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Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. Howev...

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Autores principales: Cramer, Kimberly, Bolender, Anna-Lena, Stockmar, Iris, Jungmann, Ralf, Kasper, Robert, Shin, Jae Yen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678925/
https://www.ncbi.nlm.nih.gov/pubmed/31295803
http://dx.doi.org/10.3390/ijms20143376
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author Cramer, Kimberly
Bolender, Anna-Lena
Stockmar, Iris
Jungmann, Ralf
Kasper, Robert
Shin, Jae Yen
author_facet Cramer, Kimberly
Bolender, Anna-Lena
Stockmar, Iris
Jungmann, Ralf
Kasper, Robert
Shin, Jae Yen
author_sort Cramer, Kimberly
collection PubMed
description In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems.
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spelling pubmed-66789252019-08-19 Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy Cramer, Kimberly Bolender, Anna-Lena Stockmar, Iris Jungmann, Ralf Kasper, Robert Shin, Jae Yen Int J Mol Sci Article In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems. MDPI 2019-07-10 /pmc/articles/PMC6678925/ /pubmed/31295803 http://dx.doi.org/10.3390/ijms20143376 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Cramer, Kimberly
Bolender, Anna-Lena
Stockmar, Iris
Jungmann, Ralf
Kasper, Robert
Shin, Jae Yen
Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy
title Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy
title_full Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy
title_fullStr Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy
title_full_unstemmed Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy
title_short Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy
title_sort visualization of bacterial protein complexes labeled with fluorescent proteins and nanobody binders for sted microscopy
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678925/
https://www.ncbi.nlm.nih.gov/pubmed/31295803
http://dx.doi.org/10.3390/ijms20143376
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