SR-B1 drives endothelial cell LDL transcytosis via DOCK4 to promote atherosclerosis.

Atherosclerosis, which underlies life-threatening cardiovascular disorders such as myocardial infarction and stroke1, is initiated by passage of low-density lipoprotein (LDL) cholesterol into the artery wall and its engulfment by macrophages, which leads to foam cell formation and lesion development2,3. It is unclear how circulating LDL enters the artery wall to instigate atherosclerosis. Here we show in mice that scavenger receptor class B type 1 (SR-B1) in endothelial cells mediates the delivery of LDL into arteries and its accumulation by artery wall macrophages, thereby promoting atherosclerosis. LDL particles are colocalized with SR-B1 in endothelial cell intracellular vesicles in vivo, and transcytosis of LDL across endothelial monolayers requires its direct binding to SR-B1 and an eight-amino-acid cytoplasmic domain of the receptor that recruits the guanine nucleotide exchange factor dedicator of cytokinesis 4 (DOCK4)4. DOCK4 promotes internalization of SR-B1 and transport of LDL by coupling the binding of LDL to SR-B1 with activation of RAC1. The expression of SR-B1 and DOCK4 is increased in atherosclerosis-prone regions of the mouse aorta before lesion formation, and in human atherosclerotic arteries when compared with normal arteries. These findings challenge the long-held concept that atherogenesis involves passive movement of LDL across a compromised endothelial barrier. Interventions that inhibit the endothelial delivery of LDL into artery walls may represent a new therapeutic category in the battle against cardiovascular disease.

Building momentum through networks: Bioimaging across the Americas.

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.

Soluble klotho binds monosialoganglioside to regulate membrane microdomains and growth factor signaling.

Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane's propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho.

Distinct reorganization of collagen architecture in lipopolysaccharide-mediated premature cervical remodeling.

Previous work has identified divergent mechanisms by which cervical remodeling is achieved in preterm birth (PTB) induced by hormone withdrawal (mifepristone) or lipopolysaccharide (LPS). Our current study aims to document how collagen architecture is modified to achieve premature cervical remodeling in mice treated with LPS as a model of infection-induced inflammation. Cervices were collected on gestation day (d) 15 from mice with premature cervical ripening induced by LPS and compared to d15 and d18 controls as well as a hormone withdrawal PTB model. Second harmonic generation (SHG) and electron microscopy were utilized for visualization of collagen morphology and ultrastructure. LPS-mediated premature cervical ripening is characterized by unique structural changes in collagen fiber morphology. LPS treatment increased the interfibrillar spacing of collagen fibrils. A preferential disruption of collagen fiber architecture in the subepithelial region compared to midstroma region was evidenced by increased pores lacking collagen signal in SHG images in the LPS-treated mice. Coinciding with this alteration, the infiltration of neutrophils was concentrated in the subepithelial stromal region as compared to midstromal region implicating the potential role of immune cells to extracellular matrix reorganization in inflammation-induced preterm cervical ripening. The current study demonstrates a preferential disorganization of collagen interfibrillar spacing and collagen fiber structure in LPS-mediated ripening.

Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake.

The LDL receptor (LDLR) supports efficient uptake of both LDL and VLDL remnants by binding lipoprotein at the cell surface, internalizing lipoprotein through coated pits, and releasing lipoprotein in endocytic compartments before returning to the surface for further rounds of uptake. While many aspects of lipoprotein binding and receptor entry are well understood, it is less clear where, when, and how the LDLR releases lipoprotein. To address these questions, the current study employed quantitative fluorescence imaging to visualize the uptake and endosomal processing of LDL and the VLDL remnant ß-VLDL. We find that lipoprotein release is rapid, with most release occurring prior to entry of lipoprotein into early endosomes. Published biochemical studies have identified two mechanisms of lipoprotein release: one that involves the ß-propeller module of the LDLR and a second that is independent of this module. Quantitative imaging comparing uptake supported by the normal LDLR or by an LDLR variant incapable of ß-propeller-dependent release shows that the ß-propeller-independent process is sufficient for release for both lipoproteins but that the ß-propeller process accelerates both LDL and ß-VLDL release. Together these findings define where, when, and how lipoprotein release occurs and provide a generalizable methodology for visualizing endocytic handling in situ.

Regulation of purinergic signaling in biliary epithelial cells by exocytosis of SLC17A9-dependent ATP-enriched vesicles.

ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 µm that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling.

Initiation of purinergic signaling by exocytosis of ATP-containing vesicles in liver epithelium.

