We are grateful to S. Taylor for providing the pLPCX-Histone H2-GFP plasmid. We thank R. Mertelsmann for continuous support. This work was supported by the Deutsche Forschungsgemeinschaft.

1. Seeding of U2OS-based Cyclin B-SNAP Reporter Cells (Clone 11 Cells 6) on Microscope Chamber Slides
<ol>
	<li>Trypsinize subconfluent SNAP reporter cells that were allowed to grow asynchronously in log phase for at least 48 hr.</li>
	<li>Working with 8 well microscope chambers (constant distribution of cells).</li>
</ol>
For seeding of cells onto 8 well microscope chambers at a constant distribution across the entire surface of the microscope chamber, centrifuge 10,000 cells and resuspend in 350 &mu;l of phenol red-free normal growth medium (supplemented with 10% fetal bovine serum, penicillin/streptomycin and sodium pyruvate). Transfer cell suspension to the microscope chamber (Figure 2).
Working with 8 well microscope chambers (maximum cell density in the center).
For seeding of cells at a higher density in the center of the microscope chamber, load the chamber with 300 &mu;l of phenol red-free normal growth medium. Add 5,000 cells carefully to the center of the microscope chamber (Figure 2).
Working with 96 well special optics plates (constant distribution of cells). 
For seeding of cells onto 96 well plates at a constant distribution across the entire surface of the well, centrifuge 5,000 cells and resuspend in 300 &mu;l of phenol red-free normal growth medium. Transfer the cell suspension to a 96-well plate. Depending on the total number of cell-containing wells required, adjust the cell number and the total volume of the suspension medium (Figure 2).
Working with 96 well special optics plates (maximum cell density in the center).
For seeding of cells onto 96 well plates, carefully add 1,500 cells in 15 &mu;l of phenol red-free normal growth medium in a small drop to the center of each well to achieve restriction of cell growth to the center of the well (Figure 2).
<ol start="3">
	<li>Allow seeded cells to grow for at least 18 hr under standard cell culture conditions (37 &deg;C, 100% air humidity, 5% CO2).</li>
</ol>
2. Staining of Reporter Cells with SNAP Substrate
<ol>
	<li>30 min prior to the beginning of the staining procedure allow aliquots of phenol red-free normal growth medium to warm up to 37 &deg;C.</li>
	<li>For easy handling of the SNAP substrate (in our case TMR Star) dissolve TMR Star in DMSO to obtain a concentration in the stock solution of 400 &mu;M, which can be stored at -20 &deg;C.</li>
	<li>Prior to staining, dilute 0.5 &mu;l of TMR Star stock solution in 200 &mu;l of phenol red-free normal growth medium (37 &deg;C) to obtain a final labeling concentration of 1 &mu;M.</li>
	<li>Remove normal growth medium from the asynchronously growing cells and incubate in labeling medium for 25 min under standard culture conditions.</li>
	<li>Remove labeling medium and wash cells four times with phenol red-free normal growth medium (37 &deg;C). Incubate cells in 300 &mu;l of phenol red-free normal growth medium (37 &deg;C) for 30 min. Prior to transporting to the microscope replace the medium with fresh phenol red-free normal growth medium (37 &deg;C) to remove residual unbound SNAP substrate.</li>
	<li>Transport cells to the microscope in a styrofoam box on a pre-warmed (37 &deg;C) heat block to minimize temperature variation.</li>
</ol>
3. Measurement of Fluorescence Intensity
<ol>
	<li>Two hours prior to the analysis adjust the air temperature of the climate chamber to 37 &deg;C in dry mode in order to bring the entire microscope with all its components to the desired temperature. Pre-heating before setting the humidity is important to avoid condensation and subsequent damage to the microscope.</li>
	<li>Adjust air humidity to 60% and CO2 to 5% prior to the start of the analysis.</li>
	<li>Start the Scan^R Acquisition software and define standard settings (see Table 1).</li>
	<li>Define the positions of the wells to be analyzed.</li>
	<li>Define the &Delta;t (acquisition cycle time) and the absolute number of acquisition cycles.</li>
	<li>If analysis of a higher number of wells is desired, select hardware autofocus, otherwise it is sufficient to use software autofocus alone.</li>
	<li>Start the acquisition and supervise for the first two acquisition cycles. The microscope will focus on the histone H2-GFP signal, with subsequent acquisition of a first image in that channel before the filter is changed and the corresponding TMR Star image is acquired (Figure 3). This is then repeated for all positions within a well and for each of the wells to be examined, before repeating again the next cycle.</li>
</ol>
4. Analysis of Proteolytic Profiles using Scan^R
<ol>
	<li>Start the Scan^R Analysis software and analyse the images with the cell nuclei, as visualized by histone H2-GFP, defined as the main object, using a threshold based on signal intensity and a watershed algorithm to assist in separating neighboring cells. A subobject consisting of a nucleus with cytoplasm should be created for TMRstar analysis. (Important properties of the main object are X and Y positions, time, and maximum and mean intensities of GFP for the main object and the total intensity divided by area for the TMRstar subobject.) This analysis process may take several hours due to large data quantities.</li>
	<li>Change to trace mode to visualize the subobject mean TMR Star fluorescence intensity over time (cell traces), assigned to the analyzed main objects (Figure 4). The number of cells to be examined can be narrowed down by gating on those measurements lasting at least 140 cycles and with a high maximum intensity of H2-GFP. Looking at larger numbers of cells allows a first representative and objective view (Figure 4).</li>
	<li>Select a cell trace of interest to visualize histone H2-GFP and TMR Star fluorescence simultaneously at the single cell level (Figure 5A).</li>
	<li>Using the right mouse click on a cell of interest and generate an exportable picture gallery for illustration of histone H2-GFP and cyclin B-SNAP TMR Star for every single time point.</li>
	<li>Change to the population mode of the Scan^R Analysis software and gate the region where the cell of interest is represented on the X vs. Y dot plot (Figure 5B).</li>
	<li>Apply a new dot plot window to the gated region and visualize mean TMR Star fluorescence intensity over time (Figure 5C).</li>
	<li>Export data (time and fluorescence intensity) to Microsoft Excel for further calculation.</li>
</ol>
5. Representative Results
Figure 5D and 5E depict cyclin B kinetics, represented by a TMR Star fluorescence intensity curve, of a cell that proceeds through a regular mitosis without signs of chromosomal misalignment (Figure 5E). Upon compaction of the cytoplasm following nuclear envelope breakdown (NEBD, as indicated by the red triangle), TMR Star fluorescence intensity shows an abrupt increase until the isomorphic window (brighter area in the diagram) is reached when the cell enters prometaphase 6. Fluorescence intensity remains at a stable level as long as the cell proceeds through prophase and metaphase and then starts to drop rapidly once all of the chromosomes have established a stable metaphase plate (Figure 5D and 5E). This drop precedes chromosome separation during anaphase (blue dot on the curve). In late mitosis the chromatin starts to decondense (blue bars) and the cell adopts interphase morphology while the fluorescence intensity curve approaches a plateau which is lower than the plateau before mitosis (Figure 5D and 5E).
<table border="1">
	<tbody>
		<tr>
			<td>Autofocus settings</td>
			<td>Histone H2-GFP (main object acquisition settings)</td>
			<td>Cyclin B-SNAP labeled with TMR Star 
			(acquisition settings)</td>
			<td>Acquisition cycle time Repetitions</td>
		</tr>
		<tr>
			<td>Coarse autofocus +/-39 &mu;m 24 Layers 
			Fine autofocus 
			+/-5.4 &mu;m 14 Layers</td>
			<td>GFP filter set: 
			Exposure time: 100 msec 
			Light intensity: 25%</td>
			<td>TRITC filter set: 
			Exposure time: 150 msec 
			Light intensity: 33.3%</td>
			<td>2 to 5 min.</td>
		</tr>
		<tr>
			<td>GFP filter set: 
			Exposure time: 12 msec 
			Light intensity: 12.5%</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Up to 48 hr of continued analysis (limited by reduced air humidity of 60%)</td>
		</tr>
	</tbody>
</table>
Table 1. Standard settings as used for analysis of cyclin B proteolysis. Histone H2-GFP was used as a reference structure to define the focus plane for measurement of cyclin B-SNAP fluorescence intensity. Light intensities during the focusing procedure are lower as compared to image acquisition settings to avoid cumulative phototoxicity.
<img alt="Figure 1" src="/files/ftp_upload/4239/4239fig1.jpg" /> 
 Figure 1. Schematic of the measurement of cyclin B proteolysis through expression of a chimeric cyclin B derivative with degradation characteristics similar to the endogenous protein. The decline in fluorescence intensity after pulse-chase labeling is a measure of APC/C-activity.
<img alt="Figure 2" src="/files/ftp_upload/4239/4239fig2.jpg" /> 
 Figure 2. Analysis of cells on 8 well slides or 96 well plates. Regular distribution of the cells across the surface of the well is achieved by resuspension in a final volume of 300 &mu;l and is recommended, if only a single or few positions are manually defined for analysis. Enrichment of cells in the center region is achieved by addition of cells to the center of prefilled or empty wells. This technique is recommended in case of automated image acquisition in different wells.
<img alt="Figure 3" src="/files/ftp_upload/4239/4239fig3.jpg" /> 
 Figure 3. Sequence of data acquisition. A) Detection of histone H2-GFP is used for focus plane definition and monitoring of chromosomal alignment during mitosis. B) Fluorescence intensity of the TMR Star fluorescence intensity is a measure for the absolute amount of pulse-chase labeled cyclin B-SNAP.
<img alt="Figure 4" src="/files/ftp_upload/4239/4239fig4.jpg" /> 
 Figure 4. Representative Scan^R Analysis software-based depiction of mean TMR Star fluorescence intensities (total TMRstar intensity divided by area) over time. Selection of a representative cell trace (blue). The corresponding images (shown on the right) allow the simultaneous visualization of histone H2-GFP (green) and cyclin B-SNAP (red). <a href="https://www.jove.com/files/ftp_upload/4239/4239fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" src="/files/ftp_upload/4239/4239fig5.jpg" /> 
 Figure 5. A+B) Example for gating of dots representing a cell of interest on the XY-dot plot. C) Visualization of the mean TMRstar fluorescence intensity (total TMRstar intensity divided by area) over time using the dot plot. D+E) Representative fluorescence intensity curve of cyclin B-SNAP with isomorphic window (brigher field on the diagram) between NEBD (nuclear envelope breakdown) and chromatin decondensation (blue bar) as generated in Microsoft Excel. Anaphase is indicated by the blue dot on the curve. <a href="https://www.jove.com/files/ftp_upload/4239/4239fig5large.jpg" target="_blank"> Click here to view larger figure</a>.
Movie 1. <a href="https://www.jove.com/files/ftp_upload/4239/4239movie1.avi" target="_blank"> Click here to view supplemental movie</a>.

<ol>
	<li>Musacchio, A., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spindle-assembly+checkpoint+in+space+and+time.">The spindle-assembly checkpoint in space and time.</a> Nat. Rev. Mol. Cell Biol. 8, 379-393 (2007).</li><li>Nasmyth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregating+sister+genomes:+the+molecular+biology+of+chromosome+separation.">Segregating sister genomes: the molecular biology of chromosome separation.</a> Science. 297, 559-565 (2002).</li><li>Cahill, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+of+mitotic+checkpoint+genes+in+human+cancers.">Mutations of mitotic checkpoint genes in human cancers.</a> Nature. 392, 300-303 (1998).</li><li>Lengauer, C., Kinzler, K. W., Vogelstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+instabilities+in+human+cancers.">Genetic instabilities in human cancers.</a> Nature. 396, 643-649 (1998).</li><li>Fletcher, D. A., Mullins, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics+and+the+cytoskeleton.">Cell mechanics and the cytoskeleton.</a> Nature. 463, 485-4892 (2010).</li><li>Schnerch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+APC/C+activity+in+the+presence+of+chromosomal+misalignment+in+unperturbed+cell+populations.">Monitoring APC/C activity in the presence of chromosomal misalignment in unperturbed cell populations.</a> Cell Cycle. 11, (2012).</li><li>Keppler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+method+for+the+covalent+labeling+of+fusion+proteins+with+small+molecules+in+vivo.">A general method for the covalent labeling of fusion proteins with small molecules in vivo.</a> Nat. Biotechnol. 21, 86-869 (2003).</li><li>Jansen, L. E., Black, B. E., Foltz, D. R., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+centromeric+chromatin+requires+exit+from+mitosis.">Propagation of centromeric chromatin requires exit from mitosis.</a> J. Cell. Biol. 176, 795-805 (2007).</li><li>Malureanu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc20+hypomorphic+mice+fail+to+counteract+de+novo+synthesis+of+cyclin+B1+in+mitosis.">Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis.</a> J. Cell Biol. 191, 313-329 (2010).</li><li>Mena, A. L., Lam, E. W., Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+spindle-assembly+checkpoint+response+requires+de+novo+transcription+and+translation+of+cyclin+B1.">Sustained spindle-assembly checkpoint response requires de novo transcription and translation of cyclin B1.</a> PLoS One. 5, (2010).</li><li>Wolthuis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc20+and+Cks+direct+the+spindle+checkpoint-independent+destruction+of+cyclin.">Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin.</a> A. Mol. Cell. 30, 290-302 (2008).</li></ol>

No conflicts of interest declared.

We present here a live-cell imaging-based approach facilitating the simultaneous monitoring of cyclin B proteolysis and chromosome alignment. This approach allows the study of mitotic control in unperturbed cell populations at the single cell level. Cyclin B degradation curves facilitate direct insights into the activity of the APC/C and thus indirectly reflect the status of the SAC 6.
This approach, even though established based on the Scan^R software, can easily be reproduced on ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of product</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Reporter cell line</td>
			<td>generated in-house as described 6</td>
			<td>clone 11 reporter cells 
			(U2Os-based cyclin B-SNAP expressing cells)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Retroviral cyclin B-SNAP expression vector</td>
			<td>generated in-house as described 6</td>
			<td>pLNCX2-cyclin B mut5-SNAP</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenolred-free DMEM</td>
			<td>Gibco</td>
			<td>21063-029</td>
			<td>Supplementation with FCS, sodium pyruvate, penicillin/strepto-mycin required</td>
		</tr>
		<tr>
			<td>SNAP-Cell TMR-Star</td>
			<td>New England Biolabs</td>
			<td>S9105S</td>
			<td>Stock solution 400 &mu;M in DMSO</td>
		</tr>
		<tr>
			<td>Special Opstics Plate, 96 well</td>
			<td>Costar</td>
			<td>3720</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&mu;-Slide 8 well, ibiTreat</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscopy unit</td>
			<td>Olympus</td>
			<td>IX-81 inverse microscope with climate chamber</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Olympus</td>
			<td>UPLSAPO 20x objective (N.A. 0.75)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acquisition software</td>
			<td>Olympus</td>
			<td>Scan^R Acquisition software (v.2.2.09)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>Olympus</td>
			<td>Scan^R Analysis software (v.1.2.0.6)</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

studying proteolysis of cyclin b at the single cell level in whole cell populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Watch this Scientific Journal Video about Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations at JoVE.com

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

studying-proteolysis-cyclin-b-at-single-cell-level-whole-cell

Research

JoVE Journal

Biology

10.4K Görüntüleme.  University Medical Center Freiburg. Geçiş anafaz-teşvik kompleksi (APC / C)-bağımlı ubikuitinasyon ve burada siklin B. sonraki yıkım, biz, darbe-kovalamaca etiketleme sonrasında, bir sistem kurulmuş tüm hücre popülasyonlarında siklin B proteoliz izleme sağlar ve tetiklenir anafaz için metafaz mitotik geçiş noktasından müdahale tespitini kolaylaştırır.

Video: Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Speed

Subtitles

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations