Marie-Cécile Harricane

Germinal vesicle components are not required for the cell-cycle oscillator of the early starfish embryo.

We show that certain events of the cell cycle can still occur in starfish oocytes or fertilized eggs from which the germinal vesicle (the prominent nucleus of prophase-arrested oocytes) has been removed before the induction of meiotic maturation. Two meiotic asters develop following hormonal induction of meiotic maturation in these enucleated oocytes. The asters then divide to form a transient tetrapolar figure. When enucleated oocytes are fertilized, the sperm centrosome duplicates at the times corresponding to each cleavage in control nucleated embryos. Periodic changes in the organization of the asters and in the morphology of the cell surface also occur in synchrony with controls. Decondensation of the sperm nucleus, spindle formation, and cleavage do not occur when enucleated oocytes are fertilized. Ultimately the number of asters increases to approximately 520 (about 2(9] before the pseudo-embryo arrests and cytolyzes. Fertilized eggs from which both pronuclei but not the sperm aster have been removed undergo nine cleavages and then cease cell division. The cessation of division may be related to the events that cause the midblastula transition after seven cleavages in normal nucleated embryos.

Transient down-regulation of L-type Ca2+ channel and dystrophin expression after balloon injury in rat aortic cells

OBJECTIVE Migration and proliferation of arterial smooth muscle cells are critical responses during restenosis after balloon angioplasty. We investigated the changes in the expression of Ca(2+) channels and dystrophin, two determinants of contraction, after balloon injury of rat aortas. METHODS Proliferation and migration of aortic myocytes were triggered in vivo by the passage of an inflated balloon catheter in the aortas of 12-week-old male Wistar rats. We used the whole-cell patch clamp technique to investigate Ba(2+) currents (I(Ba)) through Ca(2+) channels in single cells freshly isolated from media and neointima at various times after injury (days 2, 7, 15, 30 and 45). RESULTS No T-type Ca(2+) channel current was recorded in any cell at any time. In contrast, a dihydropyridine (DHP)-sensitive L-type I(Ba)was recorded consistently in the media of intact aorta. After aortic injury, I(Ba) decreased dramatically (at days 2 and 7) but recovered over time to reach normal amplitude on days 30 and 45. In the neointima, I(Ba) was absent on day 15 but also increased gradually over time as observed at days 30 and 45. The use of a specific antibody directed against the L-type Ca(2+) channel alpha(1C) subunit showed, both by immunostaining and by Western blotting, no expression of the Ca(2+) channel protein on day 15. Parallel immunodetection of dystrophin showed that this marker of the contractile phenotype of SMCs was also not detectable at this stage in neointimal cells. Both proteins were re-expressed at days 45 and 63. Balloon injury induces a transient down-regulation of I(Ba) in arterial cells. CONCLUSIONS Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca(2+) channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca(2+) homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.

DISTRIBUTION OF ANNEXIN I DURING NON-PATHOGEN OR PATHOGEN PHAGOCYTOSIS BY CONFOCAL IMAGING AND IMMUNOGOLD ELECTRON MICROSCOPY

Annexin I is an abundant protein in U937 cells differentiated towards a macrophagic phenotype. These cells become able to killEscherichia coli, however, the intracellular pathogenBrucella suis, known to interfere with phagosome maturation, multiply in these differentiated cells. We have analysed by confocal and electron microscopy the cellular localization of annexin I during phagocytosis of yeast, non‐pathogenicE. coliand the intracellular pathogenB. suis. Using immunocytochemical detections annexin I was found mainly as patches in the cytoplasm of uninfected cells. Upon phagocytosis of yeast orE. coliorganisms, annexin I rapidly translocated and concentrated around phagosomes. On the other hand, annexin I was never detected around liveB. suis‐containing phagosomes. However, when dead brucellae were used, annexin I did translocate to the periphagosomal region. Our results suggest that annexin I could play a role in the molecular mechanism of phagosome maturation, which is impaired by some intracellular pathogens.

A 35-kilodalton fragment from gizzard smooth muscle caldesmon that induces F-actin bundles

Specific thrombin proteolysis of native 120-kDa gizzard caldesmon gave rise to a major cleavage into an N-terminal 90-kDa and a C-terminal 35-kDa fragment. Fluorescent labeling, cosedimentation, passage through an affinity column, and carbodiimide crosslinking with actin revealed that the 35-kDa purified segment of the molecule contains the actin and the calcium-calmodulin binding regions. Electron microscopic analysis of its actin complex demonstrated that the 35-kDa segment possesses the bundling properties of the intact molecule. Thus, a possible pathway for the expression of the caldesmon regulatory function during smooth muscle contraction would be a conformational change twisting the helicoïdal structure of the actin filament, which occurs when the 35-kDa caldesmon portion binds to it.

DYSTROPHIN DOES NOT INFLUENCE REGULAR CYTOSKELETAL ARCHITECTURE BUT IS REQUIRED FOR CONTRACTILE PERFORMANCE IN SMOOTH MUSCLE AORTIC CELLS

Cultured vascular smooth muscle cells express distinct histological phenotypes due to a contractile to synthetic stage transition. In this study, we compared the behaviour of cultured aortic smooth muscle cells from young normal and mdx mice. Morphological, immunobiochemical, immunocytochemical analyses and contraction studies of these cells demonstrated that (i) the cell cytoskeleton in mdx mice is not affected by the absence of dystrophin since proteins such as caldesmon, a‐actin, and vinculin are expressed similarly in normal mice, (ii) utrophin (or dystrophin‐related protein) overexpression does not compensate for the physiological and functional role of the lacking dystrophin. These data suggested that dystrophin and utrophin cannot substitute one another and may play different or complementary roles within smooth muscle cells.

Complexes between 15 kDa caldesmon fragment and actin investigated by immuno‐electron microscopy

The regulatory system of smooth muscle thin filaments is thought to involve a major calcium‐calmodulin and actin binding protein: caldesmon. A dissective approach was used to isolate a 35 kDa C‐terminal fragment of the molecule and to produce antibodies reacting against both the intact and the 15 kDa N‐terminal end of this parental fragment. While this purified 15 kDa caldesmon fragment demonstrates a weak actin association, we observed that it cross‐links actin filaments into loose bundles. These structures were labelled with a selective antibody and showed regular periodic striation with repeats of approximately 40 nm. This work brings additional information to previous reports using an actin and calmodulin binding 25 kDa C‐terminal fragment of the caldesmon molecule [(1989) J. Biol. Chem. 264, 2869‐2875]. We demonstrate that a purified fragment corresponding to a sequence smaller than 96 amino acids, which contains no cystein residue, is able to interact with actin at a single site which is not the calmodulin modulated.

Biochemical evidence for the presence of an unconventional actin protein in a prokaryotic organism

The ubiquity of actin, like the functional diversity of many associated proteins, raises a question concerning diversification of motility mechanisms and thus the emergence of an elementary functional system. Our aim was to investigate, in particular, mobiles prokaryotics cells as Synechocystis lacking cilia and flagella, search for actin essential properties and then locate the molecular behaviours. Here we report the presence and purification of a 56-kDa (apparent molecular weight) prokaryotic protein that polymerizes to form filaments, activates myosin Mg(++)-ATPase activity, inhibits DNase-1 activity and affords close antigenic homology to skeletal actin. This protein was found to be associated with thylakoid membranes and extracted in the presence of Triton X-100.

Ultrastructural localization of dystrophin in chicken smooth muscle

We investigated the presence of dystrophin in gizzard smooth muscle by immunofluorescence assay, immunoblot detection and an immunogold electron microscopy technique. Western blot analyses, using antibodies raised against sequences 1173-1728 and 3357-3660 of the dystrophin molecule, revealed the presence of a major intact 400 kDa protein band and an immunofluorescence localization restricted to the periphery of the smooth muscle cells. We were able to precisely determine the dystrophin distribution along the plasmalemma whereas caldesmon molecules were present in the cytoplasm. The most commonly observed distance between two neighbouring dystrophin molecules suggested a self-associating arrangement. We discuss these findings in relation to the function of dystrophin in the smooth muscle cell structure.