Yeast is a popular model organism because it is easy to genetically modify, it is robust to differing environments and it is a eukaryote. Yeast can be studied under aerobic conditions when plated on agar plates. The Singer Instruments ROTOR device pins colonies of yeast, fungi or bacteria onto agar plates in 96, 384 or 1536 plate format and enables to study colonies in high-throughput. Here, different yeast clones were pinned onto a plate resulting in 384 measure points to determine the organism’s response to varying oxygen concentrations. The CLARIOstar microplate reader detected differences in yeast autoﬂuorescence, citrate synthase 2 expression reported by mCherry and a redox-sensitive roGFP2-based probe.
Yeast clones expressing mito-roGFP2-Tsa2∆CR, a mitochondrial redox-sensitive sensor1, as well as non-modiﬁed yeast was pinned onto agar plates by the ROTOR (Singer Instruments) resembling the layout of a 384 well plate. The roGFP2 probe changes its excitation spectrum in response to H2O2: the oxidized probe is excitable at 405 nm and the reduced form at 488 nm. The ratio of oxidized and reduced signal is used to report on the oxidation state of the probe. In addition to the mitochondrial redox probe, yeast cells expressed a citrate synthase 2 (Cit2) fusion protein with mCherry. Cit2 is upregulated upon activation of the retrograde pathway, a common marker of mitochondrial dysfunction. In order to correct for the growth of yeast colonies, autoﬂuorescence of yeast NAD(P)H was acquired.
Materials & Methods
- Yeast cells (strain BY4742) with with genomically integrated roGFP2-Tsa2∆CR and mCherry-fussion tag in C-terminal of endogenous Cit2
- Synthetic-deﬁned-Agar in empty plates, pinned with yeast colonies (ROTOR robot to with a 96-Pad, Singer Instruments)
- CLARIOstar microplate reader, BMG LABTECH
Yeast cells were grown at 30°C for 24 hours after pinning and then exposed to changing ambient concentrations of oxygen as displayed in Fig. 1. The experiment was performed with 24 replicates. Agar only served as blank and non-modiﬁed yeast colonies as negative control.
|Top read, number of flashes:||40|
roGFP2 (measured with filters)
|Excitation 1 (oxidized roGFP2):||400-10|
|Excitation 2 (reduced roGFP2):||482-16|
|Gains:||691 (Ex 1); 504 (Ex 2)|
NAD(P)H autofluorescence (LVF monochromator)
Cit2-mCherry (LVF monochromator)
Results & Discussion
During the initial period of oxygen decrease (0 –1.2 h) from 18 % down to 12 %, the redox state of mitochondrial H2O2 probe was not inﬂuenced by the decreasing oxygen2. This might be explained by a favored oxygen supply to mitochondria over the cytosol in case of ﬂuctuating oxygen concentrations. At lower oxygen saturation, the amount of the reduced probe increased as reported by lower roGFP2 ratios. While the oxygen is kept at 1 % (5-6.5 h), the probe per-sists in its reduced form and gets oxidized only in the phase of re-oxygenation. The immediate probe oxidation as soon as more oxygen is available further points to the high priority of mitochondrial oxygen supply.
A pathway that is induced upon inhibition of mitochondrial respiration is the retrograde pathway. It reports mitochondrial failure to the nucleus. One protein synthesized as a result of pathway activation is Cit2.
In the course of oxygen deprivation, Cit2 expression is slightly reduced whereas it remarkably increases during reperfusion (Fig. 3). Whether the Cit2 increase is due to reperfusion or if it is a delayed response to hypoxic conditions, remains to be elucidated.
The pinning of yeast or bacteria onto agar plates in a standard SBS format allows studying colonies in high throughput format. Pinned colonies can be analyzed using ﬂuorescent markers that are read at a microplate reader such as the CLARIOstar. The device is designed to meet both needs: ﬂexibility and sensitivity. Flexibility is achieved by the LVF monochromator allowing for the choice of any wavelength and bandwidth. The extraordinary sensitivity of the system can be enhanced by the use of ﬁlters. This way the output of the experiment can be maximized as demonstrated here: redox state, H2O2 levels and NAD(P)H autoﬂuorescence.
- Bruce Morgan, et al. (2016) Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes Nature Chemical Biology 12, 437–443 (2016) doi:10.1038/nchembio.2067
- Pastor-Flores D., Dick T. (2017) Monitoring yeast mitochondria with peroxiredoxin based redox probes –The Inﬂ uence of oxygen and glucose availability. Interface Focus, DOI:10.1038/nchembio.2067