Microfluidic interrogation and mathematical modeling of multi-regime calcium signaling dynamics

Abstract

Through microfluidic interrogation we analyzed real-time calcium responses of HEK293 cells stimulated with short pulses of the M3 muscarinic receptor ligand carbachol in two different concentration regimes. Lower ligand concentrations elicit oscillatory calcium signals while higher concentrations trigger a rapid rise that eventually settles down at a steady-state slightly above pre-stimulus levels, referred to as an acute signal. Cells were periodically pulsed with carbachol at these two concentration regimes using a custom-made microfluidic platform, and the resulting calcium signals were measured with a single fluorescent readout. Pulsed stimulations at these two concentration regimes resulted in multiple types of response patterns that each delivered complementary information about the M3 muscarinic receptor signaling pathway. These multiple types of calcium response patterns enabled development of a comprehensive mathematical model of multi-regime calcium signaling. The resulting model suggests that dephosphorylation of deactivated receptors is rate limiting for recovery of calcium signals in the acute regime (high ligand concentration), while calcium replenishment and IP3 production determine signal recovery in the oscillatory regime (low ligand concentration). This study not only provides mechanistic insight into multi-regime signaling of the M3 muscarinic receptor pathway, but also provides a general strategy for analyzing multi-regime pathways using only one fluorescent readout.

New insights into mammalian signaling pathways using microfluidic pulsatile inputs and mathematical modeling

“…7C (i)). They also determined that there is a minimum reset time for Smad4 amplitude recovery during pulsatile stimulation, similar to the observation of Ashall et al 46 in the NFkB signaling, and Jovic et al 43 in the GPCR signaling. This suggested that there must exist a negative feedback motif (they called it ‘adaptive mechanism’), which was yet unknown (Fig.…”

“…The analysis also revealed the importance of basal levels of PLC in generating subthreshold peaks. Pulsatile stimulation was also utilized to understand the pathway recovery properties in multiple concentration regimes 43 . In particular, we found that the reduction in phase-locking at low values of C and D and the reduction in signal amplitude at high values of C and D for a fixed R can identify the recovery properties of cytoplasmic calcium and receptor phosphorylation respectively (Fig.…”

Microfluidics: reframing biological enquiry

“…3a). Areas of recent study include cellular feedback mechanisms, noise 73 , frequency-encoded responses 74 and rate-limiting processes 19 .…”

“…The lower carbachol concentration resulted in an oscillating calcium signal, whereas the higher carbachol concentration resulted in an acute increase in the calcium signal that plateaued above pre-stimulus levels. A mathematical model of the pathway suggested mechanisms that might be responsible for these two behaviours: calcium replenishment and inositol trisphosphate production in response to the low carbachol concentration; and dephosphorylation of deactivated receptors in response to the high carbachol concentration 19 .…”

“…Coupled with spatial and temporal control of the chemical environment (for example, levels of soluble factors) and physical environment (for example, single-cell trapping), microfluidic live-cell imaging can offer insights into dynamic cellular processes (such as signalling dynamics 19 ) and can be used to study long-term developments such as cell ageing 20 , inheritance 21 and death 22 .…”