Eleanor Kable

Visualizing lipid structure and raft domains in living cells with two-photon microscopy

The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell–cell contacts and cover 10–15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 μm2), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis.

3-Dimensional imaging of collagen using second harmonic generation

Collagen is the most important structural protein of the animal body. Its unique triple-helix structure and extremely high level of crystallinity make it exceptionally efficient in generating the second harmonic of incident light, and we show here how this leads to a novel mode of microscopy of immediate practical significance in medicine and biology. In particular, it provides sensitive and high-resolution information on collagen distribution, discriminates between type I and type III collagen, and allows both a greater understanding of and a sensitive test for cirrhosis of the liver. Future research applications could include wound healing and hereditary collagen diseases such as osteogenesis imperfecta.

Mechanisms directing the nuclear localization of the CtBP family proteins

ABSTRACT The C-terminal binding protein (CtBP) family includes four proteins (CtBP1 [CtBP1-L], CtBP3/BARS [CtBP1-S], CtBP2, and RIBEYE) which are implicated both in transcriptional repression and in intracellular trafficking. However, the precise mechanisms by which different CtBP proteins are targeted to different subcellular regions remains unknown. Here, we report that the nuclear import of the various CtBP proteins and splice isoforms is differentially regulated. We show that CtBP2 contains a unique nuclear localization signal (NLS) located within its N-terminal region, which contributes to its nuclear accumulation. Using heterokaryon assays, we show that CtBP2 is capable of shuttling between the nucleus and cytoplasm of the cell. Moreover, CtBP2 can heterodimerize with CtBP1-L and CtBP1-S and direct them to the nucleus. This effect strongly depends on the CtBP2 NLS. PXDLS motif-containing transcription factors, such as BKLF, that bind CtBP proteins can also direct them to the nucleus. We also report the identification of a splice isoform of CtBP2, CtBP2-S, that lacks the N-terminal NLS and localizes to the cytoplasm. Finally, we show that mutation of the CtBP NADH binding site impairs the ability of the proteins to dimerize and to associate with BKLF. This reduces the nuclear accumulation of CtBP1. Our results suggest a model in which the nuclear localization of CtBP proteins is influenced by the CtBP2 NLS, by binding to PXDLS motif partner proteins, and through the effect of NADH on CtBP dimerization.

Dynamic and intracellular trafficking of P-glycoprotein-EGFP fusion protein: Implications in multidrug resistance in cancer

In our present study, a P‐glycoprotein‐EGFP (P‐gp‐EGFP) fusion plasmid was constructed and functionally expressed in HeLa cells to investigate the intracellular localization and trafficking of P‐glycoprotein (P‐gp). Using immunocytochemistry and fluorescent confocal microscopy techniques, colocalization studies showed that after transfection, P‐gp‐EGFP was progressively transported from the endoplasmic reticulum (ER) to the Golgi and finally to the plasma membrane within 12–48 hr. The degree of intracellular accumulation of daunorubicin was related to the particular localization of P‐gp‐EGFP. Significant daunorubicin accumulation occurred in transfected cells when P‐gp‐EGFP was localized predominantly within the ER, and accumulation remained high when P‐gp‐EGFP was mainly localized in the Golgi. However, there was little or no intracellular accumulation of daunorubicin when P‐gp‐EGFP was localized predominantly on the plasma membrane. Blocking the intracellular trafficking of P‐gp‐EGFP with brefeldin A (BFA) and monensin resulted in inhibition of traffic of P‐gp‐EGFP and retention of P‐gp‐EGFP intracellularly. Intracellular accumulation of daunorubicin also increased in the presence of BFA or monensin. Our study shows that P‐gp‐EGFP can be used to define the dynamics of P‐gp traffic in a transient expression system, and demonstrates that localization of P‐gp on the plasma membrane is associated with the highest level of resistance to daunorubicin accumulation in cells. Modulation of intracellular localization of P‐gp with agents designed to selectively modify its traffic may provide a new strategy for overcoming multidrug resistance in cancer cells.

Second-Harmonic Imaging of Collagen

Molecules that have no center of symmetry are able to convert light to its second harmonic, at twice the frequency and half the wavelength. This only happens with any efficiency at very high light intensities such as are given by a pulsed laser, and because the efficiency of the process depends on the square of the intensity, it will be focal plane selective in exactly the same way as two-photon excitation of fluorescence. Because of its unusual molecular structure and its high degree of crystallinity, collagen is, by far, the strongest source of second harmonics in animal tissue. Because collagen is also the most important structural protein in the mammalian body, this provides a very useful imaging tool for studying its distribution. No energy is lost in second-harmonic imaging, so the image will not fade, and because it is at a shorter wavelength than can be excited by two-photon fluorescence, it can be separated easily from multiple fluorescent probes. It is already proving useful in imaging collagen with high sensitivity in various tissues, including cirrhotic liver, normal and carious teeth, and surgical repair of tendons.

Intrahepatic expression of collagen and fibroblast activation protein (FAP) in hepatitis C virus infection.

Mark D. Gorrell, Xin M. Wang, Miriam T. Levy, Eleanor Kable, George Marinos , Guy Cox and Geoffrey W. McCaughan 1. A. W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Centenary Institute of Cancer Medicine and Cell Biology and the University of Sydney, NSW Australia. 2. Electron Microscope Unit, University of Sydney. 3. Gastroenterology Department, Prince of Wales Hospital, Sydney

Computer-generated, three-dimensional reconstruction of histological parallel serial sections displaying microvascular and glandular structures in human endometrium

This paper describes a technique to develop high-resolution three-dimensional (3D) images of microvasculature structures in curettage, hysterectomy or endometrial resection biopsies using parallel histological serial sections. Employing a labelled streptavidin-biotin-alkaline phosphatase (LSAB(+)) method and visualising by using DAB(+) with the primary antibody, mouse anti human Q-Bend-10, the images were directly digitised from a light microscope into the KS400 Universal Image Processing and Analysis software via a CCD colour camera; binary images of the structures were created and the binary images were exported into VoxBlast 3D rendering software to view still and rotating 3D images on a computer monitor. This in turn enabled hard copies of the full sequence to be printed.

Investigation of plant‐derived phenolic compounds as plasma membrane Ca2+‐ATPase inhibitors with potential cardiovascular activity

Various flavonoids and alkylphenols have been reported to inhibit the Ca2+‐ATPase of plasma membrane (PM) and sarcoplasmic reticulum Ca2+‐ATPase. This article describes the development of phenolic inhibitors of PM Ca2+‐ATPase as potential cardiovascular agents. The new inhibitors were obtained from the screening of alkylphenols, alkylresorcinols, and phenolic constituents of medicinal plants, followed by synthesis of analogues and analysis of their structure–activity relationships. Alkylphenols were found to inhibit the PM Ca2+‐ATPase, with 2‐nonylphenol being the most potent compound (IC50 = 30 μM) among a series of synthetic phenols. Several bis‐resorcinols were isolated from the Australian plants Grevillea robusta and Grevillea striata, including two novel compounds which were identified as 1‐(1,3‐dihydroxy‐4‐methylphenyl)‐14‐(1,3‐dihydroxyphenyl)tetradec‐8‐Z‐ene (grebustol‐A) and norstriatol‐B. Among these bis‐resorcinols, striatol was found to be the most potent inhibitor of PM Ca2+‐ATPase, with an IC50 value of 16 μM. Analogues of bis‐phenols and bis‐resorcinols were synthesised and investigated for Ca2+‐ATPase inhibitory activity. It was found that 2,2′‐(decane‐1,10‐diyl)bisphenol (decanediyl‐bisphenol) was the most potent inhibitor (IC50 = 10 μM). The synthetic bis‐phenols and alkylphenols were shown to inhibit the PM Ca2+‐ATPase in a concentration‐ and structure‐dependent fashion. The inhibitory activity of decanediyl‐bisphenol was also demonstrated in reconstituted purified Ca2+‐ATPase from human erythrocyte plasma membrane and in intact red blood cells and smooth muscle cells. The mechanism of action of these inhibitors, however, is not fully understood but presumably, due to their high lipophilic nature, they would interact with the hydrophobic interior or at the lipid–protein interface, thereby perturbing the Ca2+‐ATPase enzyme function. The inhibition was independent of calmodulin stimulation. The inhibition of Ca2+‐ATPase was selective relative to the Na+,K+‐ATPase and Mg2+‐ATPase in the same membrane preparations. These results indicate that bis‐phenolic compounds may be useful in studying the function of PM Ca2+‐ATPase and regulation of intracellular Ca2+, and may have potential cardiovascular activities. Drug Dev. Res. 46:235–249, 1999.© 1999 Wiley‐Liss, Inc.

