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“Bolstering Immunity” UGAToday – Great Commitments

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 new paper from the Lechtreck Lab: In vivo analysis of outer arm dynein transport reveals cargo-specific intraflagellar transport properties

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.

A new paper from the Gaertig Lab: Proteins that control the geometry of microtubules at the ends of cilia

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.

A recent PNAS paper from the Haltiwanger Lab: Two novel protein O-glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling

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.

A recent paper from the Haltiwanger Lab: Inhibition of Delta-induced Notch signaling using fucose analogs

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.

A recent PNAS from the Lechtreck Lab: The Bardet–Biedl syndrome protein complex is an adapter expanding the cargo range of intraflagellar transport trains for ciliary export

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.

A recent paper from the Lechtreck Lab: In vivo imaging of radial spoke proteins reveals independent assembly and turnover of the spoke head and stalk

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.

A recent paper from the Eggenschwiler Lab: Cell cycle-related kinase regulates mammalian eye development through positive and negative regulation of the Hedgehog pathway.

Cell cycle-related kinase (CCRK) is a conserved regulator of ciliogenesis whose loss in mice leads to a wide range of developmental defects, including exencephaly, preaxial polydactyly, skeletal abnormalities, and microphthalmia. Here, we investigate the role of CCRK in mouse eye development. Ccrk mutants show dramatic patterning defects, with an expansion of the optic stalk domain into the optic cup, as well as an expansion of the retinal pigment epithelium (RPE) into neural retina (NR) territory. In addition, Ccrk mutants display a shortened optic stalk. These defects are associated with bimodal changes in Hedgehog (Hh) pathway activity within the eye, including the loss of proximal, high level responses but a gain in distal, low level responses. We simultaneously removed the Hh activator GLI2 in Ccrk mutants (Ccrk-/-;Gli2-/-), which resulted in rescue of optic cup patterning and exacerbation of optic stalk length defects. Next, we disrupted the Hh pathway antagonist GLI3 in mutants lacking CCRK (Ccrk-/-;Gli3-/-), which lead to even greater expansion of the RPE markers into the NR domain and a complete loss of NR specification within the optic cup. These results indicate that CCRK functions in eye development by both positively and negatively regulating the Hh pathway, and they reveal distinct requirements for Hh signaling in patterning and morphogenesis of the eyes.

A recent paper from the Menke Lab: PITX1 promotes chondrogenesis and myogenesis in mouse hindlimbs through conserved regulatory targets

The PITX1 transcription factor is expressed during hindlimb development, where it plays a critical role in directing hindlimb growth and the specification of hindlimb morphology. While it is known that PITX1 regulates hindlimb formation, in part, through activation of the Tbx4 gene, other transcriptional targets remain to be elucidated. We have used a combination of ChIP-seq and RNA-seq to investigate enhancer regions and target genes that are directly regulated by PITX1 in embryonic mouse hindlimbs.

Esther van der Knaap has been named as one of the Presidential Extraordinary Research Faculty Hiring Initiative builds on UGA’s Signature Themes

Esther van der Knaap, a professor of horticulture in the Institute of Plant Breeding, Genetics and Genomics, is exploring the regulation of fruit shape and size in tomatoes as well as in peppers. Much of her research focuses on the molecular genetic mechanisms of cell division and cell size underlying fruit formation, and her work seeks to help boost the yield and quality of fruit and vegetable crops for the agricultural industry.

Burying beetles provide clues about genetic foundations of parenthood

A team of researchers that includes UGA scientists has identified many of the genetic changes that take place in burying beetles as they assume the role of parent. Their findings, published recently in the journal Nature Communications, may provide clues about the fundamental genetics of parenthood in insects and other animals.

Study: Blueprints for limbs encoded in snake genome

When UGA researchers examined the genome of several different snake species, they found something surprising. Embedded in the reptiles’ genetic code was DNA that, in most animals, controls the development and growth of limbs—a strange feature for creatures that are famous for their long, legless bodies and distinctive slither.

UGA scientists part of team that grew new organ in mouse

A team of scientists that includes University of Georgia genetics professor Nancy Manley have grown an organ for the first time in an animal.

Their success in inducing a mouse to grow a new thymus raises the possibility that scientists might be able to grow the organ in humans whose thymus is absent or impaired, or that their discovery might one day be used to grow important disease-fighting cells called T-cells outside the body.

Scientists grow fully functional organ from transplanted cells

The researchers created a thymus, a butterfly-shaped gland and vital component of the human immune system. Located beneath the breastbone in the upper chest, the thymus is responsible for producing T-lymphocytes, or T-cells, which help organize and lead the body’s fighting forces against threats like bacteria, viruses and even cancerous cells.

“We were all surprised by how well this works,” said Nancy Manley, professor of genetics in UGA’s Franklin College of Arts and Sciences and co-author of the paper describing their finding in Nature Cell Biology.

Growing human organs – coming soon?

A team of scientists including researchers from the University of Georgia has grown a fully functional organ from scratch in a living animal for the first time. The advance could one day aid in the development of laboratory-grown replacement organs.

Developmental biologists explore function of cancer-causing gene

Scott Dougan and his research team are discovering new roles for a specific gene known as Max’s Giant Associated protein, or MGA. A little studied protein, MGA appears to control a number of developmental processes and also may be connected to cancer development.

SE SDB Regional Meeting

Save the Date
2014 SE SDB Regional Meeting
Friday – Sunday, 16 – 18 May 2014
Callaway Gardens at Pine Mountain, GA

Researchers discover origin of unusual glands in the body

The thymus gland is a critical component of the human immune system that is responsible for the development of T-lymphocytes, or T-cells, which help organize and lead the body’s fighting forces against harmful organisms like bacteria and viruses.

Franklin College researchers discover origin of unusual glands in the body

The thymus gland is a critical component of the human immune system. It is responsible for the development of T-lymphocytes, or T-cells, which help organize and lead the body’s fighting forces against harmful organisms like bacteria and viruses. Nancy Manley, a professor of genetics in UGA’s Franklin College of Arts and Sciences and principal investigator for the project.

Biologists study part of cells linked to range of diseases

Led by Karl Lechtreck, an assistant professor in the cellular biology department, a team of researchers used total internal reflection fluorescence microscopy to analyze moving protein particles inside cilia of Chlamydomonas reinhardtii, a widely used unicellular model for the analysis of cilia.