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.

Protein Localization in the Cyanobacterium Anabaena sp. PCC7120 Using Immunofluorescence Labeling.

Techniques such as immunoflorescence are widely used to determine subcellular distribution of proteins. Here we report on a method to immunolocalize proteins in Anabaena sp. PCC7120 with fluorophore-conjugated antibodies by fluorescence microscopy. This method improves the permeabilization of cyanobacterial cells and minimizes the background fluorescence for non-specific attachments. In this protocol, rabbit antibodies were raised against the synthetic peptide of CyDiv protein ( Mandakovic et al., 2016 ). The secondary antibody conjugated to the fluorophore Alexa488 was used due to its different emission range in comparison to the autofluorescence of the cyanobacterium.

CyDiv, a Conserved and Novel Filamentous Cyanobacterial Cell Division Protein Involved in Septum Localization.

Cell division in bacteria has been studied mostly in Escherichia coli and Bacillus subtilis, model organisms for Gram-negative and Gram-positive bacteria, respectively. However, cell division in filamentous cyanobacteria is poorly understood. Here, we identified a novel protein, named CyDiv (Cyanobacterial Division), encoded by the all2320 gene in Anabaena sp. PCC 7120. We show that CyDiv plays a key role during cell division. CyDiv has been previously described only as an exclusive and conserved hypothetical protein in filamentous cyanobacteria. Using polyclonal antibodies against CyDiv, we showed that it localizes at different positions depending on cell division timing: poles, septum, in both daughter cells, but also in only one of the daughter cells. The partial deletion of CyDiv gene generates partial defects in cell division, including severe membrane instability and anomalous septum localization during late division. The inability to complete knock out CyDiv strains suggests that it is an essential gene. In silico structural protein analyses and our experimental results suggest that CyDiv is an FtsB/DivIC-like protein, and could therefore, be part of an essential late divisome complex in Anabaena sp. PCC 7120.