Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60077344%3A_____%2F19%3A00519670" target="_blank" >RIV/60077344:_____/19:00519670 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/60076658:12310/19:43899625

Výsledek na webu

<a href="https://www.frontiersin.org/articles/10.3389/fcimb.2019.00287/full" target="_blank" >https://www.frontiersin.org/articles/10.3389/fcimb.2019.00287/full</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3389/fcimb.2019.00287" target="_blank" >10.3389/fcimb.2019.00287</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes

Popis výsledku v původním jazyce

Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.-attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.

Název v anglickém jazyce

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes

Popis výsledku anglicky

Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.-attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

20201 - Electrical and electronic engineering

Projekt

<a href="/cs/project/TE01020118" target="_blank" >TE01020118: Elektronová mikroskopie</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Frontiers in Cellular and Infection Microbiology

ISSN

2235-2988

e-ISSN

—

Svazek periodika

9

Číslo periodika v rámci svazku

AUG 20 2019

Stát vydavatele periodika

CH - Švýcarská konfederace

Počet stran výsledku

14

Strana od-do

287

Kód UT WoS článku

000481779200001

EID výsledku v databázi Scopus

2-s2.0-85071773412