Extracellular ATP represents an important autocrine/paracrine signaling molecule within the liver. The mechanisms responsible for ATP release are unknown, and alternative pathways have been proposed, including either conductive ATP movement through channels or exocytosis of ATP-enriched vesicles, although direct evidence from liver cells has been lacking. Utilizing dynamic imaging modalities (confocal and total internal reflection fluorescence microscopy and luminescence detection utilizing a high sensitivity CCD camera) at different scales, including confluent cell populations, single cells, and the intracellular submembrane space, we have demonstrated in a model liver cell line that (i) ATP release is not uniform but reflects point source release by a defined subset of cells; (ii) ATP within cells is localized to discrete zones of high intensity that are approximately 1 mum in diameter, suggesting a vesicular localization; (iii) these vesicles originate from a bafilomycin A(1)-sensitive pool, are depleted by hypotonic exposure, and are not rapidly replenished from recycling of endocytic vesicles; and (iv) exocytosis of vesicles in response to cell volume changes depends upon a complex series of signaling events that requires intact microtubules as well as phosphoinositide 3-kinase and protein kinase C. Collectively, these findings are most consistent with an essential role for exocytosis in regulated release of ATP and initiation of purinergic signaling in liver cells.

Autophagy is essential for cardiac morphogenesis during vertebrate development.

Genetic analyses indicate that autophagy, an evolutionarily conserved lysosomal degradation pathway, is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryogenesis. To address this question, we examined the role of autophagy in the early development of zebrafish, a model organism for studying vertebrate tissue and organ morphogenesis. Using zebrafish that transgenically express the fluorescent autophagy reporter protein, GFP-LC3, we found that autophagy is active in multiple tissues, including the heart, during the embryonic period. Inhibition of autophagy by morpholino knockdown of essential autophagy genes (including atg5, atg7, and becn1) resulted in defects in morphogenesis, increased numbers of dead cells, abnormal heart structure, and reduced organismal survival. Further analyses of cardiac development in autophagy-deficient zebrafish revealed defects in cardiac looping, abnormal chamber morphology, aberrant valve development, and ectopic expression of critical transcription factors including foxn4, tbx5, and tbx2. Consistent with these results, Atg5-deficient mice displayed abnormal Tbx2 expression and defects in valve development and chamber septation. Thus, autophagy plays an essential, conserved role in cardiac morphogenesis during vertebrate development.

Brown adipose tissue derived VEGF-A modulates cold tolerance and energy expenditure.

We recently reported that local overexpression of VEGF-A in white adipose tissue (WAT) protects against diet-induced obesity and metabolic dysfunction. The observation that VEGF-A induces a "brown adipose tissue (BAT)-like" phenotype in WAT prompted us to further explore the direct function of VEGF-A in BAT. We utilized a doxycycline (Dox)-inducible, brown adipocyte-specific VEGF-A transgenic overexpression model to assess direct effects of VEGF-A in BAT in vivo. We observed that BAT-specific VEGF-A expression increases vascularization and up-regulates expression of both UCP1 and PGC-1α in BAT. As a result, the transgenic mice show increased thermogenesis during chronic cold exposure. In diet-induced obese mice, introducing VEGF-A locally in BAT rescues capillary rarefaction, ameliorates brown adipocyte dysfunction, and improves deleterious effects on glucose and lipid metabolism caused by a high-fat diet challenge. These results demonstrate a direct positive role of VEGF-A in the activation and expansion of BAT.

The physical chemistry of cytoplasm and its influence on cell function: an update.

From the point of view of intermolecular interactions, the cytoplasmic space is more like a crowded party in a house full of furniture than a game of tag in an empty field. Understanding the physical chemical properties of cytoplasm is thus of key importance for understanding cellular function. This article attempts to provide an entrée into the current literature on this subject and offers some general guidelines for thinking about intracellular biochemistry.

Subcellular particles tracking in time-lapse confocal microscopy images.

Automatically tracking and analyzing the mobility of live subcellular structures will expedite the understanding of signaling pathways, protein-protein interaction, drug delivery, protein synthesis and functionality. Traditional computer vision tracking methods produce yet-to-be-satisfactory results due to the complexity of the particles recorded in spatial-temporal video sequences from confocal images. The difficulties arise from diverse modalities of motion patterns (translational, Brownian, or sessile), changes in behavior during tracking, and cluttered background. In this paper, we present an effective framework to detect and track subcullular particles in different motion modalities. The methodology begins with a Divergence Filter design for motion modality detection. After that, an improved a´ trous wavelet is presented for segmenting particles. Represented by Euclidean Distance Map which contains information on object position, size, and intensity, the multiple particle tracking is carried out by solving a linear assignment problem. The proposed framework can also simultaneously evaluate particle population change by automatically counting the number of newly appeared or disappeared particles in time space.

Epicardial spindle orientation controls cell entry into the myocardium.

During heart morphogenesis, epicardial cells undergo an epithelial-to-mesenchymal transition (EMT) and migrate into the subepicardium. The cellular signals controlling this process are poorly understood. Here, we show that epicardial cells exhibit two distinct mitotic spindle orientations, directed either parallel or perpendicular to the basement membrane. Cells undergoing perpendicular cell division subsequently enter the myocardium. We found that loss of beta-catenin led to a disruption of adherens junctions and a randomization of mitotic spindle orientation. Loss of adherens junctions also disrupted Numb localization within epicardial cells, and disruption of Numb and Numblike expression in the epicardium led to randomized mitotic spindle orientations. Taken together, these data suggest that directed mitotic spindle orientation contributes to epicardial EMT and implicate a junctional complex of beta-catenin and Numb in the regulation of spindle orientation.

Tracking multiple interacting subcellular structure by sequential Monte Carlo method.

With the wide application of Green Fluorescent Proteins (GFP) in the study of live cells, there is a surging need for computer-aided analysis on the huge amount of image sequence data acquired by the advanced microscopy devices. In this paper, a framework based on Sequential Monte Carlo (SMC) is proposed for multiple interacting object tracking. The distribution of the dimension varying joint state is sampled efficiently by a Reversible Jump Markov Chain Monte Carlo (RJMCMC) algorithm with a novel height swap move. Experimental results were performed on synthetic and real confocal microscopy image sequences.

Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.

BACKGROUND: It is recognized that cancer cells exhibit highly elevated glucose metabolism compared to non-tumor cells. We have applied in vivo optical imaging to study dynamic uptake of a near-infrared dye-labeled glucose analogue, 2-deoxyglucose (2-DG) by orthotopic glioma in a mouse model. METHODOLOGY AND PRINCIPAL FINDINGS: The orthotopic glioma model was established by surgically implanting U87-luc glioma cells into the right caudal nuclear area of nude mice. Intracranial tumor growth was monitored longitudinally by bioluminescence imaging and MRI. When tumor size reached >4 mm diameter, dynamic fluorescence imaging was performed after an injection of the NIR labeled 2-DG, IRDye800CW 2-DG. Real-time whole body images acquired immediately after i.v. infusion clearly visualized the near-infrared dye circulating into various internal organs sequentially. Dynamic fluorescence imaging revealed significantly higher signal intensity in the tumor side of the brain than the contralateral normal brain 24 h after injection (tumor/normal ratio, TNR = 2.8+/-0.7). Even stronger contrast was achieved by removing the scalp (TNR = 3.7+/-1.1) and skull (TNR = 4.2+/-1.1) of the mice. In contrast, a control dye, IRDye800CW carboxylate, showed little difference (1.1+/-0.2). Ex vivo fluorescence imaging performed on ultrathin cryosections (20 microm) of tumor bearing whole brain revealed distinct tumor margins. Microscopic imaging identified cytoplasmic locations of the 2-DG dye in tumor cells. CONCLUSION AND SIGNIFICANCE: Our results suggest that the near-infrared dye labeled 2-DG may serve as a useful fluorescence imaging probe to noninvasively assess intracranial tumor burden in preclinical animal models.

Systemic fate of the adipocyte-derived factor adiponectin.

OBJECTIVE: The adipocyte-derived secretory protein adiponectin has been widely studied and shown to have potent insulin-sensitizing, antiapoptotic, and anti-inflammatory properties. While its biosynthesis is well understood, its fate, once in circulation, is less well established. RESEARCH DESIGN AND METHODS: Here, we examine the half-life of adiponectin in circulation by tracking fluorescently labeled recombinant adiponectin in the circulation, following it to its final destination in the hepatocyte. RESULTS: Despite its abundant presence in plasma, adiponectin is cleared rapidly with a half-life of approximately 75 min. A more bioactive version carrying a mutation at cysteine 39 is cleared within minutes. Even though steady-state levels of adiponectin differ between male and female mice, we failed to detect any differences in clearance rates, suggesting that differences in plasma are mostly due to differential production rates. In a metabolically challenged state (high-fat diet exposure or in an ob/ob background), adiponectin levels are reduced in plasma and clearance is significantly prolonged, reflecting a dramatic drop in adiponectin production levels. CONCLUSIONS: Combined, these results show a surprisingly rapid turnover of adiponectin with multiple fat pads contributing to the plasma levels of adiponectin and clearance mediated primarily by the liver. It is surprising that despite high-level production and rapid clearance, plasma levels of adiponectin remain remarkably constant.

Mitochondria mass is low in mouse substantia nigra dopamine neurons: implications for Parkinson's disease.

