Impact of hypotonicity on the myo-inositol permeability <i>P</i>_ino in HEK293 cells.

<p>The <i>P</i>_ino values were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e004" target="_blank">Eq. 2</a> from the rates of secondary swelling, using the volumetric data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.g002" target="_blank">Fig. 2A</a>. The fit of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e007" target="_blank">Eq. 3</a> to the data yielded a <i>C</i>₅₀ value of 144 ± 10 mOsm, i.e. the tonicity at which the myo-inositol permeability was half-activated. In the inset, the same <i>P</i>_ino data are plotted as function of the cell volume at the time point of <i>myo</i>-inositol application. Curve fitting (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e007" target="_blank">Eq. 3</a>) shows that <i>P</i>_ino was half-activated as cells swelled by about 26% (<i>v</i>₅₀ = 1.26±0.02).</p

<i>d</i>STORM imaging of immunolabeled SLC5A3 protein in the plasma membrane of HEK293 cells under isotonic and hypotonic conditions.

<p>Images of the same cells in transmitted light (TL) are also shown. From the <i>d</i>STORM images (reconstructed from 15,000 single frames), the surface membrane density of SLC5A3 localizations [loc/μm²] were identified in individual cells. The bar graph shows the impact of hypotonic stress on the surface membrane density of SLC5A3 protein localizations. The data are means (±SD) from 8–16 individual cells for each osmotic condition and hypotonic stress duration. The differences in the mean values between the isotonic control and the two hypotonic samples were statistically significant (as denoted by *; <i>P</i> < 0.05), according to the Mann-Whitney test conducted using the Software Origin 9 (Microcal, Northampton, MA). The difference between the two hypotonic samples (10 vs 20 min) was not significant (n.s.).</p

Hypotonic stress-induced upregulation of SLC5A3 at the mRNA and protein level in HEK293 cells revealed by semiquantitative RT-PCR and Western blot, respectively.

<p>Prior to RNA and protein extractions, the cells were incubated in 100-mOsm CGM for 10–30 min. Control cells were kept in isotonic CGM. The SLC5A3 mRNA level in isotonic sample was negligible, whereas hypotonicity induced substantial amounts of SLC5A3 mRNA. As with RT-PCR, Western blot analysis shows increased amounts of SLC5A3 protein (by up to ∼40%, <i>see</i> text) in hypotonic samples. For RT-PCR, β-actin was used as a template loading control (<i>see</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#sec019" target="_blank">Supporting information</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.s002" target="_blank">S2 Fig.</a>, upper image). Prior to immunoblotting, reversible Ponceau-S protein staining has been used as a loading control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.s002" target="_blank">S2 Fig.</a>, lower image).</p

A putative mechanism of SLC5A3-mediated myo-inositol release during RVD.

<p><b>A:</b> Under isotonic conditions the SLC5A3 protein is mainly localized in the cytosolic vesicles and its mRNA level is low. The plasma membrane permeability to SOOs and electrolytes is poor. <b>B:</b> Exposure of cells to a strongly hypotonic solution, e.g. 100-mOsm sucrose, causes rapid volume increase followed by cell shrinkage via RVD (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.g001" target="_blank">Fig. 1</a>). The swelling-associated increase in the plasma membrane area is achieved (i) by unfolding of microvilli and (ii) by exocytotic fusion of cytosolic vesicles [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.ref023" target="_blank">23</a>]. The fusion of vesicles carrying SLC5A3 protein leads to the incorporation of this transporter into the plasma membrane. The SLC5A3-mediated efflux of myo-inositol and related SOO contributes to cell shrinkage (RVD) and restoration of the original isotonic cell volume. <b>C:</b> During RVD, the cells recover the original membrane impermeability to myo-inositol (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.g001" target="_blank">Fig. 1</a>), presumably, via the endocytosis of excessive plasma membrane along with reinternalization and, possibly, lysosomal degradation of SLC5A3. The increased mRNA and protein expression of SLC5A3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.g006" target="_blank">Fig. 6</a>) suggest that <i>de novo</i> synthesis of SLC5A3 occurs to restore the depleted cytoplasmic pool of the transporter.</p

Confocal fluorescence imaging of HEK293 cells overexpressing the fusion protein SLC5A3-EGFP.

<p>The images <i>A</i> and <i>B</i> were taken, respectively, under isotonic conditions and 10 min after application of a strongly hypotonic 100-mOsm myo-inositol-substituted solution. Hypotonic cell swelling is clearly seen in (<i>B</i>). The insets and the intensity diagrams (<i>C</i> and <i>D</i>) illustrate the impact of hypotonic stress on the intracellular distribution of the fusion protein. Comparison of the diagrams <i>C</i> and <i>D</i> reveals a marked hypotonicity-mediated depletion of the protein in the perinuclear regions along with its increase in the peripheral cytoplasm. Together, these findings suggest that hypotonic swelling caused translocation of a large portion of SLC5A3-EGFP towards the plasma membrane.</p

Changes in the normalized volume <i>v</i> = V/V₀ of HEK293 cells in response to sequential application of sucrose and <i>myo</i>-inositol solutions of the same osmolality of 100 mOsm.

