ABSTRACT
SASE1 is the first beamline of the European XFEL that became operational in 2017. It consists of the SASE1 undulator system, the beam transport system, and the two scientific experiment stations: Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX), and Femtosecond X-ray Experiments (FXE). The beam transport system comprises mirrors to offset and guide the beam to the instruments and a set of X-ray optical components to align, manipulate and diagnose the beam. The SASE1 beam transport system is described here in its initial configuration, and results and experiences from the first year of user operation are reported.
ABSTRACT
Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.
Subject(s)
Chlamydomonas/genetics , Cilia/metabolism , Flagella/metabolism , Polycystic Kidney, Autosomal Recessive/genetics , Proteins/chemistry , Protozoan Proteins/metabolism , Tumor Suppressor Proteins , Amino Acid Sequence , Animals , Chlamydomonas/cytology , Cilia/genetics , Cilia/pathology , Cilia/ultrastructure , Cloning, Molecular , Conserved Sequence , Flagella/genetics , Flagella/pathology , Flagella/ultrastructure , Humans , Kidney/metabolism , Kidney/pathology , Meiosis , Mice , Mice, Knockout , Microscopy, Electron, Scanning , Molecular Motor Proteins/genetics , Molecular Motor Proteins/metabolism , Molecular Motor Proteins/pathology , Molecular Motor Proteins/ultrastructure , Molecular Sequence Data , Mutation/genetics , Phenotype , Plant Proteins , Polycystic Kidney, Autosomal Recessive/pathology , Polycystic Kidney, Autosomal Recessive/physiopathology , Protein Binding , Protein Subunits , Proteins/genetics , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Repetitive Sequences, Amino Acid/genetics , Repetitive Sequences, Amino Acid/physiology , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility.
Subject(s)
Chlamydomonas reinhardtii/genetics , Dyneins/genetics , Genes, Protozoan , Intracellular Signaling Peptides and Proteins , Microtubule-Associated Proteins , Protozoan Proteins/genetics , Animals , Cell Movement , Cloning, Molecular , Flagella/genetics , Flagella/ultrastructure , Mice , Microscopy, Electron , Microtubules/ultrastructure , Mutation , Nuclear Proteins/genetics , Phenotype , Protozoan Proteins/metabolism , Sequence Homology , Transformation, Genetic , Ubiquitin-Protein Ligases , t-Complex Genome RegionABSTRACT
Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.