Soft limbal niche maintains stem cell compartmentalization and function through YAP

Stem cells (SCs) decision to self-renew or differentiate largely depends on the extracellular environment and elasticity of their niche. A well-described mediator of the mechanotransduction pathway is the co-transcriptional activator Yes-associated protein (YAP), known to shuttle into the nucleus of cells grown on stiff matrices. YAP is also known to be essential for stemness, but confusingly, SCs often reside in soft niches. Furthermore, the role of matrix rigidity in niche formation and SC function in vivo is poorly understood. Here we report that the post-natal development of the murine corneal epithelium involves matrix stiffening and loss of YAP activity that is associated with the formation of differentiation compartment. Importantly, manipulating the matrix crosslinking enzyme, Lox, perturbed SC mark expression and resulted in loss of corneal transparency. In agreement, we found that YAP and mechanotransduction pathways are essential for stemness in the soft niche compartment, wound healing response, and dedifferentiation of committed cells into SCs following SC depletion. In vitro experiments revealed that stiffer substrates induced cytoplasmic YAP localization through activation of LATS1/2, facilitating SMAD2/3-mediated cell differentiation. Taken together, we propose that the soft environment of the corneal SC niche maintains YAP activity to support SC regulation during morphogenesis, adult homeostasis and regeneration by the niche.

Decreased cell stiffness facilitates cell detachment and cell migration from breast cancer spheroids in 3D collagen matrices of different rigidity

PurposeTumour-cell detachment is a critical early event in the metastatic cascade. Although several mechanisms have been reported, the role of cell mechanical properties in facilitating cell detachment and migration is not well understood. We, therefore, investigated how cells alter intracellular stiffness during these processes.MethodsMDA-MB-231 cells were embedded as 10,000-cell spheroids in 2 and 4 mg/ml collagen matrices. Using mitochondrial-based particle tracking microrheology (PTM), the intracellular stiffness of cells that have migrated different distances from the spheroid were assessed. Here, 0dC, 4dC and 6dC represented no, medium and high migration, respectively.ResultsFor 2 and 4 mg/ml collagen matrices, the MSD and cell stiffness of 0dC cells were larger than for migrated 4dC and 6dC cells. The MSD of 4dC and 6dC cells were similar; however, the cell stiffness of 4dC cells was smaller than that of 6dC cells. The stiffness of 0dC cells was lower for higher matrix concentration and rigidity compared to lower matrix rigidity, whereas matrix rigidity did not affect the stiffness of 4dC and 6dC cells.ConclusionsPTM was capable of quantifying intracellular mechanics during tumour detachment and migration in 3D environments. Based on our findings, it is proposed that decreased cell stiffness drives cellular detachment and migration. Increased matrix rigidity physically hinders migration and cells need to either soften or remodel the environment to migrate. The finding that matrix rigidity did not affect the stiffness of migrated cells suggests that cells facilitate migration by remodelling their environment through cleavage of matrix proteins.

The keratin network of intermediate filaments regulates keratinocyte rigidity sensing and nuclear mechanotransduction

The keratin network of intermediate filaments provides keratinocytes with essential mechanical strength and resilience, but the contribution to mechanosensing remains poorly understood. Here, we investigated the role of the keratin cytoskeleton in the response to altered matrix rigidity. We found that keratinocytes adapted to increasing matrix stiffness by forming a rigid, interconnected network of keratin bundles, in conjunction with F-actin stress fiber formation and increased cell stiffness. Disruption of keratin stability by overexpression of the dominant keratin 14 mutation R416P inhibited the normal mechanical response to substrate rigidity, reducing F-actin stress fibers and cell stiffness. The R416P mutation also impaired mechanotransduction to the nuclear lamina, which mediated stiffness-dependent chromatin remodeling. By contrast, depletion of the cytolinker plectin had the opposite effect and promoted increased mechanoresponsiveness and up-regulation of lamin A/C. Together, these results demonstrate that the keratin cytoskeleton plays a key role in matrix rigidity sensing and downstream signal transduction.

Glycosaminoglycan Modification of Decorin Depends on MMP14 Activity and Regulates Collagen Assembly

Proper processing of collagens COL1 and COL6 is required for normal function of adipose tissue and skeletal muscle. Proteoglycan decorin (DCN) regulates collagen fiber formation. The amino-terminus of DCN is modified with an O-linked glycosaminoglycan (GAG), the function of which has remained unclear. Previously, non-glycanated DCN (ngDCN) was identified as a marker of adipose stromal cells. Here, we identify MMP14 as the metalloprotease that cleaves DCN to generate ngDCN. We demonstrate that mice ubiquitously lacking DCN GAG (ngDCN mice) have reduced matrix rigidity, enlarged adipocytes, fragile skin, as well as skeletal muscle hypotrophy, fibrosis, and dysfunction. Our results indicate that DCN deglycanation results in reduced intracellular DCN—collagen binding and increased production of truncated COL6 chains, leading to aberrant procollagen processing and extracellular localization. This study reveals that the GAG of DCN functions to regulate collagen assembly in adipose tissue and skeletal muscle and uncovers a new mechanism of matrix dysfunction in obesity and aging.