Vertical Immobilization Method for Time-Lapse Microscopy Analysis in Filamentous Cyanobacteria

In bacteria, the study of the dynamics of cell division proteins requires a spatial orientation of the cells. For example, in the case of the Z-ring, the main component in bacterial cell division, a vertical orientation is fundamental to record the whole structure in the XY plane. In this video, we develop a method to achieve this particular orientation in the filamentous cyanobacteria Anabaena.

Anabaena is a multicellular photosynthetic organism, and share with other bacteria the ability to use atmospheric dinitrogen as nitrogen source. In the absence of this element, Anabaena differentiate cells specialized in nitrogen fixation. Therefore, it provides an excellent model in which to analyze the relationship between cellular division and multicellularity.

First, select a divisome component present in the target filamentous cyanobacterial strain. For the experiment, it is necessary to construct a filamentous cyanobacterial mutant that expresses the selected divisome component fused to a fluorescent protein. In this protocol, we use an Anabaena mutant that expresses FtsZ fused to GFP as an example.

First, make a fresh subculture of the mutant with 10 milliliters of a culture in stationary phase, and 90 milliliters of liquid medium. Then grow the new cell culture in constant light and at the optimum temperature, until reaching the exponential phase. In our example, the mutant was grown at 25 Celsius degrees for seven days.

Now take two milliliters of the growth culture and put it in a centrifuge tube. Centrifuge at 2, 500 x g for 10 minutes at room temperature. After the centrifugation, check that all the cells are on the bottom of the tube.

Be careful while handling the samples, to avoid re-suspension. Heat the 3%low-melting-point agarose solution in a microwave. It is important to do this in short time intervals, and check the solution until completely liquid.

Once it is melted, set aside to cool down. Take the centrifuge tubes of the previous step, discard 1.9 milliliters of the supernatant, and re-suspend the cells in the remaining volume by pipetting at least three times up and down. In your work place, put the tube with the cells, the melted agarose solution, a one-milliliter syringe cutted in the tip, and a scalpel with a new and clean blade.

Then mix the cells with 900 microliters of the 3%agarose solution by pipetting at least three times. It is really important to make sure that the agarose solution is not too hot, but remains liquid. With this, we are avoiding cell damage during this process.

The next step must be done fast, and before the agarose solution gelifies. Suck up the agarose matrix in a one-milliliter syringe, carefully. It is important to avoid the generation of bubbles while the sample is being sucked up.

After that, incubate the sample, putting the syringe in horizontal position at room temperature for two hours. After the incubation, carefully remove the matrix with the cells using the plunger in a clean and flat surface. Using the scalpel, cut the gel sample into slices as thin as possible, maintaining the integrity of the matrix.

Then deposit the samples in the corresponding coverslip, side by side. As shown in the video, in this step, you can use a tip to assist in the handling process of the sample. For time-lapse experiments, it is recommended the use of a cell chamber made for microscopy analysis.

In this case, we use circular coverslips that fit in these type of chambers, as you can see in the video. Finally, to prevent dehydration of the sample, add an extra volume of melted 3%agarose solution inside the chamber. Before imaging, wait until the agarose is completely gelified.

Now put the chamber with the cells in the microscope for imaging. It is recommendable to use an equipment with temperature and humidity control. Visualize the sample in bright field with maximum magnification, and search a vertically oriented filament.

You can find the division site of interest with an initial scanning using the autofluorescence signal along the Z plane. With this, use the parameters of your fluorescent protein marker to visualize the signal of your protein of interest in the division site. Now you can perform time-lapse experiments according to the biological model and the protein of interest.

In this case, we can see the whole structure of the Z-ring in the FtsZ-GFP mutant, in the XY plane. With our method, we were capable of recording the dynamics of these rings in a long period of time. Finally, with the changes in fluorescence intensity, we conclude that this structure is highly dynamic in our model.

This method is a rapid and inexpensive protocol to register the protein dynamics in the division site by means of confocal microscopy applied to complex morphologies as filamentous cyanobacteria, using Anabaena as a model.