Arash Tajik

Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal. Soft 3D gels can promote the growth of tumour-repopulating cells, a self-renewing subpopulation of cancer cells critical in cancer progression. Here, the authors investigate the mechanism behind this phenomenon and show that the histone 3 lysine residue 9 methylation and Sox2 are controlling this process.

Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell–cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell–matrix and cell–cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation. The three germ layers are formed from the inner cell mass of the mammalian embryo during gastrulation. Here, the authors present a method by which a single mouse embryonic stem cell, derived from inner cell mass, differentiates into the three germ layers in a self-organized manner when cultured in soft fibrin gel.

The ability to precisely monitor and manipulate neural circuits is essential to understand the brain. Advancements over the last decade in optical techniques such as calcium imaging and optogenetics have empowered researchers to gain insight into brain function by systematically manipulating or monitoring defined neural circuits. Combining these cutting-edge techniques enables a more direct mechanism for ascribing neural dynamics to behavior. Here, we developed a miniaturized integrated microscope that allows for simultaneous optogenetic manipulation and cellular-resolution calcium imaging within the same field of view in freely behaving mice. The integrated microscope has two LEDs, one filtered with a 435-460 nm excitation filter for imaging green calcium indicators, and a second LED filtered with a 590-650 nm excitation filter for optogenetic modulation of red-shifted opsins. We developed and tested this technology to minimize biological and optical crosstalk. We observed insignificant amounts of biological and optical crosstalk with regards to the optogenetic LED affecting calcium imaging. We observed some amounts of residual crosstalk of the imaging light on optogenetic manipulation. Despite residual crosstalk, we have demonstrated the utility of this technology by probing the causal relationship between basolateral amygdala (BLA) -to- nucleus accumbens (NAc) circuit function, behavior, and network dynamics. Using this integrated microscope we were able to observe both a significant behavioral and cellular calcium response of the optogenetic modulation on the BLA-to-NAc circuit. This integrated strategy will allow for routine investigation of the causality of circuit manipulation on cellular-resolution network dynamics and behavior.