Intracellular Ca2+ measurement with Indo-1 in substrate-attached cells: advantages and special considerations

The dual emission, Ca2+ sensitive fluorescent dye, Indo-1, offers several potential advantages over its dual excitation analogue, Fura-2. Most notable among these advantages are increased speed of measurement using dual wavelength photometry and the absence of a requirement for special quartz optics. Despite these potential advantages, only a tiny fraction of the microscopic studies of intracellular free calcium ([Ca2+]i) on substrate-attached cells has employed Indo-1. Among the reasons for the infrequent use of Indo-1 are the fact that it exhibits somewhat different spectral properties in the cytosol than it does in extracellular buffers, and the notion that it is much more sensitive to photobleaching than Fura-2. We report here that under our experimental conditions, Indo-1 photobleaching is small and does not noticeably affect the measurement of free Ca2+, even after 30 minutes of continuous illumination. We also report a new method for creating in situ standard curves that is easy, reproducible, and yields values for [Ca2+]i that are identical to those obtained with Fura-2. In addition, we have found that Indo-1 is less subject than Fura-2 to compartmentalization within subcellular organelles. These results provide baseline data to take advantage of the significant improvement afforded by Indo-1 in the measurement of rapid [Ca2+]i responses and the avoidance of compartmentalization artifacts during experiments of long duration.     

Association of cytoplasmic free Ca2+ gradients with subcellular organelles.

Previous investigations have identified gradients of intracellular free (Ca2+)i (Ca2+i) in the cytoplasm of human fibroblasts. In this study we have compared the spatial distribution of these gradients with the subcellular distribution of cytoplasmic organelles. Using the Ca(2+)-sensitive dye fura-2 and organelle-specific fluorescent dyes, we have found that the highest Ca2+ concentrations are found in the perinuclear cytoplasm and that these regions co-localize with the Golgi apparatus. The area occupied by the endoplasmic reticulum, which includes the Golgi region plus an adjacent area, is also significantly elevated above the average cellular (Ca2+)i. Most mitochondria are located in regions different from those with the highest (Ca2+)i. A variety of phenomena which could have given rise to artifactual (Ca2+)i gradients have been ruled out, including compartmentalization of fura-2 in subcellular organelles, incomplete hydrolysis of fura-2AM esters, and the presence of pH gradients which might change the Ca2+ binding characteristics of fura-2. The existence of gradients in (Ca2+)i between ER and Golgi containing regions of the cytoplasm supports the hypothesis (Sambrook: Cell 61:197-199, 1990) that the traffic of membrane bound vesicles from ER to Golgi is directed by local variations in (Ca2+)i.     

Genetic polymorphism in normal human fibroblasts as analyzed by two-dimensional polyacrylamide gel electrophoresis.

Two dimensional gel electrophoresis has been used to measure the degree of genetic polymorphism among the proteins of normal human fibroblasts. Autoradiographic analysis of the gel protein profiles from radioactively labeled cells allowed comparison of as many as 300 discrete polypeptides at a time. In addition, a newly developed technique for double label autoradiography was used to increase the sensitivity of the system for detection of small differences in the protein profiles of different cell lines. Only about 1.2% of the proteins of different cell lines were found to differ in their electrophoretic mobility. This corresponds to an average heterozygosity of approximately 0.6%. Previous studies of genetic polymorphism using different methods of one-dimensional electrophoretic analysis have estimated the average heterozygosity of the human population at about 6.7%. Detailed mathematical analysis shows the variation of the observed from the expected number of differences to be statistically highly significant. While the reasons for this difference are not clear, the observation of low levels of genetic polymorphism on two-dimensional gels should enhance the usefulness of this technique for detection of altered proteins in inherited disease.     

Double-label autoradiography on polyacrylamide gels with 3H and 14C

A method of autoradiography is deseribed which allows discrimination between ³H- and ¹⁴C-labeled materials on polyacrylamide gels. The method relies on a very simple procedure for switching from fluorography to autoradiography which results in a decrease in ¹⁴C detection sensitivity of about 10-fold but a concomitant decrease in ³H sensitivity of over 800-fold. With the proper ratio of ³H to ¹⁴C radioactivities, there is little or no “spillover” of one isotope during the detection of the other. No special equipment is required.     

