Characterization of the dual ITK/JAK3 small molecule covalent inhibitor ATI-2138.

Aclaris Therapeutics Inc (ATI)-2138 is a novel investigational covalent inhibitor of interleukin-2-inducible T cell kinase (ITK), resting lymphocyte kinase, and Janus kinase 3 (JAK3) in development for the treatment of autoimmune and inflammatory diseases. In this study, we evaluated the inhibitory effects of ATI-2138 on ITK and JAK3 signaling in cells and preclinical animal models and assessed the safety, tolerability, pharmacokinetics, and pharmacodynamics of ATI-2138 in healthy human participants. ATI-2138 potently, selectively, and irreversibly inhibited ITK, resting lymphocyte kinase, and JAK3 kinases with similar potency observed against ITK and JAK3 in biochemical and immune cell signaling assays.

CCDC61/VFL3 Is a Paralog of SAS6 and Promotes Ciliary Functions.

Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear.

RPGRIP1L helps to establish the ciliary gate for entry of proteins.

Mutations in transition zone genes change the composition of the ciliary proteome. We isolated new mutations in (denotated as in algae) that affect the localization of the transition zone protein NPHP4 in the model organism NPHP4 localization is not affected in multiple new intraflagellar transport (IFT) mutants. We compared the proteome of cilia from wild-type and mutants that affect the transition zone () or IFT ( and ) by mass spectrometry.

MAPINS, a Highly Efficient Detection Method That Identifies Insertional Mutations and Complex DNA Rearrangements.

Insertional mutagenesis, in which a piece of exogenous DNA is integrated randomly into the genomic DNA of the recipient cell, is a useful method to generate new mutants with phenotypes of interest. The unicellular green alga is an outstanding model for studying many biological processes. We developed a new computational algorithm, MAPINS (mapping insertions), to efficiently identify insertion sites created by the integration of an () cassette that confers paromomycin resistance.

Identifying RNA splicing factors using genes in .

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two genes, () and (), which lead to flagellar assembly defects in the unicellular green alga The splicing defects in these mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a deletion mutant, which suppresses the 3' splice site mutation in , and a frameshift mutant of , which suppresses the 5' splice site mutation in Surprisingly, we found and mutations suppress both splice site mutations.

The IDA3 adapter, required for intraflagellar transport of I1 dynein, is regulated by ciliary length.

We determined how the ciliary motor I1 dynein is transported. A specialized adapter, IDA3, facilitates I1 dynein attachment to the ciliary transporter called intraflagellar transport (IFT). Loading of IDA3 and I1 dynein on IFT is regulated by ciliary length.

A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures.

CCDC39 and CCDC40 were first identified as causative mutations in primary ciliary dyskinesia patients; cilia from patients show disorganized microtubules, and they are missing both N-DRC and inner dynein arms proteins. In Chlamydomonas, we used immunoblots and microtubule sliding assays to show that mutants in CCDC40 (PF7) and CCDC39 (PF8) fail to assemble N-DRC, several inner dynein arms, tektin, and CCDC39. Enrichment screens for suppression of pf7; pf8 cells led to the isolation of five independent extragenic suppressors defined by four different mutations in a NIMA-related kinase, CNK11.

Genetic and genomic approaches to identify genes involved in flagellar assembly in Chlamydomonas reinhardtii.

Flagellar assembly requires intraflagellar transport of components from the cell body to the flagellar tip for assembly. The understanding of flagellar assembly has been aided by the ease of biochemistry and the availability of mutants in the unicellular green alga, Chlamydomonas reinhardtii. In this chapter, we discuss means to identify genes involved in these processes using forward and reverse genetics.

MLT1 links cytoskeletal asymmetry to organelle placement in chlamydomonas.

Asymmetric placement of the photosensory eyespot organelle in Chlamydomonas is patterned by mother-daughter differences between the two basal bodies, which template the anterior flagella. Each basal body is associated with two bundled microtubule rootlets, one with two microtubules and one with four, forming a cruciate pattern. In wild-type cells, the single eyespot is positioned at the equator in close proximity to the plus end of the daughter rootlet comprising four microtubules, the D4.

