T. Renee Dawson1, Michelle D. Lazarus1, Martin W. Hetzer2, and Susan R. Wente1
1 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232
2 Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037
Correspondence to Susan R. Wente: firstname.lastname@example.org
Nucleocytoplasmic transport occurs exclusively through nuclearpore complexes (NPCs) embedded in pores formed by inner andouter nuclear membrane fusion. The mechanism for de novo poreand NPC biogenesis remains unclear. Reticulons (RTNs) and Yop1/DP1are conserved membrane protein families required to form andmaintain the tubular endoplasmic reticulum (ER) and the postmitoticnuclear envelope. In this study, we report that members of theRTN and Yop1/DP1 families are required for nuclear pore formation.Analysis of Saccharomyces cerevisiae prp20-G282S and nup133NPC assembly mutants revealed perturbations in Rtn1–greenfluorescent protein (GFP) and Yop1-GFP ER distribution and colocalizationto NPC clusters. Combined deletion of RTN1 and YOP1 resultedin NPC clustering, nuclear import defects, and synthetic lethalitywith the additional absence of Pom34, Pom152, and Nup84 subcomplexmembers. We tested for a direct role in NPC biogenesis usingXenopus laevis in vitro assays and found that anti-Rtn4a antibodiesspecifically inhibited de novo nuclear pore formation. We hypothesizethat these ER membrane–bending proteins mediate earlyNPC assembly steps.
Cells cycle with no poles- no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila
Julie A. Merkle1, Jamie L. Rickmyre1, Aprajita Garg2, Erin B. Loggins1, Jeanne N. Jodoin1, Ethan Lee1, Louisa P. Wu2 and Laura A. Lee1,*
1 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, U-4200 MRBIII, 465 21st Avenue South, Nashville, TN 37232, USA.
2 Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, MD 20742, USA.
* Author for correspondence (e-mail: email@example.com) Accepted 25 November 2008
In a screen for cell-cycle regulators, we identified a Drosophila maternal effect-lethal mutant that we named `no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. We identified CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, as the nopo gene. A conserved residue in the RING domain is altered in our EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. We show that mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been previously shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. We show that Bendless (BEN), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitination complex consisting of BEN-UEV1A (E2 heterodimer) and NOPO (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis. See Development 136, 304e (2009)
Insulin reverses the effects of aging and poor diet, at least on cells within the ovaries of fruit flies. Biologists Daniela Drummond-Barbosa and Hwei-Jan Hsu of Vanderbilt University in Nashville, Tennessee, found that boosting levels of insulin-like peptides slowed down the decline in stem cell numbers and function that normally occurs as flies age. The hormone does not act on the stem cells directly, but rather via the niche — the cells that form a stem cell's support system. Hormones probably influence most niches, though surprisingly little is known about it, says Erika Matunis of John Hopkins University in Baltimore, Maryland, who studies how stem cells maintain sperm production in fruit flies and was not involved in the study. "At this point the fly ovary is yielding by far the most detailed glimpse into the interactions of hormonal signaling and stem cell niches." "Normally, a lot of research on niches is concentrated on local factors produced by the niche that affect stem cells," says Drummond-Barbosa. "In this paper, we take a broader view to understand how these local microenvironments can be impacted by the environment where the organism lives."
Lu Teng1,*,, Nathan A. Mundell2,4,, Audrey Y. Frist3,4, Qiaohong Wang1 and Patricia A. Labosky1,2,3,4,
1 Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA.
2 Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-0494, USA.
3 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-0494, USA.
4 Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-0494, USA.
Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 28 February 2008
Understanding the molecular mechanisms of stem cell maintenance is crucial for the ultimate goal of manipulating stem cells for the treatment of disease. Foxd3 is required early in mouse embryogenesis; Foxd3-/- embryos fail around the time of implantation, cells of the inner cell mass cannot be maintained in vitro, and blastocyst-derived stem cell lines cannot be established. Here, we report that Foxd3 is required for maintenance of the multipotent mammalian neural crest. Using tissue-specific deletion of Foxd3 in the neural crest, we show that Foxd3flox/-; Wnt1-Cre mice die perinatally with a catastrophic loss of neural crest-derived structures. Cranial neural crest tissues are either missing or severely reduced in size, the peripheral nervous system consists of reduced dorsal root ganglia and cranial nerves, and the entire gastrointestinal tract is devoid of neural crest derivatives. These results demonstrate a global role for this transcriptional repressor in all aspects of neural crest maintenance along the anterior-posterior axis, and establish an unprecedented molecular link between multiple divergent progenitor lineages of the mammalian embryo.
Key words: Neural crest, Foxd3, Mouse embryo, Stem cell maintenance
Departmental Biannual Newletter "VCDB Binary" Fall 2008
As part of NIH's commitment to increasing opportunities for new scientists, it has created the NIH Director's New Innovator Award to support exceptionally creative new investigators who propose highly innovative projects that have the potential for unusually high impact.
Melanie Ohi recently received the highly competitive National Institutes of Health Young Innovator Award to study the structure and function of the spliceosome. Composed of both protein and RNA components this complex is responsible for removing introns from pre-mRNA to form mature message. Mistakes made by the splicing machinery, such as choosing the wrong sites to clip out the intron, can have dramatic effects on an organism, since each error can lead to a change in the protein-coding sequence of the mRNA and the possible synthesis of a defective protein. Not surprisingly, many genetic diseases and types of cancer originate with defects in pre-mRNA splicing. However, even though spliceosome activity is essential for maintaining cellular function, the extremely dynamic nature and large size of this complex has made it difficult to understand how this cellular machine actually works.
Using a structural technique called single particle cryo-electron microscopy (EM), the Ohi lab plans on generating 3D snapshots of the spliceosome stalled at distinct stages of assembly, catalysis, and disassembly. In addition, they will also begin building spliceosomal sub-complexes de novo using recombinant proteins. Studying the structures and functions of the large complexes will provide important information about overall spliceosome organization during each stage of pre-mRNA splicing, while understanding the organization and biochemical activity associated with spliceosomal sub-complexes will provide a mechanistic understanding for how the spliceosome is activated. The overall goal of this work is to generate the first structural based model for understanding how the spliceosome makes the dynamic transition from an inactive to an activated splicing machine.
When asked why she thought this project was selected for an Innovator Award, Melanie responded that “studying how the spliceosome functions has been a topic of intense research for over 20 years, but due to its large size and dynamic nature how this machine actually works still remains a mystery. Our approach is considered bold and innovative because we are proposing to focus both on mapping the overall organization of the entire 3 MDa complex using state of the art single particle EM techniques and on characterizing the function(s) associated with small sub-groups of spliceosomal proteins using biochemical and genetic approaches. I think it is the combination of taking both a top-down and bottom-up approach to understand spliceosome function and our use of a powerful combination of structural, biological, and biophysical techniques that made this proposal stand out to the reviewers.”
Departmental Biannual Newletter "VCDB Binary" Fall 2007
The Clp1-Cdc14 phosphatase contributes to the robustness of cytokinesis by association with anillin-related Mid1
Cdc14 phosphatases antagonize cyclin-dependent kinase–directed phosphorylation events and are involved in several facets of cell cycle control. We investigate the role of the fission yeast Cdc14 homologue Clp1/Flp1 in cytokinesis. We find that Clp1/Flp1 is tethered at the contractile ring (CR) through its association with anillin-related Mid1. Fluorescent recovery after photobleaching analyses indicate that Mid1, unlike other tested CR components, is anchored at the cell midzone, and this physical property is likely to account for its scaffolding role. By generating a mutation in mid1 that selectively disrupts Clp1/Flp1 tethering, we reveal the specific functional consequences of Clp1/Flp1 activity at the CR, including dephosphorylation of the essential CR component Cdc15, reductions in CR protein mobility, and CR resistance to mild perturbation. Our evidence indicates that Clp1/Flp1 must interact with the Mid1 scaffold to ensure the fidelity of Schizosaccharomyces pombe cytokinesis.