Imaging the secretory pathway: the past and future impact of live cell optical techniques

Classically, the secretory pathway has been studied using a combination of electron microscopic, biochemical and genetic approaches. In the last 20 years with the arrival of molecular biology and epitope tagging, fluorescence microscopy has become more important than previously. Moreover, with the common availability of Green Fluorescent Protein (GFP) and confocal microscopes in the last 10 years, live cell imaging has become a major experimental approach. This review highlights the impact of the recent introduction of single-cell quantitative time-lapse imaging and photobleach techniques on the study of the secretory pathway, and the potential impact of those optical techniques which may play a significant future role in the study of the Golgi apparatus and the secretory pathway. Particular attention is paid to techniques (Fluorescence Resonance Energy Transfer, Fluorescence Correlation Spectroscopy) which can monitor protein-protein interactions in living cells.     

Exploring steric constraints on protein mutations using MAGE/PROBE

When planning a mutation to test some hypothesis, one crucial question is whether the new side chain is compatible with the existing structure; only if it is compatible can the interpretation of mutational results be straightforward. This paper presents a simple way of using the sensitive geometry of all-atom contacts (including hydrogens) to answer that question. The interactive MAGE/PROBE system lets the biologist explore conformational space for the mutant side chain, with an interactively updated kinemage display of its all-atom contacts to the original structure. The Autobondrot function in PROBE systematically explores that same conformational space, outputting contact scores at each point, which are then contoured and displayed. These procedures are applied here in two types of test cases, with known mutant structures. In ricin A chain, the ability of a neighboring glutamate to rescue activity of an active-site mutant is modeled successfully. In T4 lysozyme, six mutations to Leu are analyzed within the wild-type background structure, and their Autobondrot score maps correctly predict whether or not their surroundings must shift significantly in the actual mutant structures; interactive examination of contacts for the conformations involved explains which clashes are relieved by the motions. These programs are easy to use, are available free for UNIX or Microsoft Windows operating systems, and should be of significant help in choosing good mutation experiments or in understanding puzzling results.     

The GGAs promote ARF-dependent recruitment of clathrin to the TGN

The GGAs constitute a family of modular adaptor-related proteins that bind ADP-ribosylation factors (ARFs) and localize to the trans-Golgi network (TGN) via their GAT domains. Here, we show that binding of the GAT domain stabilizes membrane-bound ARF1.GTP due to interference with the action of GTPase-activating proteins. We also show that the hinge and ear domains of the GGAs interact with clathrin in vitro, and that the GGAs promote recruitment of clathrin to liposomes in vitro and to TGN membranes in vivo. These observations suggest that the GGAs could function to link clathrin to membrane-bound ARF.GTP.     

Oxidative stress mislocalizes and retains transport factor importin-alpha and nucleoporins Nup153 and Nup88 in nuclei where they generate high molecular mass complexes

Nuclear trafficking of proteins requires the cooperation between soluble transport components and nucleoporins. As such, classical nuclear import depends on the dimeric carrier importin-alpha/beta1, and CAS, a member of the importin-beta family, which exports importin-alpha to the cytoplasm. Here we analyzed the effect of oxidative stress elicited by diethyl maleate (DEM) on classical nuclear transport. Under conditions that do not induce death in the majority of cells, DEM has little effect on the nucleocytoplasmic concentration gradient of Ran, but interferes with the nuclear accumulation of several reporter proteins. Moreover, DEM treatment alters the distribution of soluble transport factors and several nucleoporins in growing cells. We identified nuclear retention of importin-alpha, CAS as well as nucleoporins Nup153 and Nup88 as a mechanism that contributes to the nuclear concentration of these proteins. Both nucleoporins, but not CAS, associate with importin-alpha in the nuclei of growing cells and in vitro. Importin-alpha generates high molecular mass complexes in the nucleus that contain Nup153 and Nup88, whereas CAS was not detected. The formation of high molecular mass complexes containing importin-alpha, Nup153 and Nup88 is increased upon oxidant treatment, suggesting that complex formation contributes to the anchoring of importin-alpha in nuclei. Taken together, our studies link oxidative stress to the proper localization of soluble transport factors and nucleoporins and to changes in the interactions between these proteins.     

Dual-colour imaging with GFP variants

Green fluorescent protein (GFP) has become an important tool in cell biology and is widely used as a reporter for imaging intracellular proteins and structures in live cells. Recently, spectral variants of GFP with red- and blue-shifted fluorescence emissions have been characterized, opening the possibility of double labelling with two different-coloured GFP fusion proteins. This article reviews recent advances in this technique, with special emphasis on time-lapse imaging applications in living cells.     

Electron paramagnetic resonance oximetry as a quantitative method to measure cellular respiration: a consideration of oxygen diffusion interference

Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO(2)-independent respiration at higher pO(2) ranges, pO(2)-dependent respiration at low pO(2) ranges, and a static equilibrium with no change in pO(2) at very low pO(2) values. The approach here enables one to comprehensively analyze all of the three zones together-where the progression of O(2) diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO(2) data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry.     

