Mechanisms?

How does actomyosin contractility influences gene transcription?

de Lanerolle & Serebryannyy (2011) Nat Cell Biol 13: 1282-1288

a) Pol II- Pre-initiation complex assemblytogether with WASP and Arp2/3.- Actin enhances activity of chromatin-remodeling 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

Actin and myosin in transcription

Actin in the nucleus

de Lanerolle & Serebryannyy (2011) Nat Cell Biol 13: 1282-1288

Role of actin in the nucleusActin/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 intranscription.

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 nucleusGene 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

http://circres.ahajournals.org/content/103/1/16/F2.large.jpg

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: Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM) and Hutchinson-Gilford progeria syndrome.

Emerin• 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.

Lmna+/+ Lmna-/-

No strain22% strain

No strain19% strain

Increased nuclear deformation Increased nuclear fragility

Compromised survival Attenuated transcription

Lammerding (2004) J Clin Inv 113:370-378

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 lamin-deficient and mutant cells

Serum stimulation Time-lapse

Cardiac muscle

Expression of MKL1-SRF target genes* is downregulated in lamin-deficient and mutant cells

Cardiac muscleFibroblasts

*SRF and vinculin

Reduced nuclear import and increased nuclear export in lamin-deficient and mutants cells

LMB: leptomycin B inhibitor of nuclear export

IMPORT EXPORTIMPORT

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

Emerin mutant (Emd-/Y)Lamin mutants

Emerin rescue

Rescue of MKL1 translocation requires emerin-binding to actin

M151, M164 and Q133H: emerin mutants that do no binding to actin

Actin motility (FRAP)

NucleusCytoplasm

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?

StimulatedStarved

NLS-GFP-NES: GFP fused to nuclear localization signal/nuclear export signal