Narie Sasaki

Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.

Architectural role of mitochondrial transcription factor A in maintenance of human mitochondrial DNA.

ABSTRACT Mitochondrial transcription factor A (TFAM), a transcription factor for mitochondrial DNA (mtDNA) that also possesses the property of nonspecific DNA binding, is essential for maintenance of mtDNA. To clarify the role of TFAM, we repressed the expression of endogenous TFAM in HeLa cells by RNA interference. The amount of TFAM decreased maximally to about 15% of the normal level at day 3 after RNA interference and then recovered gradually. The amount of mtDNA changed closely in parallel with the daily change in TFAM while in organello transcription of mtDNA at day 3 was maintained at about 50% of the normal level. TFAM lacking its C-terminal 25 amino acids (TFAM-ΔC) marginally activated transcription in vitro. When TFAM-ΔC was expressed at levels comparable to those of endogenous TFAM in HeLa cells, mtDNA increased twofold, suggesting that TFAM-ΔC is as competent in maintaining mtDNA as endogenous TFAM under these conditions. The in organello transcription of TFAM-ΔC-expressing cells was no more than that in the control. Thus, the mtDNA amount is finely correlated with the amount of TFAM but not with the transcription level. We discuss an architectural role for TFAM in the maintenance of mtDNA in addition to its role in transcription activation.

Live-Cell Imaging Reveals the Dynamics of Two Sperm Cells during Double Fertilization in Arabidopsis thaliana

Flowering plants have evolved a unique reproductive process called double fertilization, whereby two dimorphic female gametes are fertilized by two immotile sperm cells conveyed by the pollen tube. The two sperm cells are arranged in tandem with a leading pollen tube nucleus to form the male germ unit and are placed under the same genetic controls. Genes controlling double fertilization have been identified, but whether each sperm cell is able to fertilize either female gamete is still unclear. The dynamics of individual sperm cells after their release in the female tissue remain largely unknown. In this study, we photolabeled individual isomorphic sperm cells before their release and analyzed their fate during double fertilization in Arabidopsis thaliana. We found that sperm delivery was composed of three steps. Sperm cells were projected together to the boundary between the two female gametes. After a long period of immobility, each sperm cell fused with either female gamete in no particular order, and no preference was observed for either female gamete. Our results suggest that the two sperm cells at the front and back of the male germ unit are functionally equivalent and suggest unexpected cell-cell communications required for sperm cells to coordinate double fertilization of the two female gametes.

Species Preferentiality of the Pollen Tube Attractant Derived from the Synergid Cell of Torenia fournieri

The synergid cell of Torenia fournieri attracts pollen tubes by a diffusible but yet unknown chemical attractant. Here we investigated the species difference of the attractant using five closely related species in two genera, namely T. fournieri, Torenia baillonii, Torenia concolor, Lindernia (Vandellia) crustacea, and Lindernia micrantha. These five species have an exserted embryo sac, and ablation experiments confirmed that their synergid cells attracted the pollen tube. When ovules of T. fournieri and one of the other species were cultivated together with pollen tubes of each species, pollen tubes were significantly more attracted to synergid cells of the corresponding species. The attraction was not affected by the close proximity of embryo sacs of different species. This suggests that the attractant is a species-preferential molecule that is likely synthesized in the synergid cell. The calcium ion, long considered a potential attractant, could not serve as the sole attractant in these species, because elevation of the calcium ion concentration did not affect the observed attraction. In vivo crossing experiments also showed that the attraction of the pollen tube to the embryo sac was impaired when pollen tubes of different species arrived around the embryo sac, suggesting that the species preferentiality of the attractant may serve as a reproductive barrier in the final step of directional control of the pollen tube.

Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c

Mammalian cells typically contain thousands of copies of mitochondrial DNA assembled into hundreds of nucleoids. Here we analyzed the dynamic features of nucleoids in terms of mitochondrial membrane dynamics involving balanced fusion and fission. In mitochondrial fission GTPase dynamin-related protein (Drp1)-deficient cells, nucleoids were enlarged by their clustering within hyperfused mitochondria. In normal cells, mitochondrial fission often occurred adjacent to nucleoids, since localization of Mff and Drp1 is dependent on the nucleoids. Thus, mitochondrial fission adjacent to nucleoids should prevent their clustering by maintaining small and fragmented nucleoids. The enhanced clustering of nucleoids resulted in the formation of highly stacked cristae structures in enlarged bulb-like mitochondria (mito-bulbs). Enclosure of proapoptotic factor cytochrome c, but not of Smac/DIABLO, into the highly stacked cristae suppressed its release from mitochondria under apoptotic stimuli. In the absence of nucleoids, Drp1 deficiency failed to form mito-bulbs and to protect against apoptosis. Thus, mitochondrial dynamics by fission and fusion play a critical role in controlling mitochondrial nucleoid structures, contributing to cristae reformation and the proapoptotic status of mitochondria.

Environment-Sensitive Fluorescent Probe: A Benzophosphole Oxide with an Electron-Donating Substituent†

Electron-donating aryl groups were attached to electron-accepting benzophosphole skeletons. Among several derivatives thus prepared, one benzophosphole oxide was particularly interesting, as it retained high fluorescence quantum yields even in polar and protic solvents. This phosphole-based compound exhibited a drastic color change of its fluorescence spectrum as a function of the solvent polarity, while the absorption spectra remained virtually unchanged. Capitalizing on these features, this phosphole-based compound was used to stain adipocytes, in which the polarity of subcellular compartments could then be discriminated on the basis of the color change of the fluorescence emission.

Early weaning induces anxiety and precocious myelination in the anterior part of the basolateral amygdala of male Balb/c mice.

Weaning is one of the most important events that occur during the early stages of life. For example, precocious weaning is known to increase anxiety-related behaviors in rodents. Here, we demonstrate that in addition to increasing anxiety, early-weaning manipulations alter the accumulation of galactosylceramide, a specific myelin constituent, and the axonal structure of myelinated fibers in the amygdala of male Balb/c mice. We found that early-weaned male mice entered the open arms of an elevated plus-maze less frequently than normally weaned mice at 3 and 5 weeks of age, which indicates persistently higher anxiety levels. However, early-weaned females exhibited fewer entries into the open arms only at 5 weeks of age. Lipid analysis of mice amygdalas showed the early accumulation of galactosylceramide in early-weaned male, but not female, mice at 5 weeks. The precocious accumulation of galactosylceramide was observed only in the amygdala; galactosylceramide accumulation was not observed in the prefrontal cortex or hippocampus of early-weaned male mice. Electron microscopy showed an increase in the number and a decrease in the diameter of myelinated axons in the anterior part of the basolateral amygdala in early-weaned male mice at 5 weeks. These results suggest that the higher anxiety levels observed in early-weaned male mice could be related to precocious myelin formation in the anterior part of the basolateral amygdala.

Detection and localization of a chloroplast-encoded HU-like protein that organizes chloroplast nucleoids

Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids). The structure of cpDNA is thought to be important for its maintenance and regulation. In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization. However, a primary structural protein has yet to be found in cp-nucleoids. Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae. The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants. The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C. merolae chloroplast genome. Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid. The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA. These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid.

Mitochondrial Nucleoid and Transcription Factor A

Abstract: Nuclear DNA is tightly packed into nucleosomal structure. In contrast, human mitochondrial DNA (mtDNA) had long been believed to be rather naked because mitochondria lack histone. Mitochondrial transcription factor A (TFAM), a member of a high mobility group (HMG) protein family and a first‐identified mitochondrial transcription factor, is essential for maintenance of mitochondrial DNA. Abf2, a yeast counterpart of human TFAM, is abundant enough to cover the whole region of mtDNA and to play a histone‐like role in mitochondria. Human TFAM is indeed as abundant as Abf2, suggesting that TFAM also has a histone‐like architectural role for maintenance of mtDNA. When human mitochondria are solubilized with non‐ionic detergent Nonidet‐P40 and then separated into soluble and particulate fractions, most TFAM is recovered from the particulate fraction together with mtDNA, suggesting that human mtDNA forms a nucleoid structure. TFAM is tightly associated with mtDNA as a main component of the nucleoid.

The complete DNA sequence of the mitochondrial genome of Physarum polycephalum

Abstract. The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarum polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14–19 are transcribed.