Experimental Screening Protocols, Immunocytochemistry and Microscopy-based Imaging Techniques for Penium margaritaceum

Summary

This report describes the fundamental methods used to culture and experimentally manipulate the unicellular streptophyte alga, Penium margaritaceum. It also provides fundamental protocols of microscopy-based imaging, including live cell labeling with monoclonal antibodies and other fluorescent probes and scanning electron microscopy.

Abstract

The cell wall is the first component of signal reception/transduction for a plant cell during development and when responding to environmental abiotic and biotic stressors. The cell constantly monitors the integrity of its cell wall and modulates it in response to stress. Elucidating the specific structural and biochemical modulations occurring in the cell wall is a difficult task especially when employing multicellular plants and their organs/tissues. This is due to limits as to what can be resolved in an individual cell that is part of a complex multicellular network. The unicellular streptophyte alga, Penium margaritaceum, has recently been used in investigations of pectin dynamics, cell wall-based phenotypic plasticity and multiple aspects of algal cell biology. Its simple phenotype, distinct cell wall that has many components notably similar to land plant cell walls, and ease in immunocytochemical and experimental studies make it a powerful model organism in plant cell wall biology. The goal of this study is to provide the basic techniques for culturing, experimental manipulation and screening of applied stressors. Screening protocols for immunocytochemistry, confocal laser scanning microscopy imaging and scanning electron microscopy imaging of cell wall structure. Likewise, many of the described techniques may be modified for a wide array of other cell and molecular studies.

Introduction

The cell wall of a plant is a complex polymeric network that has multiple roles in the life of a plant cell¹. The integrity of the cell wall is constantly monitored by the cell during development and in response to environmental stress and, modulates in chemistry and structure accordingly. Penium margaritaceum is a unicellular green alga that has been recently used in studies of Streptophyte algae (Streptophyta, the group of green algae most closely related and ancestral to land plants²).

Over the past two decades, P. margaritaceum has been an important organism in investigations of the cell wall and extracellular matrix dynamics, endomembrane system activities, cell shape manifestation, and plant evolution^{3,4,5,6,7,8,9,10,11}. The goal of this work is to provide plant cell wall researchers with the fundamental methods of culturing P. margaritaceum, experimentally manipulating it using microplate-based techniques, and monitoring the structure of its cell wall using live-cell immunocytochemical labeling and imaging with light and electron microscopy techniques. P. margaritaceum has many similarities in cell wall biochemistry to the primary cell walls of land plants. We have devised multiple protocols that take advantage of this alga's unique unicellular phenotype and provide a rapid means of studying cell wall dynamics that are often difficult to monitor in multicellular plants. These techniques will be of help to plant cell wall biologists who want to elucidate detailed cell wall dynamics, especially those dealing with pectin, and serve as a starting point for studies dealing with plant and streptophyte algal cell biology.

Protocol

NOTE: Penium margaritaceum is obtained at the Sammlung von Algenkulturen der Universität Göttingen - Culture Collection of Algae at Göttingen University, SAG; strain #2640.

1. Maintaining cultures

1. Maintain the alga in liquid Woods Hole medium (WH¹²) that may also be supplemented with soil water extract (i.e., 40 mL per L of medium). Maintain cultures at 20-25 °C with a 16 h:8 h light: dark cycle with 3.5 klux (74 µmol photons/m²/s) of cool white fluorescent light.
2. Prepare subcultures weekly and use cell cultures when they are 10-14 days old. Penium cultures will be viable for 6 months and can be used to start subcultures during this time. The alga will also grow on 1%-2% agar solidified WHM.
3. For routine cell cycle synchronization, place cells from 10-14-day-old cultures in the dark for 2-6 weeks at 20-25 °C. After this time, wash the cells with fresh WHM (see below) and culture as described above.

2. Labeling cell wall with monoclonal antibodies

NOTE: P. margaritaceum is covered with a primary cell wall that has many of the same constituents found in land plant primary cell walls¹¹. Many of the monoclonal antibodies (mAbs) raised against land plant cell wall epitopes recognize components of the P. margaritaceum cell wall. Sources of these antibodies include the Complex Carbohydrate Research Center of the University of Georgia (ccrc@uga.edu) or Kerafast (kerafast.com). Following cell wall labeling of live cells with primary mAbs specific for cell wall epitopes and fluorophore-conjugated secondary antibodies (e.g., FITC, TRITC), the cells can be placed back into culture without affecting the health of the cell or cell wall deposition. The fluorescent labeling of the cell wall remains indefinitely, and the newly secreted cell wall presents as dark (i.e., unlabeled) zones that can be measured to determine cell expansion rates and/or labeled again with mAbs or other probes.

1. Remove 5 mL of actively growing liquid cell culture (10-14 days old) and place it in a 15 mL plastic centrifuge tube. Centrifuge on a tabletop centrifuge at 1,000 x g for 1 min.
2. Pour off and discard the supernatant. Resuspend the pellet in 5 mL of fresh WHM. Place the cap tightly and vigorously shake the tube for 10 s to resuspend the pellet and remove any extracellular polymeric substance or EPS from the cell wall surface.
3. Centrifuge at 1,000 x g for 1 min. Repeat step 2.2 and centrifuge. Resuspend the pellet in 1 mL of fresh WHM and transfer 200 µL aliquots of the cell suspension to 1.5 mL microcentrifuge tubes.
4. Cap the tubes and centrifuge in a microcentrifuge at 1,000 x g. Remove the supernatant and resuspend the pellet in 400 µL of fresh WHM.
5. To the suspension, add 20 µL of mAb (e.g., JIM5, a rat mAb with specificity to low methyl esterified homogalacturonan; final dilution is 1/20 with WHM). Vortex the tube, wrap it in aluminum foil, and place it on a laboratory rotator for 90 min. For best results, vortex the tube 2x during the 90 min incubation step.
6. Centrifuge the cell suspension at 1,000 x g for 1 min. Remove the supernatant and resuspend the pellet in 500 µL of fresh WHM. Cap the tube and vortex the cell suspension for 10 s.
7. Repeat step 2.6 2x. After centrifugation, resuspend the pellet in 400 µL of WHM and 8 µL of goat anti-rat TRITC or FITC (diluted 1/75 with WHM). Vortex, wrap the tube in aluminum foil, and place it on a laboratory rotator for 90 min.
8. Repeat steps 2.6 and 2.7. Resuspend the pellet in 100 µL of growth medium, cap the tube, and wrap it in aluminum foil till ready to image.
   NOTE: Incubating the cells in WHM containing a blocking agent like No Fat Carnation Instant milk (1%) or bovine serum albumin (1%) prior to incubation in the mAbs may also be used but in our experience does nothing to label quality or intensity. Penium does not expand its cell wall or produce large amounts of EPS in the dark. The labeled antibody remains intact for at least 3-4 days. So, imaging does not have to be performed immediately. Other mAbs can be applied here, but concentrations will need to be tested. For chemical and physical screening studies, one can label several mL of cell suspension, kept in the dark for several days and used in microplate studies. Cells can also be labeled with the primary antibody only prior to treatment, followed by the secondary antibody later.

3. Measuring cell wall and cell expansion rates over time

1. Take 1 mL of cells labeled with JIM5-TRITC and place 1 mL of WHM in a 1.5 mL microcentrifuge tube. Centrifuge at 1,000 x g for 1 min and discard the supernatant. Resuspend the pellet in 250 µL of WHM and vortex to mix the cells.
2. To each well of a 12-welled Petri dish add 1 mL of WHM. At this time, specific inhibitors and growth regulators can be added to each well. In this paper, we demonstrate the effects of increasing calcium levels during incubation.
3. Take 30 µL of labeled cell suspension (step 1) and add to each well of the microplate. Gently swirl the plate to mix. Seal with transparent film.
4. Culture as above (step 1.1) for 24 h, 48 h, or 72 h. At specified times, remove 250 mL of cell suspension from each well and place it in a 1.5 µL microcentrifuge tube.
5. Centrifuge at 1,000 x g for 1 min. Remove the supernatant. Resuspend the pellet in 50 µL of growth medium and vortex.
6. Place a 15 µL drop of the cell suspension onto a glass slide and cover with a 22 x 22 coverslip (thickness 1.5). Observe cells with a fluorescence microscope using a TRITC filter at 10x -20x.
7. Capture images of at least 50-100 cells. For each cell, measure and record the total cell length and the length of the dark zone (i.e., the new wall produced during incubation after labeling).
8. Determine the average % of new cell walls in the culture. Repeat for cells collected after 48 h and 72 h to obtain information on cell wall expansion over time.
   NOTE: Steps 3.5-3.8 yield a dense cell suspension that allows for imaging of many cells in one field of view. This makes manual measuring much more convenient. Many camera software programs provide convenient and/or automatic measuring of cell dimensions that can be used with this alga. One can also collect the cells and label them as above with JIM5 but substitute anti-rat FITC as the secondary antibody. Using a confocal laser scanning microscope (CLSM), one can image cells with both the FITC and TRITC filters. The fluorescent signals can then be assigned to different pseudocolors. This also allows a mean of distinguishing the structure of the new cell wall produced during various treatments (labeled with JIM5-FITC) compared with pre-labeling cell walls (labeled with JIM5-TRITC).

4. Timelapse imaging of cell wall expansion

1. Label the cell wall with JIM5-TRITC as described above (steps 2.1-2.8). Dilute cells 10x in WHM and add a 50 µL drop of cells onto either a coverslip or glass bottom Petri dish.
2. Allow cells to adhere to glass for 2 min in the dark. Gently rinse away cells that have not stuck to the glass with 1 mL of WHM. Use a micropipette and carefully add WHM dropwise to the glass.
3. Add 30 µL of WHM on top of the cells attached to the glass. Add 30 µL of warm 4% agarose/WHM on top of the droplet of WHM/cells. Allow the agarose to cool to room temperature and solidify.
4. For samples in a Petri dish, add enough WHM to completely cover the agarose-embedded cells. For cells on a coverslip, invert the coverslip and gently place it over a depression slide filled with WHM.
5. Mount cells on a fluorescent microscope. External lighting (lamp) can be set up, or light from the microscope itself can be used to provide energy for cell growth and movement. On an Olympus Ix83 or Ix63 microscope, a power of 5-6 V for the trans lamp works well. This may require trial and error for your system.
6. Image every 10-30 min using the TRITC filter set for tracking of cell wall expansion. Add chemicals/enzymes to WHM used in step 3.8, if any.

5. Observing extracellular polymeric substance (EPS) production

1. Preparation of cells: Obtain 5 mL cells from 10-14-day-old cultures and wash them as in steps 1-4 above.
2. Preparation of fluorescent beads: To a 1.5 mL microcentrifuge tube, add 1 drop (approximately 100 µL) of 0.75 µm fluorescent beads (Polysphere). Add 1 mL of WHM to the tube and vigorously shake to resuspend the beads. Centrifuge at 10,000 x g for 3 min. Remove the supernatant. Add 1 mL of WHM to the pellet and shake. Centrifuge at 10,000 x g for 3 min. Resuspend the pellet in 500 µL of WHM.
3. Preparation of wells: Add 1 mL of WHM medium to the wells of a 12-well plate. Add inhibitors or growth regulators to the desired concentration and gently swirl. Add 10 µL of the bead solution and gently swirl the plate to mix the beads. Add 10 µL of washed cells to each well and swirl the plate gently.
4. Culture the plate of cells as above (1.1). After 24 h, gently place the plate (be careful not to mix) onto an inverted fluorescence microscope equipped with a FITC filter. The beads adhere to the EPS and reveal the patterns of EPS release. Photograph the cells at 4x, 10x, or 20x.

6. Timelapse imaging of EPS trail formation

1. Perform time-lapse imaging in a 12-well plate (step 3.2), as prepared above, or in an individual Petri dish. Follow steps 1-4. Rather than culture the cells under fluorescent lighting, the cells can be grown while mounted on an inverted fluorescent microscope.
2. Image cells every 5-10 min to best capture cell movement. Fluorescent beads are visible using a FITC filter set, but for concentrated beads use the brightfield channel. External lighting (lamp) can be set up, or light from the microscope itself can be used to provide energy for cell growth and movement. On an Olympus Ix83 microscope, a power of 5-6 V for the trans lamp works well. This may require trial and error for your system.

7. Correlative structural analysis of the cell wall with Scanning electron microscopy (SEM)

NOTE: Altered features of the cell wall observed in live cells labeled with cell-specific antibodies can be imaged for detailed features using SEM. This correlative approach allows for obtaining ultrastructural data that can be compared with the fluorescence data.

1. Obtain 1 mL of a cell suspension (control or experimentally treated) in a 1.5 mL microcentrifuge tube. Centrifuge at 4,000 x g for 1 min.
2. Discard the supernatant. Plunge the tube containing the pellet in liquid nitrogen, or if unavailable, place it in a -80 °C freezer. Frozen cells can be stored at -80 °C for several months.
3. At the time of cell wall processing, remove the tube from the freezer and allow it to thaw for 15 min. Resuspend the pellet in 20 µL of WHM and place a drop of dense cell suspension onto a 45 mm x 50 mm coverslip.
4. Place a second coverslip on top of the drop to create a sandwich. Place on a laboratory table and continuously press down on the sandwich to rupture the cells (e.g. 30 s).
5. Carefully separate the glass coverslips and wash the ruptured cells into a 15 mL centrifuge tube. Centrifuge at 500 x g for 1 min.
6. Pour off the supernatant. The pellet should be white or slightly green. If subsequent imaging shows that a sufficient number of cells have not been ruptured, repeat step 3 with the pellet here.
7. Resuspend the pellet that contains cell walls in D-H₂O and repeat steps 7.4 and 7.5. Resuspend the pellet in 100 µL of D-H₂O and place it in a 1.5 mL centrifuge tube.
8. Obtain a Cambridge stub (6 mm or 8 mm radius) and adhere carbon tape (EMS) to its surface. Next, place 5 µL drops of the resuspended cell wall suspension (step 7) onto the carbon tape. Observe the number of cell walls using a dissecting microscope. If the suspension is too dense, dilute it with D-H2O.
9. Place the stub in a covered container and allow it to dry (2 h to overnight). Sputter coat the stub for 50 s using a palladium target (other targets may be employed as well). Observe the cells at 5 kV, spot size, at 10 cm from the secondary electron detector.
   NOTE: Diluting the cell wall suspension before depositing drops onto the carbon tape will limit cell walls depositing on each other.

Results

The labeling of the cell wall of P. margaritaceum with anti-pectin mAbs (e.g., JIM5) reveals a network of calcium-complexed fibers and projections that form a regular pattern or lattice (Figure 1). The pectin is deposited in the cell center or isthmus, where it displaces older pectin toward the poles (Figure 2). Labeling with a different pectin antibody-like JIM7 highlights the initial secretion of high methyl-esterified pectin in a narrow band at the i...

Discussion

P. margaritaceum is an efficacious organism for elucidating the dynamics of cell wall development and ECM secretion in plants and streptophyte algae. The main attributes include a unicellular habit and ease in culture maintenance and experimental manipulation, a primary cell wall with a distinct outer pectin lattice and other polymers, ease in live cell labeling with cell wall-directed mAbs that can be followed in time for subsequent developmental and/or experimental studies and the production of large amounts o...

Materials

Name	Company	Catalog Number	Comments
1.5 mL microcent. Tubes	Fisher Scientific	01-549-740
12 welled microplate	Fisher Scientific	50-233-6077
22 x 22 mm coverslips	Fisher Scientific	12-541-016
45x 50 cm coverslips	Brain Research	4550-1.5D
Agar	Sigma Aldrich	A9414
anti-rat FITC	Sigma Aldrich	F6258
anti-rat TRITC	Sigma Aldrich	T4280
calcium chloride	Sigma Aldrich	C4981
Cambbridge stubs	EMS	75183-65
Fluoview CLSM	Evident	Fluoview 1200
JIM5	Kerafast	ELD004
JIM7	Kerafast	ELD005
Microcentrifuge	Fisher Scientific	13-100-675
Micropipetors	BioRad	1660499EDU
Penium margaritaceum	Sammlung von Algenkulturen der Universität Göttingen - Culture Collection of Algae at Göttingen University	2640
Polysphere kit	Polysciences	18336
SEM	ThermoFisher	Quattro SEM
sputter coater	EMS	Q150V
Vortex mixer	Fisher Scientific	02-215-414