CSAP localizes to polyglutamylated microtubules and promotes proper cilia function and zebrafish development

The diverse populations of microtubule polymers in cells are functionally distinguished by different posttranslational modifications, including polyglutamylation. Polyglutamylation is enriched on subsets of microtubules including those found in the centrioles, mitotic spindle, and cilia. However, whether this modification alters intrinsic microtubule dynamics or affects extrinsic associations with specific interacting partners remains to be determined. Here we identify the microtubule-binding protein centriole and spindle–associated protein (CSAP), which colocalizes with polyglutamylated tubulin to centrioles, spindle microtubules, and cilia in human tissue culture cells. Reducing tubulin polyglutamylation prevents CSAP localization to both spindle and cilia microtubules. In zebrafish, CSAP is required for normal brain development and proper left–right asymmetry, defects that are qualitatively similar to those reported previously for depletion of polyglutamylation-conjugating enzymes. We also find that CSAP is required for proper cilia beating. Our work supports a model in which polyglutamylation can target selected microtubule-associated proteins, such as CSAP, to microtubule subpopulations, providing specific functional capabilities to these populations.National Institutes of Health (U.S.) (Grant no. GM074746)American Cancer Society. Research Scholar Grant (121776)National Institute of General Medical Sciences (U.S.) (GM088313

DNA content of a functioning chicken kinetochore

© The Author(s) 2014. In order to understand the three-dimensional structure of the functional kinetochore in vertebrates, we require a complete list and stoichiometry for the protein components of the kinetochore, which can be provided by genetic and proteomic experiments. We also need to know how the chromatin-containing CENP-A, which makes up the structural foundation for the kinetochore, is folded, and how much of that DNA is involved in assembling the kinetochore. In this MS, we demonstrate that functioning metaphase kinetochores in chicken DT40 cells contain roughly 50 kb of DNA, an amount that corresponds extremely closely to the length of chromosomal DNA associated with CENP-A in ChIP-seq experiments. Thus, during kinetochore assembly, CENP-A chromatin is compacted into the inner kinetochore plate without including significant amounts of flanking pericentromeric heterochromatin. © 2014 The Author(s).Wellcome Trust [grant number 073915]; Wellcome Trust Centre for Cell Biology (core grant numbers 077707 and 092076); Darwin Trust of Edinburg

A two-step mechanism for epigenetic specification of centromere identity and function

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.National Institutes of Health grant: (GM 074150); Ludwig Institute for Cancer Research; European Molecular Biology Organization (EMBO) long-term fellowship

Molecular markers of anti-malarial drug resistance in Central, West and East African children with severe malaria.

BACKGROUND: The Plasmodium falciparum multidrug resistance 1 (PfMDR1), P. falciparum Ca(2+)-ATPase (PfATP6) and Kelch-13 propeller domain (PfK13) loci are molecular markers of parasite susceptibility to anti-malarial drugs. Their frequency distributions were determined in the isolates collected from children with severe malaria originating from three African countries. METHODS: Samples from 287 children with severe malaria [(Gabon: n = 114); (Ghana: n = 89); (Kenya: n = 84)] were genotyped for pfmdr1, pfatp6 and pfk13 loci by DNA sequencing and assessing pfmdr1 copy number variation (CNV) by real-time PCR. RESULTS: Pfmdr1-N86Y mutation was detected in 48, 10 and 10% in Lambaréné, Kumasi and Kisumu, respectively. At codon 184, the prevalence of the mutation was 73% in Lambaréné, 63% in Kumasi and 49% Kisumu. The S1034C and N1042D variants were absent at all three sites, while the frequency of the D1246Y mutation was 1, 3 and 13% in Lambaréné, Kumasi and Kisumu, respectively. Isolates with two pfmdr1 gene copy number predominantly harboured the N86Y wild-type allele and were mostly found in Kumasi (10%) (P < 0.0001). Among the main pfmdr1 haplotypes (NFD, NYD and YFD), NYD was associated with highest parasitaemia (P = 0.04). At the pfatp6 locus, H243Y and A623E mutations were observed at very low frequency at all three sites. The prevalence of the pfatp6 E431K variant was 6, 18 and 17% in Lambaréné, Kumasi and Kisumu, respectively. The L263E and S769N mutations were absent in all isolates. The pfk13 variants associated with artemisinin resistance in Southeast Asia were not observed. Eleven novel substitutions in the pfk13 locus occurring at low frequency were observed. CONCLUSIONS: Artemisinins are still highly efficacious in large malaria-endemic regions though declining efficacy has occurred in Southeast Asia. The return of chloroquine-sensitive strains following the removal of drug pressure is observed. However, selection of wild-type alleles in the multidrug-resistance gene and the increased gene copy number is associated with reduced lumefantrine sensitivity. This study indicates a need to constantly monitor drug resistance to artemisinin in field isolates from malaria-endemic countries

Drug Resistance in Eukaryotic Microorganisms

Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies

A Kinase-Phosphatase Network that Regulates Kinetochore-Microtubule Attachments and the SAC

The KMN network (for KNL1, MIS12 and NDC80 complexes) is a hub for signalling at the outer kinetochore. It integrates the activities of two kinases (MPS1 and Aurora B) and two phosphatases (PP1 and PP2A-B56) to regulate kinetochore-microtubule attachments and the spindle assembly checkpoint (SAC). We will first discuss each of these enzymes separately, to describe how they are regulated at kinetochores and why this is important for their primary function in controlling either microtubule attachments or the SAC. We will then discuss why inhibiting any one of them individually produces secondary effects on all the others. This cross-talk may help to explain why all enzymes have been linked to both processes, even though the direct evidence suggests they each control only one. This chapter therefore describes how a network of kinases and phosphatases work together to regulate two key mitotic processes.</p

Roles for the Conserved Spc105p/Kre28p Complex in Kinetochore-Microtubule Binding and the Spindle Assembly Checkpoint

Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain approximately 70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores.We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells.Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes

Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain

The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis