The dynamin-like protein ADL1C is essential for plasma membrane maintenance during pollen maturation

Dynamin-related GTPases regulate a wide variety of dynamic membrane processes in eukaryotes. Here, we investigated the function of ADL1C, a member of the Arabidopsis 68 kDa dynamin-like protein family. Analysis of heterozygous adl1C-1 indicates that the mutation specifically affects post-meiotic male gametogenesis. Fifty percent of the mature pollen from heterozygous adl1C-1 androecia are shriveled and fail to germinate in vitro. During microspore maturation, adl1C-1 pollen grains display defects in the plasma membrane and intine morphology, suggesting that ADL1C is essential for the formation and maintenance of the pollen cell surface and viability during desiccation. Consistent with a role in cell-surface dynamics, immunofluorescence microscopy indicates that ADL1C is localized to the cell plate of dividing somatic cells and to the tip of expanding root hairs. We propose that ADL1C functions in plasma membrane dynamics, and we discuss the role of the ADL1 family in plant growth and development.     

GRIM-19 is essential for maintenance of mitochondrial membrane potential

GRIM-19 was found to copurify with complex I of mitochondrial respiratory chain and subsequently was demonstrated to be involved in complex I assembly and activity. To further understand its function in complex I, we dissected its functional domains by generating a number of deletion, truncation, and point mutants. The mitochondrial localization sequences were located at the N-terminus. Strikingly, deletion of residues 70-80, 90-100, or the whole C-terminal region (70-144) led to a loss of mitochondrial transmembrane potential (DeltaPsim). However, similar deletions of another two complex I subunits, NDUFA9 and NDUFS3, did not show such effect. We also found that deletion of the last 10 residues affected GRIM-19's ability to be assembled to complex I. We constructed a dominant-negative mutant containing the N-terminal 60 and the last C-terminal 10 residues, which could be assembled into complex I, but failed to maintain normal DeltaPsim. Cells overexpressing this mutant did not spontaneously undergo cell death, but were sensitized to apoptosis induced by cell death agents. Our results demonstrate that GRIM-19 is required for electron transfer activity of complex I, and disruption of DeltaPsim by GRIM-19 mutants enhances the cells' sensitivity to apoptotic stimuli.     

Excitatory glutamate is essential for development and maintenance of the piloneural mechanoreceptor

The piloneural collar in mammalian hairy skin comprises an intricate pattern of circumferential and longitudinal sensory afferents that innervate primary and secondary pelage hairs. The longitudinal afferents tightly associate with terminal Schwann cell processes to form encapsulated lanceolate nerve endings of rapidly adapting mechanoreceptors. The molecular basis for piloneural development, maintenance and function is poorly understood. Here, we show that Nefh-expressing glutamatergic neurons represent a major population of longitudinal and circumferential sensory afferents innervating the piloneural collar. Our findings using a VGLUT2 conditional-null mouse model indicate that glutamate is essential for innervation, patterning and differentiation of NMDAR(+) terminal Schwann cells during piloneural collar development. Similarly, treatment of adult mice with a selective NMDAR antagonist severely perturbed piloneural collar structure and reduced excitability of these mechanosensory neurons. Collectively, these results show that DRG-derived glutamate is essential for the proper development, maintenance and sensory function of the piloneural mechanoreceptor.     

DHTKD1 is essential for mitochondrial biogenesis and function maintenance

Maintaining the functional integrity of mitochondria is crucial for cell function, signal transduction and overall cell activities. Mitochondrial dysfunctions may alter energy metabolism and in many cases are associated with neurological diseases. Recent studies have reported that mutations in dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1), a mitochondrial protein encoding gene, could cause neurological abnormalities. However, the function of DHTKD1 in mitochondria remains unknown. Here, we report a strong correlation of DHTKD1 expression level with ATP production, revealing the fact that DHTKD1 plays a critical role in energy production in mitochondria. Moreover, suppression of DHTKD1 leads to impaired mitochondrial biogenesis and increased reactive oxygen species (ROS), thus leading to retarded cell growth and increased cell apoptosis. These findings demonstrate that DHTKD1 contributes to mitochondrial biogenesis and function maintenance.     

O-GlcNAcase is essential for embryonic development and maintenance of genomic stability

Dysregulation of O-GlcNAc modification catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, type 2 diabetes, and Alzheimer's disease. Here we found that natural aging in wild-type mice was marked by a decrease in OGA and OGT protein levels and an increase in O-GlcNAcylation in various tissues. Genetic disruption of OGA resulted in constitutively elevated O-GlcNAcylation in embryos and led to neonatal lethality with developmental delay. Importantly, we observed that serum-stimulated cell cycle entry induced increased O-GlcNAcylation and decreased its level after release from G2/M arrest, indicating that O-GlcNAc cycling by OGT and OGA is required for precise cell cycle control. Constitutively, elevated O-GlcNAcylation by OGA disruption impaired cell proliferation and resulted in mitotic defects with downregulation of mitotic regulators. OGA loss led to mitotic defects including cytokinesis failure and binucleation, increased lagging chromosomes, and micronuclei formation. These findings suggest an important role for O-GlcNAc cycling by OGA in embryonic development and the regulation of the maintenance of genomic stability linked to the aging process.     

Rac1 is crucial for hair follicle integrity but is not essential for maintenance of the epidermis

Rac1 is a small GTPase that regulates the actin cytoskeleton but also other cellular processes. To investigate the function of Rac1 in skin, we generated mice with a keratinocyte-restricted deletion of the rac1 gene. Rac1-deficient mice lost nearly all of their hair within a few weeks after birth. The nonpermanent part of mutant hair follicles developed constrictions; lost expression of hair follicle-specific keratins, E-cadherin, and alpha6 integrin; and was eventually removed by macrophages. The permanent part of hair follicles and the sebaceous glands were maintained, but no regrowth of full-length hair follicles was observed. In the skin of mutant mice, epidermal keratinocytes showed normal differentiation, proliferation, cell-cell contacts, and basement membrane deposition, demonstrating no obvious defects of Rac1-deficient epidermis in vivo. In vitro, Rac1-null keratinocytes displayed a strong spreading defect and slightly impaired adhesion. These data show that Rac1 plays an important role in sustaining the integrity of the lower part of hair follicles but not in maintenance of the epidermis.     

Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells

Yeast Mre11 functions with Rad50 and Xrs2 in a complex that has pivotal roles in homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA double-strand break (DSB) repair pathways. Vertebrate Mre11 is essential. Conditionally, MRE11 null chicken DT40 cells accumulate chromosome breaks and die upon Mre11 repression, showing frequent centrosome amplification. Mre11 deficiency also causes increased radiosensitivity and strongly reduced targeted integration frequencies. Mre11 is, therefore, crucial for HR and essential in mitosis through its role in chromosome maintenance by recombinational repair. Surprisingly perhaps, given the role of Mre11 in yeast NHEJ, disruption of NHEJ by deletion of KU70 greatly exacerbates the effects of MRE11 deficiency, revealing a significant Mre11-independent component of metazoan NHEJ.     

The Sm-protein methyltransferase, dart5, is essential for germ-cell specification and maintenance

BACKGROUND: The C-terminal tails of spliceosomal Sm proteins contain symmetrical dimethylarginine (sDMA) residues in vivo. The precise function of this posttranslational modification in the biogenesis of small nuclear ribonucleoproteins (snRNPs) and pre-mRNA splicing remains largely uncharacterized. Here, we examine the organismal and cellular consequences of loss of symmetric dimethylation of Sm proteins in Drosophila. RESULTS: Genetic disruption of dart5, the fly ortholog of human PRMT5, results in the complete loss of sDMA residues on spliceosomal Sm proteins. Similarly, valois, a previously characterized grandchildless gene, is also required for sDMA modification of Sm proteins. In the absence of dart5, snRNP biogenesis is surprisingly unaffected, and homozygous mutant animals are completely viable. Instead, Dart5 protein is required for maturation of spermatocytes in males and for germ-cell specification in females. Embryos laid by dart5 mutants fail to form pole cells, and Tudor localization is disrupted in stage 10 oocytes. Transgenic expression of Dart5 exclusively within the female germline rescues pole-cell formation, whereas ubiquitous expression rescues sDMA modification of Sm proteins and male sterility. CONCLUSIONS: We have shown that Dart5-mediated methylation of Sm proteins is not essential for snRNP biogenesis. The results uncover a novel role for dart5 in specification of the germline and in spermatocyte maturation. Because disruption of both dart5 and valois causes the specific loss of sDMA-modified Sm proteins and studies in C. elegans show that Sm proteins are required for germ-granule localization, we propose that Sm protein methylation is a pivotal event in germ-cell development.     

Puromycin-sensitive aminopeptidase is essential for the maternal recognition of pregnancy in mice

Maternal recognition of pregnancy in rodents requires semicircadian surges of hypophyseal PRL secretion during early gestation, which are required for the formation of the corpus luteum of pregnancy (CLP). Here we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice display female infertility that results from impaired formation of CLP. Transplantation of mutant ovaries into normal females restored fertility but not vice versa. Psa-deficient females revealed no semicircadian surges of PRL induced after mating stimuli. Pregnancy in the mutant females was restored by grafting intact pituitaries to elevate circulating levels of PRL. Psa is thus required for the appearance of the semicircadian surges of PRL secretion that are crucial for maintaining pregnancy in rodents.     

Cebpa is essential for the embryonic myeloid progenitor and neutrophil maintenance in zebrafish

In vertebrates, myeloid cells arise from multiple waves of development: the first or embryonic wave of myelopoiesis initiates early from non-hematopoietic stem cell(HSC) precursors and gives rise to myeloid cells transiently during early development; whereas the second or adult wave of myelopoiesis emerges later from HSCs and produces myeloid cells continually during fetal and adult life. In the past decades, a great deal has been learnt about the development of myeloid cells from adult myelopoiesis, yet the genetic network governing embryonic myelopoiesis remains poorly defined. In this report, we present an in vivo study to delineate the role of Cebpa during zebrafish embryonic myelopoiesis. We show that embryonic myelopoiesis in cebpa-deficient zebrafish mutants initiates properly but fails to produce macrophages and neutrophils. The lack of macrophages and neutrophils in the mutants is largely attributed to the cell cycle arrest of embryonic myeloid progenitors, resulting in the impairment of their maintenance and subsequent differentiation. We further show that Cebpa, perhaps acting cooperatively with Runx1, plays a critical role in embryonic neutrophil maintenance. Our findings reveal a new role of Cebpa in embryonic myelopoiesis.     

The Rcs phosphorelay system is essential for pathogenicity in Erwinia amylovora

The Rcs phosphorelay system is a modified two-component signal transduction system found exclusively in Enterobacteriaceae . In this study, we characterized the roles of the Rcs system in Erwinia amylovora , a highly virulent and necrogenic enterobacterium causing fire blight disease on rosaceous plants. Our results showed that rcsB , rcsC , rcsD and rcsBD mutants were non-pathogenic on immature pear fruit. The bacterial growth of these mutants was also greatly reduced compared with that of the wild-type strain in immature pear fruit. In an in vitro amylovoran assay, rcsB and rcsD mutants were deficient in amylovoran production, whereas the rcsC mutant exhibited higher amylovoran production than that of the wild-type. Consistent with amylovoran production, expression of the amylovoran biosynthetic gene amsG , using green fluorescent protein as a reporter, was not detectable in rcsB , rcsD and rcsBD mutants both in vitro and in vivo . The expression of amsG in vitro was higher in the rcsC mutant than in the wild-type, whereas its expression in vivo was higher in the wild-type than in the rcsC mutant. In addition, rcs mutants were more susceptible to polymyxin B treatment than the wild-type, suggesting that the Rcs system conferred some level of resistance to polymyxin B. Furthermore, rcs mutants showed irregular and slightly reduced motility on swarming plates. Together, these results indicate that the Rcs system plays a major role in virulence and survival of E. amylovora in immature pear fruit.     

The MutS C terminus is essential for mismatch repair activity in vivo

An Escherichia coli K-12 strain was constructed with a chromosomal deletion (mutSdelta800) in the mutS gene that produced the removal of the C-terminal 53 amino acids which are not present in the MutS crystal structure. This strain has a MutS null phenotype for mutation avoidance, anti-recombination, and sensitivity to cytotoxic agents in a dam mutant background.     

The kinesin related motor protein, Eg5, is essential for maintenance of pre-implantation embryogenesis

Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5-3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.     

The Schizosaccharomyces pombe Pfh1p DNA helicase is essential for the maintenance of nuclear and mitochondrial DNA

Schizosaccharomyces pombe Pfh1p is an essential member of the Pif family of 5′-3′ DNA helicases. The two Saccharomyces cerevisiae homologs, Pif1p and Rrm3p, function in nuclear DNA replication, telomere length regulation, and mitochondrial genome integrity. We demonstrate here the existence of multiple Pfh1p isoforms that localized to either nuclei or mitochondria. The catalytic activity of Pfh1p was essential in both cellular compartments. The absence of nuclear Pfh1p resulted in G₂ arrest and accumulation of DNA damage foci, a finding suggestive of an essential role in DNA replication. Exogenous DNA damage resulted in localization of Pfh1p to DNA damage foci, suggesting that nuclear Pfh1p also functions in DNA repair. The absence of mitochondrial Pfh1p caused rapid depletion of mitochondrial DNA. Despite localization to nuclei and mitochondria in S. pombe, neither of the S. cerevisiae homologs, nor human PIF1, suppressed the lethality of pfh1Δ cells. However, the essential nuclear function of Pfh1p could be supplied by Rrm3p. Expression of Rrm3p suppressed the accumulation of DNA damage foci but not the hydroxyurea sensitivity of cells depleted of nuclear Pfh1p. Together, these data demonstrate that Pfh1p has essential roles in the replication of both nuclear and mitochondrial DNA.