Channelrhodopsins act as photoreceptors for control of motility behavior in flagellates and are widely used as genetically targeted tools to optically manipulate the membrane potential of specific cell populations (“optogenetics”). The first two channelrhodopsins were obtained from the model organism Chlamydomonas reinhardtii (CrChR1 and CrChR2). By homology cloning we identified three new channelrhodopsin sequences from the same genus, CaChR1, CyChR1 and CraChR2, from C. augustae, C. yellowstonensis and C. raudensis, respectively. CaChR1 and CyChR1 were functionally expressed in HEK293 cells, where they acted as light-gated ion channels similar to CrChR1. However, both, which are similar to each other, differed from CrChR1 in current kinetics, inactivation, light intensity dependence, spectral sensitivity, and dependence on the external pH. These results show that extensive channelrhodopsin diversity exists even within the same genus, Chlamydomonas. The maximal spectral sensitivity of CaChR1 was at 520 nm at pH 7.4, about 40 nm red-shifted as compared to that of CrChR1 under the same conditions. CaChR1 was successfully expressed in Pichia pastoris and exhibited an absorption spectrum identical to the action spectrum of CaChR1-generated photocurrents. The red-shifted spectra and the lack of fast inactivation in CaChR1- and CyChR1-generated currents are features desirable for optogenetics applications.

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...

Pollen tube tip growth is a widely used model ideally suited to study cellular processes underlying polarized cell expansion. Local secretion supplying material for plasma membrane (PM) and cell wall extension is essential for this process. Cell wall biogenesis requires fusion of secretory vesicles with the PM at an about 10× higher rate than PM extension. Excess material is therefore incorporated into the PM, which needs to be reinternalized through endocytosis. The classical model of tip growth proposes that exocytosis occurs at the apex and that newly incorporated PM material is transported to adjacent lateral regions, where excess material is endocytically recycled. This model was recently challenged based on studies indicating that lateral exocytosis may be balanced by apical endocytosis. This review provides an overview of published data pertaining to exocytosis, endocytosis and vesicular trafficking in pollen tubes. Its key aim is to present classical and alternative models of tip growth in the light of available experimental data. By necessity, the review focusses on pollen tubes of angiosperm models (Nicotiana tabacum, Arabidopsis, Lilium longiflorum), which have been studied far more extensively and grow much faster than structurally strikingly different gymnosperm pollen tubes. Only major transport pathways are considered, which substantially contribute to the mass-flow of membrane material at the pollen tube tip. Growth oscillation, which may be displayed in particular by fast-growing pollen tubes, are not discussed as their influence on the spatial organization of apical membrane traffic is not understood.