Tetraspanin CD82 regulates bone marrow homing of acute myeloid leukemia by modulating the molecular organization of N-cadherin.

Communication between acute myeloid leukemia (AML) and the bone marrow microenvironment is known to control disease progression. Therefore, regulation of AML cell trafficking and adhesion to the bone marrow is of significant interest. In this study, we demonstrate that differential expression of the membrane scaffold CD82 modulates the bone marrow homing of AML cells. By combining mutational analysis and super-resolution imaging, we identify membrane protein clustering by CD82 as a regulator of AML cell adhesion and bone marrow homing. Cluster analysis of super-resolution data indicates that N-linked glycosylation and palmitoylation of CD82 are both critical modifications that control the microdomain organization of CD82 as well as the nanoscale clustering of associated adhesion protein, N-cadherin. We demonstrate that the inhibition of CD82 glycosylation increases the molecular packing of N-cadherin and promotes the bone marrow homing of AML cells. In contrast, we find that the inhibition of CD82 palmitoylation disrupts the formation and organization of N-cadherin clusters and significantly diminishes bone marrow trafficking of AML. Taken together, these data establish a mechanism where the membrane organization of CD82, through specific posttranslational modifications, regulates N-cadherin clustering and membrane density, which impacts the in vivo trafficking of AML cells. As such, these observations provide an alternative model for targeting AML where modulation of protein organization within the membrane may be an effective treatment therapy to disrupt the bone marrow homing potential of AML cells.

Dual-color superresolution microscopy reveals nanoscale organization of mechanosensory podosomes.

Podosomes are multimolecular mechanosensory assemblies that coordinate mesenchymal migration of tissue-resident dendritic cells. They have a protrusive actin core and an adhesive ring of integrins and adaptor proteins, such as talin and vinculin. We recently demonstrated that core actin oscillations correlate with intensity fluctuations of vinculin but not talin, suggesting different molecular rearrangements for these components. Detailed information on the mutual localization of core and ring components at the nanoscale is lacking. By dual-color direct stochastic optical reconstruction microscopy, we for the first time determined the nanoscale organization of individual podosomes and their spatial arrangement within large clusters formed at the cell-substrate interface. Superresolution imaging of three ring components with respect to actin revealed that the cores are interconnected and linked to the ventral membrane by radiating actin filaments. In core-free areas, αMß2 integrin and talin islets are homogeneously distributed, whereas vinculin preferentially localizes proximal to the core and along the radiating actin filaments. Podosome clusters appear as self-organized contact areas, where mechanical cues might be efficiently transduced and redistributed. Our findings call for a reevaluation of the current "core-ring" model and provide a novel structural framework for further understanding the collective behavior of podosome clusters.

Double-pass Fourier transform imaging spectroscopy.

Fourier-Transform Imaging Spectroscopy (FTIS) has recently become a widely used tool for spectral imaging of biological fluorescent samples. Here we report on a novel double-pass FTIS system, that is capable of obtaining an excitation as well as an emission spectrum of the fluorescent sample with only a single sweep of the interferometer. This is achieved by modifying an existing FTIS system, placing the excitation source before the interferometer so as to spectrally modulate the excitation as well as the detection. An analysis of the acquired signal allows for the reconstruction of the excitation as well as the emission spectrum of each fluorophore assuming an independence of the two spectra for each fluorophore. Due to the patterned excitation generated by the Sagnac interferometer a substantial degree of optically sectioning is achieved at excitation wavelengths. Further analysis of the acquired data also enables the generation of optically sectioned emission images. A theoretical analysis and experimental data based on fluorescent beads are presented.

Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells.

In this work, we characterized streptavidin-conjugated quantum dots (QDs) manufactured by Quantum Dot Corporation. We present data on: (1) two-photon excitation; (2) fluorescence lifetimes; (3) ensemble and single QD emission anisotropy; (4) QDs as donors for Forster resonance energy transfer (FRET); and (5) spectral conversion of QDs exposed to high-intensity illumination. We also demonstrate the utility of QDs for (1) imaging the binding and uptake of biotinylated transferrin on living cells, and (2) resolving by fluorescence lifetime imaging microscopy (FLIM) signals originating from QDs from those of spatially and spectrally overlapping visible fluorescent proteins (VFPs).

Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET).

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.

Dynamics of low-energy helium vapor pulses.

We report results of experiments in which pulses of helium vapor are produced by a current pulse in a chromium film covered with superfluid helium at around 0.3 K. The pulses were detected by a titanium bolometer operating at 0.47 K. The shape of the detected signal is a strong function of the power of the initiating current pulse. For low powers the signal from a single current pulse also contains a single peak, but for higher powers, a single current pulse produces two and then at the highest powers, three peak signals. To analyze the origin of these phenomena we report results of hybrid gas-dynamics and hydrodynamics simulations, which demonstrate that the signals arise from shock waves formed in the vapor. The shock waves form due to the presence of a gradient in the small ambient background of helium vapor in the chamber and are extremely sensitive to the pulse power.