Developmental Biology Alliance
University of Georgia
University of Georgia faculty member Tania Rozario has received a $2 million grant from the National Institutes of Health Director’s New Innovator Award Program, which supports early-career investigators of exceptional creativity who propose high-risk, high-reward research projects.
Chromatin modifications function as critical regulators of gene expression and cellular identity, especially in the regulation and maintenance of the pluripotent state. However, many studies of chromatin modification in stem cells—and pluripotent stem cells in particular—are performed in mammalian stem cell culture, an in vitro condition mimicking a very transient state during mammalian development. Thus, new models for studying pluripotent stem cells in vivo could be helpful for understanding the roles of chromatin modification, for confirming prior in vitro studies, and for exploring evolution of the pluripotent state.
The most important organ for your body’s immune system isn’t working well anymore. The thymus is an organ most people have never heard of, but it serves a vital purpose. The thymus produces the body’s T cells, which serve as the immune system’s front line against disease.
A group of University of Georgia researchers led by geneticist Douglas Menke has become the first in the world to successfully produce a genetically modified reptile—specifically, four albino lizards—using the CRISPR-Cas9 gene-editing tool. The team’s results, which appeared online March 31, have been submitted for peer review.
Abstract
Outer dynein arms (ODAs) are multiprotein complexes that drive flagellar beating. Based on genetic and biochemical analyses, ODAs preassemble in the cell body and then move into the flagellum by intraflagellar transport (IFT). To study ODA transport in vivo, we expressed the essential intermediate chain 2 tagged with mNeonGreen (IC2-NG) to rescue the corresponding Chlamydomonas reinhardtii mutant oda6. IC2-NG moved by IFT; the transport was of low processivity and increased in frequency during flagellar growth. As expected, IFT of IC2-NG was diminished in oda16, lacking an ODA-specific IFT adapter, and in ift46 IFT46ΔN lacking the ODA16-interacting portion of IFT46. IFT loading appears to involve ODA16-dependent recruitment of ODAs to basal bodies followed by handover to IFT. Upon unloading from IFT, ODAs rapidly docked to the axoneme. Transient docking still occurred in the docking complex mutant oda3 indicating that the docking complex stabilizes rather than initiates ODA–microtubule interactions. In full-length flagella, ODAs continued to enter and move inside cilia by short-term bidirectional IFT and diffusion and the newly imported complexes frequently replaced axoneme-bound ODAs. We propose that the low processivity of ODA-IFT contributes to flagellar maintenance by ensuring the availability of replacement ODAs along the length of flagella.
Abstract
Cilia, essential motile and sensory organelles, have several compartments: the basal body, transition zone, and the middle and distal axoneme segments. The distal segment accommodates key functions, including cilium assembly and sensory activities. While the middle segment contains doublet microtubules (incomplete B-tubules fused to complete A-tubules), the distal segment contains only A-tubule extensions, and its existence requires coordination of microtubule length at the nanometer scale. We show that three conserved proteins, two of which are mutated in the ciliopathy Joubert syndrome, determine the geometry of the distal segment, by controlling the positions of specific microtubule ends. FAP256/CEP104 promotes A-tubule elongation. CHE-12/Crescerin and ARMC9 act as positive and negative regulators of B-tubule length, respectively. We show that defects in the distal segment dimensions are associated with motile and sensory deficiencies of cilia. Our observations suggest that abnormalities in distal segment organization cause a subset of Joubert syndrome cases.
Significance
The Notch-signaling pathway is normally activated by receptor–ligand interactions. Extracellular domains (ECDs) of Notch receptors are heavily modified with O-linked glycans, such as O-glucose (O-Glc), O-fucose (O-Fuc), and O-GlcNAc. The significance of multiple types of O-glycans on Notch is not understood. NOTCH1 ECD interacts with ligands at multiple points, including an O-Glc monosaccharide on the 11th Epidermal Growth Factor (EGF) repeat (EGF11). Here, we identify two novel protein O-glucosyltransferases that modify NOTCH1 EGF11 with O-Glc. Combined deletion of the O-Glc site on EGF11 with O-Fuc modification sites on EGF8 or EGF12 markedly reduced NOTCH1 cell-surface expression or activation of NOTCH1 by Delta-like ligand 1, respectively. This study identifies a cooperative mechanism for fine-tuning the Notch-signaling pathway by different types of O-glycans.
