The Arabidopsis homologue of Xrcc3 plays an essential role in meiosis

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified from yeast (Rad55, Rad57 and Dmc1) to vertebrates (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3 and Dmc1). These Rad51-like proteins are all members of the genetic recombination and DNA damage repair pathways. The sequenced genome of Arabidopsis thaliana encodes putative homologues of all six vertebrate Rad51-like proteins. We have identified and characterized an Arabidopsis mutant defective for one of these, AtXRCC3, the homologue of XRCC3. atxrcc3 plants are sterile, while they have normal vegetative development. Cytological observation shows that the atxrcc3 mutation does not affect homologous chromosome synapsis, but leads to chromosome fragmentation after pachytene, thus disrupting both male and female gametogenesis. This study shows an essential role for AtXrcc3 in meiosis in plants and possibly in other higher eukaryotes. Furthermore, atxrcc3 cells and plants are hypersensitive to DNA-damaging treatments, supporting the involvement of this Arabidopsis Rad51-like protein in recombinational repair.     

The autophagy gene ATG5 plays an essential role in B lymphocyte development

Macroautophagy (herein autophagy) is an evolutionarily conserved process, requiring the gene ATG5, by which cells degrade cytoplasmic constituents and organelles. Here we show that ATG5 is required for efficient B cell development and for the maintenance of B-1a B cell numbers. Deletion of ATG5 in B lymphocytes using Cre-LoxP technology or repopulation of irradiated mice with ATG5-/- fetal liver progenitors resulted in a dramatic reduction in B-1 B cells in the peritoneum. ATG5-/- progenitors exhibited a significant defect in B cell development at the pro- to pre-B cell transition, although a proportion of pre-B cells survived to populate the periphery. Inefficient B cell development in the bone marrow was associated with increased cell death, indicating that ATG5 is important for B cell survival during development. In addition, B-1a B cells require ATG5 for their maintenance in the periphery. We conclude that ATG5 is differentially required at discrete stages of development in distinct, but closely related, cell lineages.     

The autophagy gene ATG5 plays an essential role in B lymphocyte development

Macroautophagy (herein autophagy) is an evolutionarily conserved process, requiring the gene ATG5, by which cells degrade cytoplasmic constituents and organelles. Here we show that ATG5 is required for efficient B cell development and for the maintenance of B-1a B cell numbers. Deletion of ATG5 in B lymphocytes using Cre-LoxP technology or repopulation of irradiated mice with ATG5^-/- fetal liver progenitors resulted in a dramatic reduction in B-1 B cells in the peritoneum. ATG5^-/- progenitors exhibited a significant defect in B cell development at the pro- to pre-B cell transition, although a proportion of pre-B cells survived to populate the periphery. Inefficient B cell development in the bone marrow was associated with increased cell death, indicating that ATG5 is important for B cell survival during development. In addition, B-1a B cells require ATG5 for their maintenance in the periphery. We conclude that ATG5 is differentially required at discrete stages of development in distinct, but closely related, cell lineages.     

Light plays an essential role in intracellular distribution of auxin efflux carrier PIN2 in Arabidopsis thaliana

Background

Light plays a key role in multiple plant developmental processes. It has been shown that root development is modulated by shoot-localized light signaling and requires shoot-derived transport of the plant hormone, auxin. However, the mechanism by which light regulates root development is not largely understood. In plants, the endogenous auxin, indole-3-acetic acid, is directionally transported by plasma-membrane (PM)-localized auxin influx and efflux carriers in transporting cells. Remarkably, the auxin efflux carrier PIN proteins exhibit asymmetric PM localization, determining the polarity of auxin transport. Similar to PM-resident receptors and transporters in animal and yeast cells, PIN proteins undergo constitutive cycling between the PM and endosomal compartments. Auxin plays multiple roles in PIN protein intracellular trafficking, inhibiting PIN2 endocytosis at some concentrations and promoting PIN2 degradation at others. However, how PIN proteins are turned over in plant cells is yet to be addressed.

