vertical-immobilization-method-for-time-lapse-microscopy-analysis

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 ...

Pontificia Universidad Católica de Chile

Universidad Espíritu Santo

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.

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

Techniques such as immunoflorescence are widely used to determine subcellular distribution of proteins. Here we report on a method to immunolocalize proteins in  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 , 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.

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

The largest wave of infection with SARS-CoV-2 virus in Ecuador was observed in mid-December 2021 and early January 2022, driven by B.1.1.529/BA (Omicron) variant. During the second half of March, an increase in the number of daily cases was observed and coincided with the emergence of the BA.2 variant, which we describe in the present study. The first sequenced five cases of SARS-CoV-2 21L/BA.2 in Ecuador were identified using variant specific genotyping by qPCR and confirmed by whole genome sequencing (WGS). The first sequenced Ecuadorian BA.2 isolate was obtained from a person with international travel history who became symptomatic 3 days after travelling, whereas in the other cases no travel history was recorded.

Derly Andrade

Departamento de Genética Molecular y Microbiología,
Facultad de Ciencias Biológicas,
Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas

Pontificia Universidad Católica de Chile

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

Derly Andrade

Departamento de Genética Molecular y Microbiología,Facultad de Ciencias Biológicas,Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas

Pontificia Universidad Católica de Chile

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

Departamento de Genética Molecular y Microbiología,
Facultad de Ciencias Biológicas,
Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas