This work was supported by NIH grant R01 GM104010. Thanks for assistance with fluorescence microscopy to Vytas Bindokas and Christine Labno at the Integrated Microscopy Core Facility, which is supported by the NIH-funded Cancer Center Support Grant P30 CA014599.

1. Preparation
<ol>
	<li>Make nonfluorescent minimal NSD medium2. The absence of riboflavin is expected to reduce background intracellular green fluorescence and associated phototoxicity.</li>
	<li>Grow the yeast strain overnight at ~23 &#176;C to logarithmic phase in 5 mL NSD in a 50 mL baffled flask with good aeration. About 3-4 h prior to analysis, dilute the yeast culture in fresh NSD&#160;so that the final OD600 will be 0.5-0.8 at the time of imaging.</li>
	<li>Prepare a 2 mg/mL solution of concanavalin A (ConA). If desired, freeze aliquots of this solution in liquid nitrogen and store at -80 &#176;C.</li>
	<li>Spin the ConA solution for 5 min at full speed in a microcentrifuge to remove particulate matter that may interfere with imaging. Then add 250 &#181;L of the supernatant to a clean 35 mm coverglass bottom microscopy dish. After 15 min, wash 2-3 times with 2 mL of dH2O and let dry.</li>
	<li>Add 250 &#181;L of the yeast culture to the ConA-coated dish, wait 10 min to allow the cells to adhere, and gently wash 2-3 times with 2 mL NSD. Cover the cells with 2 mL fresh NSD.</li>
</ol>
2. Imaging
<ol>
	<li>Use a 63x or 100x oil immersion lens. A numerical aperture (NA) of 1.40 is sufficient, but a higher NA lens can also be used.</li>
	<li>Format the frame size to 256 x 128 (width x height). If a larger frame size is needed, increasing the width will not reduce the scan speed as long as the confocal microscope is equipped with a resonant scanner.</li>
	<li>Adjust the zoom factor to make the pixel size ~80 nm.</li>
	<li>Use the maximum scan speed, which is typically on the order of 8 kHz. Turn on bidirectional X scanning if it is available, and if control experiments confirm that the scans from the two directions are in register.</li>
	<li>Set the line accumulation to 4 or 6. Be sure to use accumulation (summing) instead of averaging.</li>
	<li>Set the pinhole to 1.2 Airy units. Empirically, when imaging live yeast cells, this setting captures more photons than the standard choice of 1.0 Airy unit while causing no appreciable loss of resolution.</li>
	<li>For each fluorescence channel, set the excitation wavelength, assign a high sensitivity detector, set the emission wavelength range, and turn on photon counting mode if available. The wavelength choices will depend on the fluorophores. As an example, excite GFP fluorescence at 485&#160;nm and collect from 495-550 nm, and excite mScarlet&#160;fluorescence at 555&#160;nm and collect from 562-625&#160;nm.</li>
	<li>Set the intensity of each laser to be as low as possible. This setting must be determined empirically. An appropriate intensity will result in capture of a noisy but interpretable image sequence, with the fluorescence signal bleaching no more than 50% by the end of a 5 min movie. On the confocal microscope used here (see Table of Materials), this intensity corresponds to a laser power setting on the order of 5%.</li>
	<li>Use notch filters or time gating to avoid capturing reflected light from the coverslip. If time gating is available, set the gating window to 0.6-10.0 ns.</li>
	<li>Turn on brightfield imaging and use a low sensitivity detector for data collection. Set the gain to a level that makes the cells clearly visible. Do not use differential interference contrast (DIC) because the prism will interfere with the capture of reliable fluorescence data.</li>
	<li>Set the Z-step interval to 0.25-0.35 &#181;m. Image the entire volume of the yeast cells by collecting about 20-25 optical sections per Z-stack. Specify the directionality of imaging such that &#34;down&#34; moves toward the coverslip.</li>
	<li>For a typical movie, set the time interval between Z-stacks to 2 s and set the movie duration to 5 to 10 min. Depending on the compartment under study, reduce the interval to make shorter movies of relatively fast dynamics, or increase the interval to make longer movies of relatively slow dynamics.</li>
	<li>Save the movie as a LIF&#160;file with the brightfield images in the last channel. Higher bit depths are unnecessary at this stage, and the processing pipeline is configured to accept 8-bit TIFF data.</li>
</ol>
3. Deconvolution
<ol>
	<li>Launch the deconvolution software (see Table of Materials), using the Classic Maximum Likelihood Estimation algorithm.</li>
	<li>Open the data set generated in step 2.13. Select &#34;Deconvolution wizard&#34;. Inspect the values displayed in the &#34;Parameter wizard&#34;. The imaging parameters should be detected and correctly displayed. Under &#34;Refractive indexes&#34;, change the &#34;Embedding med.&#34; value to 1.40 to approximate the yeast cytoplasm.&#160;Under &#8220;coverslip&#8221;, select &#8220;Launch editor&#8221; and &#8220;Estimate position&#8221; and &#8220;OK&#8221; to set the coverslip position. Select &#34;Set all verified&#34; and &#34;Accept&#34;.</li>
	<li>Proceed through the &#34;Deconvolution wizard&#34; for each fluorescence channel. Select &#34;Manual&#34; as the mode for background estimation. Inspect the raw data fluorescence intensity profile to determine an estimated background value, which is typically about 0.1.</li>
	<li>In the deconvolution setup menu, enter a &#34;Maximum iterations&#34; value of 40 and turn off bleach correction. Enter an estimated SNR value, which is typically about 5.0. Click &#34;Deconvolve&#34;.</li>
	<li>If necessary, return to the original data file, adjust the background and SNR values, and repeat the deconvolution, until noise is sufficiently removed without eliminating genuine fluorescence from dim structures.</li>
	<li>Merge the brightfield and deconvolved fluorescence channels. For subsequent movie editing steps in ImageJ, arrange the channels such that red is first, green (if present) is next, blue (if present) is next, and brightfield is last. Save the image sequence as an 8-bit TIFF file.</li>
</ol>
4. Bleach Correction and Movie Generation
<ol>
	<li>Import the deconvolved image sequence into ImageJ and convert it into a hyperstack by choosing Image &#62; Hyperstacks &#62; Stack to Hyperstack. Select &#34;xyzct&#34; from the drop-down menu, and fill in the number of channels, Z-stack slices, and time frames (http://wiki.cmci.info/downloads/bleach_corrector).</li>
	<li>Choose Image &#62; Color &#62; Split Channels.</li>
	<li>Correct the fluorescence channels for photobleaching using the ImageJ plugin available from http://wiki.cmci.info/downloads/bleach_corrector. Once that plugin has been downloaded and installed, choose Plugins &#62; EMBLtools &#62; Bleach Correction. Select &#34;Exponential Fit.&#34;</li>
	<li>To merge the brightfield and bleach-corrected fluorescence channels into a hyperstack, choose Image &#62; Color &#62; Merge Channels. Save the resulting hyperstack as an 8-bit TIFF file.</li>
	<li><a href="https://www.jove.com/files/ftp_upload/58618/ImageJ.zip" target="_blank">Install the custom ImageJ plugins provided with this protocol.</a> Follow the instructions in the accompanying document to view, edit, and quantify the image sequence, and to produce a final movie of the projected Z-stacks.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+Golgi+apparatus:+insights+and+mysteries.">The yeast Golgi apparatus: insights and mysteries.</a> FEBS Letters. 583 (23), 3746-3751 (2009).</li><li>Matsuura-Tokita, K., Takeuchi, M., Ichihara, A., Mikuriya, K., Nakano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+yeast+Golgi+cisternal+maturation.">Live imaging of yeast Golgi cisternal maturation.</a> Nature. 441 (22), 1007-1010 (2006).</li><li>Day, K. J., Casler, J. C., Glick, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Budding+yeast+has+a+minimal+endomembrane+system.">Budding yeast has a minimal endomembrane system.</a> Developmental Cell. 44 (1), 56-72 (2018).</li><li>Papanikou, E., Day, K. J., Austin, J., Glick, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+selectively+drives+maturation+of+the+early+Golgi.">COPI selectively drives maturation of the early Golgi.</a> eLife. 4, 13232 (2015).</li><li>Rothstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting,+disruption,+replacement,+and+allele+rescue:+integrative+DNA+transformation+in+yeast.">Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast.</a> Methods in Enzymology. 194, 281-301 (1991).</li><li>Carlton, P. 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The authors declare that they have no competing financial interests.

4D confocal imaging of yeast organelles requires careful tuning of multiple parameters. The major concern is photobleaching and phototoxicity. A typical 4D movie involves collecting thousands of optical sections, so the laser illumination must be kept as low as possible. Tandem fluorescent protein tags can be used to boost the signal without increasing expression of the tagged protein16,17. Maximizing the scan speed helps to limit photodamage, and also allows Z-s...

Compartments of the endomembrane system are in constant flux, and their full characterization requires live cell imaging. Described here is a protocol that employs 4D (time-lapse 3D) confocal microscopy to visualize fluorescently labeled compartments in budding yeasts. The method was developed to track the dynamics of secretory compartments in Pichia pastoris and Saccharomyces cerevisiae1,2,3. This protocol focuses on S. cerevisiae, which has a nonstacked Golgi in which the individual cisternae are optically resolvable4. The unusual Golgi organization in S. cerevisiae enabled the demonstration by 4D microscopy that a Golgi cisterna initially labels with resident early Golgi proteins, and then loses those proteins while acquiring resident late Golgi proteins3,5. This transition can be visualized by creating a strain in which the early Golgi protein Vrg4 is labeled with GFP while the late Golgi protein Sec7 is labeled with a monomeric red fluorescent protein. When individual cisternae are tracked, maturation is observed as a green-to-red conversion3. This type of analysis can provide valuable information about protein localization and compartment identity. For example, two proteins with slightly offset arrival and departure times might sometimes appear to label different compartments in static images, but can be seen in 4D movies to label the same compartment at different time points6,7. Thus, 4D microscopy reveals phenomena that would not otherwise be evident.
Informative 4D microscopy of yeast compartments can be achieved with appropriate procedures and equipment2. Whenever possible, fluorescent protein tagging is performed by gene replacement8 to avoid overexpression artifacts. Because intracellular structures are often very dynamic, 4D imaging is needed to ensure that a structure is tracked reliably over time. The protocol described here employs a laser scanning confocal microscope equipped with high sensitivity detectors. With this device, the entire cell volume of S. cerevisiae can be imaged by confocal microscopy approximately every 1-3 s, with 2 s intervals being typical. Data can be collected for up to 5-15 min depending on the labeling densities of the fluorophores and their photophysical properties. The main hurdle is to minimize photobleaching. For this purpose, the laser intensities are kept as low as possible, the confocal scan speed is maximized, and the optical parameters are configured to image at the Nyquist limit in order to capture the relevant information while avoiding excessive light exposure. These settings are also expected to alleviate phototoxicity, a factor that is often overlooked during live cell imaging9,10,11. The resulting noisy data are processed with bleach correction and deconvolution algorithms to facilitate quantification of fluorescence intensities.
Even with high quality 4D movies, the analysis is tricky because yeast compartments tend to be numerous, heterogeneous, and mobile. Due to the intrinsic limitations of confocal microscopy and the non-optimal settings required for prolonged 4D imaging, fluorescent structures that are near each other are hard to resolve. This problem can be circumvented by focusing on the small number of fluorescent structures that remain optically resolvable for the duration of the labeling period, with the assumption that those structures are representative of the whole population of labeled compartments. Fluorescent compartments that can be reliably tracked are identified by viewing movies of projected Z-stacks and by creating a series of montages in which the optical sections for each time point are arrayed on a computer screen. This analysis employs custom ImageJ12 plugins, which allow an individual structure to be tracked in isolation.
Recent methods papers covered the use of fluorescent proteins in yeast13 as well as the theory and practice of 4D confocal imaging of yeast cells2. This protocol focuses on the key practical aspects of a 4D imaging experiment. It includes some enhancements to previously described procedures, as well as updated versions of the ImageJ plugin code and documentation. The example shown focuses on Golgi dynamics, but this protocol is equally suitable for imaging other yeast compartments.

<table><tbody><tr><td>35 mm glass bottom dishes No. 1.5</td><td>MatTek</td><td>P35G-0.170-14-C</td><td>Imaging dishes</td></tr><tr><td>Concanavalin A powder</td><td>Sigma-Aldrich</td><td>C2010</td><td></td></tr><tr><td>Trolox</td><td>Vector Laboratories</td><td>CB-1000</td><td></td></tr><tr><td>Leica SP8 confocal microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica Application Suite X&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td>Microscope software</td></tr><tr><td>Huygens Essential software, version 17.04</td><td>Scientific Volume Imaging</td><td></td><td>Deconvolution software</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>Image processing and analysis software</td></tr></tbody></table>

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.

The example given here documents and quantifies the maturation of two yeast Golgi cisternae as visualized by dual-color 4D confocal microscopy3. A yeast cell contains on the order of 10-15 Golgi cisternae, each of which matures over a time course of approximately 2-4 min. Maturation can be visualized by tagging the early Golgi marker Vrg4 with GFP and by tagging the late Golgi marker Sec7 with a red fluorescent protein such as mCherry or mScarlet. An individual cis...

Watch this Scientific Journal Video about 4D Microscopy of Yeast at JoVE.com

4D Microscopy of Yeast

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 ...

Research

JoVE Journal

Biology

8.6K Views.  University of Chicago. Deze methode volgt structuren in gistcellen in drie dimensies gedurende vele minuten, waardoor we de dynamiek van intracellulaire organellen en compartimenten kunnen bestuderen. 4D-beeldvorming zorgt ervoor dat we betrouwbare conclusies kunnen trekken over intracellulaire dynamiek, met name voor structuren of markers die van voorbijgaande aard kunnen zijn. Het aantonen van de procedure zal Natalie Johnson zijn, een postdoc uit mijn laboratorium.Begin met het kweken van een nachtelijke ...

Video: 4D Microscopy of Yeast

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Biochemistry

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

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.

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 ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

4D Microscopy of Yeast

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