María Teresa Alonso

An Integrated Gene Regulatory Network Controls Stem Cell Proliferation in Teeth

Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species.

Red and green aequorins for simultaneous monitoring of Ca2+ signals from two different organelles

Simultaneous control of different functions by calcium signals is possible because of subcellular compartmentalization. Targeted chemiluminescent Ca2+ probes, such as aequorins (AEQs) are optimal for detecting signals originating in different subcellular domains, but imaging is difficult because of low photon yield causing poor spatiotemporal resolution. To overcome this problem, we have co-expressed two spectrally distinct AEQs in different subcellular locations within the same cells. Seven chimeric proteins containing either green- or red-emitting AEQs, with different targeting sequences and Ca2+ affinities, have been designed and tested. We show here evidence for physical and functional independence of the different probes. Cytosolic Ca2+ signals were mirrored in the nucleus, but amplified inside mitochondria and endoplasmic reticulum, and had different time courses in the various locations. Our results demonstrate that these novel tools permit simultaneous and independent monitoring of [Ca2+] in different subcellular domains of the same cell.

Monitoring of the activation of receptor-operated calcium channels in human platelets

Activation of receptor-operated calcium channels has been monitored by measurements of the quenching of the fluorescence of intracellularly trapped fura-2 by Mn entering from the extracellular medium. Release of calcium from intracellular stores was followed simultaneously by measurements of the ratio of the fluorescence excited at 340 and 380 nm. Thrombin, ADP, platelet-activating-factor (PAF) and collagen, all produced both release of calcium from the intracellular stores and uptake of Mn from the extracellular medium. The uptake of Mn, but not the increase of (Ca2+)i, was blocked by nickel. These results suggest the existence of plasma membrane calcium channels which can be activated by the different agonists tested here. The activation of calcium channels was very fast and transient with ADP and PAF, fast and maintained with thrombin, and delayed with collagen.

Thrombin-induced changes of intracellular [Ca2+] and pH in human platelets. Cytoplasmic alkalinization is not a prerequisite for calcium mobilization

We have studied the effects of thrombin (0.1 U/ml) on intracellular Ca2+ ([Ca2+]i) and pH (pHi) in human platelets loaded with fluorescent indicators. Thrombin produced a transient decrease of pHi which reached its maximum within 15-25 seconds (s) and was followed by a sustained alkalinization which brought pHi above the resting value. [Ca2+]i increased transiently peaking at 5-10 s. The late alkalinization induced by thrombin was antagonized by ethylisopropylamiloride, an inhibitor of Na+-H+ exchange, and by sphingosine, an inhibitor of protein kinase C, with little effect on the [Ca2+]i transient. The early acidification was not inhibited by these treatments. We conclude tha the thrombin-induced changes of [Ca2+]i and pHi are mediated by different mechanisms. The late alkalinization is due to activation of Na+/H+ exchange mediated by protein kinase C and, contrarily to previous proposals (Siffert, W. and Akkerman, J.W.N. (1987) Nature 325, 456-458), it is not necessary for calcium mobilization from intracellular stores.

Bioluminescence imaging of calcium oscillations inside intracellular organelles

Ca(2+) oscillations inside intracellular organelles are important for regulation of functions such as gene expression at the nucleus, respiration at mitochondria or protein processing at the endoplasmic reticulum. Targeted aequorins are excellent calcium probes for subcellular analysis, but single-cell imaging has proven difficult because of low light yield. Here we describe a procedure that combines virus-based expression of targeted aequorins with photon-counting imaging. This methodology allows real-time resolution of changes of cytosolic, mitochondrial or nuclear Ca(2+) signals at the single-cell level.

