Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest.

Whether cell forces or extracellular matrix (ECM) can impact genome integrity is largely unclear. Here, acute perturbations (∼1 h) to actomyosin stress or ECM elasticity cause rapid and reversible changes in lamin-A, DNA damage, and cell cycle. The findings are especially relevant to organs such as the heart because DNA damage permanently arrests cardiomyocyte proliferation shortly after birth and thereby eliminates regeneration after injury including heart attack.

Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation.

Tissues can be soft like fat, which bears little stress, or stiff like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is unknown. Here, proteomics analyses revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels.

Exon-skipped dystrophins for treatment of Duchenne muscular dystrophy: mass spectrometry mapping of most exons and cooperative domain designs based on single molecule mechanics.

Force-bearing linkages between the cytoskeleton and extracellular matrix are clearly important to normal cell viability-as is evident in a disease such as Duchenne muscular dystrophy (DMD) which arises in the absence of the linkage protein dystrophin. Therapeutic approaches to DMD include antisense-mediated skipping of exons to delete nonsense mutations while maintaining reading frame, but the structure and stability of the resulting proteins are generally unclear. Here we use mass spectrometry to detect most dystrophin exons, and we express and physically characterize dystrophin "nano"-constructs based on multiexon deletions that might find use in a large percentage of DMD patients.

Upregulation of paxillin and focal adhesion signaling follows Dystroglycan Complex deletions and promotes a hypertensive state of differentiation.

Anchorage to matrix is mediated for many cells not only by integrin-based focal adhesions but also by a parallel assembly of integral and peripheral membrane proteins known as the Dystroglycan Complex. Deficiencies in either dystrophin (mdx mice) or γ-sarcoglycan (γSG(-/-) mice) components of the Dystroglycan Complex lead to upregulation of numerous focal adhesion proteins, and the phosphoprotein paxillin proves to be among the most prominent. In mdx muscle, paxillin-Y31 and Y118 are both hyper-phosphorylated as are key sites in focal adhesion kinase (FAK) and the stretch-stimulatable pro-survival MAPK pathway, whereas γSG(-/-) muscle exhibits more erratic hyper-phosphorylation.

Polymersome delivery of siRNA and antisense oligonucleotides.

siRNA and antisense oligonucleotides, AON, have similar size and negative charge and are often packaged for in vitro delivery with cationic lipids or polymers-but exposed positive charge is problematic in vivo. Here we demonstrate loading and functional delivery of RNAi and AON with non-ionic, nano-transforming polymersomes. These degradable carriers are taken up passively by cultured cells after which the vesicles transform into micelles that allow endolysosomal escape and delivery of either siRNA into cytosol for mRNA knockdown or else AON into the nucleus for exon skipping within pre-mRNA.

Shape effects of filaments versus spherical particles in flow and drug delivery.

Interaction of spherical particles with cells and within animals has been studied extensively, but the effects of shape have received little attention. Here we use highly stable, polymer micelle assemblies known as filomicelles to compare the transport and trafficking of flexible filaments with spheres of similar chemistry. In rodents, filomicelles persisted in the circulation up to one week after intravenous injection.

gamma-Sarcoglycan deficiency increases cell contractility, apoptosis and MAPK pathway activation but does not affect adhesion.

The functions of gamma-sarcoglycan (gammaSG) in normal myotubes are largely unknown, however gammaSG is known to assemble into a key membrane complex with dystroglycan and its deficiency is one known cause of limb-girdle muscular dystrophy. Previous findings of apoptosis from gammaSG-deficient mice are extended here to cell culture where apoptosis is seen to increase more than tenfold in gammaSG-deficient myotubes compared with normal cells. The deficient myotubes also exhibit an increased contractile prestress that results in greater shortening and widening when the cells are either lightly detached or self-detached.