together with the lack of mtDNA replication, segregate mtDNA into individual organelles in the Drosophila early germarium. After mtDNA segregation, mtDNA transcription begins, which activates respiration. Mitochondria harboring wild-type genomes have functional electron transport chains and propagate more vigorously than mitochondria containing deleterious mutations in hetreoplasmic cells. Therefore, mtDNA expression acts as a stress test for the integrity of mitochondrial genomes and sets the stage for replication competition. Our observations support selective inheritance at the organelle level through a series of developmentally orchestrated mitochondrial processes. We also show that the Balbiani body has a minor role in mtDNA selective inheritance by supplying healthy mitochondria to the pole plasm. These two mechanisms may act synergistically to secure the transmission of functional mtDNA through Drosophila oogenesis.

A simple empirical algorithm for optimising depletion power and resolution for dye and system specific STED imaging.

Here we show an easy method for determining an effective dye saturation factor ('PSTED ') for STED (Stimulated Emission Depletion) microscopy. We define PSTED to be a combined microscope system plus dye factor (analogous to the traditional ground truth Is measurement, which is microscope independent) that is functionally defined as the power in the depletion beam that provides a resolution enhancement of 41% compared to confocal, according to the modified Abbe's formula for STED resolution enhancement. We show that the determination of PSTED provides insight not only into the suitability of a particular dye and the best imaging parameters to be used for an experiment, but also sets the critical value for correctly determining the point spread function (PSF) used in deconvolution of STED images. PSTED can be a function of many experimental variables, both microscope and sample related. Here we show the utility of doing PSTED determinations by (1) exploiting the simple relationship between width and a threshold-defined area provided by a Gaussian PSF, for either linear or spherical objects and (2) linearising the normally inverse hyperbolic function of resolution versus power that can determine PSTED . We show that this rearrangement allows us to determine PSTED using only a few measurements: either at a few relatively low depletion powers, on traditional bead size measurements or by finding the total area of a naturally occurring sub-limit sized biological feature (in this case, microtubules). We show the derivation of these equations and methods and the utility of its use by characterising several dyes and a local imaging parameter relevant to STED microscopy. This information is used to predict the enhancement of resolution of the point spread function necessary for post-processing deconvolution. LAY DESCRIPTION: Stimulated Emission Depletion (STED) microscopy is a fluorescence imaging superresolution technique that achieves tens of nanometres resolution. This is done by utilising a depletion laser to effectively quench (deplete) fluorescence in a donut shape overlapping the normally excited fluorescence spot. The size of the remaining (undepleted) central fluorescence spot is power dependent allowing 'tunable' resolution with the power of the STED depletion laser. This depletion power versus resolution relationship is dye and instrument dependent. We have developed a method for quickly measuring this relationship to optimise experiments based on individual dyes and microscope specific parameters. This allows for quickly optimising microscope settings and for correctly postprocessing images.