Sample Preparation and Choice of Fluorophores for Single and Dual Color Photo-Activated Localization Microscopy (PALM) with Bacterial Cells

Bach, Juri Niño; Giacomelli, Giacomo; Bramkamp, Marc

doi:10.1007/978-1-4939-6810-7_9

Cited by 14 publications

(10 citation statements)

References 24 publications

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“…Spectrally distinct photoactivatable fluorescent proteins (PA-FPs) make multi-color super-resolution imaging feasible, allowing scientists to structurally describe more than one cellular component at a time 7 – 10 . Dual-color PALM using an orange and a green PA-FPs is such an example, and it has been successfully employed to describe protein distribution in various eukaryotic cells 10 – 14 , and to lesser extend in bacterial cells 15 – 18 , most likely due to challenges in employing green PA-FPs. On the other hand, the functionality of target proteins often is compromised by genetically incorporated FPs, so it can be difficult to find a suitable tag.…”

Section: Introductionmentioning

confidence: 99%

“…mNeonGreen has been widely utilized to investigate biological processes in various organisms, including bacterial and eukaryotic cells, as well as mice 15 , 19 – 24 . Although the developers of mNeonGreen have already shown its use for super-resolution PALM microscopy, and its improved performance over other green fluorescent proteins for PALM ( e .…”

Section: Introductionmentioning

confidence: 99%

“…mNeonGreen also switches back and forth between the two states, bright and dark 25 . However, contrary to Dronpa, the switching process is controlled only by one light wavelength (blue), which introduces artifacts in PALM images if post-data processing is not applied 15 (Fig. 1 ).…”

Section: Introductionmentioning

confidence: 99%

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Optimization of sample preparation and green color imaging using the mNeonGreen fluorescent protein in bacterial cells for photoactivated localization microscopy

Stockmar

Feddersen

Cramer

et al. 2018

Sci Rep

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mNeonGreen fluorescent protein is capable of photo-switching, hence in principle applicable for super-resolution imaging. However, difficult-to-control blinking kinetics that lead to simultaneous emission of multiple nearby mNeonGreen molecules impedes its use for PALM. Here, we determined the on- and off- switching rate and the influence of illumination power on the simultaneous emission. Increasing illumination power reduces the probability of simultaneous emission, but not enough to generate high quality PALM images. Therefore, we introduce a simple data post-processing step that uses temporal and spatial information of molecule localizations to further reduce artifacts arising from simultaneous emission of nearby emitters. We also systematically evaluated various sample preparation steps to establish an optimized protocol to preserve cellular morphology and fluorescence signal. In summary, we propose a workflow for super-resolution imaging with mNeonGreen based on optimization of sample preparation, data acquisition and simple post-acquisition data processing. Application of our protocol enabled us to resolve the expected double band of bacterial cell division protein DivIVA, and to visualize that the chromosome organization protein ParB organized into sub-clusters instead of the typically observed diffraction-limited foci. We expect that our workflow allows a broad use of mNeonGreen for super-resolution microscopy, which is so far difficult to achieve.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optimization of sample preparation and green color imaging using the mNeonGreen fluorescent protein in bacterial cells for photoactivated localization microscopy

Stockmar

Feddersen

Cramer

et al. 2018

Sci Rep

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“…Imaging was performed using a Zeiss ELYRA P.1 equipped with the following laser lines: a HR diode 50 mW 405 nm laser and a HR DPSS 200 mW 561 nm laser and an Andor EM‐CCD camera iXon DU 897 camera. Fluorescence was detected using a long pass 570 nm filter (LP570), similar to a procedure described before (Bach et al , ).…”

Section: Methodsmentioning

confidence: 99%

Substrate‐dependent cluster density dynamics of Corynebacterium glutamicum phosphotransferase system permeases

Martins

Giacomelli

Goldbeck

et al. 2019

Molecular Microbiology

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SummaryMany bacteria take up carbohydrates by membrane‐integral sugar specific phosphoenolpyruvate‐dependent carbohydrate:phosphotransferase systems (PTS). Although the PTS is centrally involved in regulation of carbon metabolism in different bacteria, little is known about localization and putative oligomerization of the permease subunits (EII). Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters, as well as the general components EI and HPr from Corynebacterium glutamicum using widefield and single molecule localization microscopy. PtsF and PtsG form membrane embedded clusters that localize in a punctate pattern. Size, number and fluorescence of the membrane clusters change upon presence or absence of the transported substrate, and a direct influence of EI and HPr was not observed. In presence of the transport substrate, EII clusters significantly increased in size. Photo‐activated localization microscopy data revealed that, in presence of different carbon sources, the number of EII proteins per cluster remains the same, however, the density of these clusters reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transported substrate, the EII proteins in individual clusters occupy larger membrane areas.

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“…Imaging was performed using a Zeiss ELYRA P.1 equipped with the following laser lines: a HR diode 50 mW 405 nm laser and a HR DPSS 200 mW 561 nm laser and an Andor EM-CCD camera iXon DU 897 camera. Fluorescence was detected using a long pass 570nm filter (LP570), similar to a procedure described before (62).…”

Section: Methodsmentioning

confidence: 99%

Substrate-dependent cluster density dynamics in bacterial phosphotransferase system permeases

Benevides

Giacomelli

Bramkamp

2018

Preprint

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19Bacteria take up carbohydrates by membrane-integral sugar specific 20 phosphoenolpyruvate-dependent carbohydrate:phosphotransferase systems (PTS). 21 Although PTS is at the heart of bacterial carbon uptake and centrally involved in 22 regulation of carbon metabolism, little is known about localization and putative 23 oligomerization of the permease subunits (EII) of PTS. Here, we analyzed localization 24 of the fructose specific PtsF and the glucose specific PtsG transporters from C. 25 glutamicum using widefield and single molecule localization microscopy. PtsG and 26 PtsF form membrane embedded clusters that localize in a punctate pattern within the 27 cell membrane. The size, number and fluorescence of the observed clusters changes 28 upon presence or absence of the transported substrate. In presence of the transport 29 substrate clusters significantly increased in size. Photo-activated localization 30 microscopy (PALM) data revealed that, in presence of different carbon sources, the 31 number of EII protein events per cluster remain the same, however the density of 32 PTS molecules within a cluster reduces. Our work reveals a simple mechanism for 33 efficient membrane occupancy regulation. Clusters of PTS EII transporters are 34 densely packed in absence of a suitable substrate. In presence of a transport 35 substrate the EII proteins in individual clusters occupy larger membrane areas, 36 thereby decreasing protein density in individual clusters. This mechanism allows for 37 efficient use of the limited membrane space under varying growth conditions without 38 need of protein removal and re-synthesis.39 40 Importance 41 3 The carbohydrate transport system PTS is centrally involved in the regulation of 42 sugar metabolism. Although much is known about the regulatory interaction, the 43 genetic control and the structure/function relationship of the individual PTS 44 components, we know almost nothing about the spatio-temporal organization of the 45 PTS proteins within the cell. We find dynamic clustering of PTS permeases in 46 Corynebacterium glutamicum. Using single molecule resolution photo-activated 47 localization microscopy we could show that PTS EII protein cluster are dynamically 48 changing protein density upon substrate availability. Our findings imply a novel 49 strategy of regulating limited membrane space efficiently. Furthermore, these data 50 will provide important insights in modelling carbohydrate fluxes in cells, since current 51 models assume a homogeneous distribution of PTS permeases within the 52 membrane. 53 54subunit EIIA is detached, while in B. subtilis and C. glutamicum, all three subunits are 79 fused together. In enteric bacteria EIIA (also termed EIIA crr , since the coding gene is 80 abbreviated crr), is at the heart of a complex regulatory system that governs the 81 quest for food (4-6). EIIA crr interacts with several transport proteins, such as the 82 lactose permease LacY, thereby inhibiting their activity. This leads to the inducer 83 exclusion effect (15). The accumulat...

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Sample Preparation and Choice of Fluorophores for Single and Dual Color Photo-Activated Localization Microscopy (PALM) with Bacterial Cells

Cited by 14 publications

References 24 publications

Optimization of sample preparation and green color imaging using the mNeonGreen fluorescent protein in bacterial cells for photoactivated localization microscopy

Optimization of sample preparation and green color imaging using the mNeonGreen fluorescent protein in bacterial cells for photoactivated localization microscopy

Substrate‐dependent cluster density dynamics of Corynebacterium glutamicum phosphotransferase system permeases

Substrate-dependent cluster density dynamics in bacterial phosphotransferase system permeases

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