Identifying ultrasensitive HGF dose-response functions in a 3D mammalian system for synthetic morphogenesis

Abstract

Nonlinear responses to signals are widespread natural phenomena that affect various cellular processes. Nonlinearity can be a desirable characteristic for engineering living organisms because it can lead to more switch-like responses, similar to those underlying the wiring in electronics. Steeper functions are described as ultrasensitive, and can be applied in synthetic biology by using various techniques including receptor decoys, multiple co-operative binding sites, and sequential positive feedbacks. Here, we explore the inherent non-linearity of a biological signaling system to identify functions that can potentially be exploited using cell genome engineering. For this, we performed genome-wide transcription profiling to identify genes with ultrasensitive response functions to Hepatocyte Growth Factor (HGF). We identified 3,527 genes that react to increasing concentrations of HGF, in Madin-Darby canine kidney (MDCK) cells, grown as cysts in 3D collagen cell culture. By fitting a generic Hill function to the dose-responses of these genes we obtained a measure of the ultrasensitivity of HGF-responsive genes, identifying a subset with higher apparent Hill coefficients (e.g. MMP1, TIMP1, SNORD75, SNORD86 and ERRFI1). The regulatory regions of these genes are potential candidates for future engineering of synthetic mammalian gene circuits requiring nonlinear responses to HGF signalling.

Traction microscopy with integrated microfluidics: responses of the multi-cellular island to gradients of HGF

“…Our finding using the microfluidic TFM system has important physiological implications for how MDCK cells sense secreted HGFs and efficiently activate collective cell migration and EMT. Since cells experience increases or decreases in the HGF concentration in vivo, 28,29,45 our results are consistent with the model showing that the HGF concentration and its gradient cooperate to initiate collective cell migration in the process of EMT. Immunostaining of F-actin, E-cadherin, and vinculin further provided evidence of EMT development induced by HGF.…”

“…Typically, cells migrate toward higher concentrations of soluble factors, which is known as chemotaxis; however HGF increased the expansion of MDCK cell islands without chemotactic directionality and induced EMT. 32,45,46 The highest speed of the cell groups was observed once the concentration and gradient of HGF were coapplied to the system. Traction generated by the cell groups was fairly consistent under various concentrations of HGF in solution, suggesting that the cells maintained group collectiveness.…”

Transient Turing patterns in a morphogenetic model

“…Except for the last case, the reaction-diffusion systems associated with transient Turing patterns are composed by several species, which posses the additional challenge to derive analytical predictions or to associate a phase diagram to study them. On the other hand, the present model can be associated with biological systems [25,26] and the analysis of the associated reaction-diffusion equations are feasible for some values of the Hill exponent of regulatory function. The present results show that in this model the transient patterns are due to the presence of a saddle node which have associated spatial modes with no-null amplification rate.…”

Linear‐No‐Threshold Default Assumptions are Unwarranted for Cytotoxic Endpoints Independently Triggered by Ultrasensitive Molecular Switches

“…Such switches regulate networks of intricately and homeostatically controlled processes vital to cell physiology and host survival through complex machinery that includes specific molecular mechanisms highly adapted and conserved widely throughout most higher (or all of) eukaryotic life, as well as some close (e.g., >50% homologous) molecular analogs that appear even in prokaryotes. Experimentally observed switch‐specific examples of apparent ultrasensitive dose–response nonlinearity include: switch‐like c‐Fos induction and cell proliferation in Swiss albino mouse 3T3 cells; calcium influx induced in human HeLa cervical epithelial carcinoma cells by epidermal growth factor (EGF) binding to EGF‐receptor dimers/oligomers; cannabinoid receptor‐1 agonist HU‐210 activation of Ras‐related protein 1 (Rap1) in albino mouse neuroblastoma (Neuro 2A) cells; PAI‐1 protein expression and inhibition of cell proliferation by transforming growth factor‐β (TGF‐β) in human keratinocyte (HaCaT) cells; time‐delayed cyclin‐dependent kinase‐1 (Cdk1) triggering of biochemical oscillator constituents, including securin‐CFP, cyclin B1‐CFP, and cyclin A2‐YFP degradation, and phosphorylation of cell division cycle 25 and 27 (Cdc25 and Cdc27) proteins, in Xenopus laevis embryonic cells; and gene activation by hepatocyte growth factor in Madin–Darby canine kidney cells grown in 3D collagen cell culture …”

“…As in all areas of scientific prediction beyond or between direct observations, reliable low‐dose dose–response extrapolation/interpolation will always ultimately rely on a coherent and consistent synthesis between precise and mechanistically informative empirical data and successfully predictive, mathematically expressed mechanistic theory. However, the current absence of validated, mechanistically based, switch‐specific, low‐dose extrapolation models for cytotoxic endpoints elicited by pathways independent from those shown to account for background rates does not imply that LNT extrapolation for responses triggered by switches is reasonable, let alone even plausible, given the inherently nonlinear kinetic characteristics implied and observed different types of ultrasensitive molecular switches . An alternative conservative default approach might instead be to apply, for example, quadratic low‐dose extrapolation from corresponding benchmark points of departure.…”