Irena L. Ivanovska

Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation

Introduction Tissues can be soft like brain, bone marrow, and fat, which bear little mechanical stress, or stiff like muscle, cartilage, and bone, which sustain high levels of stress. Systematic relationships between tissue stiffness, protein abundance, and differential gene expression are unclear. Recent studies of stem cells cultured on matrices of different elasticity, E, have suggested that differentiation is mechanosensitive, but the molecular mechanisms involved in particular tissues remain elusive. Tissue micromechanics correlate with abundance of collagens and nuclear lamins, which influence cell differentiation. (Left) Collagen and lamin-A levels scale with E, consistent with matching tissue stress to nuclear mechanics. (Right) Matrix stiffness in tissue culture increases cell tension and stabilizes lamin-A, regulating its own transcription and that of stress fiber genes, enhancing differentiation. RA, retinoic acid, i.e., vitamin A; RARG, YAP1, and SRF, transcription factors. Methods We developed quantitative mass spectrometry algorithms to measure protein abundance, stoichiometry, conformation, and interactions within tissues and cells in relation to stiffness of tissues and extracellular matrix. Manipulations of lamin-A levels with small interfering RNA, overexpression, and retinoic acid or antagonist were applied to stem cells cultured on different matrices to assess lamin-A’s role in mechanosensitive differentiation. To characterize molecular mechanisms, promoter analyses, transcriptional profiling, and localization of transcription factors were complemented by measurements of nuclear mechanics and by modeling of the core gene circuit. Results Proteomic profiling of multiple adult solid tissues showed that widely varied levels of collagens in extracellular matrix and of lamin-A in nuclei followed power-law scaling versus E. Scaling for mechanoresponsive lamin-A conformed to predictions from polymer physics, whereas lamin-B’s varied weakly. Tumor xenograft studies further demonstrated that matrix determined tissue E, whereas lamin-A levels responded to changes in E. In tissue culture cells, both lamin-A conformation and expression were mechanosensitive, with phosphorylation and turnover of lamin-A correlating inversely with matrix E. Lamin-A knockdown enhanced mesenchymal stem cell differentiation on soft matrix that favored a low-stress, fat phenotype. Lamin-A overexpression or transcriptional induction with a retinoic acid (RA) antagonist enhanced differentiation on stiff matrix toward a high-stress, bone phenotype. Downstream of matrix stiffness, the RA pathway regulated lamin-A transcription, but feedback by lamin-A regulated RA receptor (RARG) translocation into nuclei. High lamin-A levels physically impeded nuclear remodeling under stress but also coregulated other key factors. These factors included both serum response factor (SRF), which promoted expression of stress fiber–associated proteins involved in differentiation, and a Hippo pathway factor (YAP1) involved in growth. Discussion The characteristic stress in normal tissue favors collagen accumulation and a characteristic stiffness that cells transduce through nuclear lamin-A to enhance tissue-specific differentiation. Tension-inhibited turnover of rope-like filaments of lamin-A provides sufficient mechanochemical control of a core gene circuit to explain the steady-state scaling of lamin-A with E. High lamin-A physically stabilizes the nucleus against stress and thereby stabilizes the nuclear lamina and chromatin, with implications for epigenetic stabilization and limiting of DNA breaks. Moreover, lamin-A levels directly or indirectly regulate many proteins involved in tissue-specific gene expression, and, because lamin-A levels can vary by a factor of 10 or more downstream of tissue mechanics, an important fraction of tissue-specific gene expression depends on tissue mechanics, which changes in development, injury, and many diseases. Lamins and Tissue Stiffness Microenvironment can influence cell fate and behavior; for example, extracellular matrix (ECM) stiffness increases cell proliferation, and ECM rigidity induces disorders in tissue morphogenesis by increasing cell tension. Swift et al. (1240104; see the Perspective by Bainer and Weaver) used proteomics to identify molecules that are mechanical sensors for tissue elasticity in the nucleus and discovered that expression of lamin-A levels apparently functions as a “mechanostat.” Tissues that need to remain stiff under stress rely on lamin-A to keep the cell nucleus whole. [Also see Perspective by Bainer and Weaver] 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. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination.

Matrix elasticity, cytoskeletal forces and physics of the nucleus: how deeply do cells ‘feel’ outside and in?

Cellular organization within a multicellular organism requires that a cell assess its relative location, taking in multiple cues from its microenvironment. Given that the extracellular matrix (ECM) consists of the most abundant proteins in animals and contributes both structure and elasticity to tissues, ECM probably provides key physical cues to cells. In vivo, in the vicinity of many tissue cell types, fibrous characteristics of the ECM are less discernible than the measurably distinct elasticity that characterizes different tissue microenvironments. As a cell engages matrix and actively probes, it senses the local elastic resistance of the ECM and nearby cells via their deformation, and — similar to the proverbial princess who feels a pea placed many mattresses below — the cell seems to possess feedback and recognition mechanisms that establish how far it can feel. Recent experimental findings and computational modeling of cell and matrix mechanics lend insight into the subcellular range of sensitivity. Continuity of deformation from the matrix into the cell and further into the cytoskeleton-caged and -linked nucleus also supports the existence of mechanisms that direct processes such as gene expression in the differentiation of stem cells. Ultimately, cells feel the difference between stiff or soft and thick or thin surroundings, regardless of whether or not they are of royal descent.

Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival.

Lamins impede 3D migration but also promote survival against migration-induced stresses.

Viral capsids: Mechanical characteristics, genome packaging and delivery mechanisms

Abstract.The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its consequent confinement within the capsid. It is proposed that this pressure helps driving the genome into the host, but other mechanisms also seem to play an important role in ejection. DNA packaging and ejection strategies are obviously dependent on the mechanical properties of the capsid. This review focuses on the mechanical properties of viral capsids in general and the elucidation of the biophysical aspects of genome packaging mechanisms and genome delivery processes of double-stranded DNA bacteriophages in particular.

Internal DNA pressure modifies stability of WT phage

dsDNA in bacteriophages is highly stressed and exerts internal pressures of many atmospheres (1 atm = 101.3 kPa) on the capsid walls. We investigate the correlation between packaged DNA length in λ phage (78–100% of WT DNA) and capsid strength by using an atomic force microscope indentation technique. We show that phages with WT DNA are twice as strong as shorter genome mutants, which behave like empty capsids, regardless of high internal pressure. Our analytical model of DNA-filled capsid deformation shows that, because of DNA-hydrating water molecules, an osmotic pressure exists inside capsids that increases exponentially when the packaged DNA density is close to WT phage. This osmotic pressure raises the WT capsid strength and is approximately equal to the maximum breaking force of empty shells. This result suggests that the strength of the shells limits the maximal packaged genome length. Moreover, it implies an evolutionary optimization of WT phages allowing them to survive greater external mechanical stresses in nature.

Contractile Forces Sustain and Polarize Hematopoiesis from Stem and Progenitor Cells

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Stem cell mechanobiology: diverse lessons from bone marrow

A stem cell niche is defined by various chemical and physical features that influence whether a stem cell remains quiescent, divides, or differentiates. We review mechanical determinants that affect cell fate through actomyosin forces, nucleoskeleton remodeling, and mechanosensitive translocation of transcription factors. Current methods for physical characterization of tissue microenvironments are summarized together with efforts to recapitulate niche mechanics in culture. We focus on mesenchymal stem cells, particularly in osteogenesis and adipogenesis, and on blood stem cells - both of which reside in mechanically diverse marrow microenvironments. Given the explosion of efforts with pluripotent stem cells, the evident mechanosensitivity of clinically relevant, multipotent marrow cells underscores an increasing need to examine and understand in vivo and in vitro physical properties on length scales that cells sense.

Mechanobiology of bone marrow stem cells: From myosin-II forces to compliance of matrix and nucleus in cell forms and fates

Adult stem cells and progenitors are of great interest for their clinical application as well as their potential to reveal deep sensitivities to microenvironmental factors. The bone marrow is a niche for at least two types of stem cells, and the prototype is the hematopoietic stem cell/progenitors (HSC/Ps), which have saved many thousands of patients for several decades now. In bone marrow, HSC/Ps interact functionally with marrow stromal cells that are often referred to as mesenchymal stem cells (MSCs) or derivatives thereof. Myosin and matrix elasticity greatly affect MSC function, and these mechanobiological factors are now being explored with HSC/Ps both in vitro and in vivo. Also emerging is a role for the nucleus as a mechanically sensitive organelle that is semi-permeable to transcription factors which are modified for nuclear entry by cytoplasmic mechanobiological pathways. Since therapies envisioned with induced pluripotent stem cells and embryonic stem cells generally involve in vitro commitment to an adult stem cell or progenitor, a very deep understanding of stem cell mechanobiology is essential to progress with these multi-potent cells.

Discrete fracture patterns of virus shells reveal mechanical building blocks

Viral shells are self-assembled protein nanocontainers with remarkable material properties. They combine simplicity of construction with toughness and complex functionality. These properties make them interesting for bionanotechnology. To date we know little about how virus structure determines assembly pathways and shell mechanics. We have here used atomic force microscopy to study structural failure of the shells of the bacteriophage Φ29. We observed rigidity patterns following the symmetry of the capsid proteins. Under prolonged force exertion, we observed fracture along well-defined lines of the 2D crystal lattice. The mechanically most stable building block of the shells was a trimer. Our approach of “reverse engineering” the virus shells thus made it possible to identify stable structural intermediates. Such stable intermediates point to a hierarchy of interactions among equal building blocks correlated with distinct next-neighbor interactions. The results also demonstrate that concepts from macroscopic materials science, such as fracture, can be usefully employed in molecular engineering.

Effects of salts on internal DNA pressure and mechanical properties of phage capsids.

Based on atomic force microscopy nanoindentation measurements of phage λ, we previously proposed a minimal model describing the effect of water hydrating DNA that strengthens viral capsids against external deformation at wild-type DNA packing density. Here, we report proof of this model by testing the prediction that DNA hydration forces can be dramatically decreased by addition of multivalent ions (Mg(2+) and Sp(4+)). These results are explained using a DNA hydration model without adjustable parameters. The model also predicts the stiffness of other DNA-filled capsids, which we confirm using bacteriophage ϕ29 and herpes simplex virus type 1 particles.