Katherine Luby-Phelps

Induction of Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells by Transforming Growth Factor-β1: Potential Role in Idiopathic Pulmonary Fibrosis

The hallmark of idiopathic pulmonary fibrosis (IPF) is the myofibroblast, the cellular origin of which in the lung is unknown. We hypothesized that alveolar epithelial cells (AECs) may serve as a source of myofibroblasts through epithelial-mesenchymal transition (EMT). Effects of chronic exposure to transforming growth factor (TGF)-beta1 on the phenotype of isolated rat AECs in primary culture and a rat type II cell line (RLE-6TN) were evaluated. Additionally, tissue samples from patients with IPF were evaluated for cells co-expressing epithelial (thyroid transcription factor (TTF)-1 and pro-surfactant protein-B (pro-SP-B), and mesenchymal (alpha-smooth muscle actin (alpha-SMA)) markers. RLE-6TN cells exposed to TGF-beta1 for 6 days demonstrated increased expression of mesenchymal cell markers and a fibroblast-like morphology, an effect augmented by tumor necrosis factor-alpha (TNF-alpha). Exposure of rat AECs to TGF-beta1 (100 pmol/L) resulted in increased expression of alpha-SMA, type I collagen, vimentin, and desmin, with concurrent transition to a fibroblast-like morphology and decreased expression of TTF-1, aquaporin-5 (AQP5), zonula occludens-1 (ZO-1), and cytokeratins. Cells co-expressing epithelial markers and alpha-SMA were abundant in lung tissue from IPF patients. These results suggest that AECs undergo EMT when chronically exposed to TGF-beta1, raising the possibility that epithelial cells may serve as a novel source of myofibroblasts in IPF.

Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area.

Classical biochemistry is founded on several assumptions valid in dilute aqueous solutions that are often extended without question to the interior milieu of intact cells. In the first section of this chapter, we present these assumptions and briefly examine the ways in which the cell interior may depart from the conditions of an ideal solution. In the second section, we summarize experimental evidence regarding the physical properties of the cell cytoplasm and their effect on the diffusion and binding of macromolecules and vesicles. While many details remain to be worked out, it is clear that the aqueous phase of the cytoplasm is crowded rather than dilute, and that the diffusion and partitioning of macromolecules and vesicles in cytoplasm is highly restricted by steric hindrance as well as by unexpected binding interactions. Furthermore, the enzymes of several metabolic pathways are now known to be organized into structural and functional units with specific localizations in the solid phase, and as much as half the cellular protein content may also be in the solid phase.

Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor γ 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 γ (FcRγ) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation of FcRγ, activation of NF-κB, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor α and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases that are mediators for FcRγ chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling through FcRγ to induce innate immune responses.

Image-Based Genome-Wide siRNA Screen Identifies Selective Autophagy Factors

Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. Here, to identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide small interfering RNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to messenger RNA processing, interferon signalling, vesicle trafficking, cytoskeletal motor function and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, SMURF1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, SMURF1-deficient mice accumulate damaged mitochondria in the heart, brain and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines SMURF1 as a newly recognized mediator of both viral autophagy and mitophagy.

Tunable, Ultrasensitive pH-Responsive Nanoparticles Targeting Specific Endocytic Organelles in Living Cells†

In recent years, multifunctional nanoparticles have received considerable attention in many applications such as biosensors, diagnostic nanoprobes and targeted drug delivery.[1] These efforts have been driven to a large extent by the need to improve biological specificity in diagnosis and therapy through the precise, spatio-temporal control of agent delivery. In order to achieve this goal, continuous efforts have been dedicated to develop stimuli-responsive nanoplatforms.[2] Environmental stimuli that were exploited include pH,[3] temperature,[4] enzymatic expression,[5] redox reaction[6] and light induction.[7] Among these activating signals, pH trigger is one of the most extensively studied stimuli based on two types of pH differences: (a) pathological (e.g. tumor) vs. normal tissues and (b) acidic intracellular compartments.[8] For example, due to the unusual acidity of the tumor extracellular microenvironment (pHe ≈ 6.5), several pHe-responsive nanosystems were reported to increase the sensitivity of tumor imaging or the efficacy of therapy.[9]

Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial. HSCs are rare and few can be found in thin tissue sections or upon live imaging, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as α-catulinGFP), we discover that α-catulinGFP is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of α-catulin−GFP+c-kit+ cells give long-term multilineage reconstitution of irradiated mice, indicating that α-catulin−GFP+c-kit+ cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of α-catulin–GFP+c-kit+ cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin–GFP+c-kit+ cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr+) and Cxcl12high niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67+) and non-dividing (Ki-67−), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr+Cxcl12high cells throughout the bone marrow.

PHYSICAL PROPERTIES OF CYTOPLASM

The physical properties of cytoplasm differ considerably from dilute aqueous solutions. Recent research has improved our understanding of the properties of the fluid phase and provided a more detailed picture of cytoarchitecture and its relation to cytomechanics. Several recent holistic models indicate novel directions for future research.

A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm.

Two homologous indocyanine dyes, Cy3.18 and Cy5.18, can be used as a ratio pair for fluorometric determination of solvent viscosity. Succinimidyl ester derivatives of these dyes can be attached to inert carrier macromolecules, such as Ficoll 70, for measurement of intracellular or intravesicular solvent viscosity. When the viscosity of the solvent was varied by various methods, the fluorescence intensity ratio (Cy3/Cy5) in a mixture of Cy3.18-Ficoll 70 (Cy3F70) and Cy5.18-Ficoll 70 (Cy5F70) in solution was found to be solely a function of solvent viscosity and was insensitive to other solvent parameters such as dielectric constant, temperature, and the ability of the solvent to form hydrogen bonds. Most important, it was insensitive to the presence of large macromolecules, such as proteins, which increase the shear viscosity but have little effect on solvent viscosity. Following microinjection into the cytoplasm of living tissue culture cells, no binding of Cy3F70 or Cy5F70 to intracellular components was detected by fluorescence recovery after photobleaching. Fluorescence intensity ratio imaging of Cy3F70 and Cy5F70 in non-motile interphase CV1 and PtK1 cells showed that the solvent viscosity of cytoplasm was not significantly different from water and showed no spatial variation.

SRBC/cavin-3 is a caveolin adapter protein that regulates caveolae function

Caveolae are a major membrane domain common to most cells. One of the defining features of this domain is the protein caveolin. The exact function of caveolin, however, is not clear. One possible function is to attract adapter molecules to caveolae in a manner similar to how clathrin attracts molecules to coated pits. Here, we characterize a candidate adapter molecule called SRBC. SRBC binds PKCδ and is a member of the STICK (substrates that interact with C‐kinase) superfamily of PKC‐binding proteins. We also show it co‐immunoprecipitates with caveolin‐1. A leucine zipper in SRBC is essential for both co‐precipitation with caveolin and localization to caveolae. SRBC remains associated with caveolin when caveolae bud to form vesicles (cavicles) that travel on microtubules to different regions of the cell. In the absence of SRBC, intracellular cavicle traffic is markedly impaired. We conclude that SRBC (sdr‐related gene product that binds to c‐kinase) and two other family members [PTRF (Pol I and transcription release factor) and SDPR] function as caveolin adapter molecules that regulate caveolae function.

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 ∼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.