scholarly journals An Optogenetic Tool to Raise Intracellular pH in Single Cells and Drive Localized Membrane Dynamics

2021 ◽  
Author(s):  
Caitlin E. T. Donahue ◽  
Michael D. Siroky ◽  
Katharine A. White
Keyword(s):  
Intracellular Ph ◽  
Single Cells ◽  
Membrane Dynamics

scholarly journals An optogenetic tool to raise intracellular pH in single cells and drive localized membrane dynamics

2021 ◽  
Author(s):  
Caitlin E. T. Donahue ◽  
Michael D. Siroky ◽  
Katharine A. White
Keyword(s):  
Single Cell ◽  
Intracellular Ph ◽  
Single Cells ◽  
Cell Polarization ◽  
Membrane Dynamics ◽  
Cell Physiology ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Behaviors ◽  
Membrane Ruffling

AbstractIntracellular pH (pHi) dynamics are critical for regulating normal cell physiology. For example, transient increases in pHi (7.2-7.6) regulate cell behaviors like cell polarization, actin cytoskeleton remodeling, and cell migration. Most studies on pH-dependent cell behaviors have been performed at the population level and use non-specific methods to manipulate pHi. The lack of tools to specifically manipulate pHi at the single-cell level has hindered investigation of the role of pHi dynamics in driving single cell behaviors. In this work, we show that Archaerhodopsin (ArchT), a light-driven outward proton pump, can be used to elicit robust and physiological pHi increases over the minutes timescale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for approximately 45 minutes. We apply this spatiotemporal pHi manipulation tool to determine whether increased pHi is a sufficient driver of membrane ruffling in single cells. Using the ArchT tool, we show that increased pHi in single cells can drive localized membrane ruffling responses within seconds and increased membrane dynamics (both protrusion and retraction events) compared to control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and cell behaviors such as membrane ruffling. This tool will be transformative in facilitating the experiments required to determine if increased pHi is a driver of these cell behaviors at the single-cell level.

Fast intracellular pH determination in single cells by flow-cytometry

1981 ◽  
Vol 68 (5) ◽  
pp. 265-266 ◽  
Author(s):  
G. Valet ◽  
A. Raffael ◽  
L. Moroder ◽  
E. W�nsch ◽  
G. Ruhenstroth-Bauer
Keyword(s):  
Flow Cytometry ◽  
Intracellular Ph ◽  
Single Cells

scholarly journals Optogenetic tool to spatiotemporally manipulate intracellular pH within single cells

2020 ◽  
Author(s):  
Caitlin Donahue ◽  
Michael Siroky ◽  
Katharine White
Keyword(s):  
Intracellular Ph ◽  
Single Cells

scholarly journals Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

2009 ◽  
Vol 75 (13) ◽  
pp. 4550-4556 ◽  
Author(s):  
Vicky G. Kastbjerg ◽  
Dennis S. Nielsen ◽  
Nils Arneborg ◽  
Lone Gram
Keyword(s):  
Listeria Monocytogenes ◽  
Single Cell ◽  
Intracellular Ph ◽  
Bacterial Population ◽  
Single Cells ◽  
Viable Cell ◽  
Population Subdivision ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Counts

ABSTRACT Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pHi was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pHi 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.

scholarly journals Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium.

1993 ◽  
Vol 120 (4) ◽  
pp. 1003-1010 ◽  
Author(s):  
M A Schwartz
Keyword(s):  
Endothelial Cells ◽  
Calcium Channels ◽  
Intracellular Ph ◽  
Time Course ◽  
Single Cells ◽  
State Level ◽  
Cell Spreading ◽  
Free Calcium ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels

Intracellular calcium ([Ca2+]i) was measured in FURA 2-loaded endothelial cells plated on fibronectin or vitronectin. Average values for [Ca2+]i increased to approximately twofold above basal levels by approximately 1 h after plating, and then declined. The increase in [Ca2+]i required extracellular calcium. Substituting potassium for sodium in the medium reduced the elevation of [Ca2+]i, a result that rules out the involvement of Na-Ca exchangers or voltage-dependent calcium channels, but that is consistent with the involvement of voltage-independent calcium channels. Plating cells on an anti-integrin beta 1 subunit antibody gave a similar [Ca2+]i response, but clustering beta 1 integrins with the same antibody, or occupying integrins with RGD (arg-gly-asp) peptides had no effect. Time course measurements on single cells revealed that in each cell [Ca2+]i rose abruptly at some point during spreading, from the basal level to a higher steady-state level that was maintained for some time. The elevated [Ca2+]i was unrelated to previously observed changes in intracellular pH, because chelating the Ca2+ in the medium failed to inhibit the elevation of pHi that occurred during cell spreading. In conclusion, these results show that integrin-mediated cell spreading can regulate [Ca2+]i, and the pathways involved are distinct from those that regulate intracellular pH.