<p>The cells were bathed initially (<i>time</i> < ∼30 s) in isotonic growth medium (300 mOsm) and then exposed to a 100-mOsm sucrose solution. The strongly hypotonic sucrose solution (<i>filled symbols</i>) caused fast cell swelling to a transient maximum volume <i>v</i>_max of ∼1.6 within the first 2–3 min. After the initial swelling, the cells underwent RVD, i.e. they gradually shrank to reach the original isotonic volume (<i>v</i>₀ ≈ 1) within ∼20 min in the presence of sucrose. The replacement of sucrose by an equiosmotic amount of <i>myo</i>-inositol (<i>arrows</i>) abolished RVD and caused secondary cell swelling (<i>empty symbols</i>). The rate of secondary swelling (Δ<i>v</i>/Δ<i>t</i>_ino, <i>red fitted lines</i>) decreased with time during and after RVD (7–35 min). The addition of <i>myo</i>-inositol 40 min after hypotonic shock did not cause any significant cell swelling. Each data point represents the mean ± SE of 25–42 individual cells measured in 2–3 independent experiments. For each time point of <i>myo</i>-inositol addition, the rates of RVD Δ<i>v</i>/Δ<i>t</i>_RVD (<i>blue lines</i>) and the rates of secondary swelling Δ<i>v</i>/Δ<i>t</i>_ino (<i>red lines</i>) were determined to calculate the permeability coefficients for myo-inositol <i>P</i>_ino by applying Eqs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e004" target="_blank">2</a>. The inset illustrates the decay of <i>P</i>_ino with time during RVD.</p

Volume changes of HEK293 cells in response to solutions of varying osmolality and composition.

<p>At time ∼30 s, the cells were first transferred from isotonic growth medium to a sucrose-substituted solution having osmolality of 100, 125, … 250 or 275 mOsm. Thereafter, the hypotonic sucrose solutions were replaced at time ∼5 min with myo-inositol solutions of the same osmolalities (<b><i>A</i></b>). In contrast, the cells were exposed for 20 min to sucrose solutions only (<b><i>B</i></b>). After the initial swelling in the presence of sucrose, the cells were capable of RVD over the entire hypotonicity range (<i>B</i>). A nearly complete RVD also occurred in slightly hypotonic solutions of myo-inositol (<i>A</i>, ∼175–275 mOsm). Application of more diluted myo-inositol solutions (100–150 mOsm; <i>t</i> ≈ 5–9min) considerably inhibited cell shrinkage via RVD. Thereafter (<i>t</i> ≈ 9–20 min) the cells exhibited sustained secondary swelling (<b><i>A</i></b>), which is indicative of myo-inositol uptake by cells. For each tonicity, the rates of RVD Δ<i>v</i>/Δ<i>t</i>_RVD and secondary swelling Δ<i>v</i>/Δ<i>t</i>_ino were used to calculate the permeability coefficients for electrolytes <i>P</i>_el and inositol by applying Eqs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e004" target="_blank">2</a>, respectively.</p

FloA and FloT distribute differently within the bacterial microdomains.

<p><b>(A and B)</b> PALM images of cells labeled with FloA-mEoS2 (A) or FloT-mEoS2 (B) translational fusions grown to stationary phase and fixed with PFA (4%). With increasing localization density the color code changes from red to yellow. White arrows indicate the localization of a cluster. Scale bars are 500 nm. Detail of the right bottom of each panel shows a dashed-line decorated PALM picture as a general indicator of the cell outline. Scale bar is 500 nm. <b>(C)</b> Comparative graph of the diameter of the clusters that were generated by FloA-mEoS2 and FloT-mEoS2 fluorescence signal. The upper right corner shows a graph with the mean of the diameter of the FloA and FloT clusters. <b>(D)</b> Comparative graph of the number of clusters detected in FloA-mEoS2 and FloT-mEoS2 labeled cells. <b>(E)</b> Comparative graph of the number of FloA and FloT localizations per cluster. <b>(F)</b> Comparative graph of the percentage of localizations that organized in clusters.</p

FloA and FloT are two distinct flotillins.

<p><b>(A)</b> Comparative diagram of FloA and FloT protein structures. The membrane-anchored region is represented in blue. The PHB domain is represented in green and the coil-coiled region is magnified and EA repeats are labeled in orange. Scale bar is 100 amino acids. <b>(B)</b> Fluorescence microscopy pictures of cells labeled with FloA-GFP, FloA-mCherry (upper panel), FloT-GFP and FloT-mCherry (lower panel) translational fusions. Fluorescence signal associated with FloA is represented in green and fluorescence signal associated with FloT is represented in red. Cultures were grown in MSgg medium at 37°C until stationary phase. Scale bars are 2 μm. <b>(C)</b> Quantification of the number of foci per cell (n = 400). <b>(D)</b> Fluorescence microscopy pictures of double-labeled strains. Cultures were grown in MSgg medium at 37°C until stationary phase. GFP signal is represented in green and mCherry signal is represented in red. Right panel shows the merge of the two fluorescence signals, which is visualized as yellow fluorescence signal. Scale bars are 2 μm. <b>(E)</b> Time lapse fluorescence microscopy analysis of cells expressing FloA-GFP (green signal) and FloT-mCherry (red signal) translational fusions. Signal was monitored within the same cells at 1 sec intervals. Cultures were grown in MSgg medium at 37°C until stationary phase. Scale bar is 2 μm.</p

Tethering of signaling partners mediated by flotillins.

<p><b>(A)</b> BTH assay to quantify the interaction of PhoR (i) and ResE (ii) under different concentrations of flotillins (upper panels). Dashed line indicates the threshold limit of 700 Miller Units that defines a positive (≥ 700 Miller Units) and a negative interaction signal (≤ 700 Miller Units) according to the instructions of the manufacturer. Lower- (pSEVA-621), medium- (pSEVA-631) and high-copy (pSEVA-641) plasmids expressing His⁶-tagged FloA and FloT rendered lower (⬆), medium (⬆⬆) and higher (⬆⬆⬆) concentration of flotillin in the BTH assay, respectively, according to immunoblot analysis (lower panels). SDS-PAGE are shown as loading control. <b>(B)</b> Inhibition of the activity of a protein complex by scaffold titration. Protein assembly by scaffold proteins has potential drawbacks. At high concentrations, scaffolds may titrate enzyme and substrate away from each other.</p