Detection of channel-forming activities in subcellular fractions.

<p>Fractions 2–4 (glycosomes), 8–11 (fragments of flagella), and 15–18 (mitochondria) from Optiprep density gradients (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>) were combined and treated with Genapol X-080 to solubilize membrane proteins (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). After sedimentation of insoluble material, aliquots of the resulting supernatants were used for MCR (<b>A</b>–<b>C</b>) or SCA (<b>D</b>). (<b>A</b>) Traces of the current monitoring in the presence of glycosomal (upper panel) or mitochondrial (lower panel) preparations. The middle trace represents a timescale-expanded current recording of the upper trace. The bath solution contained 3 M KCl and the applied voltage was +10 mV. (<b>B</b>) Histograms of insertion events registered in subcellular fractions (see panel <b>A</b>). Bin size is 4.0 pA. The total number of insertion events (I.e.) is indicated. Here and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3</a> C (upper panel) all insertion events with current increments over 180 pA (for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3C</a>, lower panel −90 pA) are combined in one bin (180 pA or 90 pA, respectively). Note that the amount of insertion events in the flagella fraction (see <b>B</b>, middle panel) is lower than that observed in other fractions. This is mainly due to low channel-forming activity (per protein content) in the preparations of this fraction. For the sake of compatibility we used the same amounts of protein for measurements in different fractions. (<b>C</b>) Histograms of insertion events detected for glycosomal preparations using NH₄Cl as the electrolyte. Bin size: 4 pA (upper panel) or 2 pA (lower panel). See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figures 3A and 3B</a> for other details. (<b>D</b>) Trace of the current monitoring using the glycosomal fraction (initial holding potential +10 mV) indicating the insertion (marked by one asterisk) of a large-conductance channel that spontaneously closed (marked by two asterisks) after stepwise (each step is +10 mV) increase in the holding potential up to 50 mV.</p

SCA of a very-low-conductance channel.

<p>(<b>A</b>) Current recording of a single very-low-conductance channel. The bath solution (panels <b>A</b>, <b>B</b>, and <b>C</b>) contained 3 M KCl. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>A</b></a> for other details. (<b>B</b>) Current trace of the channel in response to the shown voltage-ramp protocol. Dotted line indicates the current level at zero holding potential. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Current traces of a single channel in response to the indicated voltage-step protocol. (<b>D</b>) Ion-selectivity of the channel. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>D</b></a> for details.</p

SCA of a high-conductance channel.

<p>(<b>A</b>) Current trace of a single high-conductance channel. The insertion event (marked by an asterisk) was registered at +10 mV and the applied voltage was then switched to −10 mV. The dashed line indicates the current level (zero) before insertion of the channel. The data in panels <b>A</b>, <b>B</b>, and <b>C</b> were collected using 3 M KCl as the electrolyte. (<b>B</b>) Current trace of the channel in response to the indicated voltage-ramp protocol. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Single channel currents in response to the indicated voltage-step protocol. (<b>D</b>) Dependence of the single channel conductance on the KCl concentration. After detection of a single channel insertion using 3 M KCl as bath solution (holding potential +10 mV), the electrolyte was diluted and registration of the current amplitudes of the same channel was conducted at 2.0 M and 1.0 M KCl, respectively. Data points are mean±SD for at least 4 independent measurements. (<b>E</b>) Current traces of a single channel in response to a low-speed linear increase (upper trace) or decrease (lower trace) of the holding potential. The bath solution contained 1.0 M NH₄Cl, 20 mM Tris-Cl, pH 7.8, and 2 mM DTT at both sides of the membrane. Note that the channel was still open even at hyperpolarizing holding potentials of ±150 mV. (<b>F</b>) Current-voltage relationship of the high-conductance channel under asymmetric salt conditions: 3.0 M KCl <i>trans</i>/1.5 M KCl <i>cis</i> compartment. The insertion of a single channel was detected at 3 M KCl at both sides of the membrane and at a voltage of +10 mV, then the electrolyte concentration in the <i>cis</i> compartment was decreased by dilution and an initial current recording was conducted at zero potential followed by stepwise (±10 mV) change of the applied voltage. Data points are mean±SD, n = 4–5. Bars in some cases are smaller than symbols.</p

SCA of a low-conductance channel.

<p>(<b>A</b>) Current traces of two low-conductance channels. The bath solution (<b>A–C</b>) contained 3 M KCl at both sides of the membrane. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>A</b></a> for other details. Note that two types of channels were registered. Most of them showed a larger current amplitude at +10 mV than at −10 mV (upper trace). In contrast, some channels displayed an opposite trend (lower trace). (<b>B</b> and <b>C</b>) Current traces of the channels in response to the indicated voltage-ramp (<b>B</b>) and voltage-step (<b>C</b>) protocols. Most detected channels displayed a current rectification at negative holding potentials (upper panels). However, in a few cases the rectification was observed at positive holding potentials (lower panels). (<b>D</b>) Dependence of the low-conductance channel activity on the electrolyte concentration. The channel insertion was registered at a holding potential of +10 mV using 3 M KCl as a bath solution. After confirming that the channel shows current rectification at negative voltages by application of a voltage-ramp protocol, the electrolyte in the chambers was diluted to 2.0 M or 1.0 M KCl and the current amplitudes were measured at +10 mV. Data are mean±SD, n = 4–5. (<b>E</b>) Ion selectivity of the low-conductance channel. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>F</b></a> for details. The current-voltage relationship of channels (rectification at negative voltages) was validated using a voltage-ramp protocol.</p

Electron microscopy of cellular organelles separated by Optiprep gradient centrifugation.

<p>Fractions enriched in glycosomes (fractions 2–5, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>), fragments of flagella (fractions 8–11) or mitochondria and other organelles (fractions 15–18) were combined and processed for EM examination (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). (<b>A</b> and <b>B</b>) Isolated glycosomes shown at lower (<b>A</b>) and higher (<b>B</b>) magnifications. The fraction consists mostly of glycosomes. Some contamination by fragments of flagella is also visible. Importantly, fragments of flagella (paraflagellar rods and axonemes) show no sign of attachment to the flagellar membrane. Note the presence of intact glycosomes as electron-dense vesicles surrounded by a single membrane (marked by arrows in panel <b>B</b>). (<b>C</b> and <b>D</b>) Fractions enriched in flagella at low (<b>C</b>) and high (<b>D</b>) magnifications. One can see many paraflagellar rods in longitudinal section (<b>C</b>) and recognize flagellar axonemes (marked by arrows in panel <b>D</b>). Some glycosomes are also visible in panel <b>C</b>. (<b>E</b> and <b>F</b>) Composition of the fraction from the top of the Optiprep gradient that is enriched with mitochondria. Several types of organelles – mitochondria, lysosomes, lipid droplets, clathrin-coated vesicles, and components from the flagellar apparatus – can be observed. Note the shrinking of the mitochondrial inner membrane (see panel <b>F</b>) apparently due to osmotic misbalance. Scale bars: 2 µm (<b>C</b> and <b>E</b>); 1 µm (<b>A</b>); 0.5 µm (<b>D</b> and <b>F</b>), and 0.1 µm (<b>B</b>).</p

Relationship of intracellular MAPK pathway to RET receptor tyrosine kinase signaling.

<p><i>In situ</i> hybridization of the RET signaling target <i>Cxcr4</i> in E13.5 (A) control kidney shows expression in UB tips (arrow) as well as in kidney mesenchyme, while expression is lost in (B) UB tip (arrow) of dko kidney. (C–D) Another RET signaling target <i>Dusp6</i>, which is a negative regulator of MAPK activity, shows no changes (arrows) in the absence of MAPK pathway activity at E13.5. (E) Table summarizing the expression results of RET signaling targets in UB of dko kidneys. Scale bar: A–D 500 µm.</p

MAPK pathway activity is required for normal E-cadherin localization and epithelial cell adhesion.

<p>(A) Merged montage of confocal image through E13.5 control and (B) dko UB stained with calbindin (green) and E-cadherin (red) to show the cell-cell contacts. (C–D) Maximal intensity Z projections of stacks shown in A and B, respectively. Arrowheads point to the baso-lateral points, which show only occasional weak E-cadherin in control UB (A, C), but strong and widespread localization in the UB lacking MAPK activity (B, D). Asterisk indicates stronger intensity at lateral membranes in dko epithelium. (E) Electron microscopy image of E12.5 control UB shows continuous cell-cell contacts (arrows) while (F) in dko UB, the connections are disrupted at several sites (arrows), but the sites where they are maintained (arrowhead) appear electron dense. Also extracellular space (asterisk) between UB epithelial cells and metanephric mesenchymal (MM) cells is enlarged in dko kidneys. Scale bars: C–D 10 µm, E–F 2 µm.</p

Localization of MAPK pathway activity in developing kidney.

<p>(A–A′) Representative cross sections of wild type E11.5 kidneys stained with anti-phospho-ERK1/2 (red) demonstrate MAPK pathway activity both in the ureteric (UB) bud, visualized by calbindin staining (green), and metanephric mesenchyme (arrow). (B–B′) In E13.5 wild type kidneys, pERK1/2 localization in the ureteric bud epithelium is concentrated to UB tips where the staining is unevenly distributed among the epithelial cells (arrowheads), which all express UB marker calbindin (green). Additional pERK1/2 staining is detected in nephron primordia (arrows). (C) Chromogenic pERK1/2 staining on E14.5 wild type kidneys shows strong but heterogeneous MAPK activity in UB tips with lack of activity in sporadic cells (red arrowheads). (D) <i>Ret</i> expression in the ureteric bud tips of E13.5 wild type kidney as detected by <i>in situ</i> hybridization of mRNA on vibratome sections. In A and B, nuclei are labeled with Hoechst. Scale bars: A–B 50 µm, C–D 500 µm.</p

Relationship of intracellular MAPK pathway to RET receptor tyrosine kinase signaling.

<p><i>In situ</i> hybridization of the RET signaling target <i>Cxcr4</i> in E13.5 (A) control kidney shows expression in UB tips (arrow) as well as in kidney mesenchyme, while expression is lost in (B) UB tip (arrow) of dko kidney. (C–D) Another RET signaling target <i>Dusp6</i>, which is a negative regulator of MAPK activity, shows no changes (arrows) in the absence of MAPK pathway activity at E13.5. (E) Table summarizing the expression results of RET signaling targets in UB of dko kidneys. Scale bar: A–D 500 µm.</p

Genetic ablation of MAPK pathway specifically in ureteric bud epithelium results in severe renal hypodysplasia.

<p>(A) Newborn kidneys with <i>Mek1</i> deleted specifically in ureteric bud (UB) derivatives are comparable to wild type kidneys. (B) Absence of three out of four <i>Mek1</i> and Mek<i>2</i> alleles is enough to support normal kidney development as shown in newborn Hoxb7CreGFP;<i>Mek1^F/+</i>;<i>Mek2^-/-</i> kidneys. (C) Simultaneous lack of <i>Mek1</i> and <i>Mek2</i> in UB results in very small kidneys with hydroureters (arrow). Histology of newborn (D) control and (E) dko kidneys demonstrates huge cysts (yellow arrowheads) and reduced nephron epithelium in double knockout newborns. Calbindin staining visualizes numerous UBs and collecting ducts in (F) newborn control kidney whereas (G) the amount of collecting duct epithelia is minimal and dilated in dko kidneys. Asterisks mark glomeruli; a, adrenal gland; b, bladder; CD, collecting duct; k, kidney. Scale bars: A–C 1 mm, D–G 100 µm.</p