Georg Kreimer

Proteomic Analysis of the Eyespot of Chlamydomonas reinhardtii Provides Novel Insights into Its Components and Tactic Movements

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. To further understand the molecular organization of the eyespot apparatus and the phototactic movement that is controlled by light and the circadian clock, a detailed understanding of all components of the eyespot apparatus is needed. We developed a procedure to purify the eyespot apparatus from the green model alga Chlamydomonas reinhardtii. Its proteomic analysis resulted in the identification of 202 different proteins with at least two different peptides (984 in total). These data provide new insights into structural components of the eyespot apparatus, photoreceptors, retina(l)-related proteins, members of putative signaling pathways for phototaxis and chemotaxis, and metabolic pathways within an algal visual system. In addition, we have performed a functional analysis of one of the identified putative components of the phototactic signaling pathway, casein kinase 1 (CK1). CK1 is also present in the flagella and thus is a promising candidate for controlling behavioral responses to light. We demonstrate that silencing CK1 by RNA interference reduces its level in both flagella and eyespot. In addition, we show that silencing of CK1 results in severe disturbances in hatching, flagellum formation, and circadian control of phototaxis.

Cell biology of phototaxis in flagellate algae

Publisher Summary This chapter describes the structural aspects of different photoreceptive organelles including their interaction with cytoskeletal elements. It also examines the mechanisms used for signal generation and modulation, the photoreceptors used and the involved photoelectrical responses, and the currently known elements of the signal transduction or processing chains toward the flagellum. The photoreceptive apparatuses of flagellate algae exhibit an enormous structural variation and result from numerous parallel evolutionary processes. The phototactic systems of flagellate algae, including the sensory transduction chain, are optimized to detect a temporal pattern of light rather than the steady-state light intensity. Proper positioning of the functional eyespot apparatus during cell division is important for signal perception or generation in algal phototaxis and for correct coupling of the photoreceptive apparatus to the signaling effector, the flagellum. Different criteria can be applied for grouping the photoreceptive organelles of flagellate algae, such as ultrastructural characteristics, the used photoreceptor pigment, the mechanisms involved in primary signal amplification, and extension to low light intensities.

An electrogenic uniport mediates light-dependent Ca2+ influx into intact spinach chloroplasts

Light‐dependent Ca2+ influx into intact spinach chloroplasts, measured with the metallochromic indicator arsenazo III, is stimulated by uncouplers (FCCP, CCCP, nigericin) and inhibited by ruthenium red. The data presented demonstrate that light‐dependent Ca2+ influx into chloroplasts is electrogenic and mediated by a uniport‐type carrier. The characteristics of the carrier system are similar to those of the Ca2+ uniport of mitochondria.

The Phosphoproteome of a Chlamydomonas reinhardtii Eyespot Fraction Includes Key Proteins of the Light Signaling Pathway

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca2+-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.

Identification of 11-cis and all-trans-retinal in the photoreceptive organelle of a flagellate green alga.

Isolation of intact photoreceptive organelles (eyespot apparatuses) involved in blue‐light mediated photoresponses in a flagellate green alga (Spermatozopsis similis) allowed for the first time the identification of both 11‐cis‐ and all‐trans‐retinal in a plant cell. Both isomers were identified by HPLC analysis in conjunction with UV spectra. Additionally, reconstitution of a distinct absorption band, centered around 540 nm, was achieved by addition of exogenous 9‐cis‐retinal to bleached, isolated eyespot apparatuses.

Light- and Ca2+-Modulated Heterotrimeric GTPases in the Eyespot Apparatus of a Flagellate Green Alga

Little is known about phototactic signal transduction in flagellate green algae; therefore, eyespot apparatuses, which are the light-sensitive “organelles” involved in photoorientation of these algae, were isolated and analyzed for the presence of heterotrimeric guanine nucleotide binding proteins (G proteins) and their coupling to the retinal-based photoreceptor. Specific high-affinity 35S-GTP-γ-S binding and GTPase activity, with sensitivity toward antibodies raised against vertebrate/invertebrate Gα subunits and fluoroaluminates, were detected. In one- and two-dimensional immunoblot analyses, an antiserum directed against Giα-type subunits exhibited cross-reactivity at 42 kD, whereas a 43-kD protein cross-reacted with antisera directed against Gqα subunits. Green light below 1 μE m−2 sec−1 suppressed cholera toxin–dependent ADP ribosylation at these apparent molecular masses and modulated a significant proportion of the GTPase activity in a reversible manner. Antisera against Chlamydomonas rhodopsin and the Gα subunits completely impaired light modulation. Both light sensitivity and dark recovery of the GTPase were affected by changes in free Ca2+. Dissociation of the putative Gα subunits from the eyespot membranes was not observed when the membranes were illuminated. Our results emphasize the regulatory potential of Gα subunits in rhodopsin-based signaling of flagellate green algae.

