A Rhodopsin Immunoanalog in the Related Photosensitive Protozoans Blepharisma Japonicum and Stentor Coeruleus

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PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences A rhodopsin immunoanalog in the related photosensitive protozoans Blepharisma japonicum and Stentor coeruleus

Hanna Fabczak, Katarzyna Sobierajska and Stanisław Fabczak*

Received 8th November 2007, Accepted 12th June 2008 First published as an Advance Article on the web 2nd July 2008 DOI: 10.1039/b717280j

Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope conﬁrmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identiﬁed two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.

Introduction responses or the time-lag of ciliary beat reversal, indicating that blepharismin and stentorin are the primary photoreceptors Blepharisma japonicum and the related ciliate Stentor coeruleus eliciting cellular photoresponses in these ciliates.15,16,24–27 The are highly sensitive to light, and in a nonuniformly illuminated nature of the primary photoprocess in photosensory transduction environment they move predominantly towards shady or dark in these organisms is unknown, although proton and/or electron 1–4 areas. This pattern of light avoidance is the result of changes transfer are believed to play a significant role.23,28–32 So far only in the motile behavior of these protists as a specific reaction to in Blepharisma and Stentor the hypericin-like protein has been 5–7 luminous stimuli. The light-induced responses have been divided identified as a chromophore of photoreceptor molecule. In some 8 into the following categories: a step-up photophobic response, other ciliate protozoa another class of photoreceptors that may positive photokinesis and negative phototaxis. The phototactic be utilized in their photobehavior have already been shown. The response appears when a moving ciliate is accidentally been colorless ciliate Paramecium bursaria has been suggested to contain exposed to a focused light beam and continues to move away a photoreceptor pigment with retinal as the chromophore.33,34 The 9,10 from the light source, along the direction of light propagation. results of investigations curried out on Fabrea salina also rise The positive photokinesis is observed in both ciliates when in area the possibility that a rhodopsin-related protein is present within of continuous illumination of higher intensity, a cell moves faster the membrane in this ciliate however they do not proved that 6,11,12 than in more shaded places. The mechanisms of phototactic this protein could be responsible for photoresponsiveness in these or photokinetic behaviors are not known in detail however in the cells.35 case of the cell phototaxis it has been supposed that it consists Considering the frequent occurrence of rhodopsin as the key 13 of a series of consecutive and short-lasting ciliary reversals. active molecule in photoreceptor cells of various higher organisms The best described motile reaction is the step-up photophobic and also some Protozoa,32,33 it is of great interest to test whether response, which occurs in a similar way in Blepharisma and in rhodopsin-like molecules exist in the photosensitive ciliates 5,7,14–16 Stentor. It consists of a change of direction of cell movement Blepharisma and the related Stentor. Therefore, this study was an as a result of temporary reversal of ciliary beating when the cells attempt to detect and localize rhodopsin-like protein which might speedily swim from the shaded area to a brightly illuminated be present in these two photoresponsive unicellular eukaryote with region. Light perception evoking motile photophobic response in standard immunological methods. both ciliates has been ascribed to their endogenous photoreceptor systems composed of pigment granules (photoreceptive units).17,18 They are located just beneath the plasma membrane, which Results and discussion contain hypericin-like pigments: blepharismin in Blepharisma and stentorin in Stentor.4,12,19–24 The absorption spectra of these The identification of rhodopsin immunoanalogs in Blepharisma pigment granules resemble the action spectra for the photophobic and the related ciliate Stentor was accomplished by immunoblotting. The reaction of blots of electrophoretically-separated proteins with a rabbit antibody raised against rhodopsin revealed Department of Cell Biology, Nencki Institute of Experimental Biology 3 Pasteur Street, 02-093, Warsaw, Poland. E-mail: [email protected]; a predominant band with a molecular mass of about 36 kDa Fax: + 48 22 822 5342; Tel: + 48 22 822 2343 in separated membrane fractions (Fig. 1A, B, lanes PB and

This journal is © The Royal Society of Chemistry and Owner Societies 2008 Photochem. Photobiol. Sci., 2008, 7, 1041–1045 | 1041 Fig. 1 Immunological detection of rhodopsin immunoanalogs in Ble- pharisma (A) and Stentor (B) cells. Proteins of cell homogenate, cytoplasmic and membrane fractions were separated by electrophoresis on 10% SDS-polyacrylamide gels then blotted onto nitrocellulose ﬁlter, reacted with rabbit antibody against rhodopsin and speciﬁc binding was detected using anti-rabbit IgG coupled to peroxidase. LB and LS, whole-cell lysates; SB and SS, supernatant (cytoplasmic) fractions; PB and PS, pellet

(membrane) fractions. Subscript characters (B)or(S) indicate lysate/cell fraction from Blepharisma or Stentor, respectively.

PS) and a much weaker band of the same size in whole-cell lysates (Fig. 1A, B, lanes LB and LS). Observations of cells labeled with the same antibody using a confocal microscope Fig. 3 Immunocytological localization of the rhodopsin immunoanalogs confirmed the immunoblotting results and demonstrated that in Blepharisma (A, B) and Stentor (C, D). Fixed ciliate cells were in both Blepharisma (Fig. 3A, B) and Stentor (Fig. 3C, D) the immunostained with antibody raised against rhodopsin and examined immunoanalog of rhodopsin was localized exclusively within the using a confocal microscope. Fluorescent staining is showing putative membrane area. localized in the submembrane layer. To confirm that the anti-rhodopsin antibody did not cross-react with other proteins present in the cell membranes of Blepharisma contain a rhodopsin-like protein, localized in the membrane area and Stentor, each membrane preparation was separated by two- and present in multiple isoelectric forms. dimensional gel electrophoresis and then immunoblotted. The Higher eukaryotic rhodopsin, a retinal receptor protein with 36 kDa membrane proteins of Blepharisma which bound the polypeptide chain that cross the cell membrane seven times antibody against rhodopsin were resolved into two distinct forms (7-TM), is the best studied example of a photoreceptor which (Fig. 2A): one robust spot was present in the more acidic quarter couples photon absorption to a cascade of biochemical reactions at an isoelectric point (pI) of 4.5 while another much weaker that amplify the input signal and terminate in the generation spot occurred at a pI of 7.0. In the case of proteins in the of a photoreceptor potential.36 Several studies have revealed Stentor membrane fraction, three different 36 kDa protein spots various types of heterogeneity present in rhodopsin from different of approximately equal intensity were present at pIs of 4.4, 5.0 species of eukaryota37–41 as well as prokaryota.42 One expla- and 6.0 (Fig. 2B). nation for this heterogeneity is phosphorylation of rhodopsin by an intrinsic rhodopsin kinase after photon absorption.43,44 The maximum number of phosphate binding sites in rhodopsin appears to be at least five and illuminated photoreceptor cells contain a mixture of phosphorylated species.39,45 The various phosphorylated forms of rhodopsin can be differentiated by their isoelectric points.46 Possibly, the multiple isoelectric forms of the rhodopsin immunoanalogs detected in Blepharisma and Stentor are the consequence of different phosphorylation states of ciliate rhodopsin-like protein (Fig. 2A,B). The presence of genes with homology to a bovine rhodopsin Fig. 2 Multiple isoelectric forms of rhodopsin immunoanalogs in Ble- cDNA has been demonstrated in DNA isolated from different or- pharisma (A) and Stentor (B) membrane fractions. Proteins in membrane ganisms, including a unicellular algae and an Archaebacterium.47 pellets were separated on two-dimensional gels, blotted onto nitrocellulose It has also been shown that the functional photoreceptor in the and reacted with antibody against rhodopsin. The only immunoreactive unicellular alga Chlamydomonas is a microbial-type rhodopsin, a polypeptides were of 36 kDa having pIs of 4.5 and 7.0 for Blepharisma, 7-TM retinal protein, however showing no sequence homology to and 4.4, 5.0 and 6.0 for Stentor. animal rhodopsins.48 Studies carried out on Euglena,49,50 Parame- cium,33,34 Peranema,51 dinoflagellates52–55 and cryptophytes53,56,57 The results of the immunoblotting and immunocytochemical have suggested that rhodopsin-like proteins may be the photore- analyses indicated that both Blepharisma and the related Stentor ceptor responsible for photobehavior in these organisms. The

1042 | Photochem. Photobiol. Sci., 2008, 7, 1041–1045 This journal is © The Royal Society of Chemistry and Owner Societies 2008 ﬁndings in this study indicate that a 36 kDa rhodopsin-like protein (TBS-Tween), the blots were incubated for 1 h at room temperature is also present in colored Blepharisma and the related ciliate with IgG-horseradish peroxidase (HRP)-conjugated secondary Stentor (Fig. 1A, B). It is an interesting challenge to determine antibody diluted 1 : 10000 in blocking buffer. Finally, after washing whether or not the detected rhodopsin immunoanalog functions the membranes several times in TBS-Tween, speciﬁc antibody as the photoreceptor and in what type of photobehavior it might binding was visualized using an Enhanced ChemiLuminescence be utilized by both ciliates. To gain a knowledge of the role of the (ECL) detection system (Amersham). Protein molecular weights rhodopsin-related molecule in the studied protists its sequencing were estimated based on their electrophoretic mobility relative to and detailed characterization of its function is the topic of future pre-stained molecular weight standards (Bio-Rad Laboratories, investigations. Hercules, USA) run on the same gels. In a set of control experiments, incubation with primary antibody was omitted. Experimental Two-dimensional gel electrophoresis Cell cultures Membrane fractions of both examined protists obtained by cell Blepharisma japonicum58 and Stentor coeruleus3 ciliates were fractionation were analyzed by two-dimensional gel electrophore- grown in Pringsheim medium (0.5 mM CaCl2, 1.0 mM MgSO4, sis (2-D PAGE) using a Protean IEF Cell (Bio-Rad Laboratories,

1.0 mM NaNO3, 0.1 mM NaH2PO4 and 1.0 mM Tris–HCl buffer Hercules, USA). Immobilized 7 cm, pH 3–10 gradient strips were to maintain pH 7.0–7.2) at room temperature in semi-darkness. run for the ﬁrst dimension and in the second dimension, proteins The ciliates were fed twice a week with Tetrahymena pyriformis were resolved on a 10% SDS-polyacrylamide gel, which was grown axenically at room temperature in a medium consisting of then analyzed by western immunoblotting with antibody against 1.0% proteose peptone with 0.1% yeast extract. For experiments, rhodopsin. cells were collected by low speed centrifugation and washed in fresh culture medium without the nutritional components. Immunocytochemistry

To study the localization of the protozoan rhodopsin, cells were Cell fractionation first fixed as described previously.61 Following two washes in To detect rhodopsin-related molecules and determine their mor- a buffer composed of 60 mM Pipes (pH 6.9), 25 mM Hepes, phological localization in Blepharisma and Stentor, the cells 10 mM EGTA, 4 mM MgCl2 (PHEM buffer), the cell preparations were first incubated in darkness in fresh culture medium for were incubated for 1 h at room temperature in the same buffer 12 h. Samples of cells were then washed and resuspended in a supplemented with 5% horse serum to block nonspecific binding buffer solution consisting of 150 mM NaCl, 1mM EDTA and (PHEM blocking buffer). The cells were then reacted overnight 20 mM Tris–HCl buffer (pH 7.4) supplemented with protease at 4 ◦C with primary anti-rhodopsin antibody diluted 1 : 100 in and phosphatase inhibitors 50 mM NaF, 2 mM phenylmethylsul- PHEM blocking buffer. Following extensive washing in TBS, fonylfluoride (PMSF), 1 lM okadaic acid, 1 lgml−1 aprotinin, the preparations were incubated for 1 h at room temperature 10 lgml−1 leupeptin and 1 lgml−1 pepstanin]. The cells were with goat anti-rabbit IgG conjugated to FITC, diluted 1 : 300 in disrupted and homogenized by passing through a 25-gauge needle PHEM blocking buffer. Finally, the cell samples were washed in at least 20 times. The resulting homogenates were centrifuged TBS and mounted in Citifluor (Citifluor Ltd, London, UK). The at 500 g for 5 min to remove nuclei and large debris. To immunostained cell preparations were examined using a confocal pellet membranes, the collected supernatants were centrifuged microscope (Leica TCS-SP2, Leica Microsystems GmbH, Wetzlar, at 100 000 g for 1 h at 4 ◦C. The obtained whole cell lysates, Germany). Nonspecific fluorescence was determined in cell sam- and cytoplasmic (supernatant) and membrane (pellet) fractions ples suspended in blocking buffer lacking the primary antibody from both tested ciliates were analyzed by gel electrophoresis and during the immunostaining process. western immunoblotting. Chemicals Electrophoresis and western immunoblotting Materials for electrophoresis and immunoblotting were purchased Cell samples were mixed with 4-fold concentrated SDS sam- from Bio-Rad Laboratories (Hercules, USA). Rabbit polyclonal ple buffer supplemented with protease and phosphatase in- antiserum raised against bovine rhodopsin was purchased from hibitors (as above) and then resolved by electrophoresis on SDS- Santa Cruz Biotechnol. Inc. (Santa Cruz, USA). Secondary polyacrylamide gels59 using a Hoefer Electrophoresis System antibody conjugated to horseradish peroxidase (HRP) was pur- (Amersham Pharm. Biotechnol., Little Chalfont, UK). The sep- chased from Calbiochem (Darmstadt, Germany), while secondary arated proteins were transferred to a nitrocellulose membrane in antibody conjugated to fluorophore AlexaFluor 488 was obtained transfer buffer using a Hoefer TE 22 System (Amersham Pharm. from Molecular Probes (Leiden, The Netherlands). All other Biotech., Piscataway, USA).60 Theblotswereblockedby2h reagents were purchased from Sigma-Aldrich Co. (Munich, Ger- incubation at room temperature in Tris-buffered saline (TBS) com- many). posed of 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, supplemented with 0.1% Tween-20 (v/v) and 5% skimmed milk (blocking buffer). Conclusions The blots were then incubated overnight at 4 ◦C with primary anti- rhodopsin polyclonal antibody diluted 1 : 1000 in blocking buffer. The results of this study indicate that both unicellular eukaryotes, After several washes in TBS supplemented with 0.1% Tween-20 Blepharisma and Stentor possess the proteins related to rhodopsin.

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