Hyperthermia-induced Hsp90·eNOS Preserves Mitochondrial Respiration in Hyperglycemic Endothelial Cells by Down-regulating Glut-1 and Up-regulating G6PD Activity

Uncoupling of NO production from NADPH oxidation by endothelial nitric-oxide synthase (eNOS) is enhanced in hyperglycemic endothelium, potentially due to dissociation of heat shock proteins 90 (Hsp90), and cellular glucose homeostasis is enhanced by a ROS-induced positive feed back mechanism. In this study we investigated how such an uncoupling impacts oxygen metabolism and how the oxidative phosphorylation can be preserved by heat shock (42 °C for 2 h, hyperthermia) in bovine aortic endothelial cells. Normal and heat-shocked bovine aortic endothelial cells were exposed to normoglycemia (NG, 5.0 mm) or hyperglycemia (30 mm). With hyperglycemia treatment, O₂ consumption rate was reduced (from V_O2max = 7.51 ± 0.54 to 2.35 ± 0.27 mm Hg/min/10⁶ cells), whereas in heat-shocked cells, O₂ consumption rate remained unaltered (8.19 ± 1.01 mm Hg/min/10 × 10⁶ cells). Heat shock was found to enhance Hsp90/endothelial NOS interactions and produce higher NO. Moreover, ROS generation in the hyperglycemic condition was also reduced in heat-shocked cells. Interestingly, glucose uptake was reduced in heat-shocked cells as a result of decrease in Glut-1 protein level. Glucose phosphate dehydrogenase activity that gives rise to NADPH generation was increased by hyperthermia, and mitochondrial oxidative metabolism was preserved. In conclusion, the present study provides a novel mechanism wherein the reduced oxidative stress in heat-shocked hyperglycemic cells down-regulates Glut-1 and glucose uptake, and fine-tuning of this pathway may be a potential approach to use for therapeutic benefit of diabetes mellitus.     

Reproductive,behavioral, and endocrine characteristics of the Dall's sheep (Ovis dalli dalli)

Failed interspecific embryo transfer between Dall's sheep (Ovis dalli dalli) and domestic ewes (Ovis aries), and a paucity of physiological data available for the Dall's sheep, provided incentive for developing a reproductive database in Dall's sheep. Urine samples were collected on a regular basis from a captive herd of Dall's sheep during the breeding to lambing interval over a 3 year period. To provide comparative endocrine data during pregnancy, samples also were collected during a single gestation period from five Suffolk ewes. All urine samples were analyzed for pregnanediol-3-glucuronide (PdG) and estrone conjugates (EC). Behavioral observations from Dall's sheep were made each year during the rut and lambing periods to calculate estrous cycle and gestation lengths. Placentas from Dall's sheep (n = 20) and Suffolk ewes (n = 10) were collected, the total number of cotyledons counted, and the length, width, area, and weight of eight cotyledons/horn from each placenta calculated. Endocrine and behavioral results indicated that the Dall's sheep is seasonally polyestrous and monovulatory, with a mean estrous cycle length of 18.2 ± 0.3 days, a mean luteal phase of 11.8 ± 0.5 days, and an average gestation length of 171.6 ± 0.9 days. While hormonal patterns for pregnancy generally were similar between the two species, there was a pronounced difference in the magnitude of hormone concentrations, particularly PdG, and an absence of a marked preparturition EC rise in domestic sheep. Cotyledon numbers, weight, and area were smaller (P < 0.05) in the Dall's sheep, compared to Suffolk ewes. These data suggest that unsuccessful interspecies embryo transfer attempts may have been due to failed fetal/maternal communications resulting from hormonal and/or placental differences. © 1996 Wiley-Liss, Inc.     

Quantitative fluorescence studies of the effects of catecholamines and hydrocortisone on endogenous amine levels in neurones and small intensely fluorescent cells of embryonic chick sympathetic ganglia in vivo and in vitro

Summary Chick embryo lumbar sympathetic ganglia (11 day) cultured for three days and uncultured (in vivo) ganglia of comparable age were freeze-dried and processed by the formaldehyde-induced fluorescence technique for the demonstration of biogenic monoamines. The catecholamine levels within principal neurone cell bodies and small intensely fluorescent (SIF) cells were then examined in plastic sections of the in vivo and in vitro ganglia by a quantitative fluorescence method under various experimental conditions. Culture of ganglia for three days in the presence of hydrocortisone acetate (10g/ml) resulted in an increased SIF cell fluorescence (P<0.001 compared to control) and a green to yellow colour shift in the fluorophore of SIF cells. No detectable alteration in the fluorescence level of neurones was observed. When neurones after three days in culture were incubated for 1 h in exogenous catecholamines, a significant increase in fluorescence levels (interpreted as an increase in catecholamine content) occurred with noradrenaline (2×10^–6 M; 2×10^–5 M). SIF cells in ganglia removed directly from 14-day old chicks similarly took up noradrenaline and dopamine, and also adrenaline (2×10^–5 M). Morphological results are presented which indicate that the cellular appearances and architecture of cultured ganglion explants are very similar to those in comparable ganglia in vivo.This work was supported by a grant from the Medical Research Council. We thank Mrs. G. O'Shea, Mr. T.T. Lee and Mr. P.F. Hire for their valuable technical assistance     

Alfalfa-derived HSP70 administered intranasally improves insulin sensitivity in mice

Heat shock protein (HSP) 70 is an abundant cytosolic chaperone protein that is deficient in insulin-sensitive tissues in diabetes and unhealthy aging, and is considered a longevity target. It is also protective in neurological disease models. Using HSP70 purified from alfalfa and administered as an intranasal solution, we tested in whether the administration of Hsp70 to diet-induced diabetic mice would improve insulin sensitivity. Both the 10 and 40 μg given three times per week for 26 days significantly improved the response to insulin. The HSP70 was found to pass into the olfactory bulbs within 4–6 hours of a single dose. These results suggest that a relatively inexpensive, plentiful source of HSP70 administered in a simple, non-invasive manner, has therapeutic potential in diabetes.