Photoactivated Adenylyl Cyclase Controls Phototaxis in the Flagellate Euglena gracilis

Abstract

Euglena gracilis, a unicellular freshwater protist exhibits different photomovement responses, such as phototaxis (oriented movement toward or away from the light source) and photophobic (abrupt turn in response to a rapid increase [step-up] or decrease [step-down] in the light fluence rate) responses. Photoactivated adenylyl cyclase (PAC) has been isolated from whole-cell preparations and identified by RNA interference (RNAi) to be the photoreceptor for step-up photophobic responses but not for step-down photophobic responses (M. Iseki, S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, C. Yoshida, M. Sugai, T. Takahashi, T. Hori, M. Watanabe [2002] Nature 415: 1047-1051). The present study shows that knockdown of PAC by RNAi also effectively suppresses both positive and negative phototaxis, indicating for the first time that PAC or a PAC homolog is also the photoreceptor for photoorientation of wild-type E. gracilis. Recovery from RNAi occurred earlier for step-up photophobic responses than for positive and negative phototaxis. In addition, we investigated several phototaxis mutant strains of E. gracilis with different cytological features regarding the stigma and paraxonemal body (PAB; believed to be the location for the phototaxis photoreceptor) as well as Astasia longa, a close relative of E. gracilis. All of the E. gracilis mutant strains had PAC mRNAs, whereas in A. longa, a different but similar mRNA was found and designated AlPAC. Consistently, all of these strains showed no phototaxis but performed step-up photophobic responses, which were suppressed by RNAi of the PAC mRNA. The fact that some of these strains possess a cytologically altered or no PAB demonstrates that at least in these strains, the PAC photoreceptor responsible for the step-up photophobic responses is not located in the PAB.The protist Euglena gracilis, a unicellular freshwater flagellate, is capable of both autotrophy and heterotrophy. The cell is characterized (Fig. 1A) by one flagellum emerging from the reservoir (invagination of the anterior plasma membrane) and a second nonemerging flagellum. The paraxonemal body (PAB) is a photosensing organelle (Ghetti et al., 1985) located inside the reservoir close to the connecting point of the two flagella. The stigma, formerly known as the eyespot, is positioned inside the cytoplasm and adjacent to the PAB. It contains carotenoids and is not involved in photosensing, as initially thought, but contributes to photoorientation (Lebert and Häder, 1997) by shading the PAB as the cell rotates around its longitudinal axis during forward swimming. A detailed description of E. gracilis can be found in Buetow (1968).E. gracilis uses light and gravity for orientation to move to and stay at optimal growth conditions in the water column. Light-induced responses can be categorized into photokinesis, a light-dependent swimming velocity; phototaxis, an oriented movement toward (positive phototaxis) or away (negative phototaxis) from the light source (Häder et al., 1981); and photophobic responses (Mikolaj...

Targeting and Readout Strategies for Fast Optical Neural ControlIn VitroandIn Vivo

“…Optical activation of intracellular signaling via modified opsins (Kim et al, 2005) and blue-light-activated adenylate cyclases (PACs) from the flagellate Euglena gracilis (Iseki et al, 2002;Ntefidou et al, 2003) can trigger rapid and reversible increases in cAMP levels in vitro and modulate behavior in Drosophila in vivo (Schroder-Lang et al, 2007). Phototropins, photo-regulated protein kinases (Huala et al, 1997;Briggs et al, 2001), might also provide useful tools for biochemistry studies of phosphorylation events within a cell that could complement ChR2/NpHR modulation of biochemical signaling, along with other lightactivated compounds yet to be identified.…”

Kinetic analysis of the activation of photoactivated adenylyl cyclase (PAC), a blue-light receptor for photomovements of Euglena

“…7 In 2003, Ntefidou et al reported that PAC itself or a molecule that is similar to PAC was also involved in positive and negative phototaxis. 4 PAC consists of alpha and beta subunits both having two flavin binding domains F1 and F2, each followed by a catalytic domain of adenylyl cyclase C1 and C2, respectively. The flavin binding domains of PAC are similar to the N-terminal region of the flavoprotein AppA from the purple bacterium Rhodobacter sphaeroides.…”

Motion of Euglena gracilis: Active fluctuations and velocity distribution

“…The motion is not purely phototactic: Euglena cells in dark conditions perform random motion with much spinning [38]. The threshold intensity of blue light required to cause the negative phototaxis is approximately 10 mW/cm 2 [39]. This is an almost two times lower limiting light intensity than that for a normal daylight [40].…”

“…Although we successfully described the population statistics, on the single cell level the mechanism of the active fluctuations and of the observed "fatigue" remains unclear. This mechanism can be investigated further if one addresses complex and not fully understood impulse-response behaviors involving the photoactivated adenylyl cyclase [39,50] and protein kinase [51].…”