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Monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in Streptococcus pneumoniae using fluorescent dyes

Clementi, Emily A; Marks, Laura R; Roche-Håkansson, Hazeline and Håkansson, Anders P LU (2014) In Journal of Visualized Experiments
Abstract

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including... (More)

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca(2+), K(+), and H(+), respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

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Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
keywords
Calcium, Cations, Cell Membrane, Cell Polarity, Fluorescent Dyes, Hydrogen, Ionophores, Membrane Potentials, Optical Imaging, Potassium, Streptococcus pneumoniae
in
Journal of Visualized Experiments
issue
84
publisher
JoVE
external identifiers
  • Scopus:84904562373
ISSN
1940-087X
DOI
10.3791/51008
language
English
LU publication?
no
id
91dfb7f2-46d2-445f-9d37-052fd1261afb
date added to LUP
2016-05-21 10:46:36
date last changed
2016-10-30 04:47:44
@misc{91dfb7f2-46d2-445f-9d37-052fd1261afb,
  abstract     = {<p>Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca(2+), K(+), and H(+), respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.</p>},
  author       = {Clementi, Emily A and Marks, Laura R and Roche-Håkansson, Hazeline and Håkansson, Anders P},
  issn         = {1940-087X},
  keyword      = {Calcium,Cations,Cell Membrane,Cell Polarity,Fluorescent Dyes,Hydrogen,Ionophores,Membrane Potentials,Optical Imaging,Potassium,Streptococcus pneumoniae},
  language     = {eng},
  number       = {84},
  publisher    = {ARRAY(0x8a3afa0)},
  series       = {Journal of Visualized Experiments},
  title        = {Monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in Streptococcus pneumoniae using fluorescent dyes},
  url          = {http://dx.doi.org/10.3791/51008},
  year         = {2014},
}