Evolution of Thermal Response Properties in a Cold-Activated TRP Channel

Animals sense changes in ambient temperature irrespective of whether core body temperature is internally maintained (homeotherms) or subject to environmental variation (poikilotherms). Here we show that a cold-sensitive ion channel, TRPM8, displays dramatically different thermal activation ranges in frogs versus mammals or birds, consistent with variations in these species' cutaneous and core body temperatures. Thus, somatosensory receptors are not static through evolution, but show functional diversity reflecting the characteristics of an organism's ecological niche

<i>X. laevis</i> TRPM8 is activated by menthol but not icilin.

<p>(A) In oocytes expressing xlTRPM8, application of menthol (1 mM) evoked inward currents (green trace) that were suppressed by a rise in bath temperature (gray trace). Holding potential was −60 mV. (B) Cell-attached patches from oocytes expressing xlTRPM8 (n = 4) but not uninjected oocytes (n = 3) displayed a strongly rectifying current (green trace) when the pipette solution contained 500 µM menthol. Formation of the inside-out configuration resulted in rapid current rundown (black trace, 90 seconds after patch excision.) (C) Concentration-response relation for menthol-evoked currents (at +80 mV) from rat (red) or <i>X. laevis</i> (green) TRPM8-expressing oocytes. (D) Application of 10 µM icilin in the presence of 2 mM extracellular Ca²⁺ failed to activate xlTRPM8, while 1 mM menthol evoked robust inward currents.</p

Cloned <i>Xenopus</i> TRPM8 channels show alterations in thermosensitivity compared to their mammalian and avian counterparts.

<p>(A) Current-voltage relation from a two-electrode voltage clamp recording of xlTRPM8-expressing oocytes under basal or cold-stimulated conditions. Note the strong outward rectification of the cold-evoked current. (B) Normalized inward (−60 mV) and outward (+80 mV) cold-evoked currents for xlTRPM8. The current obtained at a given temperature was normalized to the maximum cold-evoked current (obtained at 6°C) obtained at each potential. (C) Normalized current-temperature plots (at +80 mV) for chicken (blue), rat (red), <i>X. laevis</i> (green) and <i>X. tropicalis</i> (orange) TRPM8, yielding half-maximal activation temperatures of 29.35±0.21°C (chicken), 24.00±0.43°C (rat), 13.89±0.39°C (<i>X. laevis</i>), and 13.90±0.44°C (<i>X. trop.</i>). (D) Plot of species core body temperature (obtained from previously published measurements) versus experimentally determined temperature of half-maximal TRPM8 cold activation. Core temperatures were based on previously published values (for <i>Xenopus</i>, core temperatures were estimated as the arithmetic mean and error bars indicate the full range of tolerated temperatures) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005741#pone.0005741-Hirsch1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005741#pone.0005741-Yochim1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005741#pone.0005741-Lin1" target="_blank">[19]</a>.</p

Sequence comparison of TRPM8 species orthologs.

<p>(A) Previously described rat, human, and chicken TRPM8 sequences were aligned with the full-length sequences of <i>X. tropicalis</i> and <i>X. laevis</i> TRPM8 (this study) using MultAlin and ESPript. The locations of predicted transmembrane helices <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005741#pone.0005741-McKemy1" target="_blank">[6]</a> and the C-terminal coiled-coiled assembly domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005741#pone.0005741-Tsuruda1" target="_blank">[25]</a> are shown as black and gray bars, respectively. The asterisk indicates the polymorphic residue previously shown to determine TRPM8 icilin sensitivity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005741#pone.0005741-Chuang1" target="_blank">[17]</a>. (B) Phylogenetic tree indicating the evolutionary relationship between TRPM8 ortholog sequences.</p

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets

Correlative 3D STORM and SEM imaging of individual filamentous influenza viruses.

<p>(A) Flowchart of the major steps in the correlative 3D STORM and SEM imaging of unembedded samples. (B) Correlative 3D STORM and SEM images of Udorn virus immuno-labeled for HA. Left: STORM image. Right: SEM image. Middle: Overlaid image. (C) Magnified views of the boxed regions in (B). (D) Transverse profiles of localizations corresponding to regions boxed in red in (B). Blue bars: localization frequency measured from the STORM image. Red line: Gaussian fit of the blue bars. (E) Normalized photon numbers per switching event. The results are normalized to the average photon number obtained from samples on the No. 1.5 glass coverslip (photon number = 5233). (F) Cross-correlation between STORM and SEM images. (G) Correlative two-color STORM and SEM images of budding Udorn virus filaments immuno-labeled for M1 (red) and vRNP (green). Left: STORM image. Right: SEM image. Middle: Overlaid image. Scale bars, 500 nm in (B,C and G).</p

Correlative 3D STORM and TEM images of immunolabeled microtubules in a BS-C-1 cell.

<p>(A) 3D STORM image of microtubules in a BS-C-1 cell on a No. 1.5 glass coverslip. (B) Magnified views of the boxed regions in (A). (C) Transverse profiles of localizations corresponding to regions boxed in white in b. Blue bars: localization frequency measured from the STORM image. Red line: Gaussian fit of the blue bars. (D) Flowchart of the major steps in correlative 3D STORM and TEM imaging of unembedded samples. (E) Scheme of the SiN window and the sample mounting geometry. (F) Normalized number of photons detected on the samples mounted on the normal No. 1.5 coverslip and the SiN window. The results are normalized to the average photon number obtained from samples on the No. 1.5 glass coverslip (photon number = 5233). (G) Correlative 3D STORM and TEM images of microtubules in a BS-C-1 cell. Left: STORM image. Right: TEM image. Middle: Overlaid image. (H) Magnified views of the boxed regions in (G). Red arrows in TEM image: thick filaments corresponding to microtubules. (I) Transverse profiles of localizations corresponding to regions boxed in white in (G). Blue bars: localization frequency measured from the STORM image. Red line: Gaussian fit of the blue bars. (J) Cross-correlation between STORM and TEM images. Scale bars, 5 μm in (A,G) and 500 nm in (B,H).</p

Correlative STORM and SEM-BSE images of resin-embeded sections containing filamentous influenza viruses budding from infected cells.

<p>(A) Flowchart of the major steps in correlative 3D STORM and BSE-SEM imaging of embedded samples. (B) Correlative STORM and EM images of influenza infected A549 cells immuno-stained for HA. Left: STORM image. Right: SEM image. Middle: Overlaid image. (C) Magnified views of the boxed regions in a (B). Inset in yellow box: STORM image of an influenza virus filament, immuno-stained for HA, embedded in Ultrabed section without etching. (D) Magnified views of the boxed regions in (D). (E) Transverse profiles of localizations corresponding to regions in the white box in (D). Blue bars: localization frequency measured from the STORM image. Red line: Gaussian fit of the blue bars. (F) Cross-correlation between STORM and BSE-SEM images. Scale bars, 5 μm in (B) and 500 nm in (C, D).</p