Nuclear CaMKII inhibits neuronal differentiation of PC12 cells without affecting MAPK or CREB activation

Ca²⁺/calmodulin-regulatedprotein kinase II (CaMKII) mediates many cellular events. The fourCaMKII isoforms have numerous splice variants, three of which containnuclear localization signals. Little is known about the role of nuclearlocalized CaMKII in neuronal development. To study this process, PC12cells were transfected to produce CaMKII targeted to either thecytoplasm or the nucleus and then treated with nerve growth factor(NGF). NGF triggers a signaling cascade (MAPK) that results in thedifferentiation of PC12 cells into a neuronal phenotype, marked byneurite outgrowth. The present study found that cells expressingnuclear targeted CaMKII failed to grow neurites, whereas cellsexpressing cytoplasmic CaMKII readily produced neurites. Inhibition ofneuronal differentiation by nuclear CaMKII was independent of MAPKsignaling, as sustained Erk phosphorylation was not affected.Phosphorylation of CREB was also unaffected. Thus nuclear CaMKIImodifies neuronal differentiation by a mechanism independent of MAPKand CREB activation.

     

Rapid elevation of neuronal cytoplasmic calcium by apolipoprotein E peptide

Apolipoprotein E (apoE) and certain peptides derived from it have been shown to exert neurotoxic effects in vitro, and apoE has been linked to the etiology of Alzheimer's disease. The mechanisms underlying these toxic and pathological effects are, however, not known. To approach this question, we have studied the effects of apoE peptides on the cytoplasmic calcium ([Ca²⁺]₁) homeostasis of cultured cortical neurons. A tandem dimer repeat peptide (apoE_dp) derived from the receptor binding domain of apoE was found to have a potent effect on elevation of [Ca²⁺]₁ calcium. The pathway by which apoE_dp exerted this effect was shown to involve both the mobilization of intracellular calcium and the influx of extracellular calcium, although the effect on influx was more pronounced. Calcium mobilization occurs via a G-protein-linked phospholipase C (PLC) pathway, whereas calcium influx appears to involve a novel Co²⁺-sensitive channel. Both the mobilization and the influx of calcium require the binding of the apoE peptide to a membrane receptor because both pathways are blocked by anti-body to low-density-lipoprotein receptor-related protein. The data suggest that the neurotoxic effects of apoE may be mediated by a persistent elevation of [Ca²⁺]₁. J. Cell. Physiol. 173:73–83, 1997. © 1997 Wiley-Liss, Inc.     

Varied diet and opportunistic foraging in the Ethiopian Bush-crow Zavattariornis stresemanni,an Endangered generalist

The Ethiopian Bush-crow Zavattariornis stresemanni is an endangered, co-operatively breeding southern Ethiopian endemic with a remarkably restricted range (c. 6 000 km²). The species’ range was recently found to be almost perfectly predicted by an envelope of cooler, drier and more seasonal climate than surrounding areas, but the proximate determinants of this range restriction remain unclear. We assessed whether specialisation in diet or foraging may restrict the range of the species by conducting foraging watches to determine prey composition, augmented by observations of opportunistic foraging techniques, and by comparing our results to previously published information on diet. Prey composition comprised a range of arthropods, such as insect larvae (62.7%), beetles (Coleoptera) (15.6%), and grasshoppers and crickets (Orthoptera) (11.8%). Prey was primarily obtained by pecks above ground (74.2%) but also frequently dug up (23.8%). Prey capture was most successful during pecks and we also found chicks were preferentially fed larger prey items over smaller ones by adults. We documented opportunistic behaviours such as nest-raiding and ox-pecking. Diet and foraging are varied and unspecialised, and therefore do not appear to explain the restricted range of the Ethiopian Bush-crow.