Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form

The pigmented life of a redhead.

As a redhead I have had a personal interest in red hair, freckles and sunburns since childhood. An observation of a formaldehyde-induced fluorescence in human epidermal melanocytes initiated my scientific interest in these cells. Prota and Nicolaus demonstrated that oxidation products of cysteinyldopas are the main components of pheomelanin. Our identification of 5-S-cysteinyldopa as the source of formaldehyde-induced fluorescence of normal and pathological melanocytes started a series of investigations into this amino acid, enzymatic and non-enzymatic oxidation of catecholic compounds and the metabolism of thiols. All melanocytes with functioning tyrosinase produce cysteinyldopas and the levels of 5-S-cysteinyldopa in serum and urine are related to the size and pigment forming activity of the melanocyte population. The determination of 5-S-cysteinyldopa in serum or urine is a sensitive diagnostic method in the detection of melanoma metastasis. Some non-specific formation of cysteinyldopa is present in the body, as demonstrated by 5-S-cysteinyldopa in individuals with tyrosinase-negative albinism

Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.close816

Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy

Kottke T, van de Linde S, Sauer M, Kakorin S, Heilemann M. Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy. JOURNAL OF PHYSICAL CHEMISTRY LETTERS. 2010;1(21):3156-3159