Rapid Calcium Modulation in Cells: Direct Intracellular Access using Nanostraws

Abstract

Intracellular calcium plays a role in a host of cellular functions ranging from synaptic transmission to gene expression. As a transient, short-range signaling molecule, calcium is often presented in complex signal patterns, including oscillations and bursts. The concerted action of pumps, channels, and buffers in conjunction with the ion-impermeable cell membrane tightly regulates these calcium signals spatially and temporally but simultaneously impedes our ability to study the action of calcium. Many techniques measure changes in intracellular calcium, providing insights into the specificity of these signal patterns, but fewer are capable of controlling calcium levels in cells. Patch clamp pipette access can be used to modulate calcium in small numbers of cells, but larger scale control often requires disruption of normal calcium signaling using drugs such as thapsigargin, in order to couple intracellular calcium to an external calcium signal. Here we present a new technology to rapidly modulate intracellular calcium. Using supported nanotubes called “nanostraws,” we demonstrate direct ionic delivery into cells. The dimensions of the nanostraws (∼100 nm x 1 μm tall) allow them to spontaneously penetrate the cell, thereby allowing calcium to bypass the lipid membrane and its constituent calcium channels through the nanostraws. Calcium delivery is controlled by fluid flow, and intracellular calcium oscillations can be induced and modulated in amplitude and frequency. While previous studies have perturbed long-term cell behavior using nanostraws and similar systems, here we apply the technique to transient signaling by rapid intracellular calcium modulation and characterize the spatiotemporal delivery using calcium indicators (e.g. Fluo-4, GCaMP6). By circumventing the intrinsic calcium regulatory mechanisms, nanostraws provide direct access and the ability to mimic biological calcium oscillations, adding a new method for decoding the role of calcium signal patterns and their effects on downstream signaling.