Real-time analyses of chloroplast and mitochondrial division and differences in the behavior of their dividing rings during contraction

The time courses of chloroplast and mitochondrial division and the morphological changes in the plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) during chloroplast and mitochondrial division were studied in Cyanidioschyzon merolae De Luca, Taddei and Varano. To accomplish this, chloroplast and cell division of living cells were continuously video-recorded under light microscopy, and the morphological changes in the PD and MD rings were analyzed quantitatively and three-dimensionally by transmission electron microscopy (TEM). Under the light microscope, the diameters of the chloroplast and the cell decreased at uniform velocities, the speed depending on the temperature. To study in detail the sequential morphological change of the mitochondrion in M phase and the contractile mechanism in the divisional planes of the chloroplast and the mitochondrion, we observed the PD and MD rings, which are believed to promote contraction, under TEM, using the diameter of the chloroplast as an index of the time. Three PD rings (an outer PD ring on the cytoplasmic face of the outer envelope, a middle PD ring in the intermembrane space, and an inner PD ring on the stromal face of the inner envelope) were clearly observed, but only the outer MD ring could be observed. The PD ring started to contract soon after it formed, while the contraction of the MD ring did not occur immediately after formation, but was delayed until the contraction of the PD ring was almost complete. Once the MD ring began to contract, the rate of decrease of its circumference was 4 times as high as that of the PD ring. As the outer PD and MD rings contracted, they grew thicker and maintained a constant volume, while the thickness of the inner PD ring did not change and its volume decreased at a constant rate with contraction. In the early stage of contraction, the widths of the three PD rings increased in order, from the outer to the inner ring. With contraction, their widths changed at different rates until they came to have much the same width. In cross-section, the MD ring was wider where it was next to the chloroplast than at the opposite side, adjacent to the nucleus in the early stage of contraction. By the late stage, the widths of the two sides became equal. In our observations, the microbody elongated along the outer MD ring and touched the outer PD ring during contraction of the PD and MD rings. These results clearly revealed differences between the mode of contraction of the outer, middle, and inner PD rings, and between the PD and the MD rings. They also revealed the coordinated widening of the three PD rings, and suggested that the microbody plays a role in the contraction of the PD and MD rings. Received: 1 July 1998 / Accepted: 1 September 1998     

Distinct signaling molecules control Hoxa-11 and Hoxa-13 expression in the muscle precursor and mesenchyme of the chick limb bud.

The limb muscles, originating from the ventrolateral portion of the somites, exhibit position-specific morphological development through successive splitting and growth/differentiation of the muscle masses in a region-specific manner by interacting with the limb mesenchyme and the cartilage elements. The molecular mechanisms that provide positional cues to the muscle precursors are still unknown. We have shown that the expression patterns of Hoxa-11 and Hoxa-13 are correlated with muscle patterning of the limb bud (Yamamoto et al., 1998) and demonstrated that muscular Hox genes are activated by signals from the limb mesenchyme. We dissected the regulatory mechanisms directing the unique expression patterns of Hoxa-11 and Hoxa-13 during limb muscle development. HOXA-11 protein was detected in both the myogenic cells and the zeugopodal mesenchymal cells of the limb bud. The earlier expression of HOXA-11 in both the myogenic precursor cells and the mesenchyme was dependent on the apical ectodermal ridge (AER), but later expression was independent of the AER. HOXA-11 expression in both myogenic precursor cells and mesenchyme was induced by fibroblast growth factor (FGF) signal, whereas hepatocyte growth factor/scatter factor (HGF/SF) maintained HOXA-11 expression in the myogenic precursor cells, but not in the mesenchyme. The distribution of HOXA-13 protein expression in the muscle masses was restricted to the posterior region. We found that HOXA-13 expression in the autopodal mesenchyme was dependent on the AER but not on the polarizing region, whereas expression of HOXA-13 in the posterior muscle masses was dependent on the polarizing region but not on the AER. Administration of BMP-2 at the anterior margin of the limb bud induced ectopic HOXA-13 expression in the anterior region of the muscle masses followed by ectopic muscle formation close to the source of exogenous BMP-2. In addition, NOGGIN/CHORDIN, antagonists of BMP-2 and BMP-4, downregulated the expression of HOXA-13 in the posterior region of the muscle masses and inhibited posterior muscle development. These results suggested that HOXA-13 expression in the posterior muscle masses is activated by the posteriorizing signal from the posterior mesenchyme via BMP-2. On the contrary, the expression of HOXA-13 in the autopodal mesenchyme was affected by neither BMP-2 nor NOGGIN/CHORDIN. Thus, mesenchymal HOXA-13 expression was independent of BMP-2 from polarizing region, but was under the control of as yet unidentified signals from the AER. These results showed that expression of Hox genes is regulated differently in the limb muscle precursor and mesenchymal cells.     

Presence and expression of a novel variant form of ST2 gene product in human leukemic cell line UT-7/GM.

A novel variant cDNA from the human ST2 gene other than ST2 or ST2L was identified and tentatively named ST2V. Alternative splicing inserts a new exon which leads to a change in the C-terminal portion of ST2, causing it to gain a hydrophobic tail instead of losing the third immunoglobulin-like domain. ST2V is expressed in human leukemic cell line UT-7 and its sublines UT-7/GM, UT-7/EPO, and UT-7/TPO, in addition to human helper T cell line 5C10. The amount of ST2V mRNA is greatly diminished when UT-7/GM cells are induced to differentiate into either erythroblastic or megakaryoblastic phenotypes. The possible roles of the ST2V in growth and differentiation are intriguing.     

Removal of vegetal yolk causes dorsal deficencies and impairs dorsal-inducing ability of the yolk cell in zebrafish.

To examine the nature of cytoplasm determinants for dorsal specification in zebrafish, we have developed a method in which we remove the vegetal yolk hemisphere of early fertilized eggs (vegetal removed embryos). When the vegetal yolk mass was removed at the 1-cell stage, the embryos frequently exhibited typical ventralized phenotypes: no axial structures developed. The frequency of dorsal defects decreased when the operation was performed at later stages. Furthermore, the yolk cell obtained from the vegetal-removed embryos lost the ability to induce goosecoid in normal blastomeres while the normal yolk cell frequently did so in normal and vegetal-removed embryos. These results suggested that the vegetal yolk cell mass contains the dorsal determinants, and that the dorsal-inducing ability of the yolk cell is dependent on the determinants.     

Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.     

Telomere lengths at birth in trisomies 18 and 21 measured by Q-FISH

Trisomies 18 and 21 are genetic disorders in which cells possess an extra copy of each of the relevant chromosomes. Individuals with these disorders who survive birth generally have a shortened life expectancy. As telomeres are known to play an important role in the maintenance of genomic integrity by protecting the chromosomal ends, we conducted a study to determine whether there are differences in telomere length at birth between individuals with trisomy and diploidy, and between trisomic chromosomes and normal chromosomes. We examined samples of peripheral blood lymphocytes (PBLs) from 31 live neonates (diploidy: 10, trisomy 18: 10, trisomy 21: 11) and estimated the telomere length of each chromosome arm using Q-FISH. We observed that the telomeres of trisomic chromosomes were neither shorter nor longer than the mean telomere length of chromosomes as a whole among subjects with trisomies 18 and 21 (intra-cell comparison), and we were unable to conclude that there were differences in telomere length between 18 trisomy and diploid subjects, or between 21 trisomy and diploid subjects (inter-individual comparison). Although it has been reported that telomeres are shorter in older individuals with trisomy 21 and show accelerated telomere shortening with age, our data suggest that patients with trisomies 18 and 21 may have comparably sized telomeres. Therefore, it would be advisable for them to avoid lifestyle habits and characteristics such as obesity, cigarette smoking, chronic stress, and alcohol intake, which lead to marked telomere shortening.     

Microtubule-dependent migration of the cell nucleus toward a future leading edge in amoebae ofPhysarum polycephalum

Summary In several cell types, an intriguing correlation exists between the position of the centrosome and the direction of cell locomotion. The centrosome is positioned between the leading edge pseudopod and the nucleus. This suggests that the polarized distribution of organelles in the cytoplasm is coupled spatially with structural and functional polarity in the cell cortex. To study cellular polarization with special interest in the roles of microtubules, we have analyzed the effects of microtubule-disrupting reagents and local laser irradiation on behaviors of both the nucleus and the centrosome in living amoebae ofPhysarum polycephalum. Physarum cells often have 2–3 pseudopods. One of the pseudopods keeps extending to become a stable leading edge while the rest retracts, a crucial step that reorients cells during locomotion. The nucleus, together with the centrosome, moves specifically toward the pseudopod that will become the leading edge. Disruption of microtubules with nocodazole randomizes positions of the nucleus, indicating the involvement of microtubules in the directional migration of the nucleus toward a specific pseudopod. The migration direction of the nucleus is reversed immediately after the UV laser is irradiated at regions between the nucleus and the future leading pseudopod. In contrast, irradiation at regions between the future tail and the nucleus does not affect nuclear migration. By immunofluorescence, we confirmed fragmentation of microtubules specifically in the irradiated region. These results suggest that the nucleus is pulled together with the centrosome toward the future leading-edge pseudopod in a microtubule-dependent manner. Microtubules seem to exert the pulling force generated in the cell cortex on the centrosome. They may serve as a mediator of shape changes initiated in the cell cortex to the organelle geometry in the endoplasm.