scholarly journals Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH

2000 ◽  
Vol 66 (6) ◽  
pp. 2330-2335 ◽  
Author(s):  
Henrik Siegumfeldt ◽  
K. Björn Rechinger ◽  
Mogens Jakobsen
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactic Acid Bacterium ◽  
Intracellular Ph ◽  
Membrane Filter ◽  
Single Cells ◽  
Streptococcus Thermophilus ◽  
Extracellular Ph ◽  
Lactobacillus Delbrueckii ◽  
Rapid Drop

ABSTRACT We describe the dynamics of changes in the intracellular pH (pHi) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pHex). Strains of Lactobacillus delbrueckii subsp.bulgaricus, Streptococcus thermophilus, andLactococcus lactis were investigated. Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pHi within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pHex from 7.0 to 5.0. Under these conditions, the pHi of L. innocua remained neutral (between 7 and 8). In contrast, the pHi values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pHex was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pHi at pHex 7.0, while different kinetics of pHiregulation were observed in different species and also in different strains of S. thermophilus.

scholarly journals Single-cell intracellular pH measurements reveal cell-cycle linked pH dynamics

2021 ◽  
Author(s):  
Julia S Spear ◽  
Katharine A White
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Intracellular Ph ◽  
Cell Cycle Progression ◽  
Single Cells ◽  
Population Level ◽  
Growth Factor Signaling ◽  
Cycle Progression ◽  
Cell Behaviors ◽  
Sinusoidal Pattern

Transient changes in intracellular pH (pHi) have been shown to regulate normal cell behaviors like migration and cell-cycle progression, while dysregulated pHi dynamics are a hallmark of cancer. However, little is known about how pHi heterogeneity and dynamics influence population-level measurements or single-cell behaviors. Here, we present and characterize single-cell pHi heterogeneity distributions in both normal and cancer cells and measure dynamic pHi increases in single cells in response to growth factor signaling. Next, we measure pHi dynamics in single cells during cell cycle progression. We determined that single-cell pHi is significantly decreased at the G1/S boundary, increases from S phase to the G2/M transition, rapidly acidifies during mitosis, and recovers in daughter cells. This sinusoidal pattern of pHi dynamics was linked to cell cycle timing regardless of synchronization method. This work confirms prior work at the population level and reveals distinct advantages of single-cell pHi measurements in capturing pHi heterogeneity across a population and dynamics within single cells.

scholarly journals Optogenetic tool to spatiotemporally manipulate intracellular pH within single cells

2020 ◽  
Author(s):  
Caitlin Donahue ◽  
Michael Siroky ◽  
Katharine White
Keyword(s):  
Intracellular Ph ◽  
Single Cells

scholarly journals Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

2016 ◽  
Vol 27 (22) ◽  
pp. 3418-3435 ◽  
Author(s):  
François Aguet ◽  
Srigokul Upadhyayula ◽  
Raphaël Gaudin ◽  
Yi-ying Chou ◽  
Emanuele Cocucci ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Imaging ◽  
Single Cells ◽  
Light Sheet ◽  
Membrane Dynamics ◽  
Bottom Surface ◽  
Light Sheet Microscopy ◽  
Dividing Cells ◽  
Entire Cell ◽  
Membrane Bound

Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.

Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy

1996 ◽  
Vol 270 (5) ◽  
pp. C1438-C1446 ◽  
Author(s):  
R. Martinez-Zaguilan ◽  
M. W. Gurule ◽  
R. M. Lynch
Keyword(s):  
Intracellular Ph ◽  
Single Cells ◽  
Ion Homeostasis ◽  
Spectral Imaging ◽  
Charge Coupled Device ◽  
Fura 2 ◽  
Imaging Approach ◽  
Insulin Secreting Cells ◽  
A Cell ◽  
Cytosolic Acidification

Described is a microscopic spectral imaging approach to monitor pH and Ca2+ simultaneously from combined spectra of multiple ion indicators. Emitted light from a cell is focused onto a grating spectrograph and spectra are imaged with a cooled charge-coupled device camera. The combined spectral output of fura 2 and SNARF-1 was analyzed to follow changes in intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) simultaneously and to correct the Ca2+ signal for concurrent changes in pHi. Responses of individual hamster insulinoma (HIT-T15) cells to effectors of ion homeostasis were heterogeneous. Treatment with NH4Cl increased pHi and transiently increased [Ca2+]i. Removal of NH4Cl induced cytosolic acidification concomitant with either no change or sustained increases in [Ca2-]i. Glucose treatment generally resulted in rapid and sustained increases in both [Ca2+]i and pHi but also heterogeneous pHi and [Ca2+]i responses. Corrections of the fura 2 signal for pH were important for following Ca2+ transitions elicited by NH4Cl but were less important for glucose-induced responses. The spectral imaging microscope provides a sensitive method for simultaneous measurements of pHi and [Ca2+]i in single cells.

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