Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Mechanisms? How does actomyosin contractility influences gene transcription? Actin and myosin in transcription a) Pol II - Pre-initiation complex assembly together with WASP and Arp2/3. - Actin enhances activity of chromatinremodeling complexes and elongation machinery. - Splicing machinery (MVa) - Backward motor (MVI) counteracting motor functions (NMI). b) Pol I - Transcription initiation of rRNA - Chromatin remodeling - rRNA elongation and maturation de Lanerolle & Serebryannyy (2011) Nat Cell Biol 13: 1282-1288 Actin in the nucleus de Lanerolle & Serebryannyy (2011) Nat Cell Biol 13: 1282-1288 Role of actin in the nucleus Actin/cofilin enter the nucleus via nuclear pore (NPC), while actin/profilin is exported via exportin 6. Nuclear actin can interact with all three polymerases and engage in transcription. Nuclear actin can form uncoventional polymers (rods) that are shorter than cytoplasmic F-actin). Nuclear actin undergo post-translational modifications, such as SUMOylation (small ubiquitin-like modifier). Steroid receptors (co-activator of gene transcription) that translocate to the nucleus upon ligand binding also interact with actin, actin binding proteins and myosins. Some transcription factors (PREP2, YY1, MAL) bind to cytoplasmic actin. Changes in actin polymerization can sequester or release these factors so that they can enter the nucleus to regulate transcription. Myosin in the nucleus de Lanerolle & Serebryannyy (2011) Nat Cell Biol 13: 1282-1288 Role of myosins in the nucleus Gene transcription: NMI, MVI and actin required for transcription by RNA pol, through structural roles, motor functions or a combination of the two. Co-activators: steroid receptors (co-activator of gene transcription) translocate to the nucleus upon ligand binding also interact with actin, actin binding proteins and myosins. Transcription factors?: Heavy chain of MII and MXVIIIb have been directly implicated in the activation of genes required for differentiation. Scafolding complexes : myosins (NMI, MII) together with actin and intermediate filament proteins (lamins and emerin) form complexes that provide a link between the nucleoskeleton and chromatin. Cell cycle: MXVIb interacts with cyclin A and proliferating cell nuclear antigen (PCNA). Binding to protein phosphatase 1 (PP1) may control its activity and transport through NPC. RNA processing: MVa found in speckles and thought to be involved in RNA processing Many questions remain… • Form of actin in the nucleus and its regulation? monomer? filament? non-canonical-oligomer? Bellin et at (2013) Mol Biol Cell 24:982-04: monomer: speckles RNA-processing factors filament: submicron lengths, excluded from chromatin rich regions and part of viscoelastic mesh scaffold for organizing nuclear complexes? • How actin and myosins associate with each other and with other nuclear components? Lamin and emerin are proteins of the nuclear envelope Inner nuclear membrane proteins Lamins • IF proteins divided in Type A (Lamin A/C) and Type B (Lamin B) • bind to DNA directly or indirectly via other nuclear proteins (emerin, LINC complex) • mutations in Type A lamins are found in human laminopathies including: EmeryDreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM) and Hutchinson-Gilford progeria syndrome. Emerin • • http://circres.ahajournals.org/content/103/1/16/F2.large.jpg • • actin capping protein, promotes actin polymerization form a complex with nuclear spectrin and 4.1 proteins binds to LINC complex (nesprin/SUN) mutated in X-linked EDMD Nuclear abnormalities caused by lamin A/C deficiency • Lmna-/- cells have increase nuclear deformation, defective mechanotransduction and impaired viability under mechanical stress. • NFkB-mediated transcription in response to mechanical or cytokine stimulation (anti-apoptotic signal) is attenuated. • Tissue-specific effects of lamin A/C deficiency may arise from impaired nuclear mechanics and transcriptional activation. Increased nuclear deformation Lmna+/+ Lmna-/- No strain 22% strain No strain 19% strain Compromised survival Lammerding (2004) J Clin Inv 113:370-378 Increased nuclear fragility Attenuated transcription Background • MKL1 (aka MAL or MRTF-A) is a mechanosensitive transcription factor. • Intracellualr localization is regulated by changes in actin polymerization: - cytoplasmic: binds G-actin and has constitutive nuclear export signal. - nuclear: mitogenic/mechanical stimulation triggers RhoA-mediated actin polymerization; releases MKL1 from G-actin and exposes NLS within actin-binding domain. • MKL1 in the nucleus co-activates serum-response factor (SRF) and activates genes involved in cell motiliy/contractility: vinculin, actin and SRF itself. • Does loss/mutations of lamin A/C affect MKL1-SFR signaling? Nuclear translocation of MKL1 is impaired in lamindeficient and mutant cells Serum stimulation Time-lapse Cardiac muscle Expression of MKL1-SRF target genes* is downregulated in lamin-deficient and mutant cells Fibroblasts *SRF and vinculin Cardiac muscle Reduced nuclear import and increased nuclear export in lamin-deficient and mutants cells IMPORT IMPORT LMB: leptomycin B inhibitor of nuclear export EXPORT Impaired nuclear accumulation of MKL1 is caused by altered actin dynamics RPEL: actin-binding motif; SAP: DNA binding domain; TAD: transcription activation domain Altered actin dynamics in lamin-deficient and mutants cells Emerin expression rescues actin dynamics and restores MKL1 translocation Lamin mutants Emerin mutant (Emd-/Y) Emerin rescue Rescue of MKL1 translocation requires emerinbinding to actin Actin motility (FRAP) M151, M164 and Q133H: emerin mutants that do no binding to actin Cytoplasm Nucleus Summary • Lamin-deficient and mutant cells have impaired nuclear translocation and downstream signalling of MKL1. • Abnormal nuclear translocation of MKL1 was caused by altered actin dynamics. • Ectopic expression of emerin, which is mislocalized in lamin mutant cells, restored MKL1 nuclear translocation and rescued actin dynamics. • The findings provide insight into the etiology of laminopathies, whereby lamin A/C and emerin might regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization. Why is this experiment important? Starved Stimulated NLS-GFP-NES: GFP fused to nuclear localization signal/nuclear export signal