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/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/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/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/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

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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 un...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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. et al. 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 ...

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

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726 Views.  Rutgers University. 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. 

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Badania wizualne Wrażliwość na prędkość i kierunek ruchu w Jaszczurki

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...