310

Mitochondrial oxidant generation follows oxygen deprivation and re-oxygenation

Daniel Pastor-Flores, Tobias Dick German Cancer Research Center (DKFZ), Division of Redoxregulation (A160) 08/2017

Introduction

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.

 

Assay Principle

Yeast     clones     expressing     mito-roGFP2-Tsa2DCR,     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   mito-chondrial  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-Tsa2DCR 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

Experimental procedure

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.

 

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. These concentrations were generated in the CLARIOstar® measurement chamber by its atmospheric control unit (ACU). The microplate reader detected differences in yeast autofluorescence, citrate synthase 2 expression reported by mCherry and a redox-sensitive roGFP2-based probe.

go to top