Methodology and Principle Findings

Using laser confocal scanning microscopy, and physiological and molecular genetic approaches, here, we show that in dark-grown seedlings, the PM localization of auxin efflux carrier PIN2 was largely reduced, and, in addition, PIN2 signal was detected in vacuolar compartments. This is in contrast to light-grown seedlings where PIN2 was predominantly PM-localized. In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments. We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling. In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process.

Conclusions and Significance

Collectively, our data indicate that light plays an essential role in PIN2 intracellular trafficking, promoting PM-localization in the presence of light and, on the other hand, vacuolar targeting for protein degradation in the absence of light. Based on these results, we postulate that light regulation of root development is mediated at least in part by changes in the intracellular distribution of auxin efflux carriers, PIN proteins, in response to the light environment.     

Maspin plays an essential role in early embryonic development

Maspin (Mp) is a member of the serpin family with inhibitory functions against cell migration, metastasis and angiogenesis. To identify its role in embryonic development in vivo, we generated maspin knockout mice by gene targeting. In this study, we showed that homozygous loss of maspin expression was lethal at the peri-implantation stage. Maspin was specifically expressed in the visceral endoderm after implantation; deletion of maspin interfered with the formation of the endodermal cell layer, thereby disrupting the morphogenesis of the epiblast. In vitro, the ICM of the Mp(-/-) blastocysts failed to grow out appropriately. Data from embryoid body formation studies indicated that the Mp(-/-) EBs had a disorganized, endodermal cell mass and lacked a basement membrane layer. We showed that the embryonic ectoderm lineage was lost in the Mp(-/-) EBs, compared with that of the Mp(+/+) EBs. Re-expression of maspin partially rescued the defects observed in the Mp(-/-) EBs, as evidenced by the appearance of ectoderm cells and a layer of endoderm cells surrounding the ectoderm. In addition, a maspin antibody specifically blocked normal EB formation, indicating that maspin controls the process through a cell surface event. Furthermore, we showed that maspin directly increased endodermal cell adhesion to laminin matrix but not to fibronectin. Mp(+/-) endodermal cells grew significantly slower than Mp(+/+) endodermal cells on laminin substrate. We conclude that deletion of maspin affects VE function by reducing cell proliferation and adhesion, thereby controlling early embryonic development.     

Survivin monomer plays an essential role in apoptosis regulation

Survivin was initially described as an inhibitor of apoptosis and attracted growing attention as one of the most tumor-specific genes in the human genome and a promising target for cancer therapy. Lately, it has been shown that survivin is a multifunctional protein that takes part in several crucial cell processes. At first, it was supposed that survivin functions only as a homodimer, but now data indicate that many processes require monomeric survivin. Moreover, recent studies reveal a special mechanism regulating the balance between monomeric and dimeric forms of the protein. In this paper we studied the mutant form of survivin that was unable to dimerize and investigated its role in apoptosis. We showed that survivin monomer interacts with Smac/DIABLO and X-linked inhibitor of apoptosis protein (XIAP) both in vitro and in vivo. Due to this feature, it protects cells from caspase-dependent apoptosis even more efficiently than the wild-type survivin. We also identified that mutant monomeric survivin prevents apoptosis-inducing factor release from the mitochondrial intermembrane space, protecting human fibrosarcoma HT1080 cells from caspase-independent apoptosis. On the other hand, our results indicate that only wild-type survivin, but not the monomer mutant form, enhances tubulin stability in cells. These findings suggest that survivin partly performs its functions as a monomer and partly as a dimer. The mechanism of dimer-monomer balance regulation may also work as a "switcher" between survivin functions and thereby explain remarkable functional diversities of this protein.     

