activated when the flagellum is pointing downwards during the helical rotation of the
cell (Häder et al. 2005a). Repetitive activation of the channels results in a bending of
the flagellum mediating upward steering. The influx of Ca
2+ ions during gravitactic
orientation results in a change in the membrane potential which can be demonstrated
using the electrochromic absorbance band shift of Oxonol VI incorporated into the
membrane (Richter et al. 2001) showing that during reorientation of the cells the
membrane potential changed accordingly (Richter et al. 2006). Disturbing the
membrane potential by application of the lipophilic cation TPMP
+ (triphenylmethyl
phosphonium) reduced the precision of gravitactic orientation in Euglena (Lebert
et al. 1996).
The identification of the mechanosensitive ion channel responsible as gravireceptor in Euglena was revealed by molecular biology. Using several primers
against the conserved regions of the corresponding gene in Saccharomyces (Häder
et al. 2003) yielded more than 1500 PCR products which were cloned in plasmids
and sequenced (Häder et al. 2009). Most of these genes coded for proteins with
different functions, but some resembled genes for a TRP (transient receptor
potential-like) channel. The large TRP protein family is involved in photoperception,
nociception, thermosensation, mechanosensing, taste, osmolar sensation, and fluid
flow detection (Nilius et al. 2012). The method of RNA interference (RNAi) was
used to identify the specific TRP involved in graviperception. For this purpose,
double-stranded RNA fragments (19–23 nucleotides) are introduced into the cell
which causes a sequence-specific post-transcriptional gene silencing. The RNA
fragment attaches itself to the mRNA coding for the protein in question and thus
blocks its translation and the synthesis of the corresponding protein. Four PCR
transcripts were found in Euglena using degenerated primers reflecting the pore
region of the mechanosensitive channel. Using RNAi showed that three of these
products were not involved in gravitaxis, but inhibition of the fourth (TRPC7)
blocked graviorientation for up to 30 days (Fig. 3.5).
The Ca
2+ ions gated into the cytoplasm during gravistimulation could bind to
calmodulins, a group of proteins with usually four binding sites for Ca
2+ , which
function as universal messenger in many organisms from bacteria to vertebrates
(Adler 2013). Calmodulin can be inhibited by trifluoperazine, fluphenazine or W7
(Naccache 1985; Russo et al. 2014; Son et al. 2014). Application of these drugs to
Euglena strongly impaired gravitaxis (Häder et al. 2006). In order to test the
hypothesis that a calmodulin is involved in the gravitaxis sensory transduction
chain we searched for corresponding genes in Euglena. Surprisingly we found and
sequenced five genes (CaM.1–CaM.5; Daiker et al. 2010). One of these (CaM.1) was
already known in E. gracilis and shown to be located under the pellicula (Toda et al.
1992). Applying again RNAi we blocked the protein synthesis of each calmodulin in
separate populations. Using RT-PCR confirmed that blocking the individual mRNA
suppressed the synthesis of the corresponding calmodulin. Inhibiting CaM.1 caused
strong abnormalities of the cell form and inhibited swimming motility even though
the flagellum was visible. RNAi against CaM.3–CaM.5 did not affect graviorientation but inhibition of CaM.2 effectively impaired gravitaxis.
3.6 Sensory Transduction Chain for Gravitaxis
37
cell (Häder et al. 2005a). Repetitive activation of the channels results in a bending of
the flagellum mediating upward steering. The influx of Ca
2+ ions during gravitactic
orientation results in a change in the membrane potential which can be demonstrated
using the electrochromic absorbance band shift of Oxonol VI incorporated into the
membrane (Richter et al. 2001) showing that during reorientation of the cells the
membrane potential changed accordingly (Richter et al. 2006). Disturbing the
membrane potential by application of the lipophilic cation TPMP
+ (triphenylmethyl
phosphonium) reduced the precision of gravitactic orientation in Euglena (Lebert
et al. 1996).
The identification of the mechanosensitive ion channel responsible as gravireceptor in Euglena was revealed by molecular biology. Using several primers
against the conserved regions of the corresponding gene in Saccharomyces (Häder
et al. 2003) yielded more than 1500 PCR products which were cloned in plasmids
and sequenced (Häder et al. 2009). Most of these genes coded for proteins with
different functions, but some resembled genes for a TRP (transient receptor
potential-like) channel. The large TRP protein family is involved in photoperception,
nociception, thermosensation, mechanosensing, taste, osmolar sensation, and fluid
flow detection (Nilius et al. 2012). The method of RNA interference (RNAi) was
used to identify the specific TRP involved in graviperception. For this purpose,
double-stranded RNA fragments (19–23 nucleotides) are introduced into the cell
which causes a sequence-specific post-transcriptional gene silencing. The RNA
fragment attaches itself to the mRNA coding for the protein in question and thus
blocks its translation and the synthesis of the corresponding protein. Four PCR
transcripts were found in Euglena using degenerated primers reflecting the pore
region of the mechanosensitive channel. Using RNAi showed that three of these
products were not involved in gravitaxis, but inhibition of the fourth (TRPC7)
blocked graviorientation for up to 30 days (Fig. 3.5).
The Ca
2+ ions gated into the cytoplasm during gravistimulation could bind to
calmodulins, a group of proteins with usually four binding sites for Ca
2+ , which
function as universal messenger in many organisms from bacteria to vertebrates
(Adler 2013). Calmodulin can be inhibited by trifluoperazine, fluphenazine or W7
(Naccache 1985; Russo et al. 2014; Son et al. 2014). Application of these drugs to
Euglena strongly impaired gravitaxis (Häder et al. 2006). In order to test the
hypothesis that a calmodulin is involved in the gravitaxis sensory transduction
chain we searched for corresponding genes in Euglena. Surprisingly we found and
sequenced five genes (CaM.1–CaM.5; Daiker et al. 2010). One of these (CaM.1) was
already known in E. gracilis and shown to be located under the pellicula (Toda et al.
1992). Applying again RNAi we blocked the protein synthesis of each calmodulin in
separate populations. Using RT-PCR confirmed that blocking the individual mRNA
suppressed the synthesis of the corresponding calmodulin. Inhibiting CaM.1 caused
strong abnormalities of the cell form and inhibited swimming motility even though
the flagellum was visible. RNAi against CaM.3–CaM.5 did not affect graviorientation but inhibition of CaM.2 effectively impaired gravitaxis.
3.6 Sensory Transduction Chain for Gravitaxis
37