Small lipid droplets are rigid enough to indent a nucleus, dilute the lamina, and cause rupture.

The nucleus in many cell types is a stiff organelle, but fat-filled lipid droplets (FDs) in cytoplasm are seen to indent and displace the nucleus. FDs are phase-separated liquids with a poorly understood interfacial tension γ that determines how FDs interact with other organelles. Here, micron-sized FDs remain spherical as they indent peri-nuclear actomyosin and the nucleus, while causing local dilution of Lamin-B1 independent of Lamin-A,C and sometimes triggering nuclear rupture. Focal accumulation of the cytosolic DNA sensor cGAS at the rupture site is accompanied by sustained mislocalization of DNA repair factors to cytoplasm, increased DNA damage, and delayed cell cycle. Macrophages show FDs and engulfed rigid beads cause similar indentation dilution. Spherical shapes of small FDs indicate a high γ, which we measure for FDs mechanically isolated from fresh adipose tissue as ∼40 mN/m. This value is far higher than that of protein condensates, but typical of oils in water and sufficiently rigid to perturb cell structures including nuclei.

Rescue of DNA damage after constricted migration reveals a mechano-regulated threshold for cell cycle.

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation.

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. Embryonic hearts, cardiac-differentiated iPS cells (induced pluripotent stem cells), and various nonmuscle cell types all show that actomyosin-driven nuclear rupture causes cytoplasmic mis-localization of DNA repair factors and excess DNA damage. Binucleation and micronuclei increase as telomeres shorten, which all favor cell-cycle arrest. Deficiencies in lamin-A and repair factors exacerbate these effects, but lamin-A-associated defects are rescued by repair factor overexpression and also by contractility modulators in clinical trials. Contractile cells on stiff ECM normally exhibit low phosphorylation and slow degradation of lamin-A by matrix-metalloprotease-2 (MMP2), and inhibition of this lamin-A turnover and also actomyosin contractility are seen to minimize DNA damage. Lamin-A is thus stress stabilized to mechano-protect the genome.

Manipulating the mechanics of extracellular matrix to study effects on the nucleus and its structure.

Tissues such as brain, muscle, and bone differ greatly not only in their biological functions but also in their mechanical properties. Brain is far softer than muscle while bone is the stiffest tissue. Stiffness of extracellular microenvironments affects fundamental cell biological processes such as polarization and DNA replication, which affect nuclear size, shape, and levels of nuclear proteins such as the lamins that modulate gene expression. Reductionist approaches have helped dissect the effects of matrix mechanics away from confounding biochemical signals. Here, we summarize materials and methods for synthesizing and characterizing soft and stiff synthetic hydrogels widely used for mechanobiological studies. Such gels are also easily made to mimic the mechanical heterogeneity of fibrotic tissues. We further describe a nano-thin collagen fiber system, which enables control of anisotropy in addition to stiffness. With the different systems, we illustrate the effects of matrix mechanics on nuclear size, shape, and proteins including the lamins.

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors.

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage.

Progerin phosphorylation in interphase is lower and less mechanosensitive than lamin-A,C in iPS-derived mesenchymal stem cells.

Interphase phosphorylation of lamin-A,C depends dynamically on a cell's microenvironment, including the stiffness of extracellular matrix. However, phosphorylation dynamics is poorly understood for diseased forms such as progerin, a permanently farnesylated mutant of LMNA that accelerates aging of stiff and mechanically stressed tissues. Here, fine-excision alignment mass spectrometry (FEA-MS) is developed to quantify progerin and its phosphorylation levels in patient iPS cells differentiated to mesenchymal stem cells (MSCs). The stoichiometry of total A-type lamins (including progerin) versus B-type lamins measured for Progeria iPS-MSCs prove similar to that of normal MSCs, with total A-type lamins more abundant than B-type lamins. However, progerin behaves more like farnesylated B-type lamins in mechanically-induced segregation from nuclear blebs. Phosphorylation of progerin at multiple sites in iPS-MSCs cultured on rigid plastic is also lower than that of normal lamin-A and C. Reduction of nuclear tension upon i) cell rounding/detachment from plastic, ii) culture on soft gels, and iii) inhibition of actomyosin stress increases phosphorylation and degradation of lamin-C > lamin-A > progerin. Such mechano-sensitivity diminishes, however, with passage as progerin and DNA damage accumulate. Lastly, transcription-regulating retinoids exert equal effects on both diseased and normal A-type lamins, suggesting a differential mechano-responsiveness might best explain the stiff tissue defects in Progeria.

Cross-linked matrix rigidity and soluble retinoids synergize in nuclear lamina regulation of stem cell differentiation.

Synergistic cues from extracellular matrix and soluble factors are often obscure in differentiation. Here the rigidity of cross-linked collagen synergizes with retinoids in the osteogenesis of human marrow mesenchymal stem cells (MSCs). Collagen nanofilms serve as a model matrix that MSCs can easily deform unless the film is enzymatically cross-linked, which promotes the spreading of cells and the stiffening of nuclei as both actomyosin assembly and nucleoskeletal lamin-A increase. Expression of lamin-A is known to be controlled by retinoic acid receptor (RAR) transcription factors, but soft matrix prevents any response to any retinoids. Rigid matrix is needed to induce rapid nuclear accumulation of the RARG isoform and for RARG-specific antagonist to increase or maintain expression of lamin-A as well as for RARG-agonist to repress expression. A progerin allele of lamin-A is regulated in the same manner in iPSC-derived MSCs. Rigid matrices are further required for eventual expression of osteogenic markers, and RARG-antagonist strongly drives lamin-A-dependent osteogenesis on rigid substrates, with pretreated xenografts calcifying in vivo to a similar extent as native bone. Proteomics-detected targets of mechanosensitive lamin-A and retinoids underscore the convergent synergy of insoluble and soluble cues in differentiation.

Nuclear constriction segregates mobile nuclear proteins away from chromatin.

As a cell squeezes its nucleus through adjacent tissue, penetrates a basement membrane, or enters a small blood capillary, chromatin density and nuclear factors could in principle be physically perturbed. Here, in cancer cell migration through rigid micropores and in passive pulling into micropipettes, local compaction of chromatin is observed coincident with depletion of mobile factors. Heterochromatin/euchromatin was previously estimated from molecular mobility measurements to occupy a volume fraction f of roughly two-thirds of the nuclear volume, but based on the relative intensity of DNA and histones in several cancer cell lines drawn into narrow constrictions, f can easily increase locally to nearly 100%. By contrast, mobile proteins in the nucleus, including a dozen that function as DNA repair proteins (e.g., BRCA1, 53BP1) or nucleases (e.g., Cas9, FokI), are depleted within the constriction, approaching 0%. Such losses-compounded by the occasional rupture of the nuclear envelope-can have important functional consequences. Studies of a nuclease that targets a locus in chromosome-1 indeed show that constricted migration delays DNA damage.

Nuclear lamins in cancer.

Dysmorphic nuclei are commonly seen in cancers and provide strong motivation for studying the main structural proteins of nuclei, the lamins, in cancer. Past studies have also demonstrated the significance of microenvironment mechanics to cancer progression, which is extremely interesting because the lamina was recently shown to be mechanosensitive. Here, we review current knowledge relating cancer progression to lamina biophysics. Lamin levels can constrain cancer cell migration in 3D and thereby impede tumor growth, and lamins can also protect a cancer cell's genome. In addition, lamins can influence transcriptional regulators (RAR, SRF, YAP/TAZ) and chromosome conformation in lamina associated domains. Further investigation of the roles for lamins in cancer and even DNA damage may lead to new therapies or at least to a clearer understanding of lamins as bio-markers in cancer progression.

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.

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

Cell migration through solid tissue often involves large contortions of the nucleus, but biological significance is largely unclear. The nucleoskeletal protein lamin-A varies both within and between cell types and was shown here to contribute to cell sorting and survival in migration through constraining micropores. Lamin-A proved rate-limiting in 3D migration of diverse human cells that ranged from glioma and adenocarcinoma lines to primary mesenchymal stem cells (MSCs). Stoichiometry of A- to B-type lamins established an activation barrier, with high lamin-A:B producing extruded nuclear shapes after migration. Because the juxtaposed A and B polymer assemblies respectively conferred viscous and elastic stiffness to the nucleus, subpopulations with different A:B levels sorted in 3D migration. However, net migration was also biphasic in lamin-A, as wild-type lamin-A levels protected against stress-induced death, whereas deep knockdown caused broad defects in stress resistance. In vivo xenografts proved consistent with A:B-based cell sorting, and intermediate A:B-enhanced tumor growth. Lamins thus impede 3D migration but also promote survival against migration-induced stresses.

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.

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. 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.

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.

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.

Failure of viral shells.

We report a combined theoretical and experimental study of the structural failure of viral shells under mechanical stress. We find that discontinuities in the force-indentation curve associated with failure should appear when the so-called Föppl-von Kármán (FvK) number exceeds a critical value. A nanoindentation study of a viral shell subject to a soft-mode instability, where the stiffness of the shell decreases with increasing pH, confirms the predicted onset of failure as a function of the FvK number.