A role for Gle1, a regulator of DEAD-box RNA helicases, at centrosomes and basal bodies.

Control of organellar assembly and function is critical to eukaryotic homeostasis and survival. Gle1 is a highly conserved regulator of RNA-dependent DEAD-box ATPase proteins, with critical roles in both mRNA export and translation. In addition to its well-defined interaction with nuclear pore complexes, here we find that Gle1 is enriched at the centrosome and basal body. Gle1 assembles into the toroid-shaped pericentriolar material around the mother centriole. Reduced Gle1 levels are correlated with decreased pericentrin localization at the centrosome and microtubule organization defects. Of importance, these alterations in centrosome integrity do not result from loss of mRNA export. Examination of the Kupffer's vesicle in Gle1-depleted zebrafish revealed compromised ciliary beating and developmental defects. We propose that Gle1 assembly into the pericentriolar material positions the DEAD-box protein regulator to function in localized mRNA metabolism required for proper centrosome function

Isolation and characterization of new Saccharomyces cerevisiae mutants perturbed in nuclear pore complex assembly

BACKGROUND: Nuclear pore complexes (NPCs) are essential for facilitated, directional nuclear transport; however, the mechanism by which ~30 different nucleoporins (nups) are assembled into NPCs is unknown. We combined a genetic strategy in Saccharomyces cerevisiae with Green Fluorescence Protein (GFP) technology to identify mutants in NPC structure, assembly, and localization. To identify such mutants, a bank of temperature sensitive strains was generated and examined by fluorescence microscopy for mislocalization of GFP-tagged nups at the non-permissive temperature. RESULTS: A total of 121 mutant strains were isolated, with most showing GFP-Nic96 and Nup170-GFP mislocalized to discrete, cytoplasmic foci. By electron microscopy, several mutants also displayed an expansion of the endoplasmic reticulum (ER). Complementation analysis identified several mutant groups with defects in components required for ER/Golgi trafficking (sec13, sec23, sec27, and bet3). By directed testing, we found that mutant alleles of all COPII components resulted in altered GFP-Nup localization. Finally, at least nine unknown complementation groups were identified that lack secretion defects. CONCLUSION: The isolation of sec mutants in the screen could reflect a direct role for vesicle fusion or the COPII coat during NPC assembly; however, only those sec mutants that altered ER structure affected Nup localization. This suggests that the GFP-Nup mislocalization phenotypes observed in these mutants were the indirect result of overproliferation of the ER and connected outer nuclear envelope. The identification of potentially novel mutants with no secretory defects suggests the distinct GFP-Nup localization defects in other mutants in the collection will provide insights into NPC structure and assembly

Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures—one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency

Inositol Polyphosphates Regulate Zebrafish Left-Right Asymmetry

SummaryVertebrate body plans have a conserved left-right (LR) asymmetry manifested in the position and anatomy of the heart, visceral organs, and brain. Recent studies have suggested that LR asymmetry is established by asymmetric Ca2+ signaling resulting from cilia-driven flow of extracellular fluid across the node. We report here that inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), which generates inositol hexakisphosphate, is critical for normal LR axis determination in zebrafish. Zebrafish embryos express ipk1 symmetrically during gastrulation and early segmentation. ipk1 knockdown by antisense morpholino oligonucleotide injection randomized LR-specific gene expression and organ placement, effects that were associated with reduced intracellular Ca2+ flux in cells surrounding the ciliated Kupffer’s vesicle, a structure analogous to the mouse node. Our data suggest that the pathway for inositol hexakisphosphate production is a key regulator of asymmetric Ca2+ flux during LR specification

