Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species

By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques

Atomic force microscopy (AFM).

<p>Trypomastigotes were AFM imaged by tapping mode. Upper panels show height traces and bottom panels show 3D transformation. Special focus was done over the flagellum where heterogeneous and irregular domains following parallel structures along the flagellum could be observed. Arrows indicate neighboring domains to highlight size distribution and domain separation. Flag: flagellum.</p

Sialylated mucins and TS are included in segregated membrane microdomains.

<p>A) Live trypomastigotes were sialylated and FLAG-tagged. Sialylated mucins and TS rendered a dotted pattern on the trypomastigotes surface that did not co-localize (confocal microscopy). Line profiles for sialic acid and TS signal in the flagellum (boxed area) showed that mucins and TS do not co-localize but were rather out-of-phase with each other. GSDIM superresolution fluorescence microscopy performed for sialylated mucins (B) and TS (C) independently showed that mucins were included in domains 90 nm wide and separated 120-500nm from each other. Results for TS were equivalent. Size and distribution of trypomastigote membrane domains explain why under a confocal microscope, restricted to classical light diffraction limits, the domains for TS and mucins were not fully resolved.</p

The membrane of trypomastigotes is complex to the nanometer scale.

<p>Membrane model for <i>T</i>. <i>cruzi</i> trypomastigotes. The surface is packed in microdomains of different size, shape, lipid composition and embedded proteins. Some of these domains are detergent resistant, however this does not imply a functional profile. Mucins are included in DRMs whereas TS is not, thus being segregated in the membrane of trypomastigotes. This challenges the membrane bound TS as the sialylating factor for mucins, a role proposed for the shed TS instead. DRMs embed different proteins, many of them localized to the flagellum. Flagellum domains tend to be smaller and closer together than those in the cell body and suggest an association to the flagellar cytoskeleton. Mucins and TS are shed to the extracellular environment included in microvesicles probably resulting from membrane budding and fission events. Furthermore, TS is shed associated to vesicles instead of as a soluble protein. No hydrolysis of the GPI-anchors occurs in the trypomastigote stage.</p

Distribution of proteins contained in DRMs.

<p>Proteins identified by mass spectrometry were analyzed by immunofluorescence. KMP, TolT and CLCP were located in domains of the flagellum. ADK-1 displayed a dotted pattern in the cell body and in the flagellum. No co-localization with mucins or with TS was found. Arrow points to an amastigote, this stage remains unsialylated. Bar: 5μm.</p

Mucin sialylation and characterization.

<p>A) Mucins (or any glycoconjugate bearing a terminal β-galactose) may be tagged by the TS using a Neu5Az donor such as Neu5Azα(2–3)LacβOMe. Then the Azide group may be coupled via the Staudinger-Bertozzi chemistry or the Cu²⁺-free click chemistry to obtain a FLAG or biotin tag ready for detection. B) Western blot of Neu5Az <i>trans</i>-sialylated trypomastigote lysates revealing the relative molecular mass distribution of acceptor molecules. A line profile of the blot is also plotted. Neg Ctrl: Negative control. C) Neu5Az <i>trans</i>-sialylated parasites were submitted to organic solvents extraction as described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005559#ppat.1005559.ref020" target="_blank">20</a>] to determine their mucin nature. Extracted material was subjected to Western blot. F1, F2 and F3 refer to the different purification fractions (for details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005559#sec010" target="_blank">M&M</a>). D) Neu5Az <i>trans</i>-sialylated trypomastigotes (900x10⁶) were lysed and sialylated proteins pulled-down with anti-FLAG antibodies. Western blots of this material were revealed with anti-TcMUC II antibodies. E-F) Confocal images displaying partial colocalization of anti-FLAG and anti-TcMUC II (E) or anti-αGal (F) labeling at the parasite surface.</p

Shedding kinetics of sialylated mucins.

<p>Living parasites were incubated with Neu5Az donor. A) Acquired Neu5Az was followed by flow cytometry to determine its shedding kinetics. B) Western blot and C) immunofluorescence of the parasites from (A). Western blots indicate that Neu5Az was readily accepted into diffuse bands between 80-150kDa corresponding to the trypomastigote mucins [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005559#ppat.1005559.ref007" target="_blank">7</a>]. The shed material was recovered in the conditioned media indicating that the residue was not hydrolyzed but conserved in the shed material. C) Fluorescence microscopy. Sialylated mucins fluorescence intensity fades while TS labeling remains. Raw images are shown to avoid biased observations. Bar: 10μm.</p

Sialylated mucins are included in lipid rafts whereas TS is not.

<p>(A) Cold Triton X-100 partition. Sialylated trypomastigotes were lysed at 4°C. Mucins were predominantly recovered in the pellet whereas TS and HSP70, a cytosolic protein, were recovered in the supernatant. (B) Triton X-114 extraction for GPI-anchored proteins. Parasites were lysed at 4°C and detergent and aqueous phases separated at 37°C and analyzed by Western blots. Mucins and TS partitioned in the detergent phase due to their GPI-anchoring. Glutamate Dehydrogenase, a cytosolic protein, was recovered in the aqueous phase. (C) Purification of DRMs by step-gradient ultracentrifugation. Trypomastigotes were lysed in Triton X-100 at 4°C or 37°C and centrifuged in an Optiprep gradient. Mucins floated to the 35%-5% interface (lane 6) only when lysis was done at 4°C indicating its DRM nature in contrast to TS. (D) Living parasites were sialylated from a Neu5Az donor, then treated for membrane fluidization with 1% diethyl ether in phosphate-buffered saline (PBS) and fixed with <i>p</i>-formaldehyde (PFA). Doted labeling for TS and mucins was disrupted only after 90sec treatment even showing colocalization.</p

Selected Hits from Mass Spectrometry Assays from Lipid Rafts Proteins.

<p>Selected Hits from Mass Spectrometry Assays from Lipid Rafts Proteins.</p