Imaging the spatial dynamics of calmodulin activation during mitosis.

BACKGROUND: Calcium is an important and ubiquitous signalling ion. In most cell types, changes in intracellular calcium concentrations are sensed by calmodulin, a signal transduction protein that regulates cell function through its interactions with kinases and phosphatases. Calcium signals show complex spatiotemporal patterning, but little, if anything, is known about the patterns of calmodulin activation inside cells. RESULTS: We have measured calmodulin activation continuously during mitosis in living cells with a new probe, a fluorescent adduct of calmodulin termed TA-calmodulin. We found that calmodulin was activated locally and episodically in the nucleus and mitotic spindle. The pattern of calmodulin activation was different from the pattern of calcium signals and could not be predicted from the pattern of calcium increase. Calmodulin activation was essential for mitotic progression: both entry into mitosis and exit from mitosis were blocked by a novel peptide that bound to calmodulin with high affinity and so prevented the interaction of calmodulin with its target proteins. CONCLUSIONS: These data suggest that calmodulin regulates mitotic transitions and demonstrate the utility of fluorescent adducts for studying protein activation in living cells with good temporal and spatial resolution.

Expression pattern of insulin receptor mRNA during Xenopus laevis embryogenesis.

We have identified a member of the insulin receptor (InsR)/insulin-like growth factor-1 receptor (IGF-1R) family. The Xenopus insulin receptor (Xe-InsR) is present as a maternal 6.6 kb transcript. Northern blot analysis reveals the presence of this transcript until the mid-blastula transition (MBT), when levels decrease. At neurulation, two distinct transcripts of 6.6 and 7.7 kb are detected, both of which persist throughout embryogenesis. In situ hybridization analysis shows that InsR expression is restricted to regions of ectodermal and mesodermal origin, notably the encephalon, otic vesicles, optic vesicles, gills, somites and the pronephros.

Insulin-like growth factor I receptor messenger expression during oogenesis in Xenopus laevis.

To study Xenopus insulin-like growth factor I (IGF-I) receptor messenger expression during oogenesis, we isolated a complementary DNA (cDNA) corresponding to the beta-subunit of the receptor. There is a high degree of conservation between the deduced polypeptide and the three mammalian sequences previously described for the IGF-I receptor (75% homology) even though it is lower than the homology among mammals themselves (95% homology). IGF-I receptor messenger RNAs were specifically detected by reverse transcription-PCR in oocytes, embryos, and adult liver and muscle. By in situ hybridization, these messenger RNAs could be visualized only in oocytes. Quantification showed that they accumulated from the previtellogenic stage until early vitellogenesis. No specific labeling could be detected in oocytes after stage IV of vitellogenesis. Thus, the IGF-I receptor messenger stock does not seem to increase further beyond this point or may even decrease. The long 3'-untranslated sequence (1.8 kilobases) included in the cDNA presents no homology with those of mammalian receptor cDNAs and could be longer, as no polyadenylated tail is observed. Some motifs corresponding to sequence described as cytoplasmic polyadenylation element or that have been described in unstable messengers could be observed. Moreover, a deadenylation of this RNA occurs in the postvitellogenic stage. These results suggest that translation occurred very early during oogenesis. Therefore, IGF-I receptor could play a role early on, during oocyte growth, in addition to its involvement in the maturation process.

Characterization of the insulin-like growth factor type 1 receptor messenger in two teleost species.

The insulin-like growth factor type 1 receptor (IGF-1R) is a tyrosine kinase which plays essential role in the regulation of growth and development. In this study, we have cloned cDNAs encoding the tyrosine kinase domain of the IGF-1R from two species of fish. The turbot and trout nucleotide sequences share 82% identity. Moreover, the deduced polypeptides are also highly conserved (> 90% identity) compared with the IGF-1R sequences described in other vertebrates, particularly within domains involved in the catalytic activity and in the transduction pathway. Northern blot analyses have revealed a unique 13-kb mRNA transcript. Using an RT-PCR approach, we have also shown that the polyadenylation status seems to vary according to the developmental stage in turbot: polyadenylated in oocytes and in the first larval stages, the mRNA becomes undetectable in the polyadenylated fraction in later stages or in adult somatic tissues. These results suggest that IGF-1R mRNA undergoes complex post-transcriptional regulation.

An anaphase calcium signal controls chromosome disjunction in early sea urchin embryos.

A transient increase in intracellular calcium concentration [Ca2+]i occurs throughout the cell as sea urchin embryos enter anaphase of the first cell cycle. The transient just precedes chromatid disjunction and spindle elongation. Microinjection of calcium chelators or heparin, an InsP3 receptor antagonist, blocks chromosome separation. Photorelease of calcium or InsP3 can reverse the block. Nuclear reformation is merely delayed by calcium antagonists at concentrations that block chromatid separation. Thus, the calcium signal triggers the separation of chromatids, while calcium-independent pathways can bring about the alterations in microtubule dynamics and nuclear events associated with anaphase progression. That calcium triggers chromosome disjunction alone is unexpected. It helps explain previous conflicting results and allows the prediction that calcium plays a similar role at anaphase in other cell types.

Activation of protein kinase C alters p34(cdc2) phosphorylation state and kinase activity in early sea urchin embryos by abolishing intracellular Ca2+ transients.

The p34(cdc2) protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34(cdc2) are induced by Ca(2+) and prevented by Ca(2+) chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca(2+) transients may play an important physiological role in the control of p34(cdc2) kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca(2+) concentration ([Ca(2+)](i)) in early sea urchin embryos without altering the normal resting level of Ca(2+). We have used this finding to investigate whether [Ca(2+)](i) transients control p34(cdc2) kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34(cdc2). In the present study we show that the elimination of [Ca(2+)](i) transients during interphase blocks p34(cdc2) activation and entry into mitosis, while the elimination of mitotic [Ca(2+)](i) transients prevents p34(cdc2) inactivation and exit from mitosis. Moreover, we find that [Ca(2+)](i) transients are not required for the synthesis of cyclin B, its binding to p34(cdc2) or its destruction during anaphase. However, in the absence of interphase [Ca(2+)](i) transients p34(cdc2) does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca(2+)](i) transients p34(cdc2) does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca(2+)](i) transients trigger the dephosphorylation of p34(cdc2) at key regulatory sites, thereby controlling the timing of mitosis entry and exit.