To determine the role of intracellular Ca 2؉ in compaction, the first morphogenetic event in embryogenesis, we analyzed preimplantation mouse embryos under several decompacting conditions, including depletion of extracellular Ca 2؉ , blocking of Ca 2؉ channels, and inhibition of microfilaments, calmodulin, and intracellular Ca 2؉ release. Those treatments induced decompaction of mouse morulae and simultaneously induced changes in cytosolic free Ca 2؉ concentration and deregionalization of E-cadherin and fodrin. When morulae were allowed to recompact, the location of both proteins recovered. In contrast, actin did not change its cortical location with compaction nor with decompaction-recompaction. Calmodulin localized in areas opposite to cell-cell contacts in eight-cell stage embryos before and after compaction. Inhibition of calmodulin with trifluoperazine induced its delocalization while morulae decompacted. A nonspecific rise of intracellular free Ca 2؉ provoked by ionomycin did not affect the compacted shape. Moreover, the same decompacting treatments when applied to uncompacted embryos did not produce any change in intracellular Ca 2؉ . Our results demonstrate that in preimplantation mouse embryos experimentally induced stage-specific changes of cell shape are accompanied by changes of intracellular free Ca 2؉ and redistribution of the cytoskeleton-related proteins E-cadherin, fodrin, and calmodulin. We conclude that intracellular Ca 2؉ specifically is involved in compaction and probably regulates the function and localization of cytoskeleton elements.Extracellular calcium is crucial during the preimplantation development of mammalian embryos because of its role in cell-cell adhesion (1-5). Intracellular free calcium ([Ca 2ϩ ] i ) plays a pivotal role in triggering early events related to fertilization, namely, oocyte maturation, cortical granule exocytosis, and egg activation (6-19). However, little is known about the role of [Ca 2ϩ ] i during the following steps of mammalian development. Indirect evidence for a regulatory role was provided in experiments where development of mouse embryos was delayed by treatments with calcium channel blockers or with an inhibitor of calmodulin (20). It was only recently that [Ca 2ϩ ] i changes were studied directly during early mouse development. An endogenous increase in Ca 2ϩ -releasing activity leading to Ca 2ϩ transients was observed during the first mitotic division (21). Moreover, the rate of cavitation and cell division was either accelerated or delayed by experimentally induced elevation or reduction of [Ca 2ϩ ] i , respectively (22-24). Stacheki and Armant (24) suggested that in mouse embryos, inositol 1,4,5-trisphosphate-sensitive and ryanodinesensitive Ca 2ϩ stores exist at the morula stage and that calmodulin is involved in mediating the Ca 2ϩ signaling effects to its distal targets.Despite these temporal effects, no other developmental events have been examined so far under conditions where [Ca 2ϩ ] i has been manipulated and therefore t...

It has been demonstrated that microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. However, there is no clear evidence on the possible roles of the interactions of MAPs in vivo with other cytoskeletal components in maintaining the integrity of the cell architecture. To address this question we extracted the neuronal cytoskeleton from brain cells and studied the selective dissociation of specific molecular isospecies of tau protein under various experimental conditions. Tau, and in some cases MPA-2, were analysed by the use of anti-idiotypic antibodies that recognize epitopes on their tubulin binding sites. Fractions of microtubule-bound tau isoforms were extracted with 0.35 M NaCl or after the addition of nocodazole to allow microtubule depolymerization. Protein eluted with this inhibitor contained most of the assembled tubulin dimer pool and part of the remaining tau and MAP-2. When the remaining cytoskeletal pellet was treated with cytochalasin D to allow depolymerization of actin filaments, only tau isoforms were extracted. Immunoprecipitation studies along with immunolocalization experiments in cell lines containing tau-like components supported the findings on the roles of tau isospecies as linkers between tubulin in the microtubular structure with actin filaments. Interestingly, in certain types of cells, antibody-reactive tau isospecies were detected by immunofluorescence with a discrete distribution pattern along actin filaments, which was affected by cytochalasin disruption of the actin filament network. These results suggest the possible in vivo roles of subsets of tau protein in modulating the interactions between microtubules and actin filaments.

The microtubule-associated protein Tau, a major component of brain microtubules, shares common repeated C-terminal sequences with the high molecular-weight protein MAP-2. It has been shown that tau peptides V187-G204 and V218-G235, representing two main repeats, induced brain tubulin assembly in a concentration-dependent fashion. The specific roles of these repeats in the interaction of tau with microtubules, and its antigenic nature were investigated using synthetic tau peptides and site-directed monoclonal antibodies. Tau peptides appeared to compete with MAP-2 incorporation into assembled microtubules. The interactions of the tau fragments with beta-tubulin peptides bearing the tau binding domain on tubulin were analyzed by fluorescence spectroscopy. The specificity of the binding was further demonstrated by the reactivity of tau and the tau peptides with a monoclonal anti-idiotypic antibody produced after immunization with the beta-II(422-434) tubulin peptide, as assessed by enzyme-linked immunoassay. Western blots confirmed the interaction of tau with the monoclonal antibody. In addition, immunoassays revealed a competition between the MAP-reacting monoclonal antibody and the tubulin peptide beta-II(422-434) for their interaction with the tau molecule.