1. Yeast Preparations
<ol>
	<li>Transform yeast strains with a plasmid carrying a GAL1-GFP-Y68L (UBC9ts) cassette.</li>
	<li>Grow yeast in synthetic media containing 2% raffinose for 24 hr and back diluted to 2% galactose containing media for 16 hr or O/N. Incubate cells at 30 &deg;C while shaking at 200 rpm.</li>
	<li>The following morning, dilute the query strain to OD600= 0.2. Shake for 4-6 hr at 30 &deg;C (200 rpm) until the culture reaches OD600= 0.8-1.0.</li>
	<li>To repress expression (so as to monitor only the already-translated and folded pool of GFP-Ubc9ts), change media to synthetic media supplemented with 2% glucose (SD) 30 min prior to imaging.</li>
	<li>Treatment - Heat shock the cells for 20 min at 37 &deg;C to induce misfolding, or continuously in microscope incubator to follow protein aggregation in heat-shock conditions. Note - if you are using the microscope at any temperature above RT, pre-heat for about 2 hr to equilibrate the temperature along the body of the microscope and the objectives (see below).</li>
</ol>
2. Plate\Slide Preparations
<ol>
	<li>Choose appropriate plate. The main consideration is the ability to maintain focus during imaging acquisition. The following points are critical for 4D imaging and not for 2D time lapses or 3D imaging.
	<ol>
		<li>Multiple wells plate- the bottom of the different wells may not be homogenous (i.e. the wells will be at different heights relative to the objective). This will make it difficult to maintain focus across x y points and time points, regardless of the autofocusing technique being used.</li>
		<li>Slide material: glass vs. plastic. Glass bottomed slides are more z-homogenous between wells, but are more expensive.</li>
		<li>Thickness of the plate's bottom: we usually use coverslip-bottom plates index 1.5, but index 1 is also acceptable depending on the objective.</li>
	</ol>
	</li>
	<li>Cover bottom of the plate\slide with ConA (0.25 mg/ml) for 10 min. ConA is used to adhere cells to the slide, which enables following a single cell over time.</li>
	<li>Remove ConA and incubate the slide in a chemical hood so that excess ConA will evaporate.</li>
	<li>Plate 200 &mu;l of yeast sample (OD600= 0.5) into the ConAed well.</li>
	<li>Incubate for 15 min, as to enable cells stick to the surface of the plate.</li>
	<li>Extract the medium, and wash three times with new medium to get one layer of cells. Note: if a long time lapse is planned, seed the cells sparsely so that new buds won't fill and interrupt the region of interest.</li>
</ol>
3. Microscope Preparations
<ol>
	<li>We use a Nikon A1Rsi confocal microscope with a few non-standard modifications. For yeast imaging we use up to 4 lasers (405 nm, 50 mW CUBE laser; 457-514 nm, 65 mW Argon Ion laser; 561 nm, 50 mW Sapphire laser; and 640 nm, 40 mW CUBE laser), and up to 4 PMTs equipped with filters. Most of our imaging is done with a green filter set for EGFP and a red filter set for mCherry and tdTomato. With GFP and mCherry there is almost no bleedthrough, therefore we often use simultaneous scanning. However, line-scanning can also be used to prevent bleedthrough when it does occur. Our confocal is also equipped with a PInano Piezo stage (MCL), which can make 3 msec steps in z, enabling 2-3 z-stacks (30-50 sections) per second. The systems also has a spectral detector, Perfect Focus (laser offset), and two scanners - a galvano scanner and a resonant scanner.</li>
	<li>Choose appropriate objective. Main considerations:
	<ol>
		<li>Water/Oil/Air objective: the main consideration is the refractive index of the imaging medium vs the medium of the sample.
		<ol>
			<li>Oil objective advantages:
			<ol>
				<li>Oil objectives can have higher numerical apertures (oil breaks light more than water, and therefore more photons go back to the objective). Oil objectives can have up to NA 1.49, compared to 1.27 for water. (However, this is not actually a huge difference in resolution).</li>
				<li>The refractive index of oil matches the refractive index of glass, therefore no photons are lost going from the sample, through the glass, to the objective. (Note - chose an immersion oil that has a refractive index that is appropriate for your objective and your coverslip).</li>
				<li>Oil enables hassle-free long time-lapses since it does not evaporate.</li>
			</ol>
			Oil Objective disadvantages:
			<ol>
				<li>The higher resolution enabled by the higher NA of oil objectives degrades rapidly if the light has to travel through an aqueous medium (such as a cell).</li>
				<li>Images typically have more spherical aberrations and a "stretched out" effect in 3D.</li>
				<li>Oil is sticky, messy, and a hazard to objectives.</li>
			</ol>
			</li>
			<li>Water objective advantages
			<ol>
				<li>The cell itself is aqueous, therefore there are fewer distortions in z, and the resolution remains high deep into an aqueous sample.</li>
				<li>Clean and user friendly. Water objective disadvantages: evaporates quickly, therefore not suitable for long movies without special arrangements being made to pump water continuously to the objective.</li>
			</ol>
			</li>
			<li>Air objective advantages: 1. No material is lost/evaporates during multiple point acquisition or a long movie. 2. Clean and user friendly. Disadvantage: low resolution and low signal sensitivity.</li>
		</ol>
		</li>
		<li>Room and incubator temperature: changing temperature affects matter properties, and in delicate systems changes the focus. The temperature should be adjusted before choosing points for acquisition. Changing the temperature during acquisition will disrupt imaging and will result in loss of focus. We let our microscope system equilibrate for 2 hr at the desired temperature before imaging.</li>
		<li>Correction collars: Many objectives come with a correction ring enabling the objective to be adjusted for a given cover-slip thickness. We recommend adjusting the correction collar while using fluorescent beads to visualize the point spread function. This will ensure correct settings for every sample.</li>
		<li>Stable sample holders: we find that most commercially available sample holders are the weakest part of the microscope. They are often wobbly and not straight. This is a disaster for high-resolution, multi-point 4D imaging. We design our own sample holders that are straight and fit tightly onto the stage. They can also be bolted down when needed.</li>
		<li>Multi-point acquisition: our imaging system is equipped with the Nikon "Perfect focus" system, which is basically a laser-based auto-focus mechanism. However, autofocusing is not necessarily necessary with 4D imaging, especially if the system does not drift a lot.</li>
	</ol>
	</li>
</ol>
4. Imaging
<ol>
	<li>Turn on the system: lasers, stage, controller, camera and computer software.</li>
	<li>Place plate on stage holder, properly secured and stabilized.</li>
	<li>Use eye port to determine location and orientation of yeast.</li>
	<li>Using brightfield, assess cell health and viability according to shape and texture.</li>
	<li>Turn on epifluorescent light according to the relevant fluorophore (e.g. FITC filter for GFP), and focus on cells displaying the phenotype which is subject to your research. Cells which are highly fluorescent in more than one wavelength may be dead and therefore autofluorescent. We visualize the nucleus with a tdTomato fluorophore fused to an SV40 NLS signal (NLS-TFP). TFP is twice the size of GFP, and is therefore above the diffusion limit of the nucleus, therefore it works very well as a nuclear marker. We can also excite TFP with a green (488 nm) laser simultaneously as GFP, but collect the green and red emissions into two separate PMTs for spectral resolution.</li>
	<li>Adjust the following settings to minimize noise and oversaturation:
	<ol>
		<li>Laser power - photobleaching and phototoxicity vs. brightness of image should be considered.</li>
		<li>Gain: affects camera sensitivity, thus Signal to Noise ratio.</li>
		<li>Pinhole diameter - should be adjusted according to the laser with the shorter wavelength. The pinhole diameter determines the thickness (height) of the optical section imaged. Thus, opening the pinhole will collect more light (let more photons in), but will decrease resolution in z (less confocality).</li>
	</ol>
	</li>
	<li>Useful tips:
	<ol>
		<li>If different fluorophores emit congruent wavelength, use the Spectral Detector feature which enables the choice of virtual filters. Keep in mind that spectral detectors are less sensitive than regular PMTs.</li>
		<li>If different fluorophores are excited by the same laser, use the line scanning feature.</li>
		<li>A Galvano scanner enables more sensitivity, but has a higher risk for photobleaching. A Resonant scanner enables faster acquisition, thus lowering the risk for photobleaching, but is less sensitive.</li>
		<li>Averaging between 2 and 16 images, it enhances signal to noise ratio, but makes acquisition slower and, of course, involves more exposure of the sample to the laser.</li>
		<li>Duration of acquisition- depends on the biological question (e.g JUNQ and IPOD formation takes ~2 hr). We have imaged yeast with the resonant scanner for up to 30 hr in 3D time-lapse.</li>
		<li>Time lapse intervals- Smaller intervals will create a more coherent movie but might cause photo bleaching and therefore loss of signal.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gershenson, A., Gierasch, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+folding+in+the+cell:+challenges+and+progress.">Protein folding in the cell: challenges and progress.</a> Current opinion in structural biology. 21, 32-41 (2011).</li><li>Aguzzi, A., Calella, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions:+protein+aggregation+and+infectious+diseases.">Prions: protein aggregation and infectious diseases.</a> Physiological reviews. 89, 1105-1152 (2009).</li><li>Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteotoxic+stress+and+inducible+chaperone+networks+in+neurodegenerative+disease+and+aging.">Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging.</a> Genes &amp; development. 22, 1427-14 (2008).</li><li>Cohen, E., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insulin+paradox:+aging,+proteotoxicity+and+neurodegeneration.">The insulin paradox: aging, proteotoxicity and neurodegeneration.</a> Nature reviews. Neuroscience. , (2008).</li><li>Hershko, A., Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+system.">The ubiquitin system.</a> Annual review of biochemistry. 67, (1998).</li><li>Tyedmers, J., Mogk, A., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+strategies+for+controlling+protein+aggregation.+Nature+reviews.">Cellular strategies for controlling protein aggregation. Nature reviews.</a> Molecular cell biology. 11, 777-788 (2010).</li><li>Treusch, S., Cyr, D. M., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+deposits:+Protection+against+toxic+protein+species?.">Amyloid deposits: Protection against toxic protein species?.</a> Cell cycle (Georgetown, Tex.). 8, 1668-1674 (2009).</li><li>Spokoini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confinement+to+Organelle-Associated+Inclusion+Structures+Mediates+Asymmetric+Inheritance+of+Aggregated+Protein+in+Budding+Yeast.">Confinement to Organelle-Associated Inclusion Structures Mediates Asymmetric Inheritance of Aggregated Protein in Budding Yeast.</a> Cell Rep. , (2012).</li><li>Weisberg, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+superoxide+dismutase+1+(SOD1G93A)+aggregates+determines+their+toxicity.">Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity.</a> Proc Natl Acad Sci U S A. 109, 15811-15816 (2012).</li><li>Kaganovich, D., Kopito, R., Frydman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Misfolded+proteins+partition+between+two+distinct+quality+control+compartments.">Misfolded proteins partition between two distinct quality control compartments.</a> Nature. 454, 1088 (2008).</li><li>Betting, J., Seufert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+Ubc9+mutant+protein+with+temperature-sensitive+in+vivo+function+is+subject+to+conditional+proteolysis+by+a+ubiquitin-+and+proteasome-dependent+pathway.">A yeast Ubc9 mutant protein with temperature-sensitive in vivo function is subject to conditional proteolysis by a ubiquitin- and proteasome-dependent pathway.</a> The Journal of biological chemistry. 271, 25790 (1996).</li><li>Tongaonkar, P., Beck, K., Shinde, U. P., Madura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+temperature-sensitive+mutant+of+a+ubiquitin-conjugating+enzyme+and+its+use+as+a+heat-inducible+degradation+signal.">Characterization of a temperature-sensitive mutant of a ubiquitin-conjugating enzyme and its use as a heat-inducible degradation signal.</a> Analytical biochemistry. 272, 263 (1999).</li></ol>

No conflicts of interest declared.

Our intuitions about biochemical processes derive from bench top experiments in which a well-mixed solution of reactants and products is allowed to reach equilibrium in a beaker. In such a setting, the concentration of a given chemical species may be expressed as a single number, which is the ratio of a molar quantity of molecules to a macroscopic volume. Much of what we know about protein structure and function derives from using methods that reflect the classic, bulk reaction picture: western blots, centrifugations, and spectrophotometric measurements carried out on extracts from homogenates of whole populations of cells.
As the technology we use to look at cells under magnification improves by leaps and bounds, it becomes ever clearer that the conditions in which most biochemical reactions take place in vivo bear only the slightest resemblance to those of the classic bench top scenario. Not only is the interior of the cell a densely packed environment, in which crowding effects substantially alter the activities of various reactants, it is also quite the opposite of well-mixed. This accounts for the frequent disparity between in vitro and in vivo efficiencies of a wide range of complex macromolecular reactions.
Nowhere are intuitions stemming from classical in vitro biochemical experiments more prone to mislead as in questions pertaining to the in vivo folding, misfolding, and aggregation of proteins. Whereas studies of protein chemistry in bulk reactions can treat the issue of folding for a given protein as a simple yes or no question, any attempt to track the dynamics of whole populations of macromolecules in a live cell must be sensitive to the whole distribution of possible conformational outcomes available to a polypeptide chain, and in particular to the risk of misfolding and aggregation. For example, we might examine a bulk cell lysate of an aggregating protein by western blotting, and determine that the protein is mostly insoluble and not ubiquitinated. However, in the living cell a discrete sub-population of the protein, difficult to detect when averaging over many cells, may be soluble and ubiquitinated in a particular compartment where the local concentration of the species is extremely high. The latter scenario may have more important consequences for the viability of the cell than the larger bulk sub-population. Furthermore, whereas chaperones display a variety of pleiotropic behaviors and functions in vitro, it is becoming evident that in the cell their discrete functions are spatially and temporally confined.
In the newly emerging paradigm for understanding biochemistry, concentration becomes a variable property of each specific nano-environment in the cell, and the molecular events that underlie biological processes must be assayed not only in time, but also in space. The 4D imaging approach presented here enables sensitive modeling of protein misfolding in live cells, though it can be used to study any number of other biological processes and how they are regulated in space, time, and following changes in environmental conditions. In this paper we use the Ubc9ts folding sensor, which effectively demonstrates the stages and options for dealing with the onset of protein aggregation in the cytosol. In addition to illustrating the cell biology of aggregation quality control, this approach can serve as a powerful tool for deciphering the effect of specific perturbations or genetic mutations on proteostasis (for example Ubc9ts can be used to measure protein folding stress in response to oxidation, the expression of a toxic aggregate, or mutations in the quality control pathway).
4D imaging is also essential for accurately determining protein localization or colocalization between two different proteins, and for detecting phenomena which maybe be transient but important. For example, especially in a small spherical organism such as yeast, it may appear to be the case that a structure or aggregate has juxtanuclear localization, whereas 4D imaging may reveal that this is simply an artifact of the angle of inspection.
In the example experiment we present here, we demonstrate the use of a model misfolded protein, Ubc9ts, to follow aggregation quality control over time and space in the cytosol. At the permissive temperature, Ubc9ts is folded and diffused in the nucleus and cytosol. Upon heat-induced misfolding, it initially forms rapidly diffusing small aggregate Stress Foci that are processed for proteasomal degradation. When the proteasome is partially inhibited, these Stress Foci are converted into JUNQ and IPOD inclusions over the course of about 2 hr. If ubiquitin-mediated degradation is not available as a quality control option, Ubc9ts is immediately re-routed to the IPOD inclusion for protective aggregation. These tools offer incredible opportunities to discover novel genetic factors involved in aggregation quality control, and to explore their spatial and temporal regulation in the cell.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MG132</td>
			<td>Mercury</td>
			<td>mbs474790</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>con A</td>
			<td>Sigma</td>
			<td>C2010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass bottom plates</td>
			<td>ibidi</td>
			<td>ibd81158</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4D Fluorescence Imaging of Protein Aggregation 
			Confocal 3D movies were acquired using a Nikon A1R-si microscope equipped with a PInano Piezo stage (MCL), using a 60X water objective NA 1.27, 0.3 micron slices, 0.5% laser power (from 65 mW 488 laser and 50 mW 561 laser). z-stacks were acquired every 5 min for 90 min. Each z-series was acquired with 0.5 micron step size and 30 total steps. Image processing was performed using NIS-Elements software.</td>
		</tr>
	</tbody>
</table>

4d imaging of protein aggregation in live cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

<img alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50083/50083fig1highres.jpg" src="/files/ftp_upload/50083/50083fig1.jpg" /> 
 Figure 1. Model: Sub-cellular compartmentalization of misfolded proteins. Quality control machinery directs misfolded proteins into distinct compartments with distinct functions: Soluble proteins, targeted for degradation, undergo poly-ubiquitination and are sent to the Juxta-NuclearQuality control compartment (JUNQ). Insoluble proteins that can't be ubiquitinated are sent for protective sequestration to the InsolubleProteinDeposit (IPOD), adjacent to the vacuole, where they undergo active aggregation.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50083/50083fig2highres.jpg" src="/files/ftp_upload/50083/50083fig2.jpg" /> 
 Figure 2. Modeling protein misfolding with GFP-Ubc9ts. Under normal conditions, GFP-Ubc9ts (green) is natively folded, and is localized diffusely in the nucleus and the cytosol. The nucleus is labeled by NLS-TFP (red). Expression of Ubc9ts was shut off by addition of 2% glucose before imaging in all experiments. <a href="https://www.jove.com/files/ftp_upload/50083/50083fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<ol>
	<li>Upon temperature shift to 37 &deg;C, GFP-Ubc9ts (green) is misfolded and forms cytosolic Stress Foci. The nucleus is labeled by NLS-TFP (red).</li>
	<li>Upon recovery from heat shock at 23 &deg;C, the thermally denaturated GFP-Ubc9ts is degraded, as indicated by decreased fluorescence level.</li>
	<li>Upon temperature shift to 37 &deg;C and proteasome inhibition with 80 Mm MG132, GFP-Ubc9ts is misfolded and processed into JUNQ and IPOD inclusions. The nucleus is labeled by NLS-TFP (red).</li>
	<li>Upon temperature shift to 37 &deg;C and ubiquitination inhibition, GFP-Ubc9ts is misfolded and processed into the IPOD inclusion. The nucleus is labeled by NLS-TFP (red). The Ubiquitin Protease 4 (Ubp4) is overexpressed to block Ubc9ts ubiquitination.</li>
</ol>
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50083/50083fig3highres.jpg" src="/files/ftp_upload/50083/50083fig3.jpg" /> 
 Figure 3. Time lapse of JUNQ and IPOD formation. Upon temperature shift to 37 &deg;C and proteasome inhibition with 80 Mm MG132, GFP-Ubc9ts Stress Foci are processed into JUNQ and IPOD inclusions. The nucleus is labeled by NLS-TFP (red). 3D images were acquired at 4 min intervals. (also see Movie 1). <a href="https://www.jove.com/files/ftp_upload/50083/50083fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about 4D Imaging of Protein Aggregation in Live Cells at JoVE.com

4D Imaging of Protein Aggregation in Live Cells

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

4d-imaging-of-protein-aggregation-in-live-cells

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17.2K Views.  Hebrew University of Jerusalem. Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

Video: 4D Imaging of Protein Aggregation in Live Cells

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

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 ...

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 ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...