Dystrophin is a microtubule-associated protein

Cytolinkers are giant proteins that can stabilize cells by linking actin filaments, intermediate filaments, and microtubules (MTs) to transmembrane complexes. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. Although no direct link between dystrophin and MTs has been documented, costamere-associated MTs are disrupted when dystrophin is absent. Using tissue-based cosedimentation assays on mice expressing endogenous dystrophin or truncated transgene products, we find that constructs harboring spectrinlike repeat 24 through the first third of the WW domain cosediment with MTs. Purified Dp260, a truncated isoform of dystrophin, bound MTs with a K_d of 0.66 µM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, α- and β-tubulin expression is increased ∼2.5-fold in mdx skeletal muscle without altering the tubulin–MT equilibrium. Collectively, these data suggest dystrophin directly organizes and/or stabilizes costameric MTs and classifies dystrophin as a cytolinker in skeletal muscle.     

Differential binding of Escherichia coli DNA polymerases to the beta-sliding clamp

Escherichia coli strains expressing the mutant beta159-sliding clamp protein (containing both a G66E and a G174A substitution) are temperature sensitive for growth and display altered DNA polymerase (pol) usage. We selected for suppressors of the dnaN159 allele able to grow at 42 degrees C, and identified four intragenic suppressor alleles. One of these alleles (dnaN780) contained only the G66E substitution, while a second (dnaN781) contained only the G174A substitution. Genetic characterization of isogenic E. coli strains expressing these alleles indicated that certain phenotypes were dependent upon only the G174A substitution, while others required both the G66E and G174A substitutions. In order to understand the individual contributions of the G66E and the G174A substitution to the dnaN159 phenotypes, we utilized biochemical approaches to characterize the purified mutant beta159 (G66E and G174A), beta780 (G66E) and beta781 (G174A) clamp proteins. The G66E substitution conferred a more pronounced effect on pol IV replication than it did pol II or pol III, while the G174A substitution conferred a greater effect on pol III and pol IV than it did pol II. Taken together, these findings indicate that pol II, pol III and pol IV interact with distinct, albeit overlapping surfaces of the beta clamp.     

Diversification of andromonoecy in Solanum section Lasiocarpa (Solanaceae): the roles of phenotypic plasticity and architecture

Quantitative analyses of sexual expression show extensive interspecific variation in the strength of andromonoecy (proportions of hermaphroditic and staminate flowers) among Solanum species in the monophyletic section Lasiocarpa. The roles of phenotypic plasticity and inter- and intra-inflorescence architecture in the diversification of andromonoecy within this small clade were analyzed. Four species that represent a range of expression of andromonoecy were examined. Staminate flowers produced within inflorescences ranged from 3% (S. candidum) to 7% (S. ferox) in weakly andromonoecious species and from 39% (S. pseudolulo) to 60% (S. quitoense) in more strongly andromonoecious species. Manipulation of fruit set on clonal replicates of multiple genotypes demonstrated variation among species for phenotypic plasticity. The strongly andromonoecious species, S. pseudolulo and S. quitoense, were not plastic and produced a large proportion of staminate flowers regardless of fruiting treatment, whereas S. candidum and S. ferox were phenotypically plastic and produced significantly more staminate flowers in the presence of developing fruit. Staminate flower production of all four species varied both within and among inflorescences. A greater proportion of staminate flowers were produced in distal (later produced) inflorescences. Within inflorescences, hermaphroditic flowers occurred in basal positions, whereas staminate flowers, when produced, occurred more distally. This pattern of staminate flower production is qualitatively the same in all species investigated; however, quantitative variation in the transition from hermaphroditic to staminate flower production within and among inflorescences is associated with variation in the strength of andromonoecy. At least three factors have contributed to the diversification of andromonoecy in section Lasiocarpa including the presence or absence of phenotypic plasticity in response to fruit set, quantitative variation in intra- and inter-inflorescence architectural effects, and total flower production.     

Mutations in CKAP2L,the Human Homolog of the Mouse Radmis Gene,Cause Filippi Syndrome

Filippi syndrome is a rare, presumably autosomal-recessive disorder characterized by microcephaly, pre- and postnatal growth failure, syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals have intellectual disability, seizures, undescended testicles in males, and teeth and hair abnormalities. We performed homozygosity mapping and whole-exome sequencing in a Sardinian family with two affected children and identified a homozygous frameshift mutation, c.571dupA (p.Ile191Asnfs^∗6), in CKAP2L, encoding the protein cytoskeleton-associated protein 2-like (CKAP2L). The function of this protein was unknown until it was rediscovered in mice as Radmis (radial fiber and mitotic spindle) and shown to play a pivotal role in cell division of neural progenitors. Sanger sequencing of CKAP2L in a further eight unrelated individuals with clinical features consistent with Filippi syndrome revealed biallelic mutations in four subjects. In contrast to wild-type lymphoblastoid cell lines (LCLs), dividing LCLs established from the individuals homozygous for the c.571dupA mutation did not show CKAP2L at the spindle poles. Furthermore, in cells from the affected individuals, we observed an increase in the number of disorganized spindle microtubules owing to multipolar configurations and defects in chromosome segregation. The observed cellular phenotypes are in keeping with data from in vitro and in vivo knockdown studies performed in human cells and mice, respectively. Our findings show that loss-of-function mutations in CKAP2L are a major cause of Filippi syndrome.     

De Novo Truncating Mutations in AHDC1 in Individuals with Syndromic Expressive Language Delay,Hypotonia, and Sleep Apnea

Clinical whole-exome sequencing (WES) for identification of mutations leading to Mendelian disease has been offered to the medical community since 2011. Clinically undiagnosed neurological disorders are the most frequent basis for test referral, and currently, approximately 25% of such cases are diagnosed at the molecular level. To date, there are approximately 4,000 “known” disease-associated loci, and many are associated with striking dysmorphic features, making genotype-phenotype correlations relatively straightforward. A significant fraction of cases, however, lack characteristic dysmorphism or clinical pathognomonic traits and are dependent upon molecular tests for definitive diagnoses. Further, many molecular diagnoses are guided by recent gene-disease association discoveries. Hence, there is a critical interplay between clinical testing and research leading to gene-disease association discovery. Here, we describe four probands, all of whom presented with hypotonia, intellectual disability, global developmental delay, and mildly dysmorphic facial features. Three of the four also had sleep apnea. Each was a simplex case without a remarkable family history. Using WES, we identified AHDC1 de novo truncating mutations that most likely cause this genetic syndrome.