Immunocytochemical Visualization of Proteins from Cyanobacterial Cells with High Autofluorescence of Phycoerythrin and Phycourobilin

Summary

This protocol outlines the visualization and quantification of a particular protein within cells at the cellular level for the phycoerythrin-containing cyanobacterium, Crocosphaera watsonii.

Abstract

Presented is a protocol for visualizing and quantifying a specific protein in cells at the cellular level for the marine cyanobacterium Crocosphaera watsonii, a crucial primary producer and nitrogen fixer in oligotrophic oceans. One of the challenges for marine autotrophic N₂ fixers (diazotrophs) is distinguishing probe-derived fluorescence signals from autofluorescence. C. watsonii was selected to represent chlorophyll-, phycoerythrin- and phycourobilin-containing cyanobacteria. The protocol allows for simple and semi-quantitative visualization of proteins in C. watsonii at a single-cell level, enabling investigation of protein production under different environmental conditions to evaluate the metabolic activities of the target cyanobacteria. Furthermore, the fixation and permeabilization methods are optimized to enhance the fluorescence signals from target proteins to distinguish them from autofluorescence, especially from phycoerythrin and phycourobilin. The enhanced signal can be visualized using confocal or widefield fluorescence microscopy. Additionally, fluorescence intensity was semi-quantified using Fiji software. This single-cell analysis workflow allows the evaluation of cell-to-cell variations of specific protein content. The protocol can be performed in any life science laboratory as it requires only standard equipment and can also be easily adapted to other phycoerythrin-containing cyanobacterial cells.

Introduction

The physiological variation from cell to cell (commonly referred to as "heterogeneity") in metabolic activities within microorganisms, including cyanobacteria, has been documented through studies on clone cultures^1,2,3,4. This heterogeneity encompasses diverse metabolic activities such as cell division⁵, carbon assimilation^6,7,8, and nitrogen assimilation^9,10.....

Protocol

1. Cyanobacteria cultivation

1. Cultivate Crocosphaera watsonii PS0609 cells in Erlenmeyer flasks or photobioreactors in YBCII medium²⁷. To determine the culture density, measure the cell density using a method of choice (e.g., microscopy, flow cytometry, cell counter, etc.).
2. Maintain the cell densities between ~1 × 10⁴ to ~5 × 10⁶ cells mL^-1.

2. Preparation of reagents

1. Keep 100% methanol at -20 °C.
2. Phosphate-buffered saline (PBS): Dissolve 1.36 g of KH₂PO₄, ....

Results

The fluorescence signal was observed from extracellular substances in the negative control, where the 1^st antibody was not used (Figure 1A-C). The fluorescence signal of the tyramide-boosted reagent, conjugated to the large subunit of the Rubisco protein (RbcL), was successfully detected in C. watsonii under a fluorescence microscope using a DAPI filter with UV excitation (Figure 1D-F.......

Discussion

For cyanobacteria, the TSA system has found widespread use in TSA-fluorescence in situ hybridization (TSA-FISH, CARD-FISH), targeting specific rRNA. However, its application for proteins remains limited²⁶. In this study, a TSA procedure was applied to enable whole-cell immunodetection of the N₂-fixing cyanobacterium C. watsonii, incorporating modifications based on a previous reference²⁰. Notable revisions encompassed permeabilization, acco.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Achromopeptidase	FUJIFILM	014-09661
Alexa Fluor350	Thermo Scientific	B40952	Tyramide-350
Alexa Fluor405	Thermo Scientific	B48254	Tyramide-405
Alexa Fluor488 Tyramide SuperBoost Kit	Thermo Scientific	B40922	Goat anti-rabbit IgG
Bovine serum albumin	Sigma-Aldrich	A2153
Centrifuge	Eppendorf	5804 R
Centrifuge tubes (15 mL)	VWR	525-1085	For harvesting cells
Confocal microscope	Zeiss	LSM880	Equipped with Airyscan
Fluorescence microscope	Olympus	BX51	DAPI filter: Ex.360-370 nm, Em. 420-460 nm
Gelatine	Merk	4070
High precision microscope cover glasses for confocal microscope	Deckgläser	No. 1.5H
Liquid Blocker Regular/Mini	Daido Sangyo Co., Ltd.	Part 6505	For keeping the cells on the slide glass
Lysozyme	ITW Reagents	A4972
Methanol	Carl Roth	67-56-1
Monopotassium Phosphate	Penta	12290
Monunting medium	Sigma-Aldrich	345789-20ML	FluorSave Reagent
Mounting medium	Vectashild	H-1300
Objective lens used in the confocal microscope	Zeiss	Plan-Apochromat 63x/1.4 Oil DIC M27
Paraformaldehyde	Sigma-Aldrich	158127
Poly-lysine coated slide glass	Sigma-Aldrich	P0425-72EA
Potassium chloride	Lach-Ner
Safe lock tube (1.5 mL)	Eppendorf	0030 120.086	For treating cells and storing chemicals
Sodium chloride	Penta	16610
Sodium hydrogen phosphate	Penta	15130
Triton X-100	Sigma-Aldrich	X100