Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells.

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

Targeting KDM4A epigenetically activates tumor-cell-intrinsic immunity by inducing DNA replication stress.

Developing strategies to activate tumor-cell-intrinsic immune response is critical for improving tumor immunotherapy by exploiting tumor vulnerability. KDM4A, as a histone H3 lysine 9 trimethylation (H3K9me3) demethylase, has been found to play a critical role in squamous cell carcinoma (SCC) growth and metastasis. Here we report that KDM4A inhibition promoted heterochromatin compaction and induced DNA replication stress, which elicited antitumor immunity in SCC. Mechanistically, KDM4A inhibition promoted the formation of liquid-like HP1γ puncta on heterochromatin and stall DNA replication, which activated tumor-cell-intrinsic cGAS-STING signaling through replication-stress-induced cytosolic DNA accumulation. Moreover, KDM4A inhibition collaborated with PD1 blockade to inhibit SCC growth and metastasis by recruiting and activating CD8+ T cells. In vivo lineage tracing demonstrated that KDM4A inhibition plus PD1 blockade efficiently eliminated cancer stem cells. Altogether, our results demonstrate that targeting KDM4A can activate anti-tumor immunity and enable PD1 blockade immunotherapy by aggravating replication stress in SCC cells.

Three-dimensional virtual refocusing of fluorescence microscopy images using deep learning.

We demonstrate that a deep neural network can be trained to virtually refocus a two-dimensional fluorescence image onto user-defined three-dimensional (3D) surfaces within the sample. Using this method, termed Deep-Z, we imaged the neuronal activity of a Caenorhabditis elegans worm in 3D using a time sequence of fluorescence images acquired at a single focal plane, digitally increasing the depth-of-field by 20-fold without any axial scanning, additional hardware or a trade-off of imaging resolution and speed. Furthermore, we demonstrate that this approach can correct for sample drift, tilt and other aberrations, all digitally performed after the acquisition of a single fluorescence image. This framework also cross-connects different imaging modalities to each other, enabling 3D refocusing of a single wide-field fluorescence image to match confocal microscopy images acquired at different sample planes. Deep-Z has the potential to improve volumetric imaging speed while reducing challenges relating to sample drift, aberration and defocusing that are associated with standard 3D fluorescence microscopy.

Deep learning enables cross-modality super-resolution in fluorescence microscopy.

We present deep-learning-enabled super-resolution across different fluorescence microscopy modalities. This data-driven approach does not require numerical modeling of the imaging process or the estimation of a point-spread-function, and is based on training a generative adversarial network (GAN) to transform diffraction-limited input images into super-resolved ones. Using this framework, we improve the resolution of wide-field images acquired with low-numerical-aperture objectives, matching the resolution that is acquired using high-numerical-aperture objectives. We also demonstrate cross-modality super-resolution, transforming confocal microscopy images to match the resolution acquired with a stimulated emission depletion (STED) microscope. We further demonstrate that total internal reflection fluorescence (TIRF) microscopy images of subcellular structures within cells and tissues can be transformed to match the results obtained with a TIRF-based structured illumination microscope. The deep network rapidly outputs these super-resolved images, without any iterations or parameter search, and could serve to democratize super-resolution imaging.

Mouse infection and pathogenesis by Trypanosoma brucei motility mutants.

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream-form motility mutant in 427-derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared with wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. Wesurprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time-course, maximum parasitaemia, number of waves of parasitaemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host.

Polyunsaturated Fatty Acids Mend Macrophage Transcriptome, Glycome, and Phenotype in the Patients with Neurodegenerative Diseases, Including Alzheimer's Disease.

BACKGROUND: Macrophages of healthy subjects have a pro-resolution phenotype, upload amyloid-ß (Aß) into endosomes, and degrade Aß, whereas macrophages of patients with Alzheimer's disease (AD) generally have a pro-inflammatory phenotype and lack energy for brain clearance of Aß. OBJECTIVE: To clarify the pathogenesis of sporadic AD and therapeutic effects of polyunsaturated fatty acids (PUFA) with vitamins B and D and antioxidants on monocyte/macrophage (MM) migration in the AD brain, MM transcripts in energy and Aß degradation, MM glycome, and macrophage clearance of Aß. METHODS: We followed for 31.3 months (mean) ten PUFA-supplemented neurodegenerative patients: 3 with subjective cognitive impairment (SCI), 2 with mild cognitive impairment (MCI), 3 MCI/vascular cognitive impairment, 2 with dementia with Lewy bodies, and 7 non-supplemented caregivers. We examined: monocyte migration in the brain and a blood-brain barrier model by immunochemistry and electron microscopy; macrophage transcriptome by RNAseq; macrophage glycome by N-glycan profiling and LTQ-Orbitrap mass spectrometry; and macrophage phenotype and phagocytosis by immunofluorescence. RESULTS: MM invade Aß plaques, upload but do not degrade Aß, and release Aß into vessels, which develop cerebrovascular amyloid angiopathy (CAA); PUFA upregulate energy and Aß degradation enzyme transcripts in macrophages; PUFA enhance sialylated N-glycans in macrophages; PUFA reduce oxidative stress and increase pro-resolution MM phenotype, mitochondrial membrane potential, and Aß phagocytosis (p < 0.001). CONCLUSION: Macrophages of SCI, MCI, and AD patients have interrelated defects in the transcriptome, glycome, Aß phagocytosis, and Aß degradation. PUFA mend macrophage transcriptome, enrich glycome, enhance Aß clearance, and benefit the cognition of early-stage AD patients.

Bat teeth illuminate the diversification of mammalian tooth classes.

Tooth classes are an innovation that has contributed to the evolutionary success of mammals. However, our understanding of the mechanisms by which tooth classes diversified remain limited. We use the evolutionary radiation of noctilionoid bats to show how the tooth developmental program evolved during the adaptation to new diet types. Combining morphological, developmental and mathematical modeling approaches, we demonstrate that tooth classes develop through independent developmental cascades that deviate from classical models. We show that the diversification of tooth number and size is driven by jaw growth rate modulation, explaining the rapid gain/loss of teeth in this clade. Finally, we mathematically model the successive appearance of tooth buds, supporting the hypothesis that growth acts as a key driver of the evolution of tooth number and size. Our work reveal how growth, by tinkering with reaction/diffusion processes, drives the diversification of tooth classes and other repeated structure during adaptive radiations.

Features of endogenous cardiomyocyte chromatin revealed by super-resolution STED microscopy.

Despite the extensive knowledge of the functional unit of chromatin-the nucleosome-for which structural information exists at the atomic level, little is known about the endogenous structure of eukaryotic genomes. Chromosomal capture techniques and genome-wide chromatin immunoprecipitation and next generation sequencing have provided complementary insight into global features of chromatin structure, but these methods do not directly measure structural features of the genome in situ. This lack of insight is particularly troublesome in terminally differentiated cells which must reorganize their genomes for large scale gene expression changes in the absence of cell division. For example, cardiomyocytes, which are fully committed and reside in interphase, are capable of massive gene expression changes in response to physiological stimuli, but the global changes in chromatin structure that enable such transcriptional changes are unknown. The present study addressed this problem utilizing super-resolution stimulated emission depletion (STED) microscopy to directly measure chromatin features in mammalian cells. We demonstrate that immunolabeling of histone H3 coupled with STED imaging reveals chromatin domains on a scale of 40-70 nm, several folds better than the resolution of conventional confocal microscopy. An analytical workflow is established to detect changes in chromatin structure following acute stimuli and used to investigate rearrangements in cardiomyocyte genomes following agonists that induce cellular hypertrophy. This approach is readily adaptable to investigation of other nuclear features using a similar antibody-based labeling technique and enables direct measurements of chromatin domain changes in response to physiological stimuli.

Correlative nanomechanical profiling with super-resolution F-actin imaging reveals novel insights into mechanisms of cisplatin resistance in ovarian cancer cells.

