All-Optical Detection of Neuronal Membrane Depolarization in Live Cells Using Colloidal Quantum Dots.

Luminescent semiconductor quantum dots (QDs) have recently been suggested as novel probes for imaging and sensing cell membrane voltages. However, a key bottleneck for their development is a lack of techniques to assess QD responses to voltages generated in the aqueous electrolytic environments typical of biological systems. Even more generally, there have been relatively few efforts to assess the response of QDs to voltage changes in live cells. Here, we develop a platform for monitoring the photoluminescence (PL) response of QDs under AC and DC voltage changes within aqueous ionic environments. We evaluate both traditional CdSe/CdS and more biologically compatible InP/ZnS QDs at a range of ion concentrations to establish their PL/voltage characteristics on chip. Wide-field, few-particle PL measurements with neuronal cells show the QDs can be used to track local voltage changes with greater sensitivity (ΔPL up to twice as large) than state-of-the-art calcium imaging dyes, making them particularly appealing for tracking subthreshold events. Additional physiological observation studies showed that while CdSe/CdS dots have greater PL responses on membrane depolarization, their lower cytotoxicity makes InP/ZnS far more suitable for voltage sensing in living systems. Our results provide a methodology for the rational development of QD voltage sensors and highlight their potential for imaging changes in cell membrane voltage.

Effective cell membrane tension is independent of polyacrylamide substrate stiffness.

Most animal cells are surrounded by a cell membrane and an underlying actomyosin cortex. Both structures are linked, and they are under tension. In-plane membrane tension and cortical tension both influence many cellular processes, including cell migration, division, and endocytosis. However, while actomyosin tension is regulated by substrate stiffness, how membrane tension responds to mechanical substrate properties is currently poorly understood. Here, we probed the effective membrane tension of neurons and fibroblasts cultured on glass and polyacrylamide substrates of varying stiffness using optical tweezers. In contrast to actomyosin-based traction forces, both peak forces and steady-state tether forces of cells cultured on hydrogels were independent of substrate stiffness and did not change after blocking myosin II activity using blebbistatin, indicating that tether and traction forces are not directly linked. Peak forces in fibroblasts on hydrogels were about twice as high as those in neurons, indicating stronger membrane-cortex adhesion in fibroblasts. Steady-state tether forces were generally higher in cells cultured on hydrogels than on glass, which we explain by a mechanical model. Our results provide new insights into the complex regulation of effective membrane tension and pave the way for a deeper understanding of the biological processes it instructs.

Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain.

Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that during development local tissue stiffness changes significantly within tens of minutes. Within this time frame, a stiffness gradient arose in the developing Xenopus brain, and retinal ganglion cell axons turned to follow this gradient. Changes in local tissue stiffness were largely governed by cell proliferation, as perturbation of mitosis diminished both the stiffness gradient and the caudal turn of axons found in control brains. Hence, we identified a close relationship between the dynamics of tissue mechanics and developmental processes, underpinning the importance of time-resolved stiffness measurements.

Mechanosensing is critical for axon growth in the developing brain.

During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signaling. However, growing neurons interact not only chemically but also mechanically with their environment. Here we identify mechanical signals as important regulators of axon pathfinding. In vitro, substrate stiffness determined growth patterns of Xenopus retinal ganglion cell axons. In vivo atomic force microscopy revealed a noticeable pattern of stiffness gradients in the embryonic brain. Retinal ganglion cell axons grew toward softer tissue, which was reproduced in vitro in the absence of chemical gradients. To test the importance of mechanical signals for axon growth in vivo, we altered brain stiffness, blocked mechanotransduction pharmacologically and knocked down the mechanosensitive ion channel piezo1. All treatments resulted in aberrant axonal growth and pathfinding errors, suggesting that local tissue stiffness, read out by mechanosensitive ion channels, is critically involved in instructing neuronal growth in vivo.