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

Our research focuses on the role of the cell wall in maintaining cell shape and responding to external stress. We use a range of light microscopy and confocal laser scanning microscopy techniques for imaging cell wall structure in live cells and electron microscopy for high-resolution imaging of cell walls.

The current lack of transformed cell lines for streptophyte algae expressing specific subcellular proteins for highlighting cell wall dynamics are lacking. This then requires using antibodies and other fluorescent probes for study. The cell wall of Penium responds to various forms of abiotic and biotic stress, and this leads to phenotypic plasticity. We have found that cell wall pectins are an important part of this process. The unicellular phenotype and ability to perform live cell labeling with Penium offers plant biologists the opportunity to delve into the structure and development of the cell wall.

[Instructor] To begin, remove five milliliters of actively growing liquid cell cultures of Penium margaritaceum. Transfer into a 15 milliliter plastic centrifuge tube, then centrifuge. After pouring out the supernatant, resuspend the pellet in five milliliters of fresh WHM. Secure the tube cap tightly and vigorously shake the tube for 10 seconds to resuspend the pellet and remove any extracellular polymeric substance from the cell wall surface. After the final wash, resuspend the pellet in one milliliter of fresh WHM, then transfer 200 microliter aliquots of the cell suspension into 1.5 milliliter microcentrifuge tubes. Centrifuge the cap tubes in the microcentrifuge at 1000 G for one minute, then aspirate the supernatant and resuspend the pellet in 400 microliters of fresh WHM. Next, add 20 microliters of diluted monoclonal antibody to the cell suspension and vortex well. Then, wrap the tube in aluminum foil and incubate it on a laboratory rotator for 90 minutes. Centrifuge the suspension, aspirate the supernatant, and resuspend the pellet in 500 microliters of fresh WHM before vortexing for 10 seconds. After the final centrifugation, resuspend the pellet in 400 microliters of WHM and add eight microliters of goat anti-rat TRITC or FITC. Finally, resuspend the pellet in 100 microliters of growth medium. Cap the tube and wrap it in aluminum foil until ready for imaging. Dilute the cells labeled with JIM5 antibody tenfold in WHM. Add a 50 microliter drop of the diluted cell suspension onto a cover slip. Incubate the cells for two minutes in the dark to allow for cell adherence, then carefully pipette one milliliter of WHM dropwise to rinse away any unadhered cells. Add 30 microliters of WHM on top of the attached cells. Overlay the droplet with 30 microliters of warm 4% agarose in WHM and let it solidify. For samples in a Petri dish, add enough WHM to completely cover the agarose-embedded cells. For cells on a cover slip, invert the cover slip and gently place it over a depression slide filled with WHM. Mount the prepared cells onto a fluorescent microscope. Set up an external lamp or use the microscope's built-in illumination to support cell growth and movement. Capture images every 10 to 30 minutes using the TRITC filter set to track cell wall expansion. Prepare a tube containing five milliliters of washed 10 to 14 day old cell cultures. Next, add about 100 microliters of 0.75 micrometer fluorescent beads into a 1.5 milliliter microcentrifuge tube. Pipette one milliliter of WHM into the tube and vigorously shake to resuspend the beads. Centrifuge the tube at 10,000 G for three minutes. Resuspend the final pellet in 500 microliters of WHM. Next, pipette one milliliter of WHM medium to each well of a 12-well plate. Add inhibitors or growth regulators to achieve the desired concentration and gently swirl the plate. Add 10 microliters of the bead solution and gently swirl again to mix, then add 10 microliters of washed cells to each well before mixing. Incubate the plate for 24 hours in the light. Without disturbing the plate, place it onto an inverted fluorescence microscope equipped with a FITC filter. Pipette out one milliliter of a cell suspension into a 1.5 milliliter microcentrifuge tube. Centrifuge the tube at 4,000 G for one minute. After discarding the supernatant, plunge the tube containing the pellet into liquid nitrogen or freeze at minus 80 degrees Celsius. Resuspend the thawed pellet in 20 microliters of WHM. Place a drop of the dense cell suspension onto a cover slip measuring 45 by 50 millimeters. Place a second cover slip on top of the drop to create a sandwich. Press down continuously on the sandwich for 30 seconds to rupture the cells. Carefully separate the cover slips. Add WHM over the cover slips to wash the ruptured cells into a 15 milliliter centrifuge tube and centrifuge. Examine the white or slightly green pellet after discarding the supernatant. Resuspend the pellet containing the cell walls in deionized water. After cell rupture and centrifugation, resuspend the pellet in 100 microliters of deionized water before transferring it to a 1.5 milliliter centrifuge tube. Next, attach carbon tape to the surface of a Cambridge stub. Pipette five microliter drops of the resuspended cell wall suspension onto the carbon tape. Use palladium target to sputter coat the stub for 50 seconds after letting it dry overnight. Observe the cells at five kilovolts, with an appropriate spot size positioned 10 centimeters from the secondary electron detector. The labeling of the cell wall of P. margaritaceum with anti-pectin monoclonal antibodies revealed a network of calcium complexed fibers forming irregular lattice pattern. The pectin was deposited in the cell center or isthmus, pushing older pectin toward the poles. JIM7 labeling showed that high methyl-esterified pectin was initially secreted in a narrow band at the isthmus. Other polymers in the cell wall, such as arabinogalactan protein, were detected with the monoclonal antibody JIM13. Large amounts of extracellular polymeric substances were secreted outside the cell wall, enabling gliding and cell aggregation. Labeling techniques allowed for quantitative cell wall and expansion studies, integrating developmental imaging approaches. Correlative structural studies revealed that the typical pectin lattice was composed of a mesh of fibers terminating externally in distinct projections. Treatment with high concentrations of calcium transformed the pectin lattice into irregular deposits, as observed in JIM5 labeled cells. When calcium-treated cell walls were examined under scanning electron microscopy, the disorganized pectin structure was observed in detail.