Simultaneous Optogenetics and Cellular Resolution Calcium Imaging During Active Behavior Using a Miniaturized Microscope.

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.

Soft matrices downregulate FAK activity to promote growth of tumor-repopulating cells.

Tumor-repopulating cells (TRCs) are a tumorigenic sub-population of cancer cells that drives tumorigenesis. We have recently reported that soft fibrin matrices maintain TRC growth by promoting histone 3 lysine 9 (H3K9) demethylation and Sox2 expression and that Cdc42 expression influences H3K9 methylation. However, the underlying mechanisms of how soft matrices induce H3K9 demethylation remain elusive. Here we find that TRCs exhibit lower focal adhesion kinase (FAK) and H3K9 methylation levels in soft fibrin matrices than control melanoma cells on 2D rigid substrates. Silencing FAK in control melanoma cells decreases H3K9 methylation, whereas overexpressing FAK in tumor-repopulating cells enhances H3K9 methylation. Overexpressing Cdc42 or RhoA in the presence of FAK knockdown restores H3K9 methylation levels. Importantly, silencing FAK, Cdc42, or RhoA promotes Sox2 expression and proliferation of control melanoma cells in stiff fibrin matrices, whereas overexpressing each gene suppresses Sox2 expression and reduces growth of TRCs in soft but not in stiff fibrin matrices. Our findings suggest that low FAK mediated by soft fibrin matrices downregulates H3K9 methylation through reduction of Cdc42 and RhoA and promotes TRC growth.

Transcription upregulation via force-induced direct stretching of chromatin.

Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green fluorescent protein (GFP)-tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

E-cadherin-mediated force transduction signals regulate global cell mechanics.

This report elucidates an E-cadherin-based force-transduction pathway that triggers changes in cell mechanics through a mechanism requiring epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), and the downstream formation of new integrin adhesions. This mechanism operates in addition to local cytoskeletal remodeling triggered by conformational changes in the E-cadherin-associated protein α-catenin, at sites of mechanical perturbation. Studies using magnetic twisting cytometry (MTC), together with traction force microscopy (TFM) and confocal imaging identified force-activated E-cadherin-specific signals that integrate cadherin force transduction, integrin activation and cell contractility. EGFR is required for the downstream activation of PI3K and myosin-II-dependent cell stiffening. Our findings also demonstrated that α-catenin-dependent cytoskeletal remodeling at perturbed E-cadherin adhesions does not require cell stiffening. These results broaden the repertoire of E-cadherin-based force transduction mechanisms, and define the force-sensitive signaling network underlying the mechano-chemical integration of spatially segregated adhesion receptors.

Matrix softness regulates plasticity of tumour-repopulating cells via H3K9 demethylation and Sox2 expression.

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.

Generation of organized germ layers from a single mouse embryonic stem cell.

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.

Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins.

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 ß1 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.

Intrinsically high-Q dynamic AFM imaging in liquid with a significantly extended needle tip.

Atomic force microscope (AFM) probe with a long and rigid needle tip was fabricated and studied for high Q factor dynamic (tapping mode) AFM imaging of samples submersed in liquid. The extended needle tip over a regular commercially available tapping-mode AFM cantilever was sufficiently long to keep the AFM cantilever from submersed in liquid, which significantly minimized the hydrodynamic damping involved in dynamic AFM imaging of samples in liquid. Dynamic AFM imaging of samples in liquid at an intrinsic Q factor of over 100 and an operational frequency of over 200 kHz was demonstrated. The method has the potential to be extended to acquire viscoelastic material properties and provide truly gentle imaging of soft biological samples in physiological environments.