The ATPase SRCAP is associated with the mitotic apparatus, uncovering novel molecular aspects of Floating-Harbor syndrome

Background A variety of human genetic diseases is known to be caused by mutations in genes encoding chromatin factors and epigenetic regulators, such as DNA or histone modifying enzymes and members of ATP-dependent chromatin remodeling complexes. Floating-Harbor syndrome is a rare genetic disease affecting human development caused by dominant truncating mutations in the SRCAP gene, which encodes the ATPase SRCAP, the core catalytic subunit of the homonymous chromatin-remodeling complex. The main function of the SRCAP complex is to promote the exchange of histone H2A with the H2A.Z variant. According to the canonical role played by the SRCAP protein in epigenetic regulation, the Floating-Harbor syndrome is thought to be a consequence of chromatin perturbations. However, additional potential physiological functions of SRCAP have not been sufficiently explored. Results We combined cell biology, reverse genetics, and biochemical approaches to study the subcellular localization of the SRCAP protein and assess its involvement in cell cycle progression in HeLa cells. Surprisingly, we found that SRCAP associates with components of the mitotic apparatus (centrosomes, spindle, midbody), interacts with a plethora of cytokinesis regulators, and positively regulates their recruitment to the midbody. Remarkably, SRCAP depletion perturbs both mitosis and cytokinesis. Similarly, DOM-A, the functional SRCAP orthologue in Drosophila melanogaster, is found at centrosomes and the midbody in Drosophila cells, and its depletion similarly affects both mitosis and cytokinesis. Conclusions Our findings provide first evidence suggesting that SRCAP plays previously undetected and evolutionarily conserved roles in cell division, independent of its functions in chromatin regulation. SRCAP may participate in two different steps of cell division: by ensuring proper chromosome segregation during mitosis and midbody function during cytokinesis. Moreover, our findings emphasize a surprising scenario whereby alterations in cell division produced by SRCAP mutations may contribute to the onset of Floating-Harbor syndrome.

Structure-based virtual screening for new lead compounds targeted Plasmodium α-tubulin

Aim. Search for new dinitroaniline and phosphorothioamide compounds, capable of selective binding with Plasmodium α-tubulin, affecting its mitotic apparatus. Methods. Structural biology methods of computational prediction of protein-ligand interaction: molecular docking, molecular dynamics and pharmacophore analysis. Selection of compounds based on pharmacophore characteristics and virtual screening results. Results. The protocol and required structural conditions for target (α-tubulin of P. falciparum) preparation and correct modeling of the ligand-protein interaction (docking and virtual screening) were developed. The generalized pharmacophore model of ligand-protein interaction and key functional groups of ligands responsible for specific binding were identified. Conclusions. Based on results of virtual screening, 22 commercial compounds were selected. Identified compounds proposed as potential inhibitors of Plasmodium mitotic machinery and the base of new antimalarial drugs. Keywords: malaria, Plasmodium, intermolecular interaction, dinitroaniline derived, phosphorothioamidate derived.

Analysis of interaction mechanisms between main objects of tuberculosis-target therapy and corresponding selective inhibitors

Aim. Search for new inhibitors of the mitotic apparatus of mycobacterium and a number of enzymatic targets. Methods. 3D models of key targets reconstruction and geometry optimization and analysis of biologically active conformations of inhibitors were performed according to a previously developed technique. Results. A revision of mycobacterial inhibitors, which exhibit antimicrobial action against representatives of the genus Mycobacterium, was carried out, which made it possible to create an appropriate reference library of compounds. The complete spatial structure of a number of the main targets of targeted therapy for tuberculosis was reconstructed and verified, and the features of their interaction with selective inhibitors were established. Chemogenomic profiling was performed, which made it possible to draw conclusions regarding the uniqueness of the studied sites and the potential toxicity of compounds related to these sites for humans. Conclusions. A well-developed search algorithm for known inhibitors of proteins with M. tuberculosis allows further study of the features of their interaction with the corresponding homologues of M. bovis and the development of new, more selective compounds using molecular dynamics and docking methods. Keywords: tuberculosis, in silico, anti-tuberculosis drugs.

Genetic Instability Due to Spindle Anomalies Visualized in Mutants of Dictyostelium

Aberrant centrosome activities in mutants of Dictyostelium discoideum result in anomalies of mitotic spindles that affect the reliability of chromosome segregation. Genetic instabilities caused by these deficiencies are tolerated in multinucleate cells, which can be produced by electric-pulse induced cell fusion as a source for aberrations in the mitotic apparatus of the mutant cells. Dual-color fluorescence labeling of the microtubule system and the chromosomes in live cells revealed the variability of spindle arrangements, of centrosome-nuclear interactions, and of chromosome segregation in the atypical mitoses observed.

Cell Models for Chromosome 20q11.21 Amplification and Drug Sensitivities in Colorectal Cancer

Background and objectives: The chromosome locus 20q11.21 is a commonly amplified locus in colorectal cancer, with a prevalence of 8% to 9%. Several candidate cancer-associated genes are transcribed from the locus. The therapeutic implications of the amplification in colorectal cancer remain unclear. Materials and Methods: Preclinical cell line models of colorectal cancer included in the Cancer Cell Line Encyclopedia (CCLE) collection were examined for the presence of amplifications in 20q11.21 genes. Correlations of the presence of 20q11.21 amplifications with gene essentialities and drug sensitivities were surveyed on salient databases for determination of therapeutic leads. Results: A significant subset of colorectal cancer cell lines in the CCLE (12 of 63 cell lines, 19%) bear amplifications of genes located at 20q11.21. Cancer-associated genes of the locus include ASXL1, DNMT3B, BCL2L1, TPX2, KIF3B and POFUT1. These genes are all amplified in the 12 cell lines, but they are variably over-expressed at the mRNA level, compared to non-amplified lines. 20q11.21 amplified cell lines are sensitive to various tyrosine kinase inhibitors and are resistant to chemotherapy drugs targeting the mitotic apparatus and microtubules. CRISPR and RNAi dependencies screening revealed, besides the β-catenin and KRAS genes, a few recurrent gene dependencies in more than one cell line, including YAP1 and JUP. Conclusions: Cell line models of colorectal cancer with 20q11.21 gene amplifications display dependencies on the presence of specific genes and resistance or sensitivity to specific drugs and drug categories. Observations from in vitro models may form the basis for clinical drug development in this subtype of colorectal cancer. Genetic lesions conferring synthetic lethality to certain drugs or categories of drugs could be discovered with this approach.

The Nuclear Mitotic Apparatus (NuMA) Protein: A Key Player for Nuclear Formation, Spindle Assembly, and Spindle Positioning

The nuclear mitotic apparatus (NuMA) protein is well conserved in vertebrates, and dynamically changes its subcellular localization from the interphase nucleus to the mitotic/meiotic spindle poles and the mitotic cell cortex. At these locations, NuMA acts as a key structural hub in nuclear formation, spindle assembly, and mitotic spindle positioning, respectively. To achieve its variable functions, NuMA interacts with multiple factors, including DNA, microtubules, the plasma membrane, importins, and cytoplasmic dynein. The binding of NuMA to dynein via its N-terminal domain drives spindle pole focusing and spindle positioning, while multiple interactions through its C-terminal region define its subcellular localizations and functions. In addition, NuMA can self-assemble into high-ordered structures which likely contribute to spindle positioning and nuclear formation. In this review, we summarize recent advances in NuMA’s domains, functions and regulations, with a focus on human NuMA, to understand how and why vertebrate NuMA participates in these functions in comparison with invertebrate NuMA-related proteins.

Establishment of international autoantibody reference standards for the detection of autoantibodies directed against PML bodies, GW bodies, and NuMA protein

ObjectivesReference materials are important in the standardization of autoantibody testing and only a few are freely available for many known autoantibodies. Our goal was to develop three reference materials for antibodies to PML bodies/multiple nuclear dots (MND), antibodies to GW bodies (GWB), and antibodies to the nuclear mitotic apparatus (NuMA).MethodsReference materials for identifying autoantibodies to MND (MND-REF), GWB (GWB-REF), and NuMA (NuMA-REF) were obtained from three donors and validated independently by seven laboratories. The sera were characterized using indirect immunofluorescence assay (IFA) on HEp-2 cell substrates including two-color immunofluorescence using antigen-specific markers, western blot (WB), immunoprecipitation (IP), line immunoassay (LIA), addressable laser bead immunoassay (ALBIA), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation–mass spectrometry (IP-MS).ResultsMND-REF stained 6–20 discrete nuclear dots that colocalized with PML bodies. Antibodies to Sp100 and PML were detected by LIA and antibodies to Sp100 were also detected by ELISA. GWB-REF stained discrete cytoplasmic dots in interphase cells, which were confirmed to be GWB using two-color immunofluorescence. Anti-Ge-1 antibodies were identified in GWB-REF by ALBIA, IP, and IP-MS. All reference materials produced patterns at dilutions of 1:160 or greater. NuMA-REF produced fine speckled nuclear staining in interphase cells and staining of spindle fibers and spindle poles. The presence of antibodies to NuMA was verified by IP, WB, ALBIA, and IP-MS.ConclusionsMND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.