Optogenetics allows to manipulate cells by light. Using this technique, cells are genetically modified to produce light-sensitive proteins. One of these proteins is the photo-activated adenylate cyclase bPAC. It allows to control the synthesis of the intracellular messenger cAMP by light. We introduced bPAC into mouse sperm in order to manipulate sperm function by light.
To analyze the complex signal processing that takes place inside cells it is necessary to manipulate cellular signaling with high temporal and spatial precision. Light is particularly well suited to this task, as it can be switched on and off quickly and does not affect the regular signal processing of a cell. Using optogenetics, cells are genetically modified to produce light-sensitive proteins and, thereby, become susceptible to light. The most widely used optogenetic tools are the so-called channelrhodopsins. These light-activated ion channels from the green algaeChlamydomonas were first identified in 2002, and are a well-established tool in neuroscience: the activity of neurons expressing channelrhodopsins can be precisely triggered by light. In addition to the membrane potential, other signaling events within a cell can be manipulated using light. Photo-activated adenylate cyclases (PACs) synthesize the intracellular messenger 3’-5’-cyclic adenosine monophosphate (cAMP). One member of this protein family is bPAC, from the bacterium Beggiatoa, which was discovered in Peter Hegemann’s laboratory at the Humboldt University in Berlin.
With our first experiments, we determined whether the concentration of intracellular cAMP in HEK293 cells could be manipulated with bPAC. HEK293 is a cell line derived from human embryonic kidney cells and has been used as a model system in cell biology research for many years. We generated cells expressing bPAC together with a cAMP-gated ion channel. An increase in intracellular cAMP opens the ion channel, allowing calcium ions to enter the cell (figure 1a). This calcium influx can be visualized by fluorescent indicators. bPAC only remains active as long as the light is switched on; when it is switched off, the cAMP concentration declines (figure 1b). The amplitude of the cAMP signal can be regulated by the duration of the light pulse: more light produces more cAMP (figure 1c).
Figure 1: a. Assay to analyze the intracellular cAMP concentration. Opening of the CNG channel by cAMP causes an influx of calcium (Ca2+) into the cell. Fluorescent indicators convert this increase in calcium into a light signal. b. Light stimulation (gray bar) of cells expressing bPAC causes cAMP synthesis. In non-bPAC cells (control) cAMP levels do not change upon light stimulation. c. The amplitude of a light- induced cAMP signal can be regulated by the exposure time.
bPAC is therefore well-suited to manipulate the intracellular cAMP concentration by light. But is it also possible to use bPAC as an optogenetic tool in vivo?
cAMP is a common cellular messenger that controls a whole range of important physiological processes. In particular, it is indispensable for sperm function: cAMP regulates sperm development, maturation, and motility. cAMP synthesis in sperm is controlled by the SACY enzyme – a soluble adenylate cyclase regulated by bicarbonate (HCO3-). However, the cAMP-dependent signaling processes that occur in sperm are poorly understood.
Analyzing cAMP-dependent processes in sperm is hindered by the fact that pharmacological substances used to manipulate cAMP give rise to unwanted side effects. Optogenetics allows to analyze these signaling events without side effects. Therefore, we generated a transgenic mouse line, which expresses bPAC in sperm. In sperm of these mice cAMP synthesis is activated by light (figure 2a). An increase in cAMP accelerates the flagellar beat of sperm. In bPAC sperm, the flagellar beat can be directly controlled by light: A single 200-millisecond light pulse is sufficient to increase the flagellar beat frequency (figure 2b).
FIGURE 2: a. Sperm were prepared under red light (dark) to avoid bPAC activation. When the sperm samples were illuminated with a halogen lamp (light), the cAMP concentration in transgenic sperm increased. In wild-type sperm, cAMP concentration were not affected by light. The numbers in parentheses indicate the number of experiments. b. The flagellar beat frequency of a single bPAC sperm cell was recorded. Stimulating the sperm with a flash of UV light (dotted line) caused the sperm to beat faster. This effect is reversible: bPAC deactivates in the absence of light.
The importance of cAMP for sperm function is illustrated by the knockout mouse model sNHE-KO, which lacks active SACY. In sperm of sNHE-KO males, cAMP synthesis is abolished. Their sperm are unable to move; therefore the sNHE-KO males are infertile. To restore the motility in sperm by light, we crossed the sNHE-KO mice with bPAC mice.
In the dark, sperm of these double-transgenic mice are immotile as well. However, light stimulation restores the flagellar beating and the sperm starts to swim (figure 3a). We also could restore fertilization in sNHE-KO/bPAC mice in vitro using light. In this experiment, sperm and egg cells are incubated together and the number of fertilized eggs (two-cell stages) is determined. In the dark, no two-cell stages are found, but if the experiment is carried out in the light, sNHE-KO/bPAC sperm can successfully fertilize egg cells (figure 3b).
FIGURE 3: a. sNHE-KO/bPAC sperm are immotile in the dark. A flash of light restores the flagellar beating. b. When stimulated by light, sNHE-KO/bPAC sperm are able to fertilize the egg in vitro.
The results show that fundamental processes like fertilization can be controlled by light. Thus, optogenetics has conquered a new field of research in addition to neuroscience.
Jansen, V., Alvarez, L., Balbach, M., Strünker, T., Hegemann, P., Kaupp, U.B. & Wachten, D. (2015) "Controlling fertilization and cAMP signaling in sperm by optogenetics" eLife 4, e05161