The overall goal of this imaging technique is to observe heat stress-induced cellular perturbations in Dictyostelium discoideum cells. This method can help to understand the mechanisms of heat stress response and protein quality control in the amoeba Dictyostelium discoideum. The main advantage of this technique is the tight and precise control of the temperature applied to the cells'file imaging.
To begin the procedure, prepare Dictyostelium cells as detailed in the text protocol. The day before imaging, split the cells into low fluorescence medium to 10, 000 cells per milliliter. Before imaging, harvest the cells from the tissue plate in LFM, then transfer an adequate number of cells into glass bottom dishes.
Allow the cells to settle for 20 minutes then carefully replace the used medium with fresh medium, to remove non-settled cells. Image cells under non-stressed conditions at 23 degrees Celsius, acquire a minimum of six fields of view. Acquire Z-stacks of a maximum of seven micrometers in one to 0.25 micrometer steps to capture the whole volume of the cell.
Then acquire a reference bright field image to assess the total number of cells. Next, adjust the temperature of the cooling chamber to 30 degrees Celsius. After the temperature display has reached the desired value, refocus manually in the bright field channel.
Check all recorded FOVs before starting the time-lapse, then start a time-lapse for four hours of heat stress with a time interval of five to 10 minutes between time points. Check to ensure the focusing is correct during the acquisition of the first two time points. After the heat stress is complete, reduce the temperature back to 23 degrees Celsius.
Wait for the temperature display to adjust and refocus manually. Then record time-lapse movies in the recovery phase for eight to 10 hours in 10 to 20 minute time intervals. Check to ensure the focusing is correct during the acquisition of the first time point.
Begin by opening the image processing package equipped with the Cell Counter plugin. Load the bright field image stack by clicking File, then Open. Next, open the Cell Counter plugin, load the image stack by clicking Initialize.
Choose the type of counters by clicking the appropriate box. After this, count the total number of cells, save the markers by clicking Save Markers. Once the markers are saved, load the maximum projection of the fluorescence image hyperstack.
Open the Cell Counter plugin and load the markers by clicking Load Markers. Next, choose the marker type for cytosolic foci and count the cells. The existing markers can be used as guidance for the position of individual cells.
Retrieve the number of counts per slice by clicking on Results, then save the counts elsewhere for further calculations. In this study, the distribution GFP-tagged polyglutamine protein Q103 was observed in Dictyostelium cells. Under normal growth conditions, Q103-GFP is diffusely distributed in the cytoplasm.
During heat stress, the Q103-GFP marker coalesces to punctate structures. Upon stress release and growth at normal growth conditions, these structures dissolve. The redistribution of Q103-GFP and formation of heat stress-induced cytosolic foci is quantified using image analysis.
The representative results of the heat stress response can be seen here. The number of cells with cytosolic Q103-GFP foci increases in number with continuing heat stress. During heat stress, the protein quality control system is overwhelmed, thus aggregation-prone proteins cannot be maintained in a soluble state.
After heat stress, the number of cells with cytosolic foci decreases, suggesting that the aggregated proteins dissolve. After watching this video, you should have a good understanding how to monitor heat stress-induced perturbations in the amoeba Dictyostelium discoideum. Throughout imaging, it is important to remember to ensure that the focus is properly adjusted, if your microscope does not posses a hardware autofocus.
The temperature control showed in this protocol can be used beyond the field of heat stress response. Imaging under normal growth conditions using temperature control can notably reduce phototoxic effects.
