The authors have no Acknowledgements.

1. Cell Preparation
<ol>
	<li>Growing cells
	<ol>
		<li>Grow D. discoideum cells in AX medium at 23 &#176;C under light in tissue culture plates. Avoid keeping cells at high densities above 75% confluency. 
		NOTE: For an optimal response to heat stress, cells need to be in the exponential growth phase and should not have reached stationary phase.</li>
		<li>The day before imaging, split cells into low fluorescence medium (LFM) to 1 x 104 cells/ml. 
		NOTE: This step is necessary to reduce background fluorescence caused by the AX medium. The incubation time can be reduced to a minimum of 2 hr. However, vesicles taken up in AX medium can still generate some background signal after 2 hr.</li>
	</ol>
	</li>
	<li>Preparing cells for microscopy
	<ol>
		<li>Before imaging, harvest cells from the tissue plate in LFM and transfer an adequate number of cells into glass bottom dishes. 
		NOTE: Usually, 1 x 105 cells/ml is sufficient; however, if the experimental setup requires long imaging periods (longer than 8 hr), cell numbers need to be carefully adjusted, as D. discoideum cells will transition to the developmental cycle and start to stream and aggregate if cell numbers are high while nutrients are low.</li>
		<li>Allow the cells to settle for 20 min. Remove non-settled cells by carefully replacing the used medium with fresh medium.</li>
	</ol>
	</li>
</ol>
2. Imaging
NOTE: The protocol can be used on wide-field systems as well as in confocal microscopy setups. The usage of a temperature-controlled incubation box covering the objectives and the microscope stage is helpful, yet not essential for temperature control. The temperature of the incubation box is set to the highest temperature used in the experiment, e.g., to 40 &#176;C if heat stress is performed. Otherwise, the temperature is set to 25 &#176;C. Temperature changes should be made in advance to allow the system to adapt, as changes in temperature during imaging can affect the focus. 
NOTE: During imaging, the temperature is controlled using a cooling chamber. The default temperature for imaging non-stressed cells is 23 &#176;C. For heat stress conditions, the temperature is increased accordingly to the desired level. The piezo element in the cooling chamber can respond very quickly to the temperature shift. However, the focus of the images will change slightly (it can deviate to up to 20 &#181;m depending on the temperature shift). Thus manual refocusing is required within the first minutes of image acquisition, unless the used microscope is equipped with a hardware auto-focus.
<ol>
	<li>Image acquisition under non-stressed conditions
	<ol>
		<li>Image cells under non-stressed conditions at 23 &#176;C, acquire a minimum of 6 fields of view (FOVs). 
		NOTE: Non-stress condition can be visualized either by recording a short time-lapse (5-30 min) or acquisition of representative stills. For each condition or cell line acquire a minimum of 6 fields of view (FOVs). 
		NOTE: In case individual cells are of interest, position the respective cells in the middle of the FOV and record a 3 x 3 tile series with the FOV containing the cells of interest in the middle. D. discoideum cells are motile and might migrate out of the FOV. However, the 3 x 3 tile series increases the area potentially explored by the cell of interest and it remains traceable for time-lapse periods of ~6 hr.</li>
		<li>Acquire z-stacks of maximum 7 &#181;m in 1-0.25 &#181;m steps to capture the whole volume of the cell.</li>
		<li>Acquire a reference bright field image to assess the total number of cells.</li>
	</ol>
	</li>
	<li>Image acquisition for heat stress
	<ol>
		<li>Adjust the temperature of the cooling chamber to 30 &#176;C according to the manufacturer&#39;s instructions.</li>
		<li>After the temperature display has reached the desired value, refocus manually in the bright field channel and check all recorded FOVs before starting the time-lapse.</li>
		<li>Start a time-lapse for 4 hr of heat stress with a time interval of 5-10 min between time points. Check for correct focusing during the acquisition of the first two time points.</li>
	</ol>
	</li>
	<li>Image acquisition during recovery
	<ol>
		<li>After heat stress, reduce the temperature back to 23 &#176;C, wait for the temperature display to adjust and refocus manually.</li>
		<li>Record time-lapse movies in the recovery phase for 8-10 hr in 10-20 min time intervals. Check for correct focusing during the acquisition of the first time point.</li>
	</ol>
	</li>
</ol>
3. Image Analysis
NOTE: To quantify the number of cells harboring cytosolic foci, assess the total number of cells and the number of cells with cytosolic foci in each time frame. Due to the difficulty of segmenting cells from bright field images, the quantification needs to be done manually. In the following, data analysis is described for the free image processing package ((http://fiji.sc/Fiji). It requires the plugin &#8220;Cell Counter&#8221; (http://fiji.sc/Cell_Counter).
<ol>
	<li>Assessing total number of cells
	<ol>
		<li>Load the bright field image stack (XYT) by clicking &#34;File&#34; and &#34;Open&#34;.</li>
		<li>Open the &#34;Cell Counter&#34; plugin (&#34;Plugins&#34; -&#34;Cell Counter&#34;). Load the image stack by clicking &#34;Initialize&#34;.</li>
		<li>Choose the appropriate type of counters by ticking the appropriate box. 
		NOTE: use type 1 as counter for total number of cells and type 2 as counter for cells with cytosolic foci. Count all cells.</li>
		<li>Save the markers by clicking &#34;Save Markers&#34;.</li>
	</ol>
	</li>
	<li>Assessing the number of cells with cytosolic foci
	<ol>
		<li>Load the fluorescence image hyperstack (XZYT) (&#34;File&#34; - &#34;Open&#34;) and make a maximum intensity projection using the &#34;Z-Projector&#34; (Image/Stacks).</li>
		<li>Open the &#34;Cell Counter&#34; plugin. Load the resulting image stack (XYT) by clicking &#34;Initialize&#34;.</li>
		<li>Load the markers by clicking &#34;Load Markers&#34;. 
		CAUTION: The file names of the reference image and the fluorescence stack must be identical. If necessary, rename accordingly before initializing the image stack.</li>
		<li>Choose the appropriate marker type (see 3.1.3) and count cells with cytosolic foci. The existing markers can be used as guidance for the position of individual cells.</li>
		<li>Retrieve the number of counts per slice by clicking on &#34;Results&#34; and save the counts elsewhere for further calculations.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Bonner, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+Formation+of+Cell+Aggregates+by+Chemotaxis+in+the+Development+of+the+Slime+Mold+Dictyostelium+Discoideum.">Evidence for the Formation of Cell Aggregates by Chemotaxis in the Development of the Slime Mold Dictyostelium Discoideum.</a> Journal of Experimental Zoology. 106 (1), 1-26 (1947).</li><li>Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+during+development+of+Dictyostelium.">Cell signaling during development of Dictyostelium.</a> Developmental Biology. 391 (1), 1-16 (2014).</li><li>Nichols, J. M. E., Veltman, D., Kay, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+of+a+model+organism:+progress+with+Dictyostelium.">Chemotaxis of a model organism: progress with Dictyostelium.</a> Current Opinion in Cell Biology. 36, 7-12 (2015).</li><li>Williams, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+finds+new+roles+to+model.">Dictyostelium finds new roles to model.</a> Genetics. 185 (3), 717-726 (2010).</li><li>M&#252;ller-Taubenberger, A., Kortholt, A., Eichinger, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+system+-+substantial+share:+The+use+of+Dictyostelium+in+cell+biology+and+molecular+medicine.">Simple system - substantial share: The use of Dictyostelium in cell biology and molecular medicine.</a> European Journal of Cell Biology. 92 (2), 45-53 (2013).</li><li>Malinovska, L., Palm, S., Gibson, K., Verbavatz, J. M., Alberti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+discoideum+has+a+highly+Q/N-rich+proteome+and+shows+an+unusual+resilience+to+protein+aggregation.">Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation.</a> Proc Natl Acad Sci U S A. 112 (20), E2620-E2629 (2015).</li><li>Malinovska, L., Alberti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding+in+Dictyostelium:+Using+a+freak+of+nature+to+gain+insight+into+a+universal+problem.">Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem.</a> Prion. 9 (5), 339-346 (2015).</li><li>Schneider, K., Bertolotti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+protein+quality+control+catastrophes+-+from+cells+to+organisms.">Surviving protein quality control catastrophes - from cells to organisms.</a> Journal of Cell Science. 128 (21), 3861-3869 (2015).</li><li>Dobson, C. 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+and+misfolding.">Protein folding and misfolding.</a> Nature. 426 (6968), 884-890 (2003).</li><li>Fukui, Y., Yumura, S., Yumura, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar-overlay+immunofluorescence:+high-resolution+studies+of+cytoskeletal+components+and+their+changes+during+chemotaxis.">Agar-overlay immunofluorescence: high-resolution studies of cytoskeletal components and their changes during chemotaxis.</a> Methods in cell biology. 28, 347-356 (1987).</li><li>Graf, R., Rietdorf, J., Zimmermann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+spinning+disk+microscopy.">Live cell spinning disk microscopy.</a> Microscopy Techniques. 95, 57-75 (2005).</li><li>Samereier, M., Meyer, I., Koonce, M. P., Graef, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Cell-Imaging+Techniques+for+Analyses+of+Microtubules+in+Dictyostelium.">Live Cell-Imaging Techniques for Analyses of Microtubules in Dictyostelium.</a> Methods in cell biology. 97, 341-357 (2010).</li><li>Herman, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stationary+phase+in+yeast.">Stationary phase in yeast.</a> Current opinion in microbiology. 5 (6), 602-607 (2002).</li><li>Loomis, W. F., Wheeler, S., Wheeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+response+of+Dictyostelium.">Heat shock response of Dictyostelium.</a> Developmental Biology. 79 (2), 399-408 (1980).</li></ol>

The authors have nothing to disclose.

The protocol described here can be used to study the behavior of a particular protein of interest in response to heat-induced stress. Temperature increase to 30 &#176;C has been reported to trigger heat stress response and under these conditions the viability of D. discoideum is markedly reduced.
Modifications 
The protocol can be modified to compare the behavior of different proteins under the same stress conditions. For this, cells expressing different proteins with the same fluorescent tag are transferred into multi-well dishes, such as four chamber dishes (section 1.3.1). The protocol can also be applied on cells co-expressing proteins with different markers, such as GFP or RFP. This can be for example used to monitor the behavior of different components of the protein quality control (PQC) system. Cells expressing GFP-tagged aggregation marker and different RFP-tagged PCQ components can be observed using multi-well dished for imaging. This ensures the same stress conditions (speed of temperature increase/decrease, duration of the temperature increase/decrease) and allows for comparative studies.
Moreover, the protocol can be used to study the influence of the PQC system on the heat stress response. The activity of the components can be modulated by changing expression levels using genetic tools such as knock-out or overexpression or by using commercially available specific inhibitors6. The proteasome can be inhibited by adding MG132 (100 &#181;M) or lactacystin (10 &#181;M) to the growth medium. The chaperone Hsp90 can be inhibited using geldanamycin (6 &#181;M) or radicicol (10 &#181;M). The chaperone Hsp70 can be inhibited with VER-155088, although we could not confirm the efficacy of the inhibition in our experimental settings so far. Inhibitors should be added one day before imaging and the cells are incubated for 14 hr.
Critical Steps within the Protocol 
The critical step for the assessment of heat-induced perturbation is the state of the cells prior to the imaging experiment. Studies in yeast showed that cells acquire resistance to a variety of environmental stresses during stationary phase13. We also observed minimal responsiveness to applied heat stress if D. discoideum cells had reached stationary phase prior to imaging. Therefore, maintaining a constant cell number &#60;5 x 105 cells/ml is critical.
Moreover, high cell numbers can induce transition from the vegetative cycle of D. discoideum to the developmental cycle, thus triggering starvation pathways, which might lead to a different response to heat stress. If high cell numbers are reached in the recovery phase after heat stress, streaming and aggregating cells can interfere with data analysis as the cells move out of focus (see Figure 4).
Limitation of the Technique 
The loss of focus is the bottleneck of the protocol. During temperature changes, the focal plane can shift drastically, which requires manual refocusing in a time period immediately after changing the temperature. The jump in the focus can sometimes be too extreme to be overcome by software autofocus, especially if the time between the time points is high. However, if the used microscope is equipped with a hardware autofocus, this bottleneck can be circumvented.
Significance of the Technique with Respect to Existing Methods 
In comparison with existing setups such as the use of air-conditioned rooms, temperature-controlled incubation boxes covering the objectives and the microscope stage or temperature-controlled stages and objective collars, the use of a cooling chamber provides a more precise control of the ambient temperature. The Peltier-element in the cooling chamber can maintain a constant and uniform temperature throughout the experimental setup. In classical setups, local temperature differences might lead to different observation outcomes. Moreover, it can respond quickly and rapidly to induced changes in the temperature, while classical setups adapt very slowly to induced temperature changes, especially lowering the temperature. The Peltier-element in the cooling chamber can also cover a wider temperature range (15-40 &#176;C) than classical setups, with which reaching temperatures such as 15 &#176;C or 40 &#176;C is very difficult.
Dictyostelium discoideum is particularly sensitive to photo-toxicity. Previous studies used ascorbate as scavenger to reduce photo-toxicity. However, long imaging periods require additional supplementation. Moreover, the use of ascorbate is limited to studies where the mechanism of interest is not affected by the antioxidant supplement. We propose that temperature-controlled imaging can be used as an alternative approach to minimize photo-toxicity and could be combined with addition of ascorbate to further decrease toxicity.
Phototoxic effects can be minimized by providing a constant and uniform temperature of 23 &#176;C using a cooling chamber. Cells imaged using temperature control show fewer signs of photo-toxicity, such cell rounding, for a longer time period. This also allows imaging with higher intensities, smaller time intervals or more fields of view (FOVs).
General Application 
Heat stress at higher temperatures has been shown to trigger a different heat stress response14. Therefore, increasing the temperature to 34 &#176;C or 37 &#176;C might induce a different response. In addition to the applied temperature stress, the duration of a particular stress situation can be modified to study the immediate response or long-term adaptation to heat stress.
In general, the described protocol can be expanded to a wide set of applications. Because the precise temperature control considerably reduces photo-toxic effects, the protocol can be used in settings which require high exposure times and/or higher excitation light intensities, e.g., for visualizing proteins with a low expression level, or in combination with short time intervals between imaging, e.g., during time-lapse imaging. It can also be advantageous for imaging objects spanning the whole cell, e.g., microtubules, as these settings require a high number of optical z-sections.

Dictyostelium discoideum are solitary soil living amoebae that feed on bacteria and other microorganisms, which are taken up by phagocytosis. It has a unique and remarkable life cycle that has been a major area of research since its discovery1. The early interest in multicellular development2 and the molecular basis of chemotaxis3 was soon complemented by studies focusing on cell motility, cell polarity, innate immunity. In addition, D. discoideum was introduced as a model system for biomedical research4,5.
Recently, we established D. discoideum as a new system to study the protein quality control (PQC) system6,7. Its proteome is enriched in aggregation-prone prion-like proteins, which poses a challenge to protein quality control8. To investigate whether D. discoideum has developed special molecular mechanisms to control its highly aggregation-prone proteome, we studied the behavior of aggregation-prone marker proteins both under normal growth conditions and during stress. Stress conditions, such as heat stress, can be used to increase the rate of protein misfolding9. Therefore, we sought a system where we could induce temperature changes and simultaneously follow the behavior of marker proteins. For this purpose, we combined live-cell imaging with Peltier-controlled heating using a thermal stage insert (cooling chamber). This method allowed us to maintain a constant and uniform temperature as well as to induce a rapid, yet precise temperature change.
Live-cell imaging is used to study a variety of biological processes in D. discoideum. However, this approach faces two major limitations. First, the cells display a high motility and tend to migrate out of the field of view, thus tracking of individual cells often requires imaging of a large area. The cell mobility can be reduced by Agar overlay10, however, these conditions are not suitable for long term imaging due to a decline in viability. Second, D. discoideum cells show a particularly high sensitivity to photo-toxicity, which results in cell rounding and mitotic arrest11. Previous protocols addressed this issue by addition of ascorbate as a radical scavenger and reduction of exposure times12. The latter can be critical if the protein of interest is expressed at low levels and shows a weak fluorescence signal. The authors also suggest to provide a constant temperature of 21 &#176;C either by imaging in an air-conditioned room or by using temperature-controlled incubation boxes, which cover the objective and microscope stage12.
Here, we describe a method with improved temperature control by using a cooling chamber set at 23 &#176;C. During imaging our set-up significantly enhances the resistance to photo-toxicity. It allows the usage of higher exposure times and higher excitation light intensities. This is of particular importance during time-lapse imaging, as the time intervals need to be carefully balanced against the number of positions imaged and the exposure time used. The possibility to increase the numbers of imaged positions also allows the coverage of a wider imaging area and facilitates tracking of individual cells over a longer period of time.

<table><tbody><tr><td>AX Medium</td><td>ForMedium</td><td>AXM0102</td><td></td></tr><tr><td>LoFlo medium</td><td>ForMedium</td><td>LF1001</td><td></td></tr><tr><td>MG132</td><td>Sigma</td><td>C2211</td><td></td></tr><tr><td>Lactacystin</td><td>Sigma</td><td>L6785</td><td></td></tr><tr><td>Geldanamycin</td><td>Santa Cruz</td><td>sc-200617</td><td></td></tr><tr><td>Radicicol</td><td>Santa Cruz</td><td>sc-200620</td><td></td></tr><tr><td>MatTek disch 35 mm</td><td>MatTek corporation</td><td>P35G-1.5-14-C</td><td>glass bottom imaging dish</td></tr><tr><td>CellViell cell culture dish</td><td>Greiner</td><td>627870</td><td>4-compartment glass-bottom imaging dish</td></tr><tr><td>Thermal Stage Heater/Cooler Insert</td><td>Warner Istruments</td><td>TB-3/CCD</td><td></td></tr><tr><td>Bipolar temperature controller</td><td>Warner Istruments</td><td>CL-100</td><td></td></tr><tr><td>Liquid cooling System</td><td>Warner Instruments</td><td>LCS-1</td><td></td></tr></tbody></table>

studying the protein quality control system of d. discoideum using temperature-controlled live cell imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

The described imaging protocol can be used to analyze the behavior of aggregation-prone prion-like proteins in the amoeba D. discoideum during heat stress. Figure 1 shows the distribution of GFP-tagged polyglutamine protein Q103 in cells under normal growth conditions (Figure 1A), after 2 hours of heat stress at 30 &#176;C (Figure 1B) and after 6 hours of stress recovery and growth at normal growth conditions (Figure 1C). Upon temperature increase from 23 &#176;C to 30 &#176;C, the Q103-GFP marker coalesces from a diffuse distribution to punctate structures. Upon stress release and growth at normal temperature, these structures dissolve. The redistribution of Q103-GFP and formation of heat stress-induced cytosolic foci can be quantified using image analysis. Figure 2A shows the analysis of individual FOVs using Fiji and the plugin &#34;Cell counter&#34;. Figure 2B shows the quantification of the heat stress response. This shows that the cytosolic Q103-GFP foci increase in number with continuing heat stress. During heat stress, the protein quality control (PQC) system is overwhelmed, thus aggregation-prone proteins such as the prion-like protein Q103 cannot be maintained in a soluble state and increasingly aggregate in cytosolic foci. The cells also show a characteristic rounding, as depicted in Figure 1B. The reduction of cytosolic foci after stress removal suggests that the aggregated proteins are dissolved.
The imaging quality is sensitive to the initial cell density. Figure 3 shows an example of cells after stress recovery, comparing appropriate initial cell density (Figure 3A) and high initial cell density (Figure 3B). If the initial cell density was too high, cells started to form streams as depicted in Figure 3b. This which imposes difficulties on the subsequent quantification as the cells have moved out of the focal plane.
The described method can be used to monitor temperature-induced changes as described above. It can also be used to reduce photo-toxicity when imaging at physiological growth temperature. Figure 4 shows a comparison of cell imaged at 23 &#176;C in a cooling chamber (Figure 4A) and cells imaged without temperature control in an air conditioned room, set to 25 &#176;C (Figure 4B). Upon constant exposure to the excitation light, cells round up when not imaged under temperature-controlled conditions. This change in cell shape is indicating stress.
<img alt="Figure 1" src="/files/ftp_upload/54730/54730fig1.jpg" /> 
Figure 1: Aggregation-prone prion-like proteins change their cellular distribution during heat stress. GFP-tagged polyglutamine-rich huntingtin exon 1 (Q103) is constitutively expressed in AX2-214 D. discoideum cells (GFP signal depicted in inverted gray scale lookup table). (A) Under normal growth conditions at 23 &#176;C, Q103-GFP is diffusely distributed in the cytoplasm. (B) After 2 hr of heat stress at 30 &#176;C, Q103-GFP coalesces into cytosolic foci (red arrows). The cells show a characteristic shape change and round up. (C) Upon stress release and growth at 23 &#176;C, the cytosolic foci are dissolved. After 6 hours, no cytosolic foci can be observed. Scale bar = 10 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54730/54730fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54730/54730fig2.jpg" /> 
Figure 2: Quantification of heat-induced cytosolic foci formation. (A) Analysis of cells using the free image-processing package Fiji and the plugin &#34;Cell Counter&#34;. The total amount of cells (type 1, blue counter markers) is determined in the bright field image. The markers are loaded in the GFP-channel and the number of cells with cytosolic foci (type 2, red counter markers) is determined. (B) Quantification of foci after 3 hr of heat stress at 30 &#176;C and 3 hours of stress recovery at 23 &#176;C (n = 4 fields of view, error bar = SD). <a href="http://cloudflare.jove.com/files/ftp_upload/54730/54730fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54730/54730fig3.jpg" /> 
Figure 3: Effect of cell density on long-term time-lapse imaging. (A) Cells imaged at initial cell densities &#60;105 cells/ml. After 3 hr of heat stress at 30 &#176;C and 6 hr of stress recovery at 23 &#176;C, cells still remain in the vegetative cycle and divide. Only few cells exit the focal plane. (B) Cells imaged at initial cell densities &#62;106 cells/ml. After 9 hr of imaging (settings as in A), cells have entered the developmental cycle and started streaming and aggregating. Cells in the aggregates cannot be clearly distinguished. A large portion of cells exits the focal plane and cannot be analyzed. Scale bar = 10 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54730/54730fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54730/54730fig4.jpg" /> 
Figure 4: Effect of temperature control on phototoxic effects. (A) Cells imaged for 10 min with temperature control at 23 &#176;C: 6 fields of view, z-stack (spacing 0.5 &#181;M, sample thickness 8 &#181;M), time lapse (total time 10 min, lapse 1 min), GFP channel (0.07 msec exposure time, 10% laser intensity), RFP channel (0.1 msec exposure time, 10% laser intensity). Cells show no signs of the characteristic morphological changes associated with phototoxic stress. (B) Cells imaged for 10 min without temperature control (same conditions as in A). Cells exhibit signs of phototoxic induced rounding, indicating stress. Scale bar = 10 &#181;m. The cells were imaged on a system based on a microscope with a 100x/1.4 numerical aperture (N.A.) objective. The images were collected with a CCD camera as 1,024 x 1,024 pixel files using 1 x 1 binning. <a href="http://cloudflare.jove.com/files/ftp_upload/54730/54730fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

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6.6K Views.  Max-Planck Institute for Molecular Cell Biology and Genetics. The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Video: Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Genetics

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Immunology and Infection

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 ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, 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 ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

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 ...

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

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

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

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

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

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

Summary

Explore More Videos

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

4D Imaging of Protein Aggregation in Live Cells

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Microscopy of Fission Yeast Sexual Lifecycle

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

4D Microscopy of Yeast

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