Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity.

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H(+) -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K(i) for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.

Monitoring the metabolic state of fungal hyphae and the presence of melanin by nonlinear spectral imaging.

Label-free nonlinear spectral imaging microscopy (NLSM) records two-photon-excited fluorescence emission spectra of endogenous fluorophores within the specimen. Here, NLSM is introduced as a novel, minimally invasive method to analyze the metabolic state of fungal hyphae by monitoring the autofluorescence of NAD(P)H and flavin adenine dinucleotide (FAD). Moreover, the presence of melanin was analyzed by NLSM. NAD(P)H, FAD, and melanin were used as biomarkers for freshness of mushrooms of Agaricus bisporus (white button mushroom) that had been stored at 4°C for 0 to 17 days. During this period, the mushrooms did not show changes in morphology or color detectable by eye. In contrast, FAD/NAD(P)H and melanin/NAD(P)H ratios increased over time. For instance, these ratios increased from 0.92 to 2.02 and from 0.76 to 1.53, respectively, at the surface of mushroom caps that had been harvested by cutting the stem. These ratios were lower under the skin than at the surface of fresh mushrooms (0.78 versus 0.92 and 0.41 versus 0.76, respectively), indicative of higher metabolism and lower pigment formation within the fruiting body. Signals were different not only between tissues of the mushroom but also between neighboring hyphae. These data show that NLSM can be used to determine the freshness of mushrooms and to monitor the postharvest browning process at an early stage. Moreover, these data demonstrate the potential of NLSM to address a broad range of fundamental and applied microbiological processes.

Platelet interaction with von Willebrand factor is enhanced by shear-induced clustering of glycoprotein Ibα.

Initial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s(-1) induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s(-1). Shear-induced clustering was reversible, not accompanied by granule release or αIIbß3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation.

Highly luminescent (Zn,Cd)Te/CdSe colloidal heteronanowires with tunable electron-hole overlap.

We report the synthesis of ultranarrow (Zn,Cd)Te/CdSe colloidal heteronanowires, using ZnTe magic size clusters as seeds. The wire formation starts with a partial Zn for Cd cation exchange, followed by self-organization into segmented heteronanowires. Further growth occurs by inclusion of CdSe. The heteronanowires emit in the 530 to 760 nm range with high quantum yields. The electron-hole overlap decreases with increasing CdSe volume fraction, allowing the optical properties to be controlled by adjusting the heteronanowire composition.

Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts.

BACKGROUND: Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor. DESIGN AND METHODS: We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy. RESULTS: Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-D-glucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions. CONCLUSIONS: We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future.

Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization.

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ∼40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.

Design and application of a confocal microscope for spectrally resolved anisotropy imaging.

Biophysical imaging tools exploit several properties of fluorescence to map cellular biochemistry. However, the engineering of a cost-effective and user-friendly detection system for sensing the diverse properties of fluorescence is a difficult challenge. Here, we present a novel architecture for a spectrograph that permits integrated characterization of excitation, emission and fluorescence anisotropy spectra in a quantitative and efficient manner. This sensing platform achieves excellent versatility of use at comparatively low costs. We demonstrate the novel optical design with example images of plant cells and of mammalian cells expressing fluorescent proteins undergoing energy transfer.

Quantum dot and Cy5.5 labeled nanoparticles to investigate lipoprotein biointeractions via Förster resonance energy transfer.

The study of lipoproteins, natural nanoparticles comprised of lipids and apolipoproteins that transport fats throughout the body, is of key importance to better understand, treat, and prevent cardiovascular disease. In the current study, we have developed a lipoprotein-based nanoparticle that consists of a quantum dot (QD) core and Cy5.5 labeled lipidic coating. The methodology allows judicious tuning of the QD/Cy5.5 ratio, which enabled us to optimize Förster resonance energy transfer (FRET) between the QD core and the Cy5.5-labeled coating. This phenomenon allowed us to study lipoprotein-lipoprotein interactions, lipid exchange dynamics, and the influence of apolipoproteins on these processes. Moreover, we were able to study HDL-cell interactions and exploit FRET to visualize HDL association with live macrophage cells.

Mapping Elevated Temperatures with a Micrometer Resolution Using the Luminescence of Chemically Stable Upconversion Nanoparticles.

The temperature-sensitive luminescence of nanoparticles enables their application as remote thermometers. The size of these nanothermometers makes them ideal to map temperatures with a high spatial resolution. However, high spatial resolution mapping of temperatures >373 K has remained challenging. Here, we realize nanothermometry with high spatial resolutions at elevated temperatures using chemically stable upconversion nanoparticles and confocal microscopy. We test this method on a microelectromechanical heater and study the temperature homogeneity. Our experiments reveal distortions in the luminescence spectra that are intrinsic to high-resolution measurements of samples with nanoscale photonic inhomogeneities. In particular, the spectra are affected by the high-power excitation as well as by scattering and reflection of the emitted light. The latter effect has an increasing impact at elevated temperatures. We present a procedure to correct these distortions. As a result, we extend the range of high-resolution nanothermometry beyond 500 K with a precision of 1-4 K. This work will improve the accuracy of nanothermometry not only in micro- and nanoelectronics but also in other fields with photonically inhomogeneous substrates.

Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy.

In this work, gold nanoparticles coated with a fluorescently labelled (rhodamine B) silica shell are presented as fiducial markers for correlative light and electron microscopy (CLEM). The synthesis of the particles is optimized to obtain homogeneous, spherical core-shell particles of arbitrary size. Next, particles labelled with different fluorophore densities are characterized to determine under which conditions bright and (photo)stable particles can be obtained. 2 and 3D CLEM examples are presented where optimized particles are used for correlation. In the 2D example, fiducials are added to a cryosection of cells whereas in the 3D example cells are imaged after endocytosis of the fiducials. Both examples demonstrate that the particles are clearly visible in both modalities and can be used for correlation. Additionally, the recognizable core-shell structure of the fiducials proves to be very powerful in electron microscopy: it makes it possible to irrefutably identify the particles and makes it easy to accurately determine the center of the fiducials.

TRAK/Milton motor-adaptor proteins steer mitochondrial trafficking to axons and dendrites.

In neurons, the distinct molecular composition of axons and dendrites is established through polarized targeting mechanisms, but it is currently unclear how nonpolarized cargoes, such as mitochondria, become uniformly distributed over these specialized neuronal compartments. Here, we show that TRAK family adaptor proteins, TRAK1 and TRAK2, which link mitochondria to microtubule-based motors, are required for axonal and dendritic mitochondrial motility and utilize different transport machineries to steer mitochondria into axons and dendrites. TRAK1 binds to both kinesin-1 and dynein/dynactin, is prominently localized in axons, and is needed for normal axon outgrowth, whereas TRAK2 predominantly interacts with dynein/dynactin, is more abundantly present in dendrites, and is required for dendritic development. These functional differences follow from their distinct conformations: TRAK2 preferentially adopts a head-to-tail interaction, which interferes with kinesin-1 binding and axonal transport. Our study demonstrates how the molecular interplay between bidirectional adaptor proteins and distinct microtubule-based motors drives polarized mitochondrial transport.

EGF induces coalescence of different lipid rafts.

The suggestion that microdomains may function as signaling platforms arose from the presence of growth factor receptors, such as the EGFR, in biochemically isolated lipid raft fractions. To investigate the role of EGFR activation in the organization of lipid rafts we have performed FLIM analyses using putative lipid raft markers such as ganglioside GM1 and glycosylphosphatidylinositol (GPI)-anchored GFP (GPI-GFP). The EGFR was labeled using single domain antibodies from Llama glama that specifically bind the EGFR without stimulating its kinase activity. Our FLIM analyses demonstrate a cholesterol-independent colocalization of GM1 with EGFR, which was not observed for the transferrin receptor. By contrast, a cholesterol-dependent colocalization was observed for GM1 with GPI-GFP. In the resting state no colocalization was observed between EGFR and GPI-GFP, but stimulation of the cell with EGF resulted in the colocalization at the nanoscale level of EGFR and GPI-GFP. Moreover, EGF induced the enrichment of GPI-GFP in a detergent-free lipid raft fraction. Our results suggest that EGF induces the coalescence of the two types of GM1-containing microdomains that might lead to the formation of signaling platforms.

Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells.

Fluorescent proteins have become an invaluable tool in cell biology. The green fluorescent protein variant EGFP is especially widely applied. Use of fluorescent proteins, including EGFP, however can be hindered by inefficient protein folding, resulting in protein aggregation and reduced fluorescence. This is especially profound in prokaryotic cells. Furthermore, EBFP, a blue fluorescent variant of EGFP, is rarely used because of its dim fluorescence and fast photobleaching. Thus, efforts to improve properties such as protein folding, fluorescence brightness, and photostability are important. Strongly enhanced green fluorescent (SGFP2) and strongly enhanced blue fluorescent (SBFP2) proteins were created, based on EGFP and EBFP, respectively. We used site-directed mutagenesis to introduce several mutations, which were recently shown to improve the fluorescent proteins EYFP and ECFP. SGFP2 and SBFP2 exhibit faster and more efficient protein folding and accelerated chromophore oxidation in vitro. For both strongly enhanced fluorescent proteins, the photostability was improved 2-fold and the quantum yield of SBFP2 was increased 3-fold. The improved folding efficiency reduced the extent of protein aggregation in Escherichia coli, thereby increasing the brightness of bacteria expressing SGFP2 7-fold compared to the brightness of those expressing EGFP. Bacteria expressing SBFP2 were 16-fold more fluorescent than those expressing EBFP. In mammalian cells, the improvements were less pronounced. Cells expressing SGFP2 were 1.7-fold brighter than those expressing EGFP, which was apparently due to more efficient protein expression and/or chromophore maturation. Mammalian cells expressing SBFP2 were 3.7-fold brighter than cells expressing EBFP. This increase in brightness closely resembled the increase in intrinsic brightness observed for the purified recombinant protein. The increased maturation efficiency and photostability of SGFP2 and SBFP2 facilitate detection and extend the maximum duration of fluorescence imaging.