Myoscaffolds reveal laminin scarring is detrimental for stem cell function while sarcospan induces compensatory fibrosis.

We developed an on-slide decellularization approach to generate acellular extracellular matrix (ECM) myoscaffolds that can be repopulated with various cell types to interrogate cell-ECM interactions. Using this platform, we investigated whether fibrotic ECM scarring affected human skeletal muscle progenitor cell (SMPC) functions that are essential for myoregeneration. SMPCs exhibited robust adhesion, motility, and differentiation on healthy muscle-derived myoscaffolds. All SPMC interactions with fibrotic myoscaffolds from dystrophic muscle were severely blunted including reduced motility rate and migration. Furthermore, SMPCs were unable to remodel laminin dense fibrotic scars within diseased myoscaffolds. Proteomics and structural analysis revealed that excessive collagen deposition alone is not pathological, and can be compensatory, as revealed by overexpression of sarcospan and its associated ECM receptors in dystrophic muscle. Our in vivo data also supported that ECM remodeling is important for SMPC engraftment and that fibrotic scars may represent one barrier to efficient cell therapy.

Intranuclear HSV-1 DNA ejection induces major mechanical transformations suggesting mechanoprotection of nucleus integrity.

Maintaining nuclear integrity is essential to cell survival when exposed to mechanical stress. Herpesviruses, like most DNA and some RNA viruses, put strain on the nuclear envelope as hundreds of viral DNA genomes replicate and viral capsids assemble. It remained unknown, however, how nuclear mechanics is affected at the initial stage of herpesvirus infection-immediately after viral genomes are ejected into the nuclear space-and how nucleus integrity is maintained despite an increased strain on the nuclear envelope. With an atomic force microscopy force volume mapping approach on cell-free reconstituted nuclei with docked herpes simplex type 1 (HSV-1) capsids, we explored the mechanical response of the nuclear lamina and the chromatin to intranuclear HSV-1 DNA ejection into an intact nucleus. We discovered that chromatin stiffness, measured as Young's modulus, is increased by ∼14 times, while nuclear lamina underwent softening. Those transformations could be associated with a mechanism of mechanoprotection of nucleus integrity facilitating HSV-1 viral genome replication. Indeed, stiffening of chromatin, which is tethered to the lamina meshwork, helps to maintain nuclear morphology. At the same time, increased lamina elasticity, reflected by nucleus softening, acts as a "shock absorber," dissipating the internal mechanical stress on the nuclear membrane (located on top of the lamina wall) and preventing its rupture.

Cancer cells become less deformable and more invasive with activation of ß-adrenergic signaling.

Invasion by cancer cells is a crucial step in metastasis. An oversimplified view in the literature is that cancer cells become more deformable as they become more invasive. ß-adrenergic receptor (ßAR) signaling drives invasion and metastasis, but the effects on cell deformability are not known. Here, we show that activation of ß-adrenergic signaling by ßAR agonists reduces the deformability of highly metastatic human breast cancer cells, and that these stiffer cells are more invasive in vitro We find that ßAR activation also reduces the deformability of ovarian, prostate, melanoma and leukemia cells. Mechanistically, we show that ßAR-mediated cell stiffening depends on the actin cytoskeleton and myosin II activity. These changes in cell deformability can be prevented by pharmacological ß-blockade or genetic knockout of the ß2-adrenergic receptor. Our results identify a ß2-adrenergic-Ca2+-actin axis as a new regulator of cell deformability, and suggest that the relationship between cell mechanical properties and invasion might be dependent on context.

Stiffness of pancreatic cancer cells is associated with increased invasive potential.

Metastasis is a fundamentally physical process in which cells are required to deform through narrow gaps as they invade surrounding tissues and transit to distant sites. In many cancers, more invasive cells are more deformable than less invasive cells, but the extent to which mechanical phenotype, or mechanotype, can predict disease aggressiveness in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here we investigate the invasive potential and mechanical properties of immortalized PDAC cell lines derived from primary tumors and a secondary metastatic site, as well as noncancerous pancreatic ductal cells. To investigate how invasive behavior is associated with cell mechanotype, we flow cells through micron-scale pores using parallel microfiltration and microfluidic deformability cytometry; these results show that the ability of PDAC cells to passively transit through pores is only weakly correlated with their invasive potential. We also measure the Young's modulus of pancreatic ductal cells using atomic force microscopy, which reveals that there is a strong association between cell stiffness and invasive potential in PDAC cells. To determine the molecular origins of the variability in mechanotype across our PDAC cell lines, we analyze RNAseq data for genes that are known to regulate cell mechanotype. Our results show that vimentin, actin, and lamin A are among the most differentially expressed mechanoregulating genes across our panel of PDAC cell lines, as well as a cohort of 38 additional PDAC cell lines. We confirm levels of these proteins across our cell panel using immunoblotting, and find that levels of lamin A increase with both invasive potential and Young's modulus. Taken together, we find that stiffer PDAC cells are more invasive than more compliant cells, which challenges the paradigm that decreased cell stiffness is a hallmark of metastatic potential.

Temperature-dependent phase transitions in zeptoliter volumes of a complex biological membrane.

Phase transitions in purple membrane have been a topic of debate for the past two decades. In this work we present studies of a reversible transition of purple membrane in the 50-60 °C range in zeptoliter volumes under different heating regimes (global heating and local heating). The temperature of the reversible phase transition is 52 ± 5 °C for both local and global heating, supporting the hypothesis that this transition is mainly due to a structural rearrangement of bR molecules and trimers. To achieve high resolution measurements of temperature-dependent phase transitions, a new scanning probe microscopy-based method was developed. We believe that our new technique can be extended to other biological systems and can contribute to the understanding of inhomogeneous phase transitions in complex systems.

Protective coatings on extensible biofibres.

Formulating effective coatings for use in nano- and biotechnology poses considerable technical challenges. If they are to provide abrasion resistance, coatings must be hard and adhere well to the underlying substrate. High hardness, however, comes at the expense of extensibility. This property trade-off makes the design of coatings for even moderately compliant substrates problematic, because substrate deformation easily exceeds the strain limit of the coating. Although the highest strain capacity of synthetic fibre coatings is less than 10%, deformable coatings are ubiquitous in biological systems. With an eye to heeding the lessons of nature, the cuticular coatings of byssal threads from two species of marine mussels, Mytilus galloprovincialis and Perna canaliculus, have been investigated. Consistent with their function to protect collagenous fibres in the byssal-thread core, these coatings show hardness and stiffness comparable to those of engineering plastics and yet are surprisingly extensible; the tensile failure strain of P. canaliculus cuticle is about 30% and that of M. galloprovincialis is a remarkable 70%. The difference in extensibility is attributable to the presence of deformable microphase-separated granules within the cuticle of M. galloprovincialis. The results have important implications in the design of bio-inspired extensible coatings.

Towards local electromechanical probing of cellular and biomolecular systems in a liquid environment.

Electromechanical coupling is ubiquitous in biological systems, with examples ranging from simple piezoelectricity in calcified and connective tissues to voltage-gated ion channels, energy storage in mitochondria, and electromechanical activity in cardiac myocytes and outer hair cell stereocilia. Piezoresponse force microscopy (PFM) originally emerged as a technique to study electromechanical phenomena in ferroelectric materials, and in recent years has been employed to study a broad range of non-ferroelectric polar materials, including piezoelectric biomaterials. At the same time, the technique has been extended from ambient to liquid imaging on model ferroelectric systems. Here, we present results on local electromechanical probing of several model cellular and biomolecular systems, including insulin and lysozyme amyloid fibrils, breast adenocarcinoma cells, and bacteriorhodopsin in a liquid environment. The specific features of PFM operation in liquid are delineated and bottlenecks on the route towards nanometre-resolution electromechanical imaging of biological systems are identified.