We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons.

We have developed a suitable heterologous cell expression system to study the localization, trafficking, and site(s) of function of the human ABCG1 transporter. Increased plasma membrane (PM) and late endosomal (LE) cholesterol generated by ABCG1 was removed by lipoproteins and liposomes, but not apoA-I. Delivery of ABCG1 to the PM and LE was required for ABCG1-mediated cellular cholesterol efflux. ABCG1 LEs frequently contacted the PM, providing a collisional mechanism for transfer of ABCG1-mobilized cholesterol, similar to ABCG1-mediated PM cholesterol efflux to lipoproteins. ABCG1-mobilized LE cholesterol also trafficked to the PM by a non-vesicular pathway. Transfer of ABCG1-mobilized cholesterol from the cytoplasmic face of LEs to the PM and concomitant removal of cholesterol from the outer leaflet of the PM bilayer by extracellular acceptors suggests that ABCG1 mobilizes cholesterol on both sides of the lipid bilayer for removal by acceptors. ABCG1 increased uptake of HDL into LEs, consistent with a potential ABCG1-mediated cholesterol efflux pathway involving HDL resecretion. Thus, ABCG1 at the PM mobilizes PM cholesterol and ABCG1 in LE/LYS generates mobile pools of cholesterol that can traffic by both vesicular and non-vesicular pathways to the PM where it can also be transferred to extracellular acceptors with a lipid surface.

Cells use various mechanisms to maintain cellular cholesterol homeostasis including efflux of cholesterol from the cellular plasma membrane to cholesterol acceptors such as HDLs (high-density lipoproteins). Little is known about the transfer of cholesterol from cells into the extracellular matrix. Using a unique monoclonal antibody that detects ordered cholesterol arrays (ie, cholesterol micro[or nano]-domains), we previously identified that particles containing these cholesterol domains accumulate in the extracellular matrix during cholesterol enrichment of human monocyte-derived macrophages and are found in atherosclerotic lesions. In this study, we further investigate these deposited particles containing cholesterol microdomains and discover their unexpected morphology.

We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 μm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.

The Zebrafish Posterior Lateral Line primordium migrates in a channel between the skin and somites. Its migration depends on the coordinated movement of its mesenchymal-like leading cells and trailing cells, which form epithelial rosettes, or protoneuromasts. We describe a superficial population of flat primordium cells that wrap around deeper epithelialized cells and extend polarized lamellipodia to migrate apposed to the overlying skin. Polarization of lamellipodia extended by both superficial and deeper protoneuromast-forming cells depends on Fgf signaling. Removal of the overlying skin has similar effects on superficial and deep cells: lamellipodia are lost, blebs appear instead, and collective migration fails. When skinned embryos are embedded in Matrigel, basal and superficial lamellipodia are recovered; however, only the directionality of basal protrusions is recovered, and migration is not rescued. These observations support a key role played by superficial primordium cells and the skin in directed migration of the Posterior Lateral Line primordium.

Alterations in mitophagy have been increasingly linked to aging and age-related diseases. There are, however, no convenient methods to analyze mitophagy in vivo. Here, we describe a transgenic mouse model in which we expressed a mitochondrial-targeted form of the fluorescent reporter Keima (mt-Keima). Keima is a coral-derived protein that exhibits both pH-dependent excitation and resistance to lysosomal proteases. Comparison of a wide range of primary cells and tissues generated from the mt-Keima mouse revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima mice to analyze how mitophagy is altered by conditions including diet, oxygen availability, Huntingtin transgene expression, the absence of macroautophagy (ATG5 or ATG7 expression), an increase in mitochondrial mutational load, the presence of metastatic tumors, and normal aging. The ability to assess mitophagy under a host of varying environmental and genetic perturbations suggests that the mt-Keima mouse should be a valuable resource.

We have isolated a basic helix-loop-helix (bHLH) gene homologous to the Drosophila proneural gene atonal, termed zath3, from zebrafish. zath3 is expressed in neurons of the central nervous system and in subsets of cranial ganglia. Zebrafish mindbomb (mib) mutants have a higher density of zath3 expressing cells and narrowminded (nrd) mutants lack zath3 expression in a domain corresponding to primary sensory neurons showing that the expression of zath3 is regulated by both mib and nrd. Injection of synthetic zath3 RNA into zebrafish embryos expands the neural plate size, promotes ectopic expression of neuronal markers, and partially rescues the deficit of sensory neurons seen in nrd mutants. Interfering with zath3 function using antisense morpholino oligonucleotides (MO) has no significant effect on early neurogenesis. However, a double knock down of zath3 and neurogenin1 (ngn1), another atonal homologue, with morpholinos (MOs) leads to more severe defects in neurogenesis than are seen with ngn1 MO alone: a subtle reduction of motor and inter-neurons, and an almost complete loss all cranial ganglia. This study suggests that zath3 and ngn1 have partially overlapping roles in early neurogenesis.

