This work was supported by the National Science Foundation (IOS-2217757) (X.Q.) and the University of Arkansas for Medical Sciences (UAMS) Bronson Foundation Award (H.Z.).

1. Preparation of the solutions
<ol>
	<li>Prepare seed sterilization solution by mixing 15 mL of bleach with 35 mL of distilled water and 50 &#181;L of Triton X-100.</li>
	<li>Prepare brefeldin A (BFA) solution by dissolving the BFA powder in ethanol to a final concentration of 10 mM (stock). Prepare wortmannin (Wm) solution by dissolving the Wm powder in DMSO to a final concentration of 10 mM (stock).</li>
</ol>
2. Sowing the seeds
<ol>
	<li>Aliquot 10-50 seeds from each of the required transgenic plants into 1.5 mL microcentrifuge tubes. Add 1 mL of seed sterilization solution into each tube, and mix thoroughly by inverting the tube gently for 10 min on a shaker.</li>
	<li>Discard the seed sterilization solution, and wash the seeds with 1 mL of autoclaved distilled water five times.</li>
	<li>Add 300 &#181;L of autoclaved distilled water into each tube, and sow the seeds on half-strength MS media containing 1% (w/v) sucrose and 0.75% (w/v) agar. Supplement the medium with the corresponding antibiotics as needed.</li>
	<li>Keep the plate upside-down at 4 &#176;C in the dark for 2 days to synchronize the germination.</li>
	<li>After 2 days, transfer the plate into a growth room with a 16 h light/8 h dark cycle (80 &#181;mol/m2/s1) at 22 &#176;C. This is considered the first day post germination (1 dpg).</li>
	<li>Transplant the seedlings into soil at 10 dpg for further growth.</li>
</ol>
3. Preparation of dual-colored F1 transgenic plants
<ol>
	<li>Grow homozygous transgenic plants bearing ERL1-YFP (plant A) and homozygous transgenic plants bearing an RFP-labeled marker protein RFP-Ara7 (plant B)20 side by side until flowering in a growth room with a 16 h light/8 h dark cycle (80 &#181;mol/m2/s1) at 22 &#176;C.</li>
	<li>Choose a young inflorescence from plant A. Keep one flower that is just about to open on the inflorescence for genetic crosses. Remove all the older flowers and siliques. Additionally, carefully remove the younger flowers and floral meristems to avoid future confusion.</li>
	<li>Gently dissect the unopened flower by removing the sepals, petals, and stamen using a pair of sharp tweezers. Only leave the pistil on the inflorescence.</li>
	<li>Take a mature stamen from an opened flower on plant B, and deposit the pollen grains onto the stigma of the dissected pistil on plant A.</li>
	<li>Label this manually crossed flower by indicating its father, mother, and the date of the genetic cross. Let the flower mature, and harvest the F1 seeds when the silique turns yellow/brown (~20 days after the cross). Check for a successful F1 plant that has both YFP and RFP signals.</li>
</ol>
4. Pharmacological treatment
<ol>
	<li>For the BFA treatment, remove the cotyledons of 7-day-old seedlings, immerse the rest of the seedlings into either a mock solution (0.3% ethanol) or 30 &#181;M BFA solution, apply vacuum for 1 min, and keep the sample immersed for 30 min before imaging.</li>
	<li>For the wortmannin treatment, remove the cotyledons of 7-day-old seedlings, immerse the rest of the seedlings into either 0.25% DMSO solution or 25 mM wortmannin, apply vacuum for 1 min, and keep the sample immersed for 30 min before imaging.</li>
	<li>For the vacuum treatment of samples, in a 1.5&#160;mL microcentrifuge tube, add 500 &#181;L of the corresponding drug solution, and immerse the dissected seedlings into the solution. Tightly attach a 10 mL syringe to the microcentrifuge tube, and apply vacuum for 1 min (Figure 1). Remove the syringe, and keep the sample in the solution for the required time. Gently take out the seedlings for imaging preparation.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65257/65257fig01.jpg" /> 
Figure 1: Simple vacuum device. A 10 mL syringe is attached to a 1.5 mL microcentrifuge tube for the vacuum treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65257/65257fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Sample preparation for imaging
<ol>
	<li>Dissect the true leaf from the treated sample using a sharp razor blade. Gently put the true leaf in a drop of water on a glass slide, and keep the abaxial side up. Slowly cover the true leaf with a coverslip while avoiding trapping any bubbles.</li>
</ol>
6. Confocal imaging
NOTE: A Leica SP8 inverted scanning confocal microscope was used to image the fluorescence signal of the samples in this work.
<ol>
	<li>Beam path setup
	<ol>
		<li>Select a 514 nm laser for YFP excitation. Use a high laser power to increase the signal intensity and, thus, obtain high image quality. However, laser powers above 5% run the risk of photobleaching, and the health of the sample may be affected. If the sample signal is not too dim, start at low laser intensity.</li>
		<li>Turn on PMT/HyD for detection, and define the upper and lower emission band thresholds based on the spectrum for the YFP fluorophore. Set a detection window of 530-570 nm to collect the YFP signal.</li>
		<li>Sequential imaging for the second color: Click on the Seq button, and add a new channel. By default, the previously designed YFP beam path will be Seq 1. In Seq 2, select a 561&#160;nm laser for the RFP excitation, and set a detection window of 570-630 nm to collect the RFP signal.</li>
	</ol>
	</li>
	<li>Scan conditions setup (Figure 2)
	<ol>
		<li>To image the subcellular membrane traffic activity within stomatal precursor cells, choose a 63x/1.2 W Corr lens on the system for imaging.</li>
		<li>The format refers to the image size in pixels. Start with 1,024 pixels x 1,024 pixels, and then optimize this based on the zoom factor and the objective settings for a good resolution of publication quality.</li>
		<li>The speed refers to the speed of the scanhead as the laser passes over each pixel. Although slow scan speeds often result in a better signal-to-noise ratio, they may not efficiently capture the rapidly changing membrane trafficking events. Start with a speed of 400 Hz, and optimize this based on the specific situation of the samples.</li>
		<li>The zoom factor is used to zoom in on a region of interest without changing the objective lens. Start with a zoom factor of 1, and optimize this based on the specific needs of the experiment.</li>
		<li>The line average refers to the number of times each X-line will be scanned to obtain an average result. A greater line average reduces the noise in the resulting image, but it also increases the scanning time and exposure time to the laser light. To image membrane trafficking events, do not use a high line average. Start with a 2x line average, and optimize as needed.</li>
	</ol>
	</li>
	<li>Collecting a time series 
	NOTE: When studying membrane trafficking events, a time series is often needed to record the rapid movement of the subcellular endosomes.
	<ol>
		<li>In the Acquisition Mode, select the xyt scan mode to enable the time series utility.</li>
		<li>Decide a waiting period between the time points under Time Interval. Alternatively, click on Minimize to take the images immediately one after another. A 7 s time interval was used in the following time series experiment.</li>
		<li>Select Duration, and enter the total time for the experiment to run. Alternatively, define the number of frames to be collected by selecting Frames (Figure 3).</li>
	</ol>
	</li>
	<li>To collect the three-dimensional information regarding the membrane trafficking event within the entire cell, use the Z-stack scan mode. The maximum intensity projection of the Z-stack images is often used for the analysis.
	<ol>
		<li>Select the xyz scan mode in the&#160;Acquisition Mode, and then the Z-Stack utility will become accessible.</li>
		<li>Select the Z-wide option. Under the Scan Mode, define the top image and the bottom image of the Z-stack using the Begin and End buttons.</li>
		<li>Define the thickness of the z-step by clicking on z-step size. Alternatively, define how many images to take in order to cover the entire range of the Z-stack. To ensure consistency between the samples, define the z-step size (Figure 4).</li>
	</ol>
	</li>
	<li>Image processing
	<ol>
		<li>The maximum intensity projection of the z-stack images is generated by the confocal software (http://www.leica-microsystems.com). In the primary&#160;Process tab, first choose the interested z-stack file under the Open Projects tab on the far-left side. Then, switch to the middle tab named Process Tools, select the Projection function, choose Maximum in the dropdown panel of Method, and click on the Apply button. A z-stack projection image will be generated under the Open Projects tab.</li>
		<li>The video of the time series images is generated by Fiji (https://imagej.net/Fiji). In the&#160;File tab, use the Open function to open all the time series images in the correct order. In the Image tab, find the Stack function, and choose Images to stack to generate a video. Save the file in the .avi format with the desired frame rate (5 frames/s for Video 1).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65257/65257fig02.jpg" /> 
Figure 2: The scan mode panel of the XY dimension. The scan mode panel is used to set up the image scan conditions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65257/65257fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65257/65257fig03.jpg" /> 
Figure 3: The time series utility under the xyt scan mode. The time series utility is used to set up the imaging conditions to consecutively collect a series of images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65257/65257fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65257/65257fig04.jpg" /> 
Figure 4: The z-stack utility under the xyz scan mode. The z-stack utility is used to set up the imaging conditions to collect a series of images on the z-axis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65257/65257fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Spatiotemporal Investigation of ERL1 Dynamics via Confocal Imaging

<ol><li>Aniento, F., Sanchez de Medina Hernandez, V., Dagdas, Y., Rojas-Pierce, M., Russinova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanisms+of+endomembrane+trafficking+in+plants%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Molecular mechanisms of endomembrane trafficking in plants.</a> Plant Cell. 34 (1), 146-173 (2022).</li>
<li>Sigismund, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endocytosis+and+signaling%3A+Cell+logistics+shape+the+eukaryotic+cell+plan%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">Endocytosis and signaling: Cell logistics shape the eukaryotic cell plan.</a> Physiological Reviews. 92 (1), 273-366 (2012).</li>
<li>Lyu, Z., Genereux, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methodologies+for+measuring+protein+trafficking+across+cellular+membranes%5BTitle%5D)+AND+%22ChemPlusChem%22%5BJournal%5D)">Methodologies for measuring protein trafficking across cellular membranes.</a> ChemPlusChem. 86 (10), 1397-1415 (2021).</li>
<li>Rodriguez-Furlan, C., Raikhel, N. V., Hicks, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Merging+roads%3A+Chemical+tools+and+cell+biology+to+study+unconventional+protein+secretion%5BTitle%5D)+AND+%22Journal+of+Experimental+Botany%22%5BJournal%5D)">Merging roads: Chemical tools and cell biology to study unconventional protein secretion.</a> Journal of Experimental Botany. 69 (1), 39-46 (2017).</li>
<li>Foissner, I., Sommer, A., Hoeftberger, M., Hoepflinger, M. C., Absolonova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+wortmannin-induced+reorganization+of+the+trans-Golgi+network+the+key+to+explain+charasome+formation%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Is wortmannin-induced reorganization of the trans-Golgi network the key to explain charasome formation.</a> Frontiers in Plant Science. 7, 756(2016).</li>
<li>Qi, X., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hormonal+and+environmental+signals+guiding+stomatal+development%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">Hormonal and environmental signals guiding stomatal development.</a> BMC Biology. 16 (1), 21(2018).</li>
<li>Han, S. K., Kwak, J. M., Qi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomatal+lineage+control+by+developmental+program+and+environmental+cues%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Stomatal lineage control by developmental program and environmental cues.</a> Frontiers in Plant Science. 12, 751852(2021).</li>
<li>Bharath, P., Gahir, S., Raghavendra, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abscisic+acid-induced+stomatal+closure%3A+An+important+component+of+plant+defense+against+abiotic+and+biotic+stress%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Abscisic acid-induced stomatal closure: An important component of plant defense against abiotic and biotic stress.</a> Frontiers in Plant Science. 12, 615114(2021).</li>
<li>Becklin, K. M., Ward, J. K., Way, D. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBecklin%2C+KM+%22Photosynthesis%2C+Respiration%2C+and+Climate+Change.%2C+1st+edition%22">Photosynthesis, Respiration, and Climate Change., 1st edition</a>. , Springer. Cham, Switzerland. (2021).</li>
<li>Yang, M., Sack, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+too+many+mouths+and+four+lips+mutations+affect+stomatal+production+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">The too many mouths and four lips mutations affect stomatal production in Arabidopsis.</a> Plant Cell. 7 (12), 2227-2239 (1995).</li>
<li>Hara, K., Kajita, R., Torii, K. U., Bergmann, D. C., Kakimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+secretory+peptide+gene+EPF1+enforces+the+stomatal+one-cell-spacing+rule%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule.</a> Genes & Development. 21 (14), 1720-1725 (2007).</li>
<li>Hara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+cell+density+is+autoregulated+via+a+secretory+peptide%2C+EPIDERMAL+PATTERNING+FACTOR+2+in+Arabidopsis+leaves%5BTitle%5D)+AND+%22Plant+%26+Cell+Physiology%22%5BJournal%5D)">Epidermal cell density is autoregulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis leaves.</a> Plant & Cell Physiology. 50 (6), 1019-1031 (2009).</li>
<li>Hunt, L., Gray, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+signaling+peptide+EPF2+controls+asymmetric+cell+divisions+during+stomatal+development%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development.</a> Current Biology. 19 (10), 864-869 (2009).</li>
<li>Sugano, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomagen+positively+regulates+stomatal+density+in+Arabidopsis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Stomagen positively regulates stomatal density in Arabidopsis.</a> Nature. 463 (7278), 241-244 (2010).</li>
<li>Kondo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomatal+density+is+controlled+by+a+mesophyll-derived+signaling+molecule%5BTitle%5D)+AND+%22Plant+%26+Cell+Physiology%22%5BJournal%5D)">Stomatal density is controlled by a mesophyll-derived signaling molecule.</a> Plant & Cell Physiology. 51 (1), 1-8 (2010).</li>
<li>Hunt, L., Bailey, K. J., Gray, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+signalling+peptide+EPFL9+is+a+positive+regulator+of+stomatal+development%5BTitle%5D)+AND+%22New+Phytologist%22%5BJournal%5D)">The signalling peptide EPFL9 is a positive regulator of stomatal development.</a> New Phytologist. 186 (3), 609-614 (2010).</li>
<li>Shpak, E. D., McAbee, J. M., Pillitteri, L. J., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomatal+patterning+and+differentiation+by+synergistic+interactions+of+receptor+kinases%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Stomatal patterning and differentiation by synergistic interactions of receptor kinases.</a> Science. 309 (5732), 290-293 (2005).</li>
<li>Lin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+receptor-like+protein+acts+as+a+specificity+switch+for+the+regulation+of+stomatal+development%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">A receptor-like protein acts as a specificity switch for the regulation of stomatal development.</a> Genes & Development. 31 (9), 927-938 (2017).</li>
<li>Lee, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+interaction+of+ligand-receptor+pairs+specifying+stomatal+patterning%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Direct interaction of ligand-receptor pairs specifying stomatal patterning.</a> Genes & Development. 26 (2), 126-136 (2012).</li>
<li>Qi, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+manifold+actions+of+signaling+peptides+on+subcellular+dynamics+of+a+receptor+specify+stomatal+cell+fate%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate.</a> Elife. 9, e58097(2020).</li>
<li>MacAlister, C. A., Ohashi-Ito, K., Bergmann, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcription+factor+control+of+asymmetric+cell+divisions+that+establish+the+stomatal+lineage%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Transcription factor control of asymmetric cell divisions that establish the stomatal lineage.</a> Nature. 445 (7127), 537-540 (2007).</li>
<li>Pillitteri, L. J., Sloan, D. B., Bogenschutz, N. L., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Termination+of+asymmetric+cell+division+and+differentiation+of+stomata%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Termination of asymmetric cell division and differentiation of stomata.</a> Nature. 445 (7127), 501-505 (2007).</li>
<li>Ohashi-Ito, K., Bergmann, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arabidopsis+FAMA+controls+the+final+proliferation%2Fdifferentiation+switch+during+stomatal+development%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development.</a> Plant Cell. 18 (10), 2493-2505 (2006).</li>
<li>Kanaoka, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SCREAM%2FICE1+and+SCREAM2+specify+three+cell-state+transitional+steps+leading+to+Arabidopsis+stomatal+differentiation%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation.</a> Plant Cell. 20 (7), 1775-1785 (2008).</li>
<li>Bergmann, D. C., Lukowitz, W., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomatal+development+and+pattern+controlled+by+a+MAPKK+kinase%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Stomatal development and pattern controlled by a MAPKK kinase.</a> Science. 304 (5676), 1494-1497 (2004).</li>
<li>Wang, H., Ngwenyama, N., Liu, Y., Walker, J. C., Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomatal+development+and+patterning+are+regulated+by+environmentally+responsive+mitogen-activated+protein+kinases+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis.</a> Plant Cell. 19 (1), 63-73 (2007).</li>
<li>Ho, C. M., Paciorek, T., Abrash, E., Bergmann, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulators+of+stomatal+lineage+signal+transduction+alter+membrane+contact+sites+and+reveal+specialization+among+ERECTA+kinases%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Modulators of stomatal lineage signal transduction alter membrane contact sites and reveal specialization among ERECTA kinases.</a> Developmental Cell. 38 (4), 345-357 (2016).</li>
<li>Le, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auxin+transport+and+activity+regulate+stomatal+patterning+and+development%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Auxin transport and activity regulate stomatal patterning and development.</a> Nature Communications. 5, 3090(2014).</li>
<li>Geldner, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Arabidopsis+GNOM+ARF-GEF+mediates+endosomal+recycling%2C+auxin+transport%2C+and+auxin-dependent+plant+growth%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth.</a> Cell. 112 (2), 219-230 (2003).</li>
<li>Qi, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autocrine+regulation+of+stomatal+differentiation+potential+by+EPF1+and+ERECTA-LIKE1+ligand-receptor+signaling%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Autocrine regulation of stomatal differentiation potential by EPF1 and ERECTA-LIKE1 ligand-receptor signaling.</a> Elife. 6, 24102(2017).</li>
<li>Heilemann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subdiffraction-resolution+fluorescence+imaging+with+conventional+fluorescent+probes%5BTitle%5D)+AND+%22Angewandte+Chemie%22%5BJournal%5D)">Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.</a> Angewandte Chemie. 47 (33), 6172-6176 (2008).</li>
<li>Leighton, R. E., Alperstein, A. M., Frontiera, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+super-resolution+imaging+techniques%5BTitle%5D)+AND+%22Annual+Review+of+Analytical+Chemistry%22%5BJournal%5D)">Label-free super-resolution imaging techniques.</a> Annual Review of Analytical Chemistry. 15 (1), 37-55 (2022).</li>
<li>Oreopoulos, J., Berman, R., Browne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinning-disk+confocal+microscopy%3A+Present+technology+and+future+trends%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Spinning-disk confocal microscopy: Present technology and future trends.</a> Methods in Cell Biology. 123, 153-175 (2014).</li>
<li>Gao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+column+and+whole-brain+imaging+with+molecular+contrast+and+nanoscale+resolution%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution.</a> Science. 363 (6424), (2019).</li>
<li>Nwaneshiudu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introduction+to+confocal+microscopy%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Introduction to confocal microscopy.</a> Journal of Investigative Dermatology. 132 (12), (2012).</li>
<li>Sanderson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-photon+microscopy%5BTitle%5D)+AND+%22Current+Protocols%22%5BJournal%5D)">Multi-photon microscopy.</a> Current Protocols. 3 (1), 634(2023).</li>
</ol>

The authors declare no conflicts of interest.

The endomembrane system separates the cytoplasm of a eukaryotic cell into different compartments, which enables the specialized biological function of these organelles. To deliver cargo proteins and macromolecules to their final destination at the right time, numerous vesicles are guided to shuttle between these organelles. Highly regulated membrane trafficking events play fundamental roles in the viability, development, and growth of cells. The mechanism regulating this crucial and complicated process is still poorly understood.
Several approaches have been described here for observing the subcellular localization of membrane proteins, analyzing the co-localization between two membrane proteins tagged with different fluorescent proteins, monitoring the dynamic movement of a membrane protein using time-series images, and collecting 3D information about a membrane protein by performing z-stack imaging covering the whole cell. Besides the membrane trafficking events described here, these approaches can be easily applied to other biological processes. For instance, the time-series imaging can be conducted over a long-term period (several days) to monitor a developmental process30. The z-stack imaging can be performed to quantify the abundance of a protein within a cell30. In addition, these imaging approaches can perfectly combine with pharmacological applications, including various drugs, hormones, and even environmental cues, indicating that these imaging approaches are easily applicable to live biological samples. Besides the flexibility in the sample treatment, the methods described here require only economically affordable devices, except the confocal microscope, and only light training of the personnel in the technical skills, thus meaning these methods can be widely adopted.
Caution is needed in several critical steps of the protocol. First, the optimal concentrations of the membrane trafficking drugs vary depending upon the material and the treatment. A previous study suggests that 30 &#181;M BFA is a low concentration that can trigger characteristic BFA body formation without causing the collapse of the Golgi apparatus in meristemoids20. Likewise, 25 &#181;M Wm is a low but effective concentration that can confer Wm body formation without severely damaging the meristemoids. When a new material (different plant species, different parts of the plant, etc.) is treated, the concentrations of the drugs need to be optimized accordingly. Second, to maintain the physiological condition of the samples to a maximal extent during the treatment, the seedlings are kept partially intact (only removing the cotyledons) until the imaging step. True leaves are dissected for slide preparation, and the sample is imaged afterward. Each sample needs to be treated individually, and the slide needs to be made freshly for imaging. Lastly, both z-stack imaging and time-series imaging require repeated laser scanning of the sample. To minimize the risk of photobleaching and phototoxicity on the samples, a low laser power is strongly recommended.
The high efficiency of membrane trafficking events is ensured by the diversity of the trafficking routes and their kinetics in response to the specific developmental and environmental conditions of the cell1. The described methods have limited spatiotemporal resolution, so more sophisticated light microscopy techniques are emerging to gain detailed knowledge of membrane traffic within a eukaryotic cell. For instance, super-resolution microscopy, including stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM), greatly increase the spatial resolution of a fluorescence-tagged protein31,32, though only fixed samples can be analyzed; spinning-disk microscopy substantially accelerates the scanning speed and reduces the photobleaching of the samples33, though it is challenging to record the dynamics of proteins with low abundance using this method; light-sheet microscopy provides fast and gentle 3D imaging34,35; and two-photon microscopy enables the detection of fluorescence signals from deeper tissues35,36, though at the cost of slightly lower resolution, etc. In addition, chemical biology has identified more specific drugs interferring with certain trafficking routes. Despite the limitations, these techniques together with other emerging advanced techniques will greatly improve our knowledge and shed light on the mechanisms of the cell membrane trafficking system.

Membrane traffic is a conserved cellular process that distributes membrane components (also known as cargoes), including proteins, lipids, and other biological products, between different organelles within a eukaryotic cell or across the plasma membrane to and from the extracellular space1. This process is facilitated by a collection of membranes and organelles named the endomembrane system, which consists of the nuclear membrane, the endoplasmic reticulum, the Golgi apparatus, the vacuole/lysosomes, the plasma membrane, and multiple endosomes1. The endomembrane system enables the modification, packaging, and transport of membrane components using dynamic vesicles that shuttle between these organelles. Membrane trafficking events are crucial to cell development, growth, and environmental adaptation and, thus, are under stringent and complex regulation2. Currently, multiple approaches in molecular biology, chemical biology, microscopy, and mass spectrometry have been developed and applied to the field of membrane trafficking and have greatly advanced the understanding of the spatiotemporal regulation of the endomembrane system3,4. Molecular biology is used for classical genetic manipulations of the putative players involved in membrane trafficking, such as altering the gene expression of the protein of interest or labeling the protein of interest with certain tags. Tools in chemical biology include the usage of molecules that specifically interfere with the traffic of certain routes4,5. Mass spectrometry is powerful for identifying the components in an organelle that has been mechanically isolated by biochemical approaches3,4. However, membrane traffic is a dynamic, diverse, and complex biological process1. To visualize the membrane trafficking process in live cells under various conditions, light microscopy is an essential tool. Continuous progress has been made in advanced microscope techniques to overcome the challenges in measuring the efficiency, kinetics, and diversity of the events4. Here, this study focuses on the widely adopted methodologies in chemical/pharmacological biology, molecular biology, and microscopy to study membrane trafficking events in a naturally simplified and experimentally accessible system, the stomatal developmental process.
Stomata are micropores on plant aerial surfaces that open and close to facilitate gas exchange between the inner cells and the environment6,7,8. Hence, stomata are essential for photosynthesis and transpiration, two events that are crucial for plant survival and growth. Stomatal development is dynamically adjusted by environmental cues to optimize the plant&#39;s adaptation to the surroundings9. Dating back to studies in 2002, the identification of the receptor protein Too Many Mouths (TMM) opened the door to a new era of investigating the molecular mechanisms of stomatal development in the model plant Arabidopsis thaliana10. After just a few decades, a classical signaling pathway has been identified. From upstream to downstream, this pathway includes a group of secretory peptide ligands in the epidermal patterning factors (EFP) family, several cell-surface leucine-rich-repeat (LRR) receptor kinases in the EREECTA (ER) family, the LRR receptor protein TMM, a MAPK cascade, and several bHLH transcription factors including SPEECHLESS (SPCH), MUTE, FAMA, and SCREAM (SCRM)11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26. Previous work indicates that one of the receptor kinases, ER-LIKE 1 (ERL1), demonstrates active subcellular behaviors upon EPF perception20. ERL2 also dynamically traffics between the plasma membrane and some intracellular organelles27. Blocking the membrane trafficking steps causes abnormal stomatal patterning, resulting in stomatal clusters on the leaf surface28. These results suggest that membrane traffic plays an essential role in stomatal development. This study describes a protocol to spatiotemporally investigate the ERL1 dynamics using protein-protein subcellular co-localization analysis combined with pharmacological treatment using some membrane trafficking inhibitors.

<table><tbody><tr><td>10 mL syringes</td><td>VWR</td><td>BD309695</td><td>Vacuum samples</td></tr><tr><td>Brefeldin A (BFA)</td><td>Sigma</td><td>B7651</td><td>membrane trafficking drug</td></tr><tr><td>Confocal Microscope</td><td>Leica</td><td>Lecia SP8 TCS with LAS-X software package</td><td>Imaging</td></tr><tr><td>Dissecting Forceps</td><td>VWR</td><td>82027-402</td><td>Genetic cross</td></tr><tr><td>Fiji</td><td>NIH</td><td>https://imagej.net/Fiji</td><td>Image processing</td></tr><tr><td>Leica LAS AF software</td><td>Leica</td><td>http://www.leica-microsystems.com</td><td>Image processing</td></tr><tr><td>transgenic seeds of ERL1-YFP</td><td></td><td></td><td>Qi, X. &lt;em&gt;et al&lt;/em&gt;. The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate. Elife. 9, doi:10.7554/eLife.58097, (2020).</td></tr><tr><td>transgenic seeds of RFP-Ara7</td><td></td><td></td><td>Ebine, K. et al. A membrane trafficking pathway regulated by the plant-specific RAB GTPase ARA6. Nat Cell Biol. 13 (7), 853-859, doi:10.1038/ncb2270, (2011).</td></tr><tr><td>Wortmannin (Wm)</td><td>Sigma</td><td>W1628</td><td>membrane trafficking drug</td></tr></tbody></table>

image-based methods to study membrane trafficking events in stomatal lineage cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

A previous study indicated that ERL1 is an active receptor kinase that undergoes dynamic membrane trafficking events20. ERL1 is a transmembrane LRR-receptor kinase on the plasma membrane. Newly synthesized ERL1 in the endoplasmic reticulum is processed in the Golgi bodies and further transported to the plasma membrane. The ERL1 molecules on the plasma membrane can perceive EPF ligands using their extracellular LRR domain18. Upon activation by the inhibitory EPFs, including EPF1, ERL1 is internalized into endosomes, transported to the multi-vesicular bodies (MVB), and then transported into the vacuoles for degradation to terminate the signal20. In contrast, the antagonistic EPFL9 ligand, which promotes stomatal formation14, retains ERL1 in the endoplasmic reticulum. Alternatively, the plasma membrane-localized ERL1 can recycle between endosomes and the plasma membrane20.
Here, transgenic plants bearing ERL1 promoter::ERL1-YFP were grown for 7 days following the protocol mentioned above. When just emerging, the true leaf was dissected for imaging, as ERL1 is only expressed in stomatal lineage cells in young leaves20. As expected, scattered cells were highlighted by the YFP signal, where ERL1-YFP labeled the plasma membrane (Figure&#160;5). In addition, multiple punctate signals were also observed, suggesting that ERL1 was also localized to the endosomes.
Several organelles in plant cells present as punctate fluorescent signals when observed under a light microscope, such as the trans-Golgi network and the MVBs. When the MVB marker line bearing RFP-Ara7 was genetically crossed with the ERL1-YFP transgenic line, both marker proteins were observed using the sequential imaging approach as described above. As shown in Figure 6, RFP-Ara7 highlighted multiple punctate signals in almost all the cells, indicating the MVBs in these cells (Figure 6B). When merging with the ERL1-YFP signal, most of the ERL1-YFP-positive punctate overlapped with the RFP-Ara7-labeled punctate. This result suggests that some of the ERL1-YFP molecules are transported to the MVBs.
To monitor the dynamics of these fast-moving endosomes, time series images of the 7-day-old true leaves bearing ERL1-YFP and RFP-Ara7 were collected (Figure 7). Time intervals of 7 s were applied for 203 s, and the collected 30 images were used to generate a video in Image J (Video 1). The video showed that ERL1-YFP-labeled endosomes moved together with RFP-Ara7 within the stomatal lineage cells, thus confirming that ERL1-YFP was localized to the MVBs.
To examine the ERL1 trafficking routes using a pharmacological approach, the effect of two commonly used membrane trafficking drugs, brefeldin A (BFA) and wortmannin (Wm), were analyzed. BFA, by inhibiting the ADP-ribosylation factor guanine-nucleotide exchange factors, including GNOM, can block the endosomal recycling and endocytosis of membrane proteins29. After 30 min of exposure to the BFA solution, ERL1-YFP was detected in some large compartments known as BFA bodies (Figure 8B,&#160;C). This indicates that ERL1 trafficking can be blocked by BFA, suggesting that ERL1 undergoes recycling or endocytosis. Wm can inhibit phosphatidylinositol-3 and phosphatidylinositol-4 kinases and, thus, causes the fusion of MVBs5. When treated with Wm for 30 min, some ring-like structures highlighted by ERL1-YFP were observed, which are typically called Wm bodies (Figure 8C). This result indicates that ERL1-YFP is transported to MVBs, thus suggesting that ERL1 follows the route to vacuoles for degradation.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65257/65257fig05.jpg" /> 
Figure 5: Single plane image of ERL1-YFP. (A) The fluorescence image, (B) the bright-field image, and (C) the merged image show that ERL1-YFP labels the plasma membrane and some highly mobile punctate within stomatal lineage cells. Scale bar: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65257/65257fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65257/65257fig06.jpg" /> 
Figure 6: Sequential images of ERL1-YFP and RFP-Ara7. (A) ERL1-YFP and (B) RFP-Ara7 both label punctate signals within the cell. The white punctate in (C) the merged image and (D)&#160;the inset image indicates that the two proteins co-localize on the MVBs within stomatal lineage cells.&#160;The white arrows indicate endosomes labeled by both ERL1-YFP and RFP-Ara7. The red arrows indicate endosomes labeled by RFP-Ara7. Scale bar: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65257/65257fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65257/65257fig07.jpg" /> 
Figure 7: Time series images of ERL1-YFP and RFP-Ara7. The images were taken with a 7 s time interval. The time point of each image is indicated in the top-left corner.&#160;Scale bar: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65257/65257fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65257/65257fig08.jpg" /> 
Figure 8: Z-stack images of ERL1-YFP. (A,&#160;B) Z-stack images of the ERL1-YFP plants treated with (A) mock or (B) BFA.&#160;The numbers in the top-right corner indicate the position of the image in the z-stack from top to bottom. Scale bar: 10 &#181;m. (C) Projection of the z-stack images of ERL1-YFP treated with mock or BFA solution and mock or Wm solution, as indicated in the top-right corner. The arrows indicate the aggregation bodies of ERL1-YFP caused by the drugs. Scale bar: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65257/65257fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Video of the time series images of ERL1-YFP and RFP-Ara7. The time series images of ERL1-YFP and RFP-Ara7 are stacked into a video using Fiji at a speed of 5 frames/s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65257/Video 1.zip" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells at JoVE.com

Author Spotlight: Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

image-based-methods-to-study-membrane-trafficking-events-stomatal

Nanyang Technological University

Research

JoVE Journal

Biology

776 Views.  Rutgers University. Deze techniek bestudeert de membraanhandelgebeurtenissen in de stomatale ontwikkeling, de microporiën op het luchtoppervlak van de plant, om fotosynthese en transpiratie te vergemakkelijken. Verschillende technieken worden gebruikt om de stomatale ontwikkeling van planten te bestuderen. Dit omvat moleculaire biologie, celbiologie of microscopie, chemische biologie, massaspectrometrie, biochemie en structurele biologie.Individuele huidmondjes vormen asynchroon. Elke stoma wordt gevormd...

Video: Auteur in de schijnwerpers: op afbeeldingen gebaseerde methoden om membraantransportgebeurtenissen in huidmondjescellen te bestuderen  

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Developmental Biology

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

Environment

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally ...

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

Samenvatting

Meer video's verkennen