α-Actinin-4 drives invasiveness by regulating myosin IIB expression and myosin IIA localization.

The mechanisms by which the mechanoresponsive actin crosslinking protein α-actinin-4 (ACTN4) regulates cell motility and invasiveness remain incompletely understood. Here, we show that, in addition to regulating protrusion dynamics and focal adhesion formation, ACTN4 transcriptionally regulates expression of non-muscle myosin IIB (NMM IIB; heavy chain encoded by MYH10), which is essential for mediating nuclear translocation during 3D invasion. We further show that an indirect association between ACTN4 and NMM IIA (heavy chain encoded by MYH9) mediated by a functional F-actin cytoskeleton is essential for retention of NMM IIA at the cell periphery and modulation of focal adhesion dynamics. A protrusion-dependent model of confined migration recapitulating experimental observations predicts a dependence of protrusion forces on the degree of confinement and on the ratio of nucleus to matrix stiffness. Together, our results suggest that ACTN4 is a master regulator of cancer invasion that regulates invasiveness by controlling NMM IIB expression and NMM IIA localization. This article has an associated First Person interview with the first author of the paper.

Combined heterogeneity in cell size and deformability promotes cancer invasiveness.

Phenotypic heterogeneity is increasingly acknowledged to confer several advantages to cancer progression and drug resistance. Here, we probe the collective importance of heterogeneity in cell size and deformability in breast cancer invasion. A computational model of invasion of a heterogeneous cell aggregate predicts that combined heterogeneity in cell size and deformability enhances invasiveness of the whole population, with maximum invasiveness at intermediate cell-cell adhesion. We then show that small cells of varying deformability, a subpopulation predicted to be enriched at the invasive front, exhibit considerable overlap with the biophysical properties of cancer stem cells (CSCs). In MDA-MB-231 cells, these include CD44 hi CD24- mesenchymal CSCs, which are small and soft, and CD44 hi CD24+ hybrid CSCs, which exhibit a wide range of size and deformability. We validate our predictions by tracking the pattern of cell invasion from spheroids implanted in three-dimensional collagen gels, wherein we show temporal enrichment of CD44 hi cells at the invasive front. Collectively, our results illustrate the advantages imparted by biophysical heterogeneity in enhancing cancer invasiveness.This article has an associated First Person interview with the first author of the paper.

Nuclear plasticity increases susceptibility to damage during confined migration.

Large nuclear deformations during migration through confined spaces have been associated with nuclear membrane rupture and DNA damage. However, the stresses associated with nuclear damage remain unclear. Here, using a quasi-static plane strain finite element model, we map evolution of nuclear shape and stresses during confined migration of a cell through a deformable matrix. Plastic deformation of the nucleus observed for a cell with stiff nucleus transiting through a stiffer matrix lowered nuclear stresses, but also led to kinking of the nuclear membrane. In line with model predictions, transwell migration experiments with fibrosarcoma cells showed that while nuclear softening increased invasiveness, nuclear stiffening led to plastic deformation and higher levels of DNA damage. In addition to highlighting the advantage of nuclear softening during confined migration, our results suggest that plastic deformations of the nucleus during transit through stiff tissues may lead to bending-induced nuclear membrane disruption and subsequent DNA damage.

α-Actinin-4 confers radioresistance coupled invasiveness in breast cancer cells through AKT pathway.

Acquired radioresistance accompanied with increased metastatic potential is a major hurdle in effective radiotherapy of breast cancers. However, the nature of their inter-dependence and the underlying mechanism remains largely intangible. By employing radioresistant (RR) cell lines, we herein demonstrate that MCF-7 RR cells display phenotypic and molecular alterations evocative of epithelial to mesenchymal transition (EMT) with increased traction forces and membrane ruffling culminating in boosted invasiveness. We then show that these changes can be attributed to overexpression of alpha-actinin-4 (ACTN4), with ACTN4 knockdown near-completely abrogating both radioresistance and EMT-associated changes. We further found that in MCF-7 RR cells, ACTN4 mediates the observed effects by activating AKT, and downstream AKT/GSK3ß signalling. Though ACTN4 plays a similar role in mediating radioresistance and invasiveness in MDA-MB-231 RR cells, co-immunoprecipitation studies reveal that these changes are effected through increased association with AKT and not by overexpression of AKT. Taken together, our study identifies ACTN4/AKT/GSK3ß as a novel pathway regulating radioresistance coupled invasion which can be further explored to improve the radiotherapeutic gain.

Soft drug-resistant ovarian cancer cells migrate via two distinct mechanisms utilizing myosin II-based contractility.

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the migration mechanisms of drug-resistant cancer cells remain incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are more contractile and softer than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR cells, indicative of a protease-dependent mode of migration in CisR cells and a protease-independent mode of migration in PacR. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin II signaling pathway, regulates both modes of migration. Confined migration experiments establish the role of myosin IIA and IIB in mediating nuclear translocation and regulation of proteolytic activity. Collectively, our results highlight the importance of myosin II as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells.

MMP Secretion Rate and Inter-invadopodia Spacing Collectively Govern Cancer Invasiveness.

Invadopodia are micron-sized invasive structures that mediate extracellular matrix (ECM) degradation through a combination of membrane-bound and soluble matrix metalloproteinases (MMPs). However, how such localized degradation is converted into pores big enough for cancer cells to invade, and the relative contributions of membrane-bound versus soluble MMPs to this process remain unclear. In this article, we address these questions by combining experiments and simulations. We show that in MDA-MB-231 cells, an increase in ECM density enhances invadopodia-mediated ECM degradation and decreases inter-invadopodia spacing. ECM degradation is mostly mediated by soluble MMPs, which are activated by membrane-bound MT1-MMP. We present a computational model of invadopodia-mediated ECM degradation, which recapitulates the above observations and identifies MMP secretion rate as an important regulator of invadopodia stability. Simulations with multiple invadopodia suggest that inter-invadopodia spacing and MMP secretion rate collectively dictate the size of the degraded zones. Taken together, our results suggest that for creating pores conducive for cancer invasion, cells must tune inter-invadopodia spacing and MMP secretion rate in an ECM density-dependent manner, thereby striking a balance between invadopodia penetration and ECM degradation.

Length limitation of astral microtubules orients cell divisions in murine intestinal crypts.

Planar spindle orientation is critical for epithelial tissue organization and is generally instructed by the long cell-shape axis or cortical polarity domains. We introduced mouse intestinal organoids in order to study spindle orientation in a monolayered mammalian epithelium. Although spindles were planar, mitotic cells remained elongated along the apico-basal (A-B) axis, and polarity complexes were segregated to basal poles, so that spindles oriented in an unconventional manner, orthogonal to both polarity and geometric cues. Using high-resolution 3D imaging, simulations, and cell-shape and cytoskeleton manipulations, we show that planar divisions resulted from a length limitation in astral microtubules (MTs) which precludes them from interacting with basal polarity, and orient spindles from the local geometry of apical domains. Accordingly, lengthening MTs affected spindle planarity, cell positioning, and crypt arrangement. We conclude that MT length regulation may serve as a key mechanism for spindles to sense local cell shapes and tissue forces to preserve mammalian epithelial architecture.

Amoebal Tubulin Cleavage Late during Infection Is a Characteristic Feature of Mimivirus but Not of Marseillevirus.

Mimivirus and Marseillevirus infections of Acanthamoeba castellanii, like most other viral infections, induce cytopathic effects (CPE). The details of how they bring about CPE and to what extent and how they modify the host cytoskeletal network are unclear. In this study, we compared the rearrangement of the host cytoskeletal network induced by Mimivirus and Marseillevirus upon infection. We show that while both Mimivirus and Marseillevirus infections of A. castellanii cells cause retraction of acanthopodia and depolymerization of the host actin filament network, the Mimivirus infection also results in characteristic cleavage of the host tubulin, a phenomenon not previously reported with any intracellular pathogens. Furthermore, we show that the amoebal tubulin cleavage during Mimivirus infection is a post-replicative event. Because time-lapse microscopy showed that Mimivirus infection leads to the bursting of cells, releasing the virus, we hypothesize that tubulin cleavage together with actin depolymerization during the later stages of Mimivirus assembly is essential for cell lysis due to apoptotic/necrotic cell death. We also characterize the Mimivirus-encoded gp560, a Zn metalloprotease, however, the purified gp560 protein was unable to cleave the commercially available porcine brain tubulin. While protein synthesis is essential for causing the morphological changes in the case of Mimivirus, the proteins which are packaged in the viral capsid along with the genome are sufficient to induce CPE in the case of Marseillevirus. IMPORTANCE In general, intracellular pathogens target the cytoskeletal network to enable their life cycle inside the host. Pathogen-induced changes in the host cell morphology usually accompany global changes in the cytoskeleton resulting in cytopathic effects. While viruses have been shown to use the host actin cytoskeleton for entry and transport during early infection, the role of microtubules in the viral life cycle is only beginning to emerge. Here, we show that the giant viruses Mimivirus and Marseillevirus both induce depolymerization of the actin filament, Mimivirus also causes a characteristic cleavage of tubulin not previously reported for any intracellular pathogen. Because tubulin cleavage occurs late during infection, we hypothesize that tubulin cleavage aids in cell death and lysis rather than establishing infection. The different strategies used by viruses with similar host niches may help them survive in competition.

Geometry of a DNA Nanostructure Influences Its Endocytosis: Cellular Study on 2D, 3D, and in Vivo Systems.

Fabrication of nanoscale DNA devices to generate 3D nano-objects with precise control of shape, size, and presentation of ligands has shown tremendous potential for therapeutic applications. The interactions between the cell membrane and different topologies of 3D DNA nanostructures are crucial for designing efficient tools for interfacing DNA devices with biological systems. The practical applications of these DNA nanocages are still limited in cellular and biological systems owing to the limited understanding of their interaction with the cell membrane and endocytic pathway. The correlation between the geometry of DNA nanostructures and their internalization efficiency remains elusive. We investigated the influence of the shape and size of 3D DNA nanostructures on their cellular internalization efficiency. We found that one particular geometry, i.e., the tetrahedral shape, is more favored over other designed geometries for their cellular uptake in 2D and 3D cell models. This is also replicable for cellular processes like cell invasion assays in a 3D spheroid model, and passing the epithelial barriers in in vivo zebrafish model systems. Our work provides detailed information for the rational design of DNA nanodevices for their upcoming biological and biomedical applications.

Measuring microenvironment-tuned nuclear stiffness of cancer cells with atomic force microscopy.

Quantification of nuclear stiffness is challenging for cells encapsulated within a 3D extracellular matrix (ECM). Here, we describe an experimental setup for measuring microenvironment-dependent tuning of nuclear stiffness using an atomic force microscope (AFM). In our setup, ECM-coated polyacrylamide hydrogels mimic the stiffness of the microenvironment, enabling the measurement of nuclear stiffness using an AFM probe in live cancer cells. For complete details on the use and execution of this protocol, please refer to Das et al. (2019) (https://doi.org/10.1016/j.matbio.2019.01.001).

High ligand density drives extensive spreading and motility on soft GelMA gels.

In comparison to synthetic hydrogels where ligand density and stiffness can be independently tuned, cell responses are expected to deviate on native biopolymer networks where ligand density and stiffness are coupled. Here we probe the tensional homeostasis of fibroblasts on methacrylated gelatin (GelMA) gels, which are widely used in tissue engineering applications. On 5%-15% GelMA gels which are very soft (10-100's of Pa's in stiffness), fibroblasts were found to spread extensively and assemble prominent stress fibers and focal adhesions. Probing of contractile mechanics using trypsin-induced detachment revealed adhesive drag, but not contractility, was sensitive to GelMA concentration. Contractility-altering drugs blebbistatin and nocodazole, which exhibited opposite effects on focal adhesion size, both led to reduction in adhesive drag and cell rounding. However, cell motility was impacted only in nocodazole-treated cells. Collectively, our experiments suggest that on soft GelMA gels, contractility-independent adhesion clustering mediated by high ligand density can drive cell spreading and motility.

EpCAM-Mediated Cellular Plasticity Promotes Radiation Resistance and Metastasis in Breast Cancer.

Substantial number of breast cancer (BC) patients undergoing radiation therapy (RT) develop local recurrence over time. During RT therapy, cells can gradually acquire resistance implying adaptive radioresistance. Here we probe the mechanisms underlying this acquired resistance by first establishing radioresistant lines using ZR-75-1 and MCF-7 BC cells through repeated exposure to sub-lethal fractionated dose of 2Gy up to 15 fractions. Radioresistance was found to be associated with increased cancer stem cells (CSCs), and elevated EpCAM expression in the cell population. A retrospective analysis of TCGA dataset indicated positive correlation of high EpCAM expression with poor response to RT. Intriguingly, elevated EpCAM expression in the radioresistant CSCs raise the bigger question of how this biomarker expression contributes during radiation treatment in BC. Thereafter, we establish EpCAM overexpressing ZR-75-1 cells (ZR-75-1EpCAM), which conferred radioresistance, increased stemness through enhanced AKT activation and induced a hybrid epithelial/mesenchymal phenotype with enhanced contractility and invasiveness. In line with these observations, orthotopic implantation of ZR-75-1EpCAM cells exhibited faster growth, lesser sensitivity to radiation therapy and increased lung metastasis than baseline ZR-75-1 cells in mice. In summary, this study shows that similar to radioresistant BC cells, EpCAM overexpressing cells show high degree of plasticity and heterogeneity which ultimately induces radioresistant and metastatic behavior of cancer cells, thus aggravating the disease condition.

Nuclear softening is essential for protease-independent migration.

During amoeboidal migration, cancer cells migrate in a protease-independent manner by squeezing through pre-existing gaps in the extracellular matrix (ECM). However, the extent to which cells alter their physical properties in order to sustain this mode of migration remains unclear. Here, we address this question by documenting biophysical changes in the properties of highly invasive MDA-MB-231 and HT-1080 cells upon inhibition of pericellular proteolysis. Remarkably, treatment with the broad spectrum MMP inhibitor GM6001 not only induces cell rounding and loss of actomyosin contractility, but also induces nuclear softening via increased phosphorylation of the nuclear membrane protein lamin A/C. Though nuclear softening is necessary for sustaining migration through sub-nuclear sized transwell pores, it is not sufficient. In addition, baseline levels of contractility mediating pore entry and peri-nuclear actin inside the pores mediating pore migration are also required. Taken together, our results suggest that protease-independent migration through sub-nuclear sized pre-existing tracks is enabled by deformation of a softened nucleus by contractility and the peri-nuclear actin network.

Engineering interfacial migration by collective tuning of adhesion anisotropy and stiffness.

Interfacial migration is central to multiple processes including morphogenesis and wound healing. However, the sensitivity of interfacial migration to properties of the interfacial microenvironment has not been adequately explored. Here, we address this question by tracking motility of 3T3 fibroblasts at the interface of two hydrogels. By sandwiching cells between two adhesive gels (composed of methacrylated gelatin) or between an adhesive and a non-adhesive gel (composed of gellan), we show that cells are more motile in case of the latter. By tuning the bulk stiffness of the gellan gel, we then show that motility is tuned in a stiffness-dependent manner. Fastest motility observed in case of the stiffest gel was associated with increased cell height, suggestive of stiffness-mediated cytoskeletal assembly. Inhibition of cell motility by contractile agonists and actin depolymerizing drugs is indicative of a mode of migration wherein cells combine contractile tractions exerted at their base and actin-based pushing forces on the top surface to propel themselves forward. Together, our results suggest that dorso-ventral adhesion anisotropy and stiffness can be collectively tuned to engineer interfacial migration. STATEMENT OF SIGNIFICANCE: It is increasingly understood that cells migrate in vivo through confining spaces which typically occur as pores in the matrix and through naturally occurring interfaces that exist between neighbouring ECM fibers, or between the stroma and the vasculature. Such interfaces are also created when treating wounds on the skin surface by covering the wounds with adhesives. How multiple cues impact interfacial migration has not been adequately addressed. By studying cell migratory behaviour at the interface of two hydrogel substrates, we identify adhesivity and stiffness as two critical factors that can be tuned to maximize cell migration. We foresee a potential use of this knowledge in the design of tissue adhesives for wound healing applications.

MMP proteolytic activity regulates cancer invasiveness by modulating integrins.

Cancer invasion through dense extracellular matrices (ECMs) is mediated by matrix metalloproteinases (MMPs) which degrade the ECM thereby creating paths for migration. However, how this degradation influences the phenotype of cancer cells is not fully clear. Here we address this question by probing the function of MMPs in regulating biophysical properties of cancer cells relevant to invasion. We show that MMP catalytic activity regulates cell spreading, motility, contractility and cortical stiffness by stabilizing integrins at the membrane and activating focal adhesion kinase. Interestingly, cell rounding and cell softening on stiff gels induced by MMP inhibition is attenuated on MMP pre-conditioned surfaces. Together, our results suggest that MMP catalytic activity regulates invasiveness of cancer cells by modulating integrins.

Biophysical regulation of mouse embryonic stem cell fate and genomic integrity by feeder derived matrices.

For maintaining pluripotency, mouse embryonic stem cells (mESCs) are typically grown on mitotically inactivated mouse embryonic fibroblasts (MEFs). While the role of MEF conditioned media (MEFCM) and leukemia inhibitory factor (LIF) in regulating mESC pluripotency has led to culturing of mESCs on LIF/MEFCM supplemented gelatin-coated substrates, the role of physical interactions between MEFs and mESCs in regulating mESC pluripotency remains to be fully understood. Here, we address this question by characterizing the physicochemical properties of MEF derived matrices (MEFDMs), and probing their role in regulating mESC fate. We show that MEFDM composition and stiffness-dictated by MEF contractility-regulates mESC pluripotency by modulating mESC contractility through integrin-mediated mechanoadaptation. While baseline mESC pluripotency is maintained at early time points, activation of mESC contractility by LPA leads to drop in pluripotency levels. In contrast, addition of blebbistatin and LIF independently increases pluripotency by suppressing mechanoadaptation, highlighting the role of mechanoadaptation in regulating pluripotency and illustrating the role of LIF as a mechano-inhibitor in mESCs. Long-term culture of mESCs on MEFDMs under LIF-free conditions triggers loss of pluripotency, and induces ligand-dependent expression of the osteogenic transcription factor Runx2. Maintenance of genomic integrity (euploidy) on MEFDMs but not on gelatin-coated substrates, combined with the ability of MEFDMs in supporting LIF-free expansion and differentiation of mESCs, illustrates the suitability of MEFDMs for clinical and regenerative medicine applications.