Nitrate transporter NPF7.3/NRT1.5 plays an essential role in regulating phosphate deficiency responses in Arabidopsis

AtNPF7.3/AtNRT1.5, which is a nitrate transporter that drives root-to-shoot transport of NO₃^?, is also involved in modulating the response to K⁺ deprivation in Arabidopsis by affecting root development and K⁺ transport. However, whether NPF7.3/NRT1.5 functions in regulating plant responses to deficiencies of other nutrients remains unknown. In this study, we found that the expression of AtNPF7.3/AtNRT1.5 was predominant in the roots and was substantially induced by phosphate (Pi) starvation. The atnrt1.5 mutants displayed conspicuously longer primary roots along with a significantly reduced lateral root density under Pi-deficient conditions than did the wild-type plants, and these morphological differences in the roots were eliminated to a certain extent by the ethylene synthesis antagonist Co²⁺. Further analyses revealed that the expression of important Pi starvation-induced genes, which are directly involved in Pi transport, mobilization and distribution, were significantly higher in the atnrt1.5 mutants than that in the wild-type plants under Pi-starvation conditions; therefore, the atnrt1.5 mutants retained higher tissue Pi concentrations. Taken together, our results suggest that NPF7.3/NRT1.5 is an important component in the regulation of phosphate deficiency responses in Arabidopsis.     

gamma-tubulin plays an essential role in the coordination of mitotic events

Recent data from multiple organisms indicate that gamma-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the gamma-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (approximately 6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that gamma-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.     

NMDA receptor-mediated calcium influx plays an essential role in myoblast fusion

Ca2+ influx is known to be prerequisite for myoblast fusion during skeletal muscle differentiation. Here, we show that the N-methyl-D-aspartate (NMDA) receptor is involved in the Ca2+ influx of C2C12 myoblasts. NMDA receptor (NR) 1 and NR2D were expressed in the myoblasts during muscle differentiation. Using Ca2+ imaging analysis, Ca2+ influx through NRs was directly measured at a single-cell level. l-Glutamate increased myoblast fusion as well as intracellular Ca2+ levels, and both effects were completely blocked by MK801, a selective antagonist of NRs. Furthermore, treatment with the Ca2+ ionophore A23187 recovered MK801-mediated inhibition of myoblast fusion. These results suggest that the NRs may play an important role in myoblast fusion by mediating Ca2+ influx.     

Mitochondrial complex I plays an essential role in human respirasome assembly

The biogenesis and function of the mitochondrial respiratory chain (RC) involve the organization of RC enzyme complexes in supercomplexes or respirasomes through an unknown biosynthetic process. This leads to structural interdependences between RC complexes, which are highly relevant from biological and biomedical perspectives, because RC defects often lead to severe neuromuscular disorders. We show that in human cells, respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of complexes III and IV subunits, rather than originating from the association of preassembled individual holoenzymes. The process ends with the incorporation of complex I NADH dehydrogenase catalytic module, which leads to the respirasome activation. While complexes III and IV assemble either as free holoenzymes or by incorporation of free subunits into supercomplexes, the respirasomes constitute the structural units where complex I is assembled and activated, thus explaining the significance of the respirasomes for RC function.     

M6 membrane protein plays an essential role in Drosophila oogenesis

We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila.     

Gly-345 plays an essential role in Pyrococcus furiosus chaperonin function

Compared to the group I chaperonins, such as Escherichia coli GroEL, which facilitate protein folding, many aspects of the functional mechanism of archaeal group II chaperonins are unclear. Sequence homology between the chaperonin from Pyrococcus furiosus (PfCPN) and other group II chaperonins, together with the homo-oligomeric nature of PfCPN, suggest that PfCPN may serve as a model to clarify the role of the homologous position Gly-345 in the chaperonin-mediated protein folding. Here, we show that the purified chaperonin mutant in which the conserved residue Gly-345 is replaced by Asp (G345D) displays only about 25% ATP/ADP hydrolysis activities of the wild-type in the presence of Co²⁺ and has a reduced capacity to promote folding of denatured malate dehydrogenase in vitro. This may be a reflection that Gly-345 plays an essential role in conformational change and protein refolding by archaeal group II chaperonins.     

Iron-mediated oxidative stress plays an essential role in ferritin-induced cell death

Previously, we have demonstrated an apoptosis-inducing activity of an acidic, H-chain-rich isoferritin secreted from primary rat hepatocytes in vitro. Because this proapoptotic property may be responsible for the growth-inhibitory and immunosuppressive effects described for certain ferritin species, we aimed to address the mechanism by which ferritin can trigger cell death. Suggesting a pivotal role for iron, iron chelation by desferrioxamine significantly abrogates ferritin-mediated apoptosis and necrosis in primary rat hepatocytes and substantially lowers the extent of protein modification by 4-hydroxynonenal (HNE)—a major lipid peroxidation (LPO) product. Furthermore, supplementing the cultures with the radical-scavenging compound trolox also provided significant protection from ferritin-mediated apoptosis. Moreover, a significant increase in micronucleated cells upon exposure to ferritin indicates that ferritin also introduces damage to DNA. Based on these observations we therefore propose that endocytosis of extracellular ferritin increases the level of free ferrous iron in the lysosomal compartment, promoting Fenton chemistry-based oxidative stress involving LPO and increased lysosomal membrane permeability. Subsequently, the release of reactive lysosomal content leads to cellular damage, in particular modification of protein and DNA induced by HNE and other reactive aldehydic LPO products. Together, these effects will trigger apoptosis and necrosis based on the upregulation of p53, increased mitochondrial membrane permeability, and proapoptotic Fas signaling as described recently. In conclusion, based on their iron-storing ability, secreted acidic isoferritins may act as soluble mediators of oxidative stress under certain physiological and pathophysiological conditions.     

Plantacyanin plays a role in reproduction in Arabidopsis

Plantacyanins belong to the phytocyanin family of blue copper proteins. In the Arabidopsis (Arabidopsis thaliana) genome, only one gene encodes plantacyanin. The T-DNA-tagged mutant is a knockdown mutant that shows no visible phenotype. We used both promoter-beta-glucuronidase transgenic plants and immunolocalization to show that Arabidopsis plantacyanin is expressed most highly in the inflorescence and, specifically, in the transmitting tract of the pistil. Protein levels show a steep gradient in expression from the stigma into the style and ovary. Overexpression plants were generated using cauliflower mosaic virus 35S, and protein levels in the pistil were examined as well as the pollination process. Seed set in these plants is highly reduced mainly due to a lack of anther dehiscence, which is caused by degeneration of the endothecium. Callose deposits occur on the pollen walls in plants that overexpress plantacyanin, and a small percentage of these pollen grains germinate in the closed anthers. When wild-type pollen was used on the overexpression stigma, seed set was still decreased compared to the control pollinations. We detected an increase in plantacyanin levels in the overexpression pistil, including the transmitting tract. Guidance of the wild-type pollen tube on the overexpression stigma is disrupted as evidenced by the growth behavior of pollen tubes after they penetrate the papillar cell. Normally, pollen tubes travel down the papilla cell and into the style. Wild-type pollen tubes on the overexpression stigma made numerous turns around the papilla cell before growing toward the style. In some rare cases, pollen tubes circled up the papilla cell away from the style and were arrested there. We propose that when plantacyanin levels in the stigma are increased, pollen tube guidance into the style is disrupted.     

Arabidopsis Pht1;5 plays an integral role in phosphate homeostasis

The mobilization of inorganic phosphate (Pi) in planta is a complex process regulated by a number of developmental and environmental cues. Plants possess many Pi transporters that acquire Pi from the rhizosphere and translocate it throughout the plant. A few members of the high-affinity Pht1 family of Pi transporters have been functionally characterized and, for the most part, have been shown to be involved in Pi acquisition. We recently demonstrated that the Arabidopsis Pi transporter, Pht1;5, plays a key role in translocating Pi between tissues. Loss-of-function pht1;5 mutant seedlings accumulated more P in shoots relative to wild type but less in roots. In contrast, overexpression of Pht1;5 resulted in a lower P shoot:root ratio compared with wild type. Also, the rosette leaves of Pht1;5-overexpression plants senesced early and contained less P, whereas reproductive organs accumulated more P than those of wild type. Herein we report the molecular response of disrupting Pht1;5 expression on other factors known to modulate P distribution. The results reveal reciprocal mis-regulation of PHO1, miR399d, and At4 in the pht1;5 mutant and Pht1;5-overexpressor, consistent with the corresponding changes in P distribution in these lines. Together our studies reveal a complex role for Pht1;5 in regulating Pi homeostasis.