April 2019, written by Adrian Li & Dr. Eunkyoung Lee
BIF equipment used : Olympus FV1000 confocal microscope, PE Spinning disk confocal microscope
Plants sense and respond to changes of environmental conditions, such as drought, extreme temperatures, and salinity. At the intercellular level, highlycoordinated stress response occurs among various organelles. Endoplasmic reticulum (ER) is a highly dynamic organelle consisting of a network of continuous membrane system. The ER is distributed throughout the cytoplasm in plant cells and physically and functionally interacts with the other organelles, such as Golgi bodies, chloroplasts, and plasma membrane (PM).
Dr. Eunkyoung Lee in Rosado lab (UBC, Botany) studies the function of plant ER microdomains that interact with the PM called ER-PM contact sites. In plants, ER-PM contact sites act as signalling platforms for calcium ion homeostasis regulation and the cellular adaptation to environmental stresses. These contact sites are stabilized by membrane-tethering proteins, such as SYNAPTOTAGMIN 1 (SYT1).
In Lee et al.’s (2019) study, they generated an artificial constitutive ER-PM contact site marker, a fluorescently-tagged MAPPER protein labeling nonselectively ER–PM junctions for use in plants. And they examined whether the MAPPER-green fluorescent protein (GFP) is colocalized with red fluorescent protein (RFP) fused with SYT1, which has known as a functional plant ER-PM tether. Both markers were used to track ER-PM contact sites and ER-PM connectivity during ionic stress in live cells. With extensive confocal microscope use, the first challenge was figuring out whether or not the cover slip was causing mechanical stress to the sample and altering the appearance of the ER structures. Another challenge was to capture changes of fluorescently-tagged markers simultaneously located in either ER or PM that are present on two separate focal planes. Multiple optical sections were acquired and used for further image analyses, such as measuring changes of fluorescence intensity and colocalization analysis of marker proteins.
In response to ionic stress, the beads and strings pattern of EM-PM contact sites in normal conditions slowly expanded and became reticulated over time. Fluorescence Recovery After Photobleach (FRAP) analysis indicated that stress-induced contact site expansion occurred independent of cytoskeletons. Although the reason for the change in ER-PM connectivity is still unknown, it is thought that it facilitates the exchange of material and information, such as transfer of glycolipids, through membrane contacts between ER and PM during ionic stress. More research is currently being conducted to determine the functionof ER-PM contact sites expansion.
For more information about spinning disk and conventional confocal microscopy combined with FRAP techniques, please visit our equipment website to learn more about their capabilities.
Reference
Lee, E., Vanneste, S., Perez-Sancho, J., Benitez-Fuente, F., Strelau, M., Macho, AP., Botella, MA., Friml, J., and Rosado, A. (2019) Ionic stress enhances ER-PM connectivity via phosphoinositide-associated SYT1 contact site expansion in Arabidopsis. PNAS 116 (4), 1420-1429.
doi:10.1073/pnas.1818099116