Subcellular Ca2+ Dynamics Measured with Targeted Aequorin in Chromaffin Cells

Abstract: In the last years, intracellular organella have emerged as key components in the generation and transduction of Ca2+ signals in adrenal chromaffin cells. Therefore, accurate measurements of Ca2+ inside cytoplasmic organella are essential for a comprehensive analysis of the Ca2+ redistribution that follows cell stimulation. We have engineered the Ca2+‐sensitive photoprotein aequorin to monitor selectively Ca2+ within the endoplasmic reticulum and the mitochondria. The targeted aequorins were delivered to the appropriate organelles of bovine chromaffin cells by using a herpes simplex virus‐based amplicon vector, which permits efficient gene transfer and high levels of expression in infected cells. We have investigated the relationship between the caffeine and InsP3‐sensitive Ca2+ pools and the presence of the Ca2+‐induced Ca2+ release (CICR) mechanism in chromaffin cells. We find that ER Ca2+ pools responding to caffeine and to InsP3 mostly overlap and that CICR can be induced by Ca2+ entry elicited by high K+ depolarization. Moreover, the activation of Ca2+ channels, either the voltage‐gated Ca2+ channels on the plasma membrane or the channels on the endoplasmic reticulum (ER), generates subplasmalemmal high [Ca2+]c domains that induce Ca2+ uptake by mitochondria. Interestingly, only a subpopulation of mitochondria, the one contained in the pool located close to the plasma membrane and the ryanodine receptors, take up Ca2+ efficiently, and the [Ca2+]M reaches values of 300‐500 μM.

Effects of sodium removal on calcium mobilization and dense granule secretion induced by thrombin in human platelets

Removal of extracellular sodium decreased calcium mobilization from intracellular stores induced by thrombin in aspirin-treated human platelets. ATP and serotonin secretion were also significantly reduced. Secretion was positively correlated with calcium mobilization, but the presence or absence of sodium did not modify the slope of the regression line. Half-maximal secretion was reached when [Ca2+]i was increased by about 0.1 microM. Calcium mobilization induced by the divalent cation ionophore ionomycin was not modified by sodium removal. Secretion induced by ionomycin was much smaller than the thrombin-induced one for the same increases of [Ca2+]i. These results suggest that the presence of external sodium is required for normal thrombin-induced calcium release from the intracellular stores and hence for dense granule secretion. However, secretion cannot be only attributed to the increase of cell [Ca2+]i but also to other process(es) which are not affected by external sodium.

A Microplate-Based Bioluminescence Assay of Mitochondrial Calcium Uptake

Mitochondrial Ca2+ homeostasis is crucial for regulating vital functions such as respiration or apoptosis. Targeted aequorins are excellent probes to measure subcellular Ca2+. Ca2+ concentration in mitochondria ([Ca2+]M) is low at rest (about 10-7 M) and can increase to the micromolar or even approach the millimolar range, upon cell activation. Here we describe a new quantitative luminescent protocol to directly measure mitochondrial Ca2+ uptake, optimized for high throughput. The sensitivity of the method allows detection of changes in either the capacity or the affinity of mitochondrial Ca2+ transport.

Effects of the antithrombitic agent PCA 4230 on agonist-induced Ca2+ entry and Ca2+ release in human platelets

We have studied the effects of the antithrombitic agent PCA 4230 on the entry of Mn2+, used here as a Ca2+ surrogate for Ca2+ channels, and on the release of Ca2+ from the intracellular stores in stimulated human platelets loaded with fura-2. PCA 4230 prevented receptor-operated calcium entry activated by thrombin, ADP and collagen with no modification of the Ca2+ release from the intracellular stores. PCA 4230 also inhibited cytochrome P-450-mediated O-dealkylase activity with the same concentration-dependence as the thrombin-induced Mn2+ entry. These results suggest that the inhibitory effects of PCA 4230 on Ca2+ influx may be due to its interaction with cytochrome P-450, which has been proposed recently to be involved in the activation of receptor-operated Ca2+ channels. In addition, PCA 4230 inhibited both PAF-induced Ca2+ entry and Ca2+ release, behaving as a PAF-antagonist. All these effects contribute to explain the antithrombitic action of PCA 4230.

New Aspects of the Contribution of ER to SOCE Regulation

Apart from amplifying the cytosolic Ca2+ signals generated by Ca2+ mobilization from the endoplasmic reticulum (ER), capacitative calcium entry (CCE) is essential for quick refilling of the ER, which is necessary for repeating Ca2+ signals. SERCA is the third element of CCE. It co-localizes with STIM1 and Orai1 at puncta where it is tightly coupled to plasma membrane store-operated Ca2+ channels (SOC). This allows an extremely efficient refilling of the ER stores with the Ca2+ entering through activated SOC. Whereas ER takes up most of the store-operated Ca2+ entry, mitochondria takes up a very little part. Low mitochondrial Ca2+ uptake can be explained by longer distance to SOC and lower Ca2+ affinity of mitochondrial uptake mechanisms.