Self-assembly of pericentriolar material in interphase cells lacking centrioles

The major microtubule-organizing center (MTOC) in animal cells, the centrosome, comprises a pair of centrioles surrounded by pericentriolar material (PCM), which nucleates and anchors microtubules. Centrosome assembly depends on PCM binding to centrioles, PCM self-association and dynein-mediated PCM transport, but the self-assembly properties of PCM components in interphase cells are poorly understood. Here, we used experiments and modeling to study centriole18 independent features of interphase PCM assembly. We showed that when centrioles are lost due to PLK4 depletion or inhibition, dynein-based transport and self-clustering of PCM proteins are sufficient to form a single compact MTOC, which generates a dense radial microtubule array. Interphase self-assembly of PCM components depends on γ-tubulin, pericentrin, CDK5RAP2 and ninein, but not NEDD1, CEP152 or CEP192. Formation of a compact acentriolar MTOC is inhibited by AKAP450-dependent PCM recruitment to the Golgi or by randomly organized CAMSAP2-stabilized microtubules, which keep PCM mobile and prevent its coalescence. Linking of CAMSAP2 to a minus25 end-directed motor leads to the formation of an MTOC, but MTOC compaction requires cooperation with pericentrin-containing self-clustering PCM. Our data reveal that interphase PCM contains a set of components that can self-assemble into a compact structure and organize microtubules, but PCM self-organization is sensitive to motor- and microtubule-based rearrangement

Functional characterization of ARID1A mutations in follicular lymphoma

Background: Follicular lymphoma (FL) is one of the most common malignant lymphomas worldwide and the most common form of indolent lymphoma. FL is highly heterogeneous from both the clinical and molecular point of view. It remains a clinical challenge since advanced-stage disease is still considered incurable, and patients ultimately present with relapsed or re-fractory disease. Molecularly, FL is characterized by highly recurrent genetic mutations in genes coding for epigenetic modifiers. ARID1A mutations are among the most frequent mutations in FL (~10-20 % at the time of diagnosis). ARID1A mutations are a component of the prognostic clinic-genetic risk model m7-FLIPI (Pastore, Jurinovic et al. 2015). These mutations are primari-ly disruptive and result in protein haplodeficiency. Functionally, ARID1A is part of a SWI/SNF complex, which controls chromatin accessibility and is involved in numerous processes, includ-ing gene expression. Aim: Functionally characterize ARID1A mutations in representative human FL model systems. Methods: I used established and primary FL-like cell lines that harbor the hallmark t(14;18) translocation with or without heterozygous or homozygous ARID1A mutations (introduced by CRISPR/Cas9) or knock-down (by shRNA). I applied complementary omics approaches (RNA-Seq and ATAC-Seq) and functional assays to untangle the consequences of ARID1A loss in these FL model systems. Results: ARID1A loss profoundly altered gene expression. Across three cell lines, we observed consistent down-regulation of genes involved in cell cycle regulation and apoptosis pathways upon ARID1A loss. In functional experiments, I could show that ARID1A mutant clones are characterized by significantly slower cell proliferation and increased formation of anaphase bridges. Next, I demonstrated that ARID1A loss results in decreased FAS levels and lower sensitivity to FASLG-induced apoptosis. We discovered the underlying molecular mechanism through ad-vanced bioinformatics analyses and functional experiments. Briefly, ARID1A loss does not di-rectly affect FAS expression. Still, it results in reduced DNA accessibility and expression of the co-transcription factor RUNX3, thereby hindering RUNX3-ETS1 cooperativity and ETS1-induced FAS expression, which promotes a functionally and clinically relevant immune-evasive pheno-type. Finally, RNA-Seq analysis indicated that ARID1A loss alters the plasma membrane and cyto-skeleton functions, as well as the overall abundance of ligands and receptors. Ex vivo co-cultures of FL-like cells with T cells suggested impaired immune synapse formation with CD4 T lymphocytes upon ARID1A loss. Discussion and conclusion: Overall, our analyses provide novel insights into the functional consequences of ARID1A mutations in FL, most notably promoting immune evasion. A better understanding of mutation-specific biology, including its impact on interactions within the tumor microenvironment, holds promise for improved patient stratification and the development of personalized treatment approaches