Abstract
The Notch-signaling pathway is normally activated by Notch–ligand interactions. A recent structural analysis suggested that a novel O-linked hexose modification on serine 435 of the mammalian NOTCH1 core ligand-binding domain lies at the interface with its ligands. This serine occurs between conserved cysteines 3 and 4 of Epidermal Growth Factor-like (EGF) repeat 11 of NOTCH1, a site distinct from those modified by protein O-glucosyltransferase 1 (POGLUT1), suggesting that a different enzyme is responsible. Here, we identify two novel protein O-glucosyltransferases, POGLUT2 and POGLUT3 (formerly KDELC1 and KDELC2, respectively), which transfer O-glucose (O-Glc) from UDP-Glc to serine 435. Mass spectrometric analysis of NOTCH1 produced in HEK293T cells lacking POGLUT2, POGLUT3, or both genes showed that either POGLUT2 or POGLUT3 can add this novel O-Glc modification. EGF11 of NOTCH2 does not have a serine residue in the same location for this O-glucosylation, but EGF10 of NOTCH3 (homologous to EGF11 in NOTCH1 and -2) is also modified at the same position. Comparison of the sites suggests a consensus sequence for modification. In vitro assays with POGLUT2 and POGLUT3 showed that both enzymes modified only properly folded EGF repeats and displayed distinct acceptor specificities toward NOTCH1 EGF11 and NOTCH3 EGF10. Mutation of the O-Glc modification site on EGF11 (serine 435) in combination with sensitizing O-fucose mutations in EGF8 or EGF12 affected cell-surface presentation of NOTCH1 or reduced activation of NOTCH1 by Delta-like1, respectively. This study identifies a previously undescribed mechanism for fine-tuning the Notch-signaling pathway in mammals.
Notch is a cell-surface receptor that controls cell-fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools that inhibit Notch activity are being developed as cancer therapeutics. To this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.
Significance
Bardet–Biedl syndrome (BBS) is a rare disease caused by dysfunctional cilia. In bbs mutants, the composition of the ciliary membrane is altered due to defects in the BBSome, a conserved complex of BBS proteins. To determine the molecular function of the BBSome, we used single particle in vivo imaging. Transport of the ciliary membrane protein phospholipase D (PLD) is BBSome-dependent, and PLD comigrates with BBSomes on intraflagellar transport (IFT) trains. PLD accumulates inside cilia after removal of its ciliary export sequence (CES) or in the absence of BBSomes. In conclusion, the BBSome participates directly in ciliary protein transport by serving as an adapter allowing proteins that alone are unable to bind to IFT to be exported from cilia on IFT trains.
Abstract
Bardet–Biedl syndrome (BBS) is a ciliopathy resulting from defects in the BBSome, a conserved protein complex. BBSome mutations affect ciliary membrane composition, impairing cilia-based signaling. The mechanism by which the BBSome regulates ciliary membrane content remains unknown. Chlamydomonas bbs mutants lack phototaxis and accumulate phospholipase D (PLD) in the ciliary membrane. Single particle imaging revealed that PLD comigrates with BBS4 by intraflagellar transport (IFT) while IFT of PLD is abolished in bbs mutants. BBSome deficiency did not alter the rate of PLD entry into cilia. Membrane association and the N-terminal 58 residues of PLD are sufficient and necessary for BBSome-dependent transport and ciliary export. The replacement of PLD’s ciliary export sequence (CES) caused PLD to accumulate in cilia of cells with intact BBSomes and IFT. The buildup of PLD inside cilia impaired phototaxis, revealing that PLD is a negative regulator of phototactic behavior. We conclude that the BBSome is a cargo adapter ensuring ciliary export of PLD on IFT trains to regulate phototaxis.
Abstract
Radial spokes (RSs) are multiprotein complexes regulating dynein activity. In the cell body and ciliary matrix, RS proteins are present in a 12S precursor, which is converted into axonemal 20S spokes consisting of a head and stalk. To study RS assembly in vivo, we expressed fluorescent protein (FP)-tagged versions of the head protein RSP4 and the stalk protein RSP3 to rescue the corresponding Chlamydomonas mutants pf1, lacking spoke heads, and pf14, lacking RSs entirely. RSP3 and RSP4 mostly co-migrated by intraflagellar transport (IFT). Transport was elevated during ciliary assembly. IFT of RSP4-FP depended on RSP3. To study RS assembly independently of ciliogenesis, strains expressing FP-tagged RS proteins were mated to untagged cells with, without, or with partial RSs. RSP4-FP is added a tip-to-base fashion to preexisting pf1 spoke stalks while de novo RS assembly occurred lengthwise. In wild-type cilia, the exchange rate of head protein RSP4 exceeded that of the stalk protein RSP3 suggesting increased turnover of spoke heads. The data indicate that RSP3 and RSP4 while transported together separate inside cilia during RS repair and maintenance. The 12S RS precursor encompassing both proteins could represent transport form of the RS ensuring stoichiometric delivery by IFT.