The Ran GTPase cycle is required for yeast nuclear pore complex assembly

Here, we report the first evidence that the Ran GTPase cycle is required for nuclear pore complex (NPC) assembly. Using a genetic approach, factors required for NPC assembly were identified in Saccharomyces cerevisiae. Four mutant complementation groups were characterized that correspond to respective mutations in genes encoding Ran (gsp1), and essential Ran regulatory factors Ran GTPase–activating protein (rna1), Ran guanine nucleotide exchange factor (prp20), and the RanGDP import factor (ntf2). All the mutants showed temperature-dependent mislocalization of green fluorescence protein (GFP)-tagged nucleoporins (nups) and the pore-membrane protein Pom152. A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci. The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth. Electron microscopy analysis revealed striking membrane perturbations and the accumulation of vesicles in arrested mutants. Using both biochemical fractionation and immunoelectron microscopy methods, these vesicles were shown to contain nups. We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes

Regulation of Chromatin Remodeling by Inositol Polyphosphates

Chromatin remodeling is required for efficient transcription of eukaryotic genes. In a genetic selection for budding yeast mutants that were defective in induction of the phosphate-responsive PHO5 gene, we identified mutations inARG82/IPK2, which encodes a nuclear inositol polyphosphate kinase. In arg82 mutant strains, remodeling ofPHO5 promoter chromatin is impaired, and the adenosine triphosphate–dependent chromatin-remodeling complexes SWI/SNF and INO80 are not efficiently recruited to phosphate-responsive promoters. These results suggest a role for the small molecule inositol polyphosphate in the regulation of chromatin remodeling and transcription

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ER membrane–bending proteins are necessary for de novo nuclear pore formation

Nucleocytoplasmic transport occurs exclusively through nuclear pore complexes (NPCs) embedded in pores formed by inner and outer nuclear membrane fusion. The mechanism for de novo pore and NPC biogenesis remains unclear. Reticulons (RTNs) and Yop1/DP1 are conserved membrane protein families required to form and maintain the tubular endoplasmic reticulum (ER) and the postmitotic nuclear envelope. In this study, we report that members of the RTN and Yop1/DP1 families are required for nuclear pore formation. Analysis of Saccharomyces cerevisiae prp20-G282S and nup133Δ NPC assembly mutants revealed perturbations in Rtn1–green fluorescent protein (GFP) and Yop1-GFP ER distribution and colocalization to NPC clusters. Combined deletion of RTN1 and YOP1 resulted in NPC clustering, nuclear import defects, and synthetic lethality with the additional absence of Pom34, Pom152, and Nup84 subcomplex members. We tested for a direct role in NPC biogenesis using Xenopus laevis in vitro assays and found that anti-Rtn4a antibodies specifically inhibited de novo nuclear pore formation. We hypothesize that these ER membrane–bending proteins mediate early NPC assembly steps

Certified nurse midwives effects on catecholamine levels and maternal anxiety during labor

Studies of nurse midwifery care in the last twenty one years have reported excellent birth outcomes (Levy, Wilkenson and Marine, 1971; Platt et al. 1985; Stone et al. 1976). These outcomes are frequently attributed to the special support offered during labor and delivery by nurse midwives. This supportive style is thought to decrease catecholamine levels by reducing maternal anxiety. This prospective observational study evaluated catecholamine levels, anxiety levels, in-hospital costs, obstetrical practices and outcomes between low risk, term, labor and delivery primigravida patients managed by obstetrical residents (n = 55) or by certified nurse-midwives CNM (n = 59). The two groups were similar with regard to obstetrical risk factors present at admission. Each group was selected over the same period of time between March 23, 1994 and November 2, 1994. Specific catecholamines evaluated were epinephrine and norepinephrine. Obstetrical and newborn characteristics were also compared. This study did not prove that there is a decreased level in stress as indicated by lower levels of epinephrine and norepinephrine in nurse-midwife patients compared to obstetrical resident patients after adjusting for the use of epidural anesthesia. There was also no difference found in the perceived anxiety levels between the two groups. This study did confirm that nurse-midwives and obstetrical residents have different practice styles. Nurse-midwife patients had fewer augmented deliveries, fewer operative deliveries, less blood loss, fewer episiotomies and fewer third and fourth degree lacerations. The physician\u27s choice to utilize more interventions such as continuous fetal monitoring and epidural anesthesia did not improve outcomes. The hospital cost of the nurse-midwife patients in this study was 35 percent lower than the physician patients