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.

Nuclear mechanoprotection: From tissue atlases as blueprints to distinctive regulation of nuclear lamins.

Two meters of DNA in each of our cells must be protected against many types of damage. Mechanoprotection is increasingly understood to be conferred by the nuclear lamina of intermediate filament proteins, but very different patterns of expression and regulation between different cells and tissues remain a challenge to comprehend and translate into applications. We begin with a tutorial style presentation of "tissue blueprints" of lamin expression including single-cell RNA sequencing in major public datasets. Lamin-A, C profiles appear strikingly similar to those for the mechanosensitive factors Vinculin, Yap1, and Piezo1, whereas datasets for lamin-B1 align with and predict regulation by the cell cycle transcription factor, FOXM1, and further predict poor survival across multiple cancers. Various experiments support the distinction between the lamin types and add mechanistic insight into the mechano-regulation of lamin-A, C by both matrix elasticity and externally imposed tissue strain. Both A- and B-type lamins, nonetheless, protect the nucleus from rupture and damage. Ultimately, for mechanically active tissue constructs and organoids as well as cell therapies, lamin levels require particular attention as they help minimize nuclear damage and defects in a cell cycle.

Self-Assembled Human Skin Equivalents Model Macrophage Activation of Cutaneous Fibrogenesis in Systemic Sclerosis.

OBJECTIVE: The development of precision therapeutics for systemic sclerosis (SSc) has been hindered by the lack of models that accurately mimic the disease in vitro. This study was undertaken to design and test a self-assembled skin equivalent (saSE) system that recapitulates the cross-talk between macrophages and fibroblasts in cutaneous fibrosis. METHODS: SSc-derived dermal fibroblasts (SScDFs) and normal dermal fibroblasts (NDFs) were cultured with CD14+ monocytes from SSc patients or healthy controls to allow de novo stroma formation. Monocyte donor-matched plasma was introduced at week 3 prior to seeding keratinocytes to produce saSE with a stratified epithelium. Tissue was characterized by immunohistochemical staining, atomic force microscopy, enzyme-linked immunosorbent assay, and quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Stroma synthesized de novo from NDFs and SScDFs supported a fully stratified epithelium to form saSE. A thicker and stiffer dermis was generated by saSE with SScDFs, and more interleukin-6 and transforming growth factor ß (TGFß) was secreted by saSE with SScDFs compared to saSE with NDFs, regardless of the inclusion of monocytes. Tissue with SSc monocytes and plasma had amplified dermal thickness and stiffness relative to control tissue. Viable CD163+ macrophages were found within the stroma of saSE 5 weeks after seeding. Additionally, SSc saSE contained greater numbers of CD163+ and CD206+ macrophages compared to control saSE. TGFß blockade inhibited stromal stiffness to a greater extent in SSc saSE compared to control saSE. CONCLUSION: These data suggest reciprocal activation between macrophages and fibroblasts that increases tissue thickness and stiffness, which is dependent in part on TGFß activation. The saSE system may serve as a platform for preclinical therapeutic testing and for molecular characterization of SSc skin pathology through recapitulation of the interactions between macrophages and fibroblasts.

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.

Filomicelles Deliver a Chemo-Differentiation Combination of Paclitaxel and Retinoic Acid That Durably Represses Carcinomas in Liver to Prolong Survival.

Drug resistance and relapse is common in cancer treatments with chemotherapeutics, and while drug combinations with naturally occurring, differentiation-inducing retinoic acid (RA) provide remission-free cures for one type of liquid tumor, solid tumors present major problems for delivery. Here, inspired by filoviruses that can be microns in length, flexible filomicelles that self-assemble from an amphiphilic block copolymer (PEG-PCL) are shown to effectively deliver RA and paclitaxel (TAX) to several solid tumor models, particularly in the liver. These hydrophobic compounds synergistically load into the cores of the elongated micelles, and the coloaded micelles prove most effective at causing cell death, ploidy, and durable regression of tumors compared to free drugs or to separately loaded drugs. RA-TAX filomicelles also reduce mortality of human lung or liver derived cancers engrafted at liver, intraperitoneal, and subcutaneous sites in immunodeficient mice. In vitro studies show that the dual drug micelles effectively suppress proliferation while upregulating a generic differentiation marker. The results highlight the potency of dual-loaded filomicelles in killing cancer cells or else driving their differentiation away from growth.

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.

Evaluation of JAK3 Biology in Autoimmune Disease Using a Highly Selective, Irreversible JAK3 Inhibitor.

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.

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.

"Marker of Self" CD47 on lentiviral vectors decreases macrophage-mediated clearance and increases delivery to SIRPA-expressing lung carcinoma tumors.

Lentiviruses infect many cell types and are now widely used for gene delivery in vitro, but in vivo uptake of these foreign vectors by macrophages is a limitation. Lentivectors are produced here from packaging cells that overexpress "Marker of Self" CD47, which inhibits macrophage uptake of cells when prophagocytic factors are also displayed. Single particle analyses show "hCD47-Lenti" display properly oriented human-CD47 for interactions with the macrophage's inhibitory receptor SIRPA. Macrophages derived from human and NOD/SCID/Il2rg-/- (NSG) mice show a SIRPA-dependent decrease in transduction, i.e., transgene expression, by hCD47-Lenti compared to control Lenti. Consistent with known "Self" signaling pathways, macrophage transduction by control Lenti is decreased by drug inhibition of Myosin-II to the same levels as hCD47-Lenti. In contrast, human lung carcinoma cells express SIRPA and use it to enhance transduction by hCD47-Lenti- as illustrated by more efficient gene deletion using CRISPR/Cas9. Intravenous injection of hCD47-Lenti into NSG mice shows hCD47 prolongs circulation, unless a blocking anti-SIRPA is preinjected. In vivo transduction of spleen and liver macrophages also decreases for hCD47-Lenti while transduction of lung carcinoma xenografts increases. hCD47 could be useful when macrophage uptake is limiting on other viral vectors that are emerging in cancer treatments (e.g., Measles glycoprotein-pseudotyped lentivectors) and also in targeting various SIRPA-expressing tumors such as glioblastomas.

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.

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.

Gene signatures derived from a c-MET-driven liver cancer mouse model predict survival of patients with hepatocellular carcinoma.

Biomarkers derived from gene expression profiling data may have a high false-positive rate and must be rigorously validated using independent clinical data sets, which are not always available. Although animal model systems could provide alternative data sets to formulate hypotheses and limit the number of signatures to be tested in clinical samples, the predictive power of such an approach is not yet proven. The present study aims to analyze the molecular signatures of liver cancer in a c-MET-transgenic mouse model and investigate its prognostic relevance to human hepatocellular carcinoma (HCC). Tissue samples were obtained from tumor (TU), adjacent non-tumor (AN) and distant normal (DN) liver in Tet-operator regulated (TRE) human c-MET transgenic mice (n = 21) as well as from a Chinese cohort of 272 HBV- and 9 HCV-associated HCC patients. Whole genome microarray expression profiling was conducted in Affymetrix gene expression chips, and prognostic significances of gene expression signatures were evaluated across the two species. Our data revealed parallels between mouse and human liver tumors, including down-regulation of metabolic pathways and up-regulation of cell cycle processes. The mouse tumors were most similar to a subset of patient samples characterized by activation of the Wnt pathway, but distinctive in the p53 pathway signals. Of potential clinical utility, we identified a set of genes that were down regulated in both mouse tumors and human HCC having significant predictive power on overall and disease-free survival, which were highly enriched for metabolic functions. In conclusions, this study provides evidence that a disease model can serve as a possible platform for generating hypotheses to be tested in human tissues and highlights an efficient method for generating biomarker signatures before extensive clinical trials have been initiated.