In Parkinson's disease (PD) there is a selective loss of certain midbrain dopaminergic (DA) neurons. The most vulnerable neurons reside in the substantia nigra zona compacta (SNC), whereas the DA neurons in the ventral tegmental area (VTA) and interfascicular (IF) nucleus are less vulnerable to degeneration. Many sporadic PD patients have a defect in mitochondria respiration, and some of the genes that cause PD are mitochondrial-related (e.g., PINK1, Parkin, DJ1). The present study sought to determine whether mitochondria mass is different in SNC neurons compared to other midbrain DA neurons and to non-DA neurons in the mouse. At the electron microscopic level, mitochondria in the SN DA neurons occupy 40% less of the soma and dendritic area than in the SN non-DA neurons. The area occupied by mitochondria in the SN DA neurons is also lower than in the VTA neurons, although not different from the IF neurons. The red nucleus somata have the largest percentage of the somata occupied by mitochondria (12%). Mitochondria size is related to somata size; the largest mitochondria are found in the red nucleus neurons and the smallest mitochondria are found in the IF neurons. At the light microscopic level, SNC, VTA and IF DA neurons have <50% of the cytoplasm immunostained with the mitochondrial antibody 1D6, whereas non-DA neurons in the same midbrain regions contain mitochondria areas up to >65% of the cytoplasm area. These data indicate that mitochondria size and mass are not the same for all neurons, and the SNC DA neurons have relatively low mitochondria mass. The low mitochondria mass in SNC DA neurons may contribute to the selective vulnerability of these neurons in certain rodent models of PD.

Autophagy gene-dependent clearance of apoptotic cells during embryonic development.

Autophagy is commonly observed in metazoan organisms during programmed cell death (PCD), but its function in dying cells has been unclear. We studied the role of autophagy in embryonic cavitation, the earliest PCD process in mammalian development. Embryoid bodies (EBs) derived from cells lacking the autophagy genes, atg5 or beclin 1, fail to cavitate. This defect is due to persistence of cell corpses, rather than impairment of PCD. Dying cells in autophagy gene null EBs fail to express the "eat-me" signal, phosphatidylserine exposure, and secrete lower levels of the "come-get-me" signal, lysophosphatidylcholine. These defects are associated with low levels of cellular ATP and are reversed by treatment with the metabolic substrate, methylpyruvate. Moreover, mice lacking atg5 display a defect in apoptotic corpse engulfment during embryonic development. We conclude that autophagy contributes to dead-cell clearance during PCD by a mechanism that likely involves the generation of energy-dependent engulfment signals.

Feature selection, matching, and evaluation for subcellular structure tracking.

Understanding the motility of subcellular particles like organelles, vesicles, or mRNAs is critical to understand how cells regulate delivery of specific proteins from the site of synthesis to the site of action. The goal of this paper is to present a framework of feature selection, matching, and evaluation for the segmentation and tracking of green fluorescent protein (GFP) labeled subcellular structures. To select stable and distinctive features for small-sized subcellular particles, a grid-based minimum variance (GMV) feature selection method is proposed. To robustly keep tracking of the selected features, we propose a mean minimum to maximum ratio (MMMR) similarity measure for feature matching. In order to quantitatively evaluate the proposed methods, we define two evaluation criteria, feature convergence rate (FCVR) and feature consistence rate (FCSR), which conform with the proximity and similarity properties of Gestalt visual perception theory. Our technique was validated on real confocal video data with comparison to traditional feature selection and matching methods.

Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor gamma chain to induce innate immune responses.

Antigen presenting cells recognize pathogens via pattern recognition receptors (PRR), which upon ligation transduce intracellular signals that can induce innate immune responses. Because some C-type lectin-like receptors (e.g. dectin-1 and DCSIGN) were shown to act as PRR for particular microbes, we considered a similar role for dectin-2. Binding assays using soluble dectin-2 receptors showed the extracellular domain to bind preferentially to hyphal (rather than yeast/conidial) components of Candida albicans, Microsporum audouinii, and Trichophyton rubrum. Selective binding for hyphae was also observed using RAW macrophages expressing dectin-2, the ligation of which by hyphae or cross-linking with dectin-2-specific antibody led to protein tyrosine phosphorylation. Because dectin-2 lacks an intracellular signaling motif, we searched for a signal adaptor that permits it to transduce intracellular signals. First, we found that the Fc receptor gamma (FcRgamma) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation of FcRgamma, activation of NF-kappaB, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor alpha and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases that are mediators for FcRgamma chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling through FcRgamma to induce innate immune responses.

ERK2 enters the nucleus by a carrier-independent mechanism.

In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus. Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood. To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an in vitro import assay. GFP-ERK2 entered the nucleus in a saturable, time- and temperature-dependent manner. Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes. The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins. The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors. These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors. In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin. Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier- and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins.