Name: vertical immobilization method for time-lapse microscopy analysis in filamentous cyanobacteria
Uploaded: 2023-09-25T14:30:00
Description: Watch this Scientific Journal Video about Vertical Immobilization Method for Time-Lapse Microscopy Analysis in Filamentous Cyanobacteria at JoVE.com

We are grateful to National doctoral scholarships (ANID 21211333; 21191389) for the financing. Grant Fondecyt 1161232.
This work was supported by de Advanced Microscopy Facility UMA UC.

1. Considerations and selection of the cellular model
NOTE: Cyanobacteria have a strong autofluorescence due to the presence of photosynthetic pigments. This signal is in the red part of the spectrum, therefore, the fluorescent proteins suitable for imaging in cyanobacteria are the ones that far from the red emission. For example, GFP, YFP, Venus, Turquoise and BFP.
<ol>
	<li>Select a divisome component present in the target filamentous cyanobacterial strain. For the experim...

<ol>
	<li>L&#246;we, J., Amos, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+bacterial+cell-division+protein+FtsZ.">Crystal structure of the bacterial cell-division protein FtsZ.</a> Nature. 391 (6663), 203-206 (1998).</li><li>Erickson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FtsZ,+a+prokaryotic+homolog+of+tubulin.">FtsZ, a prokaryotic homolog of tubulin.</a> Cell. 80 (3), 367-370 (1995).</li><li>Huang, K., Mychack, A., Tchorzewski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+FtsZ+C-terminal+variable+(+CTV+)+region+in+Z-ring+assembly+and+interaction+with+the+Z-ring+stabilizer+ZapD+in+E.">Characterization of the FtsZ C-terminal variable ( CTV ) region in Z-ring assembly and interaction with the Z-ring stabilizer ZapD in E.</a> coli cytokinesis. , 1-24 (2016).</li><li>Perez, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+dynamics+of+divisome+proteins+and+PBP2x:FtsW+in+cells+of+Streptococcus+pneumoniae.">Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae.</a> Proceedings of the National Academy of Sciences. 116 (8), 3211-3220 (2019).</li><li>Bisson-Filho, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treadmilling+by+FtsZ+filaments+drives+peptidoglycan+synthesis+and+bacterial+cell+division.">Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division.</a> Science. 355 (6326), 739-743 (2017).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPase+activity-coupled+treadmilling+of+the+bacterial+tubulin+FtsZ+organizes+septal+cell+wall+synthesis.">GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis.</a> Science. 355 (6326), 744-747 (2017).</li><li>Whitley, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FtsZ+treadmilling+is+essential+for+Z-ring+condensation+and+septal+constriction+initiation+in+Bacillus+subtilis+cell+division.">FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division.</a> Nature Communications. 12 (1), (2021).</li><li>Maz&#243;n, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LexA-binding+sequences+in+Gram-positive+and+cyanobacteria+are+closely+related.">LexA-binding sequences in Gram-positive and cyanobacteria are closely related.</a> Molecular Genetics and Genomics. 271 (1), 40-49 (2004).</li><li>Mandakovic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CyDiv,+a+conserved+and+novel+filamentous+cyanobacterial+cell+division+protein+involved+in+septum+localization.">CyDiv, a conserved and novel filamentous cyanobacterial cell division protein involved in septum localization.</a> Frontiers in Microbiology. 7 (FEB), 1-11 (2016).</li><li>Camargo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZipN+is+an+essential+FtsZ+membrane+tether+and+contributes+to+the+septal+localization+of+SepJ+in+the+filamentous+cyanobacterium+Anabaena.">ZipN is an essential FtsZ membrane tether and contributes to the septal localization of SepJ in the filamentous cyanobacterium Anabaena.</a> Scientific Reports. 9 (1), 1-15 (2019).</li><li>Thiel, T., Peter Wolk, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugal+Transfer+of+Plasmids+to+Cyanobacteria.">Conjugal Transfer of Plasmids to Cyanobacteria.</a> Methods in Enzymology. 153 (C), 232-243 (1987).</li><li>Moffitt, J. R., Lee, J. B., Cluzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-cell+chemostat:+An+agarose-based,+microfluidic+device+for+high-throughput,+single-cell+studies+of+bacteria+and+bacterial+communities.">The single-cell chemostat: An agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities.</a> Lab on a Chip. 12 (8), 1487-1494 (2012).</li></ol>

The study of the dynamics of divisome proteins is without doubt a challenge. Particularly, in filamentous cyanobacteria, one the challenges is the visualization the Z-ring in the horizontal plane, for which the cells must be vertically oriented. The method we described here allows the positioning of the Z-ring in the XY plane to perform the different analyzes. This is the first simple method for filamentous cyanobacteria that allows the complete visualization of the Z-ring. 
Although this prot...

Bacterial cell division is the process in which a mother cell generates two daughter cells, in most cases by the mechanism known as binary fission. One of the earliest events in the septation process is the localization of FtsZ in the middle of the cell1. This protein, which is structurally homologous to tubulin2, is conserved and widely distributed in most bacteria, and its polymerization generates a contractile structure known as the Z-ring3. This ring serves as a scaffold for other division proteins and together they form a molecular machinery called a divisome. Several studies have shown that the Z-ring is highly dynamic and that FtsZ protofilaments move by treadmilling4,5,6. To study the Z-ring in time-lapse experiments, it is advisable to record the division site in the XY plane for better resolution and rapid sampling. In order to achieve this, it is necessary to develop vertical cell immobilization methods that commonly include the nanofabrication of microhole cell traps and complex microfluidic devices7.
Cyanobacteria are photosynthetic microorganisms that are classified as gram-negative by their cellular morphology. However, phylogenetically they are closer to gram-positive bacteria8. These organisms have cell division genes that are common within gram-positive and gram-negative bacteria, but their divisome also contains unique elements9. Anabaena sp. PCC 7120 (hereafter Anabaena sp.) is filamentous cyanobacteria with one division plane and is a model for the study of cell division in multicellular cyanobacteria. In this strain, it has been possible to determine the positioning of FtsZ in the middle of the cells10. Nevertheless, there are no studies showing the in vivo dynamics of FtsZ in this model. In our laboratory, through triparental mating and homologous recombination, we obtained a totally segregated mutant of Anabaena sp. that expresses the FtsZ protein fused to sfGFP, which replaced the complete endogenous ftsZ gene. We developed a rapid and simple cell immobilization method that favors the vertical orientation of the mutant strain filaments for time-lapse experiments to visualize the division proteins in filamentous cyanobacteria. This method does not need microfluidic devices that can be expensive and difficult to develop. As an example, we used this protocol to visualize the Z-ring in the FtsZ-sfGFP mutant by confocal microscopy.

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	<tbody>
		<tr>
			<td>1 ml syringe</td>
			<td>Qingdao Agna Medical Technology Co., Ltd.</td>
			<td>-</td>
			<td>The disposable medical plastic syringe with 1 ml needle generally used to pump liquid or injection liquid, in this experiment it is used to suck the sample and make it polymerize.</td>
		</tr>
		<tr>
			<td>Attofluor Cell Chamber</td>
			<td>ThermoFischer Scientific</td>
			<td>A7816</td>
			<td>The Attofluor Cell Camera is a durable and practical coverslip holder designed for viewing live cell samples in upright or inverted microscopes.</td>
		</tr>
		<tr>
			<td>Axygen MaxyGene II Thermal Cycler with 96 well block</td>
			<td>CORNING</td>
			<td>THERM-1001</td>
			<td>The new MaxyGene II Thermal Cycler increased speed and advanced features, providing the premium performance you have come to expect from Axygen brand products. Unique flexible programming. Rapid run times. Improved workflow over traditional gradient cyclers. Ramping rates up to 5&#176;C/sec. Adjustable heated lid accommodates strips, tubes and microplates.</td>
		</tr>
		<tr>
			<td>Fisherbrand Cover Glasses: Circles</td>
			<td>ThermoFischer Scientific</td>
			<td>12-546-2P</td>
			<td>Made of finest optical borosilicate glass, with uniform thickness and size. Circular shape. Corrosion-resistant.&#160;</td>
		</tr>
		<tr>
			<td>Low Melting Point agarose</td>
			<td>Promega</td>
			<td>V3841</td>
			<td>Agarose, Low Melting Point, Analytical Grade, is ideal for applications that require recovery of intact DNA fragments after gel electrophoresis.</td>
		</tr>
		<tr>
			<td>LSM 880 microscope from Zeiss Airyscan</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>The Zeiss Airyscan LSM 880 microscope is a laser scanning focal microscope that can acquire images under the resolution limit (lateral resolution ~ 120nm and axial resolution ~ 350nm). The detectors are highly sensitive making it possible to acquire super-resolution images at high speed.</td>
		</tr>
		<tr>
			<td>Microcentr&#237;fuga Fresco 17</td>
			<td>ThermoFischer Scientific</td>
			<td>75002402</td>
			<td>Speed up routine sample preparation processes up to 17,000 &#215; g with our standard microcentrifuge, available with refrigeration. These microcentrifuges offer productivity, versatility, safety and convenience in an easy-to-use, compact design laboratory instrument.</td>
		</tr>
		<tr>
			<td>Nikon Timelapse Microscope</td>
			<td>Nikon</td>
			<td>-</td>
			<td>The Nikon C2 laser scanning confocal microscope is an ideal microscope for long-term timelapse, because thanks to its incubation chamber it is possible to keep samples in optimal conditions for more than 8 hours.</td>
		</tr>
		<tr>
			<td>Thin razor blades Schick Super Chromium</td>
			<td>Farmazon</td>
			<td>SKU: 401146</td>
			<td>&#160;The thin razor blades is used to cut the agarose matrix, allowing us to obtain the disks with the sample while maintaining the integrity of the matrix.</td>
		</tr>
	</tbody>
</table>

vertical immobilization method for time-lapse microscopy analysis in filamentous cyanobacteria

A main event in bacterial cell division is the septation process, where the protein FtsZ is the key element. FtsZ polymerizes forming a ring-like structure (Z-ring) in the middle of the cell that serves as a scaffold for other division proteins. Super-resolution microscopy in bacterial models Escherichia coli and Bacillus subtilis showed that the Z-ring is discontinuous, while live cell imaging studies demonstrated that FtsZ moves along the ring by a mechanism known as treadmilling. To study the dynamics of FtsZ in vivo, a special cell placement in a vertical position is necessary for imaging the complete structure of the ring in the XY plane. In the case of FtsZ imaging in multicellular cyanobacteria, such as Anabaena sp. PCC7120, maintaining the filaments in a vertical position is challenging because of the size of the cells and the filaments' length. In this article, we describe a method that allows the vertical immobilization of Anabaena sp. PCC 7120 filaments using low melting point agarose and syringes, to record the Z-ring in a mutant that expresses a FtsZ-sfGFP fusion protein. This method is a rapid and inexpensive way to register protein dynamics at the division site using confocal microscopy.

Visualization of the Z-ring in Anabaena sp. using the vertical immobilization method 
To study the dynamics of Z-ring components in bacteria, it is necessary to acquire images in vertically oriented cells. In this position, it is possible to visualize the Z-ring and the main proteins of the divisome to monitor protein dynamics by time-lapse microscopy. The classic sample preparation for bacteria does not work for filamentous cyanobacteria. In the case of Anabaena sp., the filaments...

Watch this Scientific Journal Video about Vertical Immobilization Method for Time-Lapse Microscopy Analysis in Filamentous Cyanobacteria at JoVE.com

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

We present a simple and accessible method for filamentous cyanobacterial visualization in the XY plane. A low-melting point agarose matrix was used, allowing the acquisition of images of proteins involved in the division, in a vertical orientation. Therefore, this methodology can be applied to any filamentous organism and different kinds of proteins.

A main event in bacterial cell division is the septation process, where the protein FtsZ is the key element. FtsZ polymerizes forming a ring-like ...

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.

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