The exact molecular mechanisms of ovarian cancer platinum resistance are not well understood, and biomarkers to reliably predict ovarian cancer resistance to platinum and other chemotherapeutic agents are lacking. Biomechanics of cisplatin-treated ovarian cancer cells were measured quantitatively at nanoscale level using atomic force microscopy. We demonstrate that cisplatin modulates the cellular nanomechanics of ovarian cancer cells; sensitive cells show dose-dependent increase in cell stiffness, which is effected by disrupting the F-actin polymerization. In contrast, resistant cells show no significant changes in cell stiffness upon cisplatin treatment. Further, stimulated emission depletion, an emerging super-resolution microscopy, shows that at the molecular level, F-actin is indeed remodeled considerably in cisplatin-sensitive and cisplatin-resistant cells. These findings reveal a direct role of the actin remodeling mechanism in cisplatin resistance of ovarian cancer cells, suggesting potential future applications of nanomechanical profiling as a marker for cancer drug sensitivity. FROM THE CLINICAL EDITOR: In this paper, nanomechanical profiling and an emerging super-resolution microscopy method was utilized to decipher the mechanisms of cisplatin resistance in ovarian cancer cells, paving the way to future studies of this and similar other problems with drug resistance in cancer biology.

Direct synthesis of lamin A, bypassing prelamin a processing, causes misshapen nuclei in fibroblasts but no detectable pathology in mice.

Lamin A, a key component of the nuclear lamina, is generated from prelamin A by four post-translational processing steps: farnesylation, endoproteolytic release of the last three amino acids of the protein, methylation of the C-terminal farnesylcysteine, and finally, endoproteolytic release of the last 15 amino acids of the protein (including the farnesylcysteine methyl ester). The last cleavage step, mediated by ZMPSTE24, releases mature lamin A. This processing scheme has been conserved through vertebrate evolution and is widely assumed to be crucial for targeting lamin A to the nuclear envelope. However, its physiologic importance has never been tested. To address this issue, we created mice with a "mature lamin A-only" allele (Lmna(LAO)), which contains a stop codon immediately after the last codon of mature lamin A. Thus, Lmna(LAO/LAO) mice synthesize mature lamin A directly, bypassing prelamin A synthesis and processing. The levels of mature lamin A in Lmna(LAO/LAO) mice were indistinguishable from those in "prelamin A-only" mice (Lmna(PLAO/PLAO)), where all of the lamin A is produced from prelamin A. Lmna(LAO/LAO) exhibited normal body weights and had no detectable disease phenotypes. A higher frequency of nuclear blebs was observed in Lmna(LAO/LAO) embryonic fibroblasts; however, the mature lamin A in the tissues of Lmna(LAO/LAO) mice was positioned normally at the nuclear rim. We conclude that prelamin A processing is dispensable in mice and that direct synthesis of mature lamin A has little if any effect on the targeting of lamin A to the nuclear rim in mouse tissues.

Detection of mercury ion by infrared fluorescent protein and its hydrogel-based paper assay.

Mercury is a highly hazardous and widespread pollutant with bioaccumulative properties. Novel approaches that meet the criteria of desired selectivity, high sensitivity, good biocompatibility, and low background interference in natural settings are continuously being explored. We herein describe a new strategy utilizing the combination of infrared fluorescent protein (IFP) and its chromophore as an infrared fluorescence probe for mercury ion (Hg(II)) detection. Hg(II) has been validated to have specific binding affinity to a cysteine residue (C24) of IFP, thereby inhibiting the conjugation of IFP chromophore biliverdin (BV) to C24 and "turning off" the infrared emission of IFP. The IFP/BV sensor has high selectivity toward Hg(II) among other metal ions over a broad pH range. The in vitro detection limit was determined to be less than 50 nM. As a genetically encoded probe, we demonstrate the IFP/BV sensor can serve as a tool to detect Hg(II) in living organisms or tissues. Moreover, we have exploited a protein-agarose hydrogel-based paper assay to immobilize IFP for detection of Hg(II) in a portable and robust fashion.

Visualizing Pericyte Mimicry of Angiotropic Melanoma by Direct Labeling of the Angioarchitecture.

In addition to intravascular dissemination, angiotropic melanoma cells have the propensity to spread along the external surface of blood vessels in a pericytic location, or pericytic mimicry. Such continuous migration without intravasation has been termed "extravascular migratory metastasis" or EVMM. In order to visualize this mechanism of tumor propagation, we used a murine brain melanoma model utilizing green fluorescent human melanoma cells and red fluorescent lectin-tagged murine vessels. This model allows the direct microscopic visualization and mapping of the interaction of melanoma cells with the brain vasculature. In this chapter, we describe the methodology of lectin perfusion to label the entire angioarchitecture in conjunction with confocal microscopy imaging to study the pericyte mimicry of the angiotropic GFP+ melanoma cells.

Particle size, surface coating, and PEGylation influence the biodistribution of quantum dots in living mice.

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

Quantum dots for in vivo small-animal imaging.

Nanotechnology is poised to transform research, prevention, and treatment of cancer through the development of novel diagnostic imaging methods and targeted therapies. In particular, the use of nanoparticles for imaging has gained considerable momentum in recent years. This review focuses on the growing contribution of quantum dots (QDs) for in vivo imaging in small-animal models. Fluorescent QDs, which are small nanocrystals (1-10 nm) made of inorganic semiconductor materials, possess several unique optical properties best suited for in vivo imaging. Because of quantum confinement effects, the emission color of QDs can be precisely tuned by size from the ultraviolet to the near-infrared. QDs are extremely bright and photostable. They are also characterized by a wide absorption band and a narrow emission band, which makes them ideal for multiplexing. Finally, the large surface area of QDs permits the assembly of various contrast agents to design multimodality imaging probes. To date, biocompatible QD conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastatic cell tracking. Here we consider these novel breakthroughs in light of their potential clinical applications and discuss how QDs might offer a suitable platform to unite disparate imaging modalities and provide information along a continuum of length scales.

Self-Sorting Microscale Compartmentalized Block Copolypeptide Hydrogels.

Multicomponent interpenetrating network hydrogels possessing enhanced mechanical stiffness compared to their individual components were prepared via physical mixing of diblock copolypeptides that assemble by either hydrophobic association or polyion complexation in aqueous media. Optical microscopy analysis of fluorescent-probe-labeled multicomponent hydrogels revealed that the diblock copolypeptide components rapidly and spontaneously self-sort to form distinct hydrogel networks that interpenetrate at micron length scales. These materials represent a class of microscale compartmentalized hydrogels composed of degradable, cell-compatible components, which possess rapid self-healing properties and independently tunable domains for downstream applications in biology and additive manufacturing.

Human Neural Stem Cells Flown into Space Proliferate and Generate Young Neurons.

Here we demonstrate that human neural stem cells (NSCs) proliferate while in space and they express specific NSC markers after being in space. NSCs displayed both higher oxygen consumption and glycolysis than ground controls. These cells also kept their ability to become young neurons. Electrophysiological recordings of space NSC-derived neurons showed immature cell membrane properties characterized by small capacitance and very high input resistance. Current injections elicited only an incipient action potential. No spontaneous synaptic events could be detected, suggesting their immature status even though most recorded cells displayed complex morphology and numerous cell processes. Ascertaining the origin of the NSCs' increased energy requirement is of the essence in order to design effective measures to minimize health risks associated with long-duration human spaceflight missions.

Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G protein-coupled receptor G2A.

Intracellular trafficking and spatial dynamics of membrane receptors critically regulate receptor function. Using microscopic and subcellular fractionation analysis, we studied the localization of the murine G protein-coupled receptor G2A (muG2A). Evaluating green fluorescent protein-tagged, exogenously expressed as well as the endogenous muG2A, we observed that this receptor was spontaneously internalized and accumulated in endosomal compartments, whereas its surface expression was enhanced and stabilized by lysophosphatidylcholine (LPC) treatment. Monensin, a general inhibitor of recycling pathways, blocked LPC-regulated surface localization of muG2A as well as muG2A-dependent extracellular signal-regulated kinase (ERK) activation and cell migration induced by LPC treatment. Mutation of the conserved DRY motif (R-->A) enhanced the surface expression of muG2A, resulting in its resistance to monensin inhibition of ERK activation. Our data suggest that intracellular sequestration and surface expression regulated by LPC, rather than direct agonistic activity control the signaling responses of murine G2A toward LPC.

Intravital Imaging of Human Melanoma Cells in the Mouse Ear Skin by Two-Photon Excitation Microscopy.

Noninvasive imaging of reporter gene expression by two-photon excitation (2PE) laser scanning microscopy is uniquely suited to perform dynamic and multidimensional imaging down to single-cell detection sensitivity in vivo in deep tissues. Here we used 2PE microscopy to visualize green fluorescent protein (GFP) as a reporter gene in human melanoma cells implanted into the dermis of the mouse ear skin. We first provide a step-by-step methodology to set up a 2PE imaging model of the mouse ear's skin and then apply it for the observation of the primary tumor and its associated vasculature in vivo. This approach is minimally invasive and allows repeated imaging over time and continuous visual monitoring of malignant growth within intact animals. Imaging fluorescence reporter gene expression in small living animals by 2PE provides a unique tool to investigate critical pathways and molecular events in cancer biology such as tumorigenesis and metastasis in vivo with high-spatial and temporal resolutions.

Angiotropism and extravascular migratory metastasis in cutaneous and uveal melanoma progression in a zebrafish model.

Cutaneous melanoma is a highly aggressive cancer with a propensity for distant metastasis to various organs. In contrast, melanoma arising in pigmented uveal layers of the eye metastasizes mostly in the liver. The mechanisms of these metastases, which are ultimately resistant to therapy, are still unclear. Metastasis via intravascular dissemination of tumour cells is widely accepted as a central paradigm. However, we have previously described an alternative mode of tumour dissemination, extravascular migratory metastasis, based on clinical and experimental data. This mechanism is characterised by the interaction of cancer cells with the abluminal vascular surface, which defines angiotropism. Here, we employed our 3D co-culture approach to monitor cutaneous and uveal human melanoma cells dynamics in presence of vascular tubules. Using time-lapse microscopy, we evaluated angiotropism, the migration of tumour cells along vascular tubules and the morphological changes occurring during these processes. Cutaneous and uveal melanoma cells were injected in zebrafish embryos in order to develop xenografts. Employing in vivo imaging coupled with 3D reconstruction, we monitored the interactions between cancer cells and the external surface of zebrafish vessels. Overall, our results indicate that cutaneous and uveal melanoma cells spread similarly along the abluminal vascular surfaces, in vitro and in vivo.

microPET-based biodistribution of quantum dots in living mice.

UNLABELLED: This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice. METHODS: (64)Cu-Labeled 800- or 525-nm emission wavelength QD (21- or 12-nm diameter), with or without 2,000 MW (molecular weight) polyethylene glycol (PEG), were injected intravenously into mice (5.55 MBq/25 pmol QD) and studied using well counting or by serial microPET and region-of-interest analysis. RESULTS: Both methods show rapid uptake by the liver (27.4-38.9 %ID/g) (%ID/g is percentage injected dose per gram tissue) and spleen (8.0-12.4 %ID/g). Size has no influence on biodistribution within the range tested here. Pegylated QD have slightly slower uptake into liver and spleen (6 vs. 2 min) and show additional low-level bone uptake (6.5-6.9 %ID/g). No evidence of clearance from these organs was observed. CONCLUSION: Rapid reticuloendothelial system clearance of QD will require modification of QD for optimal utility in imaging living subjects. Formal quantitative biodistribution/imaging studies will be helpful in studying many types of nanoparticles, including quantum dots.