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Cellular circuitry and experimental methodology.

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posted on 2016-07-01, 11:09 authored by Javier Estrada, Natalie Andrew, Daniel Gibson, Frederick Chang, Florian Gnad, Jeremy Gunawardena

(A) Components of the calcium signalling toolkit are shown, as relevant to the models discussed in this paper. Yellow circles represent the principal small-molecule species involved in calcium dynamics, IP3, Ca2+ in the cytoplasm (Ca CY) and Ca2+ in the ER (Ca ER). Black lines denote Ca2+ fluxes, dashed blue lines denote activation. The positive and negative feedbacks between the components are illustrated in Fig 3. Abbreviations used: GPCR, G-protein coupled receptor; IP3, inositol-1,4,5-trisphosphate; IP3 R, IP3 receptor; NCX, Na+-Ca2+ exchanger; PIP2, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C; PMCA, plasma membrane Ca2+ -ATPase; SERCA, sarco/endo-plasmic reticulum Ca2+ -ATPase; mCU, mitochondrial Ca2+ uniporter; NCXm, mitochondrial Na+-Ca2+ exchanger. (B) Microfluidic platform. From left to right: first, a schematic of the two-layer PDMS device. Buffer and histamine plus buffer are provided through the indicated flow lines (blue) at 5 psig. Valves are regulated by computer through the control lines (red), operating at 25 psig, to supply histamine or buffer to the output port, thereby generating steps or pulses, as required. Second, a photograph of the device bonded to the glass-bottomed dish (Chip-In-A-Dish), showing the four tubes leading to the control lines. Third, a differential interference contrast image of HeLa cells growing in the dish next to the output port, with the device border outlined in red. Fourth, fluorescence microscopy image, showing typical Fluo4 fluorescence in response to histamine stimulation.