Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction.
The Fanconi anemia (FA) protein FANCC is essential for chromosome stability in vertebrate cells, a feature underscored by the extreme sensitivity of FANCC-deficient cells to agents that crosslink DNA. However, it is not known how this FA protein facilitates the repair of both endogenously acquired and mutagen-induced DNA damage. Here, we use the model vertebrate cell line DT40 to address this question. We discover that apart from functioning in homologous recombination, FANCC also promotes the mutational repair of endogenously generated abasic sites. Moreover in these vertebrate cells, the efficient repair of crosslinks requires the combined functions of FANCC, translesion synthesis, and homologous recombination. These studies reveal that the FA proteins cooperate with key mutagenesis and repair processes that enable replication of damaged DNA.
In flowering plants, sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize an egg and a central cell. Here, we identified the gene encoding Arabidopsis HAP2, demonstrating that it is allelic to GCS1. HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to ovules. We identified an insertion (hap2-1) that disrupts the C-terminal portion of the protein and tags mutant pollen grains with the -glucuronidase reporter. By monitoring reporter expression, we showed that hap2-1 does not diminish pollen tube length in vitro or in the pistil, but it reduces ovule targeting by twofold. In addition, we show that the hap2 sperm that are delivered to ovules fail to initiate fertilization. HAP2 is predicted to encode a protein with an N-terminal secretion signal, a single transmembrane domain and a C-terminal histidine-rich domain. These results point to a dual role for HAP2, functioning in both pollen tube guidance and in fertilization. Moreover, our findings suggest that sperm, long considered to be passive cargo, are involved in directing the pollen tube to its target.
SUMMARY Mapping neural circuits across defined synapses is essential for understanding brain function. Here we describe trans-Tango, a technique for anterograde transsynaptic circuit tracing and manipulation. At the core of trans-Tango is a synthetic signaling pathway that is introduced into all neurons in the animal. This pathway converts receptor activation at the cell-surface into reporter expression through site-specific proteolysis. Specific labeling is achieved by presenting a tethered ligand at the synapses of genetically defined neurons, thereby activating the pathway in their postsynaptic partners and providing genetic access to these neurons. We first validated trans-Tango in the Drosophila olfactory system and then implemented it in the gustatory system, where projections beyond the first-order receptor neurons are not fully characterized. We identified putative second-order neurons within the sweet circuit that include projection neurons targeting known neuromodulation centers in the brain. These experiments establish trans-Tango as a flexible platform for transsynaptic circuit analysis.
In flowering plants, the egg develops within a haploid embryo sac (female gametophyte) that is encased within the pistil. The haploid pollen grain (male gametophyte) extends a pollen tube that carries two sperm cells within its cytoplasm to the embryo sac. This feat requires rapid, precisely guided, and highly polarized growth through, between, and on the surface of the cells of the stigma, style, and ovary. Pollen tube migration depends on a series of long-range signals from diploid female cells as well as a short-range attractant emitted by the embryo sac that guides the final stage of tube growth. We developed a genetic screen in Arabidopsis thaliana that tags mutant pollen with a cell-autonomous marker carried on an insertion element. We found 32 haploid-disrupting (hapless) mutations that define genes required for pollen grain development, pollen tube growth in the stigma and style, or pollen tube growth and guidance in the ovary. We also identified genomic DNA flanking the insertion element for eleven hap mutants and showed that hap1 disrupts AtMago, a gene whose ortholog is important for Drosophila cell polarity.
The helicase-associated endonuclease for fork-structured DNA (Hef) is an archaeabacterial protein that processes blocked replication forks. Here we have isolated the vertebrate Hef ortholog and investigated its molecular function. Disruption of this gene in chicken DT40 cells results in genomic instability and sensitivity to DNA cross-links. The similarity of this phenotype to that of cells lacking the Fanconi anemia-related (FA) tumor-suppressor genes led us to investigate whether Hef functions in this pathway. Indeed, we found a genetic interaction between the FANCC and Hef genes. In addition, Hef is a component of the FA nuclear protein complex that facilitates its DNA damage-inducible chromatin localization and the monoubiquitination of the FA protein FANCD2. Notably, Hef interacts directly with DNA structures that are intermediates in DNA replication. This discovery sheds light on the origins, regulation and molecular function of the FA tumor-suppressor pathway in the maintenance of genome stability.
Whole-exome sequencing and autozygosity mapping studies, independently performed in subjects with defective combined mitochondrial OXPHOS-enzyme deficiencies, identified a total of nine disease-segregating FBXL4 mutations in seven unrelated mitochondrial disease families, composed of six singletons and three siblings. All subjects manifested early-onset lactic acidemia, hypotonia, and developmental delay caused by severe encephalomyopathy consistently associated with progressive cerebral atrophy and variable involvement of the white matter, deep gray nuclei, and brainstem structures. A wide range of other multisystem features were variably seen, including dysmorphism, skeletal abnormalities, poor growth, gastrointestinal dysmotility, renal tubular acidosis, seizures, and episodic metabolic failure. Mitochondrial respiratory chain deficiency was present in muscle or fibroblasts of all tested individuals, together with markedly reduced oxygen consumption rate and hyperfragmentation of the mitochondrial network in cultured cells. In muscle and fibroblasts from several subjects, substantially decreased mtDNA content was observed. FBXL4 is a member of the F-box family of proteins, some of which are involved in phosphorylation-dependent ubiquitination and/or G protein receptor coupling. We also demonstrate that FBXL4 is targeted to mitochondria and localizes in the intermembrane space, where it participates in an approximately 400 kDa protein complex. These data strongly support a role for FBXL4 in controlling bioenergetic homeostasis and mtDNA maintenance. FBXL4 mutations are a recurrent cause of mitochondrial encephalomyopathy onset in early infancy.
P.Pace and M.Johnson contributed equally to this workThe Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage. It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage. Here we show that the recently identi®ed FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2. Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2. Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.
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