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.

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.

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.

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.

Measuring In Vivo Mitophagy.

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.

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.

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.

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.

Mitochondrial reticulum for cellular energy distribution in muscle.

Intracellular energy distribution has attracted much interest and has been proposed to occur in skeletal muscle via metabolite-facilitated diffusion; however, genetic evidence suggests that facilitated diffusion is not critical for normal function. We hypothesized that mitochondrial structure minimizes metabolite diffusion distances in skeletal muscle. Here we demonstrate a mitochondrial reticulum providing a conductive pathway for energy distribution, in the form of the proton-motive force, throughout the mouse skeletal muscle cell. Within this reticulum, we find proteins associated with mitochondrial proton-motive force production preferentially in the cell periphery and proteins that use the proton-motive force for ATP production in the cell interior near contractile and transport ATPases. Furthermore, we show a rapid, coordinated depolarization of the membrane potential component of the proton-motive force throughout the cell in response to spatially controlled uncoupling of the cell interior. We propose that membrane potential conduction via the mitochondrial reticulum is the dominant pathway for skeletal muscle energy distribution.

Direct visualization of the arterial wall water permeability barrier using CARS microscopy.

The artery wall is equipped with a water permeation barrier that allows blood to flow at high pressure without significant water leak. The precise location of this barrier is unknown despite its importance in vascular function and its contribution to many vascular complications when it is compromised. Herein we map the water permeability in intact arteries, using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion experiments. Generation of the CARS signal is optimized for water imaging with broadband excitation. We identify the water permeation barrier as the endothelial basolateral membrane and show that the apical membrane is highly permeable. This is confirmed by the distribution of the AQP1 water channel within endothelial membranes. These results indicate that arterial pressure equilibrates within the endothelium and is transmitted to the supporting basement membrane and internal elastic lamina macromolecules with minimal deformation of the sensitive endothelial cell. Disruption of this pressure transmission could contribute to endothelial cell dysfunction in various pathologies.

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.

Macrophages Shed Excess Cholesterol in Unique Extracellular Structures Containing Cholesterol Microdomains.

OBJECTIVE: 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. APPROACH AND RESULTS: Although appearing spherical at the resolution of the conventional fluorescence microscope, super-resolution immunofluorescence and atomic force microscopy of in situ cholesterol microdomains, and immunoelectron microscopy of isolated cholesterol microdomains revealed that the microdomains are not vesicles or 3-dimensional crystals but rather appear as branching irregularly shaped deposits of varying size. These cholesterol microdomain-containing deposits are shed from the plasma membrane into the extracellular matrix. CONCLUSIONS: To date, research on cellular excretion of excess cholesterol has demonstrated cellular cholesterol efflux in the form of membranous vesicles and discoidal HDL particles released into the fluid-phase medium. Shedding of plasma membrane cholesterol microdomains provides an additional mechanism for cells such as macrophages to maintain plasma membrane cholesterol homeostasis. Furthermore, recognition that macrophages shed cholesterol microdomains into the extracellular matrix is important to our understanding of extracellular buildup of cholesterol in atherosclerosis.

Restricted mitochondrial protein acetylation initiates mitochondrial autophagy.

Because nutrient-sensing nuclear and cytosolic acetylation mediates cellular autophagy, we investigated whether mitochondrial acetylation modulates mitochondrial autophagy (mitophagy). Knockdown of GCN5L1, a component of the mitochondrial acetyltransferase machinery, diminished mitochondrial protein acetylation and augmented mitochondrial enrichment of autophagy mediators. This program was disrupted by SIRT3 knockdown. Chronic GCN5L1 depletion increased mitochondrial turnover and reduced mitochondrial protein content and/or mass. In parallel, mitochondria showed blunted respiration and enhanced 'stress-resilience'. Genetic disruption of autophagy mediators Atg5 and p62 (also known as SQSTM1), as well as GCN5L1 reconstitution, abolished deacetylation-induced mitochondrial autophagy. Interestingly, this program is independent of the mitophagy E3-ligase Parkin (also known as PARK2). Taken together, these data suggest that deacetylation of mitochondrial proteins initiates mitochondrial autophagy in a canonical autophagy-mediator-dependent program and shows that modulation of this regulatory program has ameliorative mitochondrial homeostatic effects.

Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy.

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.

Compact non-contact total emission detection for in vivo multiphoton excitation microscopy.

We describe a compact, non-contact design for a total emission detection (c-TED) system for intra-vital multiphoton imaging. To conform to a standard upright two-photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non-contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c-TED device compared to heavily optimized objective-based emission collection. The best light collection enhancement was seen from murine fat (5×-2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 µm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo-damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.

Monitoring mitochondrial calcium in cardiomyocytes during coverslip hypoxia using a fluorescent lifetime indicator.

An increase in mitochondrial calcium via the mitochondrial calcium uniporter (MCU) has been implicated in initiating cell death in the heart during ischemia-reperfusion (I/R) injury. Measurement of calcium during I/R has been challenging due to the pH sensitivity of indicators coupled with the fall in pH during I/R. The development of a pH-insensitive indicator, mitochondrial localized Turquoise Calcium fluorescence Lifetime Sensor (mito-TqFLITS), allows for quantifying mitochondrial calcium during I/R via fluorescent lifetime imaging. Mitochondrial calcium was monitored using mito-TqFLITS, in neonatal mouse ventricular myocytes (NMVM) isolated from germline MCU-KO mice and MCUfl/fl treated with CRE-recombinase to acutely knockout MCU. To simulate ischemia, a coverslip was placed on a monolayer of NMVMs to prevent access to oxygen and nutrients. Reperfusion was induced by removing the coverslip. Mitochondrial calcium increases threefold during coverslip hypoxia in MCU-WT. There is a significant increase in mitochondrial calcium during coverslip hypoxia in germline MCU-KO, but it is significantly lower than in MCU-WT. We also found that compared to WT, acute MCU-KO resulted in no difference in mitochondrial calcium during coverslip hypoxia and reoxygenation. To determine the role of mitochondrial calcium uptake via MCU in initiating cell death, we used propidium iodide to measure cell death. We found a significant increase in cell death in both the germline MCU-KO and acute MCU-KO, but this was similar to their respective WTs. These data demonstrate the utility of mito-TqFLITS to monitor mitochondrial calcium during simulated I/R and further show that germline loss of MCU attenuates the rise in mitochondrial calcium during ischemia but does not reduce cell death.

Convolutional Neural Network Transformer (CNNT) for Fluorescence Microscopy image Denoising with Improved Generalization and Fast Adaptation.

Deep neural networks have been applied to improve the image quality of fluorescence microscopy imaging. Previous methods are based on convolutional neural networks (CNNs) which generally require more time-consuming training of separate models for each new imaging experiment, impairing the applicability and generalization. Once the model is trained (typically with tens to hundreds of image pairs) it can then be used to enhance new images that are like the training data. In this study, we proposed a novel imaging-transformer based model, Convolutional Neural Network Transformer (CNNT), to outperform the CNN networks for image denoising. In our scheme we have trained a single CNNT based "backbone model" from pairwise high-low SNR images for one type of fluorescence microscope (instance structured illumination, iSim). Fast adaption to new applications was achieved by fine-tuning the backbone on only 5-10 sample pairs per new experiment. Results show the CNNT backbone and fine-tuning scheme significantly reduces the training time and improves the image quality, outperformed training separate models using CNN approaches such as - RCAN and Noise2Fast. Here we show three examples of the efficacy of this approach on denoising wide-field, two-photon and confocal fluorescence data. In the confocal experiment, which is a 5×5 tiled acquisition, the fine-tuned CNNT model reduces the scan time form one hour to eight minutes, with improved quality.

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.

Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma.

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.

Phospholipase d promotes lipid microdomain-associated signaling events in mast cells.

Initial IgE-dependent signaling events are associated with detergent-resistant membrane microdomains. Following Ag stimulation, the IgE-receptor (Fc(epsilon)RI ) accumulates within these domains. This facilitates the phosphorylation of Fc(epsilon)RI subunits by the Src kinase, Lyn, and the interaction with adaptor proteins, such as the linker for activation of T cells. Among the phospholipases (PL) subsequently activated, PLD is of interest because of its presence in lipid microdomains and the possibility that its product, phosphatidic acid, may regulate signal transduction and membrane trafficking. We find that in Ag-stimulated RBL-2H3 mast cells, the association of Fc(epsilon)RI with detergent-resistant membrane fractions is inhibited by 1-butanol, which subverts production of phosphatidic acid to the biologically inert phosphatidylbutanol. Furthermore, the knockdown of PLD2, and to a lesser extent PLD1 with small inhibitory RNAs, also suppressed the accumulation of Fc(epsilon)RI and Lyn in these fractions as well as the phosphorylation of Src kinases, Fc(epsilon)RI , linker for activation of T cells, and degranulation. These effects were accompanied by changes in distribution of the lipid microdomain component, ganglioside 1, in the plasma membrane as determined by binding of fluorescent-tagged cholera toxin B subunit and confocal microscopy in live cells. Collectively, these findings suggest that PLD activity plays an important role in promoting IgE-dependent signaling events within lipid microdomains in mast cells.