New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas.

Background: The building of a cilium or flagellum requires molecular motors and associated proteins that allow the relocation of proteins from the cell body to the distal end and the return of proteins to the cell body in a process termed intraflagellar transport (IFT). IFT trains are carried out by kinesin and back to the cell body by dynein.

Methods: We used whole genome sequencing to identify the causative mutations for two temperature-sensitive flagellar assembly mutants in Chlamydomonas and validated the changes using reversion analysis.

The Chlamydomonas mutant pf27 reveals novel features of ciliary radial spoke assembly.

To address the mechanisms of ciliary radial spoke assembly, we took advantage of the Chlamydomonas pf27 mutant. The radial spokes that assemble in pf27 are localized to the proximal quarter of the axoneme, but otherwise are fully assembled into 20S radial spoke complexes competent to bind spokeless axonemes in vitro. Thus, pf27 is not defective in radial spoke assembly or docking to the axoneme.

Whole genome sequencing identifies a deletion in protein phosphatase 2A that affects its stability and localization in Chlamydomonas reinhardtii.

Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect.

Identification of cilia genes that affect cell-cycle progression using whole-genome transcriptome analysis in Chlamydomonas reinhardtti.

Cilia are microtubule based organelles that project from cells. Cilia are found on almost every cell type of the human body and numerous diseases, collectively termed ciliopathies, are associated with defects in cilia, including respiratory infections, male infertility, situs inversus, polycystic kidney disease, retinal degeneration, and Bardet-Biedl Syndrome. Here we show that Illumina-based whole-genome transcriptome analysis in the biflagellate green alga Chlamydomonas reinhardtii identifies 1850 genes up-regulated during ciliogenesis, 4392 genes down-regulated, and 4548 genes with no change in expression during ciliogenesis.

Whole-Genome Sequencing to Identify Mutants and Polymorphisms in Chlamydomonas reinhardtii.

Whole-genome sequencing (WGS) provides a new platform for the identification of mutations that produce a mutant phenotype. We used Illumina sequencing to identify the mutational profile of three Chlamydomonas reinhardtii mutant strains. The three strains have more than 38,000 changes from the reference genome.

Synthesizing and salvaging NAD: lessons learned from Chlamydomonas reinhardtii.

The essential coenzyme nicotinamide adenine dinucleotide (NAD+) plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD+. Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD+ is synthesized from aspartate (de novo synthesis), as in plants, or nicotinamide, as in mammals (salvage synthesis).

Early sexual origins of homeoprotein heterodimerization and evolution of the plant KNOX/BELL family.

Developmental mechanisms that yield multicellular diversity are proving to be well conserved within lineages, generating interest in their origins in unicellular ancestors. We report that molecular regulation of the haploid-diploid transition in Chlamydomonas, a unicellular green soil alga, shares common ancestry with differentiation pathways in land plants. Two homeoproteins, Gsp1 and Gsm1, contributed by gametes of plus and minus mating types respectively, physically interact and translocate from the cytosol to the nucleus upon gametic fusion, initiating zygote development.

Sex determination in Chlamydomonas.

The sex-determination system of the unicellular green alga, Chlamydomonas reinhardtii, is governed by genes in the mating-type (MT) locus and entails additional genes located in autosomes. Gene expression is initiated by nitrogen starvation, and cells differentiate into plus or minus gametes within 6h. Reviewed is our current understanding of gametic differentiation and fertilization, initiation of zygote development, and the uniparental inheritance of organelle genomes.

Gametogenesis in the Chlamydomonas reinhardtii minus mating type is controlled by two genes, MID and MTD1.

In the unicellular algae Chlamydomonas reinhardtii, the plus and minus mating types are controlled by a complex locus, MT, where the dominant MID gene in the MT(-) locus has been shown to be necessary for expression of minus-specific gamete-specific genes in response to nitrogen depletion. We report studies on MID expression patterns during gametogenesis and on a second gene unique to the MT(-) locus, MTD1. Vegetative cells express basal levels of MID.

Plus and minus sexual agglutinins from Chlamydomonas reinhardtii.

Gametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins.