Name: isolating and incorporating light-harvesting antennas from diatom cyclotella meneghiniana in liposomes with thylakoid lipids
Uploaded: 2018-08-28T14:00:00
Description: Watch this Scientific Journal Video about Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids at JoVE.com

We thank Rana Adeel Ahmad for assistance in FCP purification. Prof. Claudia B&#252;chel is acknowledged for helpful discussions and reading the manuscript. This work was supported by the German Research Foundation to LD (DI1956-1/1) and the Humboldt foundation for a Feodor-Lynen fellowship to LD.

Note: Photosynthetic complexes such as FCPs are highly vulnerable to light and heat. Always work on ice and under a very dim light.
1. Isolation of FCP from Cells
<ol>
	<li>Thylakoid isolation from C. meneghiniana cells
	<ol>
		<li>Grow C. meneghiniana in five 500 mL flasks each filled with 300 mL of ASP-medium23,25 and 50 million cells. Plug the flasks with a cotton stopper and all...

FCP liposomes with natural lipid composition provide a handy, simple and reproducible tool to investigate spectroscopic properties in vitro. The lipid environment in FCP liposomes resembles the situation within the thylakoid membrane, giving rise to experimental results that are closer to natural conditions.
There are several advantages of using C. meneghiniana as a model system for FCP antenna. It grows relatively fast and is more robust in comparison to other diatom model s...

Photosynthetic organisms such as diatoms must cope with ever-changing light conditions and respond with sophisticated acclimation mechanisms that sustain high photosynthetic efficiency and protect from photo-oxidative damage caused by the excessive light. A major light-protective process in photosynthetic eukaryotes is the high energy quenching (qE) of absorbed light that occurs as the main contribution to the non-photochemical quenching (NPQ) under light stress conditions1,2,3. The light harvesting antenna complexes (LHC) are involved in the regulation of excitation energy transfer pathways. In response to high light induced low pH in the chloroplast lumen, the antenna system switches from the light harvesting state to the quenching state. This energy dissipative state protects photosystems (PS) and other complexes in the thylakoid membrane from photo-oxidation. In photosynthetic eukaryotes, the qE is usually induced by two factors1,2,3. One factor is the specialized light harvesting protein that responds to the low pH. The PsbS protein induces the qE in higher plants4. LhcSRs5, modulated by PsbS activity, induce the qE in green algae6. Diatoms possess Lhcx-like proteins which structurally related to LHCSRs7,8,9,10.
The second factor of qE is the xanthophyll cycle where carotenoids of the antenna are converted into a photo-protective form by de-epoxidation and reverted by epoxidation. In plants and green algae, violaxanthin is converted to zeaxanthin. In diatoms, diadinoxanthin is converted to diatoxanthin, which then correlates with the extent of NPQ11. The diatom light harvesting antenna possesses some peculiarities although it is evolutionary related to plant and algal LHCs. The switch from light harvesting to photo-protection is enormously fast and the NPQ capacity is higher compared to plants12. This might be one reason why diatoms are very successful in different ecological niches in a way that they are responsible for up to 45% of the oceanic net primary production13. Therefore, diatom light harvesting systems are an interesting object of photosynthesis research.
Diatoms, like the centric species Cyclotella meneghiniana, possess thylakoid intrinsic light harvesting systems named after the pigments they bind - fucoxanthin, chlorophyll (chl) a and c, hence FCP. Light harvesting proteins, such as FCPs, are embedded in the thylakoid membrane system comprising several membrane layers. Diatoms form bands of three thylakoids. This complex situation makes it difficult to study them on the molecular level in the thylakoid membrane. In addition, many components contribute to the regulation of light harvesting (see above). Therefore, in many approaches, the complexes were isolated from the membrane using mild detergents, such as n-Dodecyl-&#946;-D-maltopyranoside (&#946;-DDM), which solubilize the membrane but keep the FCP complexes intact. Many spectroscopic studies were performed using solubilized FCP to investigate intramolecular energy transfer14,15,16,17. However, this former approach was limited since the regulation of energy transfer needs excitonic interaction with other antenna complexes or photosystems. Hence, these kinds of studies cannot be carried out with solubilized complexes because the interaction between complexes is lost.
An important feature in antenna regulation is the &#34;molecular crowding&#34; of the antenna and photosystems in the thylakoid membrane18. Formerly, a simple approach was carried out to simulate this effect in vitro. The detergent was removed, which leads to random aggregation of antenna complexes. Although some reasonable data was obtained by this approach17,19, the detergent removal does not reflect the situation in vivo and has some limitations since the complexes are not interacting in their regular tertiary structure.
The use of liposomes overcomes several of the former limitations. The tertiary structure is still fully intact. The liposome membrane provides a quasi-native environment for the antenna complexes. The membrane separates the inside of the liposome from the outside environment. By these means, liposomes provide two reaction compartments for studies of ion and pH gradients as well as for transport processes. Further, the parameters of the experimental system can be controlled more easily for studies in the thylakoid membrane. Liposomes were already shown to be an excellent tool to study photosynthetic complexes. A major focus in the past was on plant LHC where the effect of altered lipid composition was tested on LHC II20. In other approaches, protein-protein interaction between different LHC II were investigated21. Also, some studies in green algae were carried out that describe spontaneous clustering between LHC22. Considering the importance of diatoms for aquatic ecosystems, relatively few studies were performed with antenna complexes of diatoms. Two studies investigated the antenna complexes of the centric Cyclotella meneghiniana, where the clustering of the FCP antenna23 and responsiveness of FCP to electrochemical gradients24 were shown. Thus, liposomes are an excellent tool to study diatom antennas and their interaction and regulation in nearly native conditions. The liposomes are versatile since many conditions such as lipid composition, liposome size, protein density and the surrounding aqueous phase can be controlled. Furthermore, the method requires low amounts of samples. The experimental system is robust and highly reproducible. The compartmentalization of liposomes allows for studying pH and ion gradients, which are important factors in the regulation of antenna complexes.
Here, we describe the isolation of FCP antenna complexes from C. meneghiniana and their incorporation in liposomes with natural thylakoid lipid composition. Also, we provide exemplary data for the spectroscopic characterization of solubilized FCP and compare them with FCP in liposomes. The method summarizes knowledge and standardized protocols obtained from the improvements of Gundermann and B&#252;chel 201223, Natali et al. 201622, and Ahmad and Dietzel 201724.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58017/58017fig1.jpg" /> 
Figure 1: Schematic representation of the workflow. (1) Refers to paragraph 1 which describes cell growth, disruption and thylakoid isolation with following FCP separation on sucrose density gradients; C. m.-Cyclotella meneghiniana cells. (2) Preparation of natural thylakoid lipid mixture (MGDG, DGDG and SQDG) described in paragraph 2 and creation of lipid-detergent micelles with octylglycoside (OG). A defined lipid-micelle size is achieved by extrusion using membranes of a defined pore diameter. FCP and lipid-micelles are unified at a predefined lipid: protein ratio and the detergents OG and &#946;-DDM are removed via controlled dialysis forming FCP proteoliposomes. <a href="https://www.jove.com/files/ftp_upload/58017/58017fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>500 ml centrifuge vials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>high speed centrifuge</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bead Mill VI 2</td>
			<td>Edmund-B&#252;hler (edmund-buehler.de)</td>
			<td></td>
			<td>newer version: Vibrogen-Zellm&#252;hle Vl 6</td>
		</tr>
		<tr>
			<td>Silibeads S 400 &#181;m</td>
			<td>Sigmund-Lindner.com</td>
			<td>5223-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Silibeads S 1,-1,3 mm</td>
			<td>Sigmund-Lindner.com</td>
			<td>4504</td>
			<td></td>
		</tr>
		<tr>
			<td>VitraPOR filter funnel - por1</td>
			<td>ROBU GmbH</td>
			<td>21121</td>
			<td></td>
		</tr>
		<tr>
			<td>polycarbonate ultracentrifuagtion vials (30 mL) for T-865</td>
			<td>Beranek Laborger&#228;te (Laborgeraete-beranek.de)</td>
			<td>314348</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge Discovery 90SE</td>
			<td>Sorvall</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor T 865</td>
			<td>ThermoFisher Scientific (thermofisher.com)</td>
			<td>51411</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer Cell Counter Chamber (improved)</td>
			<td>Carl Roth Laborbedarf (Carlroth.com)</td>
			<td>T729.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Mikroskop Primostar (7)</td>
			<td>Optik-Pro (optik-pro.de)</td>
			<td>51428</td>
			<td></td>
		</tr>
		<tr>
			<td>optical glass cuvettes (6040-OG)</td>
			<td>Hellma Analytics (hellma-analytics.com)</td>
			<td>&#34;6040-10-10&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>V-630 UV-VIS Spectrophotometer (incl. software)</td>
			<td>Jasco (jasco.de)</td>
			<td>V-630</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Dodecyl-&#946;-D-Maltopyranoside</td>
			<td>ANATRACE (anatrace.com)</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear tubes 17 ml for AH629</td>
			<td>Beranek Laborger&#228;te (Laborgeraete-beranek.de)</td>
			<td>344061</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor AH629-17-mL</td>
			<td>ThermoFisher Scientific (thermofisher.com)</td>
			<td>54285</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane concentrator_Centriprep 30 kDa cutoff</td>
			<td>Millipore (merckmillipore.com)</td>
			<td>4307</td>
			<td></td>
		</tr>
		<tr>
			<td>Biometra Minigel-Twin</td>
			<td>Analytik Jena AG (analytik-jena.de)</td>
			<td>846-010-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Stain Plus Kit</td>
			<td>Bio-Rad (bio-rad.com)</td>
			<td>1610449</td>
			<td></td>
		</tr>
		<tr>
			<td>libre office spread sheet</td>
			<td>The document foundation</td>
			<td>https://de.libreoffice.org/download/libreoffice-still/</td>
			<td></td>
		</tr>
		<tr>
			<td>special glass cuvettes for fluorescence (101-0S)</td>
			<td>Hellma Analytics (hellma-analytics.com)</td>
			<td>101-10-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrofluorometer FP-6500 (incl. Software)</td>
			<td>Jasco (jasco.de)</td>
			<td>FP-6500</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-loading buffer Roti-Load</td>
			<td>ROTH (carlroth.com)</td>
			<td>K929.1</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octyl &#946;-D-glucopyranoside</td>
			<td>ANATRACE (anatrace.com)</td>
			<td>O311</td>
			<td></td>
		</tr>
		<tr>
			<td>Monogalactosyl Diaclyglycerol (MGDG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>59-1300</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>Digalactosyl Diacylglycerol (DGDG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>59-1310</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>Sulphoquinovosyl Diacylglycerol (SQDG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>59-1230</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>L-alpha-Phosphatidylglycerol (PG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>37-0150</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>L-&#945;-Phosphatidylcholine</td>
			<td>Sigma-Aldrich (sigmaaldrich.com)</td>
			<td>P3782 SIGMA</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>sonicator bath S-50TH</td>
			<td>Sonicor (getmedonline.com</td>
			<td>SONICOR-S-50TH</td>
			<td></td>
		</tr>
		<tr>
			<td>mini-Extruder</td>
			<td>Avanti Polar Lipids (Avanti.com)</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuleopore polycarbonate membrane</td>
			<td>Avanti Polar Lipids (Avanti.com)</td>
			<td>610005</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis membrane Visking 14 kDa cutoff</td>
			<td>ROTH (carlroth.com)</td>
			<td>0653.1</td>
			<td>boil in destilled water before use</td>
		</tr>
		<tr>
			<td>Biobeads SM2 Adsorbent</td>
			<td>Biorad (Bio-rad.com)</td>
			<td>152-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose epichlorhydrin copolymer - Ficoll 400</td>
			<td>Sigma-Aldrich (sigmaaldrich.com)</td>
			<td>F4375</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate ultracentrifuagtion vials (2.7 mL) for TFT 80.4</td>
			<td>Beranek Laborger&#228;te (Laborgeraete-beranek.de)</td>
			<td>252150</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor TFT 80.4</td>
			<td>Millipore (merckmillipore.com)</td>
			<td>54356</td>
			<td></td>
		</tr>
		<tr>
			<td>material listed in order of appearance </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For specific safety instructions please refer to material safety sheets and repective manuals.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard lab material and substances are not listed.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating and incorporating light-harvesting antennas from diatom cyclotella meneghiniana in liposomes with thylakoid lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

The protocol describes the isolation of total FCP fraction from Cyclotella meneghiniana and incorporation into liposomes with native lipid composition. The thylakoid isolation is highly reproducible, but the thylakoid yield may change. The result is acceptable if more than 50% of all pigments are recovered in step 1.1.4. More than 80% is optimal.
The solubilization of the thylakoids is a critical step. Well-solubilized ...

Watch this Scientific Journal Video about Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids at JoVE.com

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

This method can help us to understand key questions in photosynthesis research, such as the fade of captured light energy and light harvesting complexes, its kinetics, and efficiency of transfer. The main advantage of this technique is that liposomes provide a membrane compartment resembling the native state of a thylakoid membrane, allowing for studies of ion and pH changes during photosynthesis. To begin the protocol, centrifuge previously-prepared C.meneghiniana cells at 4, 300 times G in a pre-cooled rotor at four degrees Celsius with 500 milliliter centrifuge vials for 15 minutes.Resuspend the cell pellets homogenization buffer by shaking and pipetting. Transfer the suspension to a single suitable plastic tube and restrict the final volume to 12 milliliters. Next, pre-cool the bead mill and equipment for about 60 minutes.Fill the 50 milliliter beaker up to 75%with a glass bead mixture, and add the cell suspension. Stir it with a spatula or glass rod and add HB if necessary. For cell disruption, use seven by 45 second pulses at full speed, with 30 seconds of cooling between each pulse.Filter the disrupted cells over a glass filter funnel and wash them by pouring the HB over the glass beads until they appear clear. Then, pool the wash fraction with the filtrate and keep the final volume lower than 150 milliliters. Centrifuge the sample for 15 minutes at 140 times G using three 50-milliliter plastic tubes to pellet the cell debris.Carefully transfer the supernatant to 20 milliliter polycarbonate ultracentrifugation vials and discard the pellet. Fill the vials with HB.Equilibrate the weight. Centrifuge in a suitable rotor for one hour at 300, 000 times G and four degrees Celsius to pellet the thylakoid membranes.Resuspend the membrane pellet with as little washing buffer as possible, using a small painter's brush. Fill the polycarbonate ultracentrifugation vials with washing buffer. Equilibrate their weight.Centrifuge them for 20 minutes at 200, 000 times G and four degrees Celsius. Resuspend the washed membranes with the painter's brush. Pool all the thylakoids in one 15 milliliter sample vial.Fill ultracentrifugation tubes with sucrose gradient solution to the top for a volume of one milliliter. Freeze the tubes at minus 20 degrees Celsius until they are completely frozen. Then, allow the tubes to thaw at four degrees Celsius.Next, use the samples corresponding to 125 micrograms of chlorophyll and adjust them with buffer B1 to a volume of 0.9 milliliters. For solubilization, add N.Dodecyl Beta D maltopyranoside to a final concentration of 20 millimolar. Invert the tube three times.Place it on ice for 20 minutes with gentle shaking to prevent foam formation. Centrifuge for five minutes at 12, 000 times G in a pre-cooled tabletop centrifuge at four degrees Celsius. Load the supernatant on the gradient, and do not load more than 125 micrograms of total chlorophyll per gradient if 17 milliliter vials are used.Centrifuge for 22 hours at 100, 000 times G and four degrees Celsius. Recover the desired brown FCP fractions from the gradient using a syringe. Estimate the volume.Then, remove a five microliter aliquot and dilute it with 995 microliters of B1A. Then, measure the volume of the recovered sample with a five milliliter pipette. Wash the FCP complexes by adding twice the recovered volume with B1 buffer that is free of detergents.Concentrate the samples in a membrane concentrator with a 30 kilodalton cutoff at 1, 000 times G and four degrees Celsius to an absorbance at 672 nanometers of at least 20. Pipette the liquids into a two milliliter reaction tube and evaporate the chloroform using a gentle nitrogen flow and spread the lipids over the whole area of the tube base. Let the nitrogen flow until all the solvent is evaporated.Solubilize the lipid mixture in 29 microliters of N Octyl beta d glucopyranoside at four degrees Celsius for four hours. Incubate the lipid mixture for 10 minutes at 30 degrees Celsius. Then, incubate the lipid mixture in a sonicator bath at 25 degrees Celsius for three times for three minutes.Place the mixture on ice for 30 seconds between each sonication. Add 221 microliters of tricine buffer and 250 microliters of four-fold dialysis buffer. Use an extruder with 0.1 micron polycarbonate membranes to obtain a defined liposome diameter of 50-70 nanometers.Assemble the extruder with the membrane and filter support. Rinse and moisten the equipment, and work carefully and slowly to prevent air bubbles, and tighten the assembly thoroughly. Fill a syringe with four-fold dialysis buffer and pre-wet the extruder until no bubbles can be seen in the second syringe.Apply the lipid detergent micelles to the extruder and press the solution from one syringe to the other back and forth. Repeat this step five times, until the solution appears homogenous. Move on to incorporation of the FCP-complexes and add FCP equal to 20 micrograms of chlorophyll a in a total volume of 500 microliters of B1A buffer to 250 microliters of the extruded lipid micelles plus 250 microliters four-fold DP.Incubate the samples for three intervals for three minutes each at 25 degrees Celsius in a thermomixer at 1, 500-3, 000 RPM, interrupted by a 30 second pause on ice.Cut the lid of four 1.5 milliliter reaction tubes just under the top, giving a ring which still fits on the lid. Prepare 1.5 by 1.5 centimeter pieces of dialysis membranes and wash them in 20 milliliters of one-fold dialysis buffer. Fill each lid with 250 microliters of the sample.Carefully lay the membrane on the lid so that the compartment is completely filled with the sample and no air bubbles occur. Tighten the reaction tube ring on the assembly so the compartment is closed. Dialyze the samples in 50 milliliters of one-fold dialysis buffer overnight for 12-16 hours on ice on a tumbling shaker.Replace the buffer with fresh dialysis buffer and add seven milligrams of adsorbent beads to remove the remaining detergents for at least six hours. Replace the dialysis buffer again, and dialyze the samples for another 12 hours. Recover the liposomes by piercing the dialysis membrane with a 200 microliter micropipette tip and aspirate all liposomes from the reaction tube lid.Centrifuge the FCP liposomes in at least two milliliters of 1X dialysis buffer for 1.5 hours at 100, 000 times G and four degrees Celsius. Recover the liposomes by turning the centrifuge tube at an angle of 45 degrees. Allow the liposomes to move down for one minute.Recover the FCP liposomes in a final volume of 25-50 microliters, and avoid disturbing the precipitate. Differences in the normalized absorbance spectra indicate pigment loss, which occurs in the spectral region between 500 and 550 nanometers, where carotenoids absorb. Pigment loss is acceptable if the complexes are still functional, indicated by similar emission and excitation spectra.The chlorophyll fluorescence yield arising from FCP in liposomes is reduced due to excitonic interaction in FCP clusters. The impact of a potassium ion gradient on FCP fluorescence yield was tested in the presence of standard buffer, which drops by 30 percent if potassium chloride is added. The potassium gradient was released by valinomycin, which restores the FCP fluorescence to the initial level and corroborates the hypothesis that FCP complexes respond to potassium ion concentration gradients in vivo.Following this procedure, other methods like tamrasol fluorescence, pump-probe, and single molecule spectroscopy may be applied in order to study additional questions in light energy transference.

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