Phototropin Influence on Eyespot Development and Regulation of Phototactic Behavior in Chlamydomonas reinhardtii

This study shows that an eyespot is a dynamic organelle and that the blue light photoreceptor phototropin is involved in the regulation of eyespot size and level of channelrhodopsin 1, which is the primary photoreceptor for photo-movement responses. This work also describes that, in addition to the C-terminal kinase domain, the N-terminal photoreceptor domains have independent signaling functions. The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity–dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.

Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt-

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt− were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500∶440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 μM), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.

Calcium modulates rapid protein phosphorylation/dephosphorylation in isolated eyespot apparatuses of the green alga Spermatozopsis similis

We present an initial characterization of protein kinase and phosphatase activities associated with isolated eyespot apparatuses, the organelle involved in blue/ green-light-mediated behavioural responses of flagellate green algae. In the presence of the phosphatase inhibitors okadaic acid and vanadate, rapid overall protein phosphorylation (t0.5 ≈ 10 s) was observed. The majority of protein kinase activities and their substrates were identified as integral or tightly-bound peripheral membrane proteins. While vanadate generally increased the phosphate incorporation into all phosphoproteins, okadaic acid specifically enhanced phosphorylation of proteins in the range of 39–43 kDa. In contrast to all other phosphoproteins in this subcellular fraction, two proteins with apparent molecular masses of 83 and 16 kDa shared remarkable similarities: (i) They exhibited a fast turnover of the 32P-label, even in the presence of phosphatase inhibitors, and (ii) their dephosphorylation was delayed at 10−8 M free Ca2+. In addition, the 16-kDa protein underwent thiophosphorylation. The general in-vitro phosphorylation pattern was strongly influenced by alterations of free Ca2+ in a concentration range known to affect responses related to phototactic and photophobic behaviour of this alga (10−8 M to 10−7 M). However, characteristics of Ca2+-calmodulin-dependent protein kinases were not observed, i.e. exogenous calmodulin and trifluoperazine had no significant effect on protein phosphorylation. Also exogenous lipids (phosphatidylserine, diacylglycerol), inhibitors of cGMP and cAMPdependent protein kinases and protein kinase C (H-7 and HA1004) as well as exogenously added cGMP and cAMP did not potentiate or inhibit protein phosphorylation. These characteristics of the kinase activity in our fraction most closely resemble those of the plant- and protist-specific group of Ca2+-dependent, calmodulin-independent protein kinases. In-situ phosphorylation experiments following electrophoretic separation revealed the presence of three putative Ca2+-dependent kinases or their catalytic subunits (77,48 and 47 kDa) in the eyespot preparation. In addition, a Ca2+-independent activity at 28 kDa was detected. Possible roles of reversible protein phosphorylation in eyespot apparatuses are discussed.

Two members of the ERabp gene family are expressed differentially in reproductive organs but to similar levels in the coleoptile of maize

A Zea mays cDNA clone, ZmERabp4, coding for a new member of the auxin-binding protein family was isolated. The primary amino acid sequence contains an N-terminal hydrophobic leader sequence, a potential glycosylation site (Asn136-Thr-Thr) and a C-terminal KDEL motif known to be responsible for retention of proteins within the lumen of the ER. The expression pattern of the ZmERabp4 gene in various organs of maize differs from the expression pattern previously observed for the ZmERabp1 gene. The ZmERabp4 gene is expressed highly in male flower organs, whereas the ZmERabp1 gene shows highest expression in female flower parts. In situ hybridization and analysis by laser scanning microscopy revealed enhanced levels of expression for both genes in the coleoptile when compared with the primary leaf of etiolated maize seedlings.