Plasma membrane calcium pump-mediated calcium efflux and bulk cytosolic free calcium in cultured aortic smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto normotensives rats

Objective To compare the efficacy of the calcium pump-mediated calcium efflux pathway in spontaneously hypertensive rats (SHR) with that in Wistar–Kyoto normotensive rats (WKY), at rest and after angiotensin II stimulation. Design The intracellular free calcium concentration and calcium-45 efflux were measured in parallel, in cultured aortic smooth muscle cells isolated from 10-week-old male SHR and WKY rats. Methods The intracellular free calcium concentration and calcium-45 efflux were studied in confluent vascular smooth muscle cells in culture. Experiments were performed in the absence of added extracellular calcium and sodium. Fura-2 was used to measure basal and angiotensin II-stimulated intracellular free calcium concentration. Effluxed calcium-45 was measured over 5 s intervals to determine basal and angiotensin II-stimulated calcium efflux rates in SHR and in WKY rats. Results No significant difference between SHR and WKY rats was observed in basal intracellular free calcium concentration or 100nmol/l angiotensin II-stimulated peak intracellular free calcium concentration. However, significantly elevated basal and 100nmol/l angiotensin II-stimulated calcium-45 efflux rates were found in SHR. The calcium-45 efflux rates in SHR were elevated when the efflux was normalized with respect to the bulk intracellular free calcium concentration. The time taken to reach the maximum calcium-45 efflux rate after angiotensin II stimulation was reduced in SHR compared with that in WKY rats and was dose-dependent in both rat strains. Conclusion The calcium-pump mediated calcium efflux pathway appears to be more efficient in SHR. This may be the result of post-translational modification, enhanced calcium pump sites in a critical region of the membrane, or the presence of a pool of calcium near the plasma membrane that is not readily detected by cytosolic Fura-2 but is higher in SHR both before and after angiotensin II stimulation.

Quantification of collagen I in airway tissues using second harmonic generation

Abstract. Extracellular matrix (ECM) remodeling contributes to the pathogenic changes in chronic obstructive pulmonary disease (COPD) and is both complex and not well understood. Collagen I, a component of the ECM altered in COPD airways, has second harmonic generation (SHG) properties. The SHG signal is coherent, propagating both forward (F) (primarily organized/mature collagen fibrils) and backward (B) (primarily disorganized/immature collagen fibrils) parallel to the incident light. The F/B SHG ratio was used to determine the proportion of organized to disorganized collagen, with lower variation in F/B ratio between sampling regions within the same patient and between patients in the same disease group compared with analyzing F and B data alone. The F/B ratio was independent of laser power drift, regions analyzed within a tissue and tissue orientation during analysis. Using this method, we identified a significant difference in collagen organization in airway tissue between COPD and nondiseased. We have developed a robust optimization and calibration methodology that will allow direct comparison of data obtained at different times and from multiple microscopes, which is directly adaptable for use with other tissue types. We report a powerful new tool for advancing our understanding of pathological ECM remodeling that may uncover new therapeutic targets in the future.