Calcium signaling and intercellular calcium waves play a pivotal role in biology, governing various essential processes within cells and coordinating communication between them. Calcium ions serve as versatile messengers, regulating diverse cellular functions such as muscle contraction, neurotransmitter release, hormone secretion, gene expression, and cell growth (1). Through precise control of the cytosolic concentration of calcium, cells can initiate specific responses and ensure proper coordination among various physiological processes. Intercellular calcium waves further enhance this communication by propagating calcium signals between neighboring cells, enabling coordinated responses and synchronized activities across tissue and organ systems (2). These waves not only facilitate intercellular coordination but also regulate crucial events during development, tissue repair, immune response, and synaptic plasticity. Understanding the intricacies of calcium signaling and intercellular calcium waves can help to unveil fundamental mechanisms in biology and holds significant potential for the development of targeted therapeutic interventions.
The first reports of calcium waves arose in 1990 when researchers from the University of California, Los Angeles reported that confluent monolayers of epithelial cells exhibited calcium waves when mechanically perturbed with a micropipette (3). For the last thirty years, mechanical stimuli like this have been used to trigger calcium waves in fundamental signaling experiments. In our work, we show that these same responses can be stimulated with mechanical force delivered by a small molecule.
Molecular machines, or MM, for short, are small molecule devices that convert light into mechanical energy through designed excited-state interactions (4). Our lab deals principally with a class of MM consisting of a rotor and a stator that actuate across a sterically overcrowded alkene in a series of photochemical and thermal steps. We have shown that these motors are capable of permeabilizing the lipid bilayer of eukaryotic cells (5), and have demonstrated various applications leveraging this effect. Eventually, we began to wonder if the forces applied by these motors could be used to control cellular activity rather than kill cells if applied in smaller doses.
We collaborated with Dr. Jacob Robinson, a neuroscientist across campus at Rice University, and got to work. Our initial experiments used simple spiking HEK cells, a subtype of the common HEK cell line that expresses various voltage-gated channels. We used these cells because initially, when the project began, our goal was to stimulate action potentials using MM. We treated these cells with fast-rotating MM and Fluo-4, a fluorescent calcium biosensor. We applied various forms of light stimulation and eventually found conditions that gave us a calcium response - a 250 ms laser stimulation, using a mode of a confocal microscope initially intended for fluorescence-recovery-after-photobleaching experiments.
Figure 1. Molecular machines induce intracellular calcium waves in a fashion that depends on their rapid, unidirectional rotation.
We were ecstatic upon reaching a response and increasingly optimistic as our control experiments showed that the responses were exclusive to cells treated with a fast-rotating MM. However, when perusing the literature, we realized that the calcium transients we were observing didn't look anything like an action potential. The elevations in cytosolic calcium arising from MM activation lasted for several minutes; our chemistry group didn't know much about biology, but we were pretty sure that action potentials didn't last more than a second. Moreover, non-spiking HEKs responded in much the same way to stimulation.
Eventually, we realized that we were not looking at action potentials of any kind - we were observing a calcium wave, the same kind of response observed by Sanderson et al. when they tapped their confluent monolayer of epithelial cells with a micropipette. We eventually showed the similarites between the two responses with a variety of pharmacology experiments.
Figure 2. Pharmacology experiments showing that calcium transients elicited by MM arise from intracellular calcium reserves.
Once we knew that our MM were causing intracellular calcium responses, we looked for model systems to show how these responses could be used to control downstream biological function. We first looked to an in vitro model of heart muscle. We showed that the stimulation of calcium waves by MM in primary rat cardiomyocytes potentiated action potential firing in the surrounding cells. The potentiated activity depended on the presence of extracellular calcium and an intact IP3 signaling circuit.
Figure 3. Molecular machines cause contraction and beating in cardiac myocytes.
Then, we moved to an in vivo model system. We used Hydra vulgaris, a freshwater cnidarian organism whose movement is primarily governed by calcium waves. We showed that local stimulation of Hydra treated with MM caused a regional calcium wave. When stimulation was targeted to the peduncle, a region containing neuronal clusters that regulate burst contraction, fast-rotating MM drove a higher rate of organism contraction, coupled with a whole-body calcium wave, than slow-rotating MM or solvent-only controls.
Figure 4. Molecular machines cause contraction in Hydra vulgaris.
These results demonstrate the ability to remotely control cell signaling using mechanical force delivered by a light-activated small molecule. While the applications of the sterically crowded alkenes demonstrated here are limited by the high light intensity needed and the poor penetration of visible light, the principle demonstrated by this work is powerful. In principle, most modern pharmaceutical drugs trigger a signaling cascade using a chemical force, a specific binding interaction. This work shows that the same can be done with a mechanical force, opening fundamental questions about the mechanosensitivity of the protein machinery driving intracellular calcium responses and demonstrating the potential for a new class of drugs leveraging a combination of chemical and mechanical forces to exact their desired effects.
1. Clapham, D. E. Calcium signaling. Cell 131, 1047–1058 (2007).
2. Leybaert, L. & Sanderson, M. J. Intercellular Ca2+ waves: mechanisms and function. Phys. Rev. 92, 1359–1392 (2012).
3. Sanderson, M. J., Charles, A. C. & Dirksen, E. R. Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells. Cell Regul. 1, 585–596 (1990).
4. Klok, M. et al. MHz unidirectional rotation of molecular rotary motors. J. Am. Chem. Soc. 130, 10484–10485 (2008).
5. García-López, V. et al. Molecular machines open cell membranes. Nature 548, 567–572 (2017).