Two-photon excitation fluorescence microscopy (TPEFM) permits the investigation of the topology of intercellular events within living animals. TPEFM was used to monitor the distribution of mitochondrial reduced nicotinamide adenine dinucleotide (NAD(P)H) in murine skeletal muscle in vivo. NAD(P)H fluorescence emission was monitored ( approximately 460 nm) using 710-720 nm excitation. High-resolution TPEFM images were collected up to a depth of 150 microm from the surface of the tibialis anterior muscle. The NAD(P)H fluorescence images revealed subcellular structures consistent with subsarcolemmal, perivascular, intersarcomeric, and paranuclear mitochondria. In vivo fiber typing between IIB and IIA/D fibers was possible using the distribution and content of mitochondria from the NAD(P)H fluorescence signal. The intersarcomeric mitochondria concentrated at the Z-line in the IIB fiber types resulting in a periodic pattern with a spacing of one sarcomere (2.34 +/- 0.17 microm). The primary inner filter effects were nearly equivalent to water, however, the secondary inner filter effects were highly significant and dynamically affected the observed emission frequency and amplitude of the NAD(P)H fluorescence signal. These data demonstrate the feasibility, and highlight the complexity, of using NAD(P)H TPEFM in skeletal muscle to characterize the topology and metabolic function of mitochondria within the living mouse.

Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell-cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSC administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies characterized by dysregulated Akt.

Schnyder corneal dystrophy (SCD) is a rare autosomal dominant disease in humans, characterized by abnormal deposition of cholesterol and phospholipids in cornea caused by mutations in the UbiA prenyltransferase domain containing 1 (UBIAD1) gene. In this study, we generated a mouse line carrying Ubiad1 N100S point mutation using the CRISPR/Cas9 technique to investigate the pathogenesis of SCD. In vivo confocal microscopy revealed hyper-reflective dot-like deposits in the anterior cornea in heterozygotes and homozygotes. No significant change was found in corneal epithelial barrier function or wound healing. Electron microscopy revealed abnormal mitochondrial morphology in corneal epithelial, stromal, and endothelial cells. Mitochondrial DNA copy number assay showed 1.27 ± 0.07 fold change in homozygotes versus 0.98 ± 0.05 variation in wild type mice (P < 0.05). Lipidomic analysis indicated abnormal metabolism of glycerophosphoglycerols, a lipid class found in mitochondria. Four (34:1, 34:2, 36:2, and 44:8) of the 11 glycerophosphoglycerols species identified by mass spectrometry showed a significant increase in homozygous corneas compared with heterozygous and wild-type mouse corneas. Unexpectedly, we did not find a difference in the corneal cholesterol level between different genotypes by filipin staining or lipidomic analysis. The Ubiad1N100S mouse provides a promising animal model of SCD revealing that mitochondrial dysfunction is a prominent component of the disease. The different phenotype in human and mouse may due to difference in cholesterol metabolism between species.

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.

We have previously shown that GFP-tagged human ABCG1 on the plasma membrane (PM) and in late endosomes (LE) mobilizes sterol on both sides of the membrane lipid bilayer, thereby increasing cellular cholesterol efflux to lipid surfaces. In the present study, we examined ABCG1-induced changes in membrane cholesterol distribution, organization, and mobility. ABCG1-GFP expression increased the amount of mobile, non-sphingomyelin(SM)-associated cholesterol at the PM and LE, but not the amount of SM-associated-cholesterol or SM. ABCG1-mobilized non-SM-associated-cholesterol rapidly cycled between the PM and LE and effluxed from the PM to extracellular acceptors, or, relocated to intracellular sites of esterification. ABCG1 increased detergent-soluble pools of PM and LE cholesterol, generated detergent-resistant, non-SM-associated PM cholesterol, and increased resistance to both amphotericin B-induced (cholesterol-mediated) and lysenin-induced (SM-mediated) cytolysis, consistent with altered organization of both PM cholesterol and SM. ABCG1 itself resided in detergent-soluble membrane domains. We propose that PM and LE ABCG1 residing at the phase boundary between ordered (Lo) and disordered (Ld) membrane lipid domains alters SM and cholesterol organization thereby increasing cholesterol flux between Lo and Ld, and hence, the amount of cholesterol available for removal by acceptors on either side of the membrane bilayer for either efflux or esterification.

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.

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.

Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in trans) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in cis). As a result, forms of DeltaD that have the MID can activate Notch in trans and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in trans fails but ability to inhibit in cis is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.

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+.

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.

To better define the nature of the transient neutropenia shortly following granulocyte-colony stimulating factor (G-CSF) administration.