Abstract
Fluorescence-tags, commonly used to visualize the spatial distribution of proteins within cells, can influence the localization of the tagged proteins by affecting their stability, interaction with other proteins or the induction of oligomerization artifacts. To circumvent these obstacles, a protocol was developed to generate 50 nm thick serial sections suitable for immunogold labeling and subsequent reconstruction of the spatial distribution of immuno-labeled native proteins within individual bacterial cells. Applying this method, we show a cellular distribution of the staphylococcal alkaline shock protein 23 (Asp23), which is compatible with filament formation, a property of Asp23 that we also demonstrate in vitro.
Highlights
Introduction of fluorescently labeled protein tags, such as green-fluorescent protein (GFP), and the development of super-resolution fluorescence microscopy techniques have led to a renaissance of microbial cell biology appreciating the remarkably high degree of spatial-temporal organization of the bacterial cell physiology[1,2,3]
We have developed a non-invasive protocol enabling the three-dimensional (3D-) visualization of proteins in serial sections of bacterial cells by immunofluorescence microscopy or immunogold labeling envisioned by electron microscopy
Asp[23] is the eponym of the poorly investigated Asp[23] protein family. Members of this protein family are exclusively present in Gram-positive bacteria[8], where they are functionally linked to lipid metabolism (Bacillus subtilis)[9], survival at low pH and during nutrient limitation (Streptococcus agalactiae)[10], the bile stress response (Enterococcus faecium)[11], cell morphology control (Enterococcus faecalis)[12] and nutrient sensing (Streptococcus pneumoniae)[13]
Summary
Introduction of fluorescently labeled protein tags, such as green-fluorescent protein (GFP), and the development of super-resolution fluorescence microscopy techniques have led to a renaissance of microbial cell biology appreciating the remarkably high degree of spatial-temporal organization of the bacterial cell physiology[1,2,3]. One way to achieve this goal is the detection of proteins by immunofluorescence This approach typically requires permeabilization or removal of the bacterial cell envelope in order to allow antibody penetration into bacterial cells. This experimental step provides a major source of artifact generation because removal of the cell wall significantly affects the integrity and structure of the cell To circumvent this problem, we have developed a non-invasive protocol enabling the three-dimensional (3D-) visualization of proteins in serial sections of bacterial cells by immunofluorescence microscopy or immunogold labeling envisioned by electron microscopy. Using our serial section electron microscopy and immune-labeling protocol, we show a distribution pattern of Asp[23] compatible with the formation of Asp[23] filaments, a property of Asp[23] that we demonstrate in vitro These results provide a framework for future investigations analyzing the relationship between Asp[23] oligomer formation and function
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