Martin Straub

KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: measurements in living cells using FRET.

How the occupied KDEL receptor ERD2 is sorted into COPI vesicles for Golgi-to-ER transport is largely unknown. Here, interactions between proteins of the COPI transport machinery occurring during a wave of transport of a KDEL ligand were studied in living cells. FRET between CFP and YFP fusion proteins was measured by multifocal multiphoton microscopy and bulk-cell spectrofluorimetry. Ligand binding induces oligomerization of ERD2 and recruitment of ARFGAP to the Golgi, where the (ERD2)n/ARFGAP complex interacts with membrane-bound ARF1. During KDEL ligand transport, interactions of ERD2 with beta-COP and p23 decrease and the proteins segregate. Both p24a and p23 interact with ARF1, but only p24 interacts with ARFGAP. These findings suggest a model for how cargo-induced oligomerization of ERD2 regulates its sorting into COPI-coated buds.

Live cell imaging by multifocal multiphoton microscopy.

Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.

Multifocal multiphoton microscopy: a fast and efficient tool for 3-D fluorescence imaging

Multifocal multiphoton microscopy (MMM) is an efficient and technically simple method for generating multiphoton fluorescence images. Featuring the high axial resolution of confocal and multiphoton scanning microscopes, MMM also achieves high speed in 3-D microscopy. In this paper, examples of fast-mode 3-D microscopy are given including imaging of fixed brain tissue as well as living PC12 cells. The imaging speed of MMM is solely determined by the fluorescence photon flux, so that in practice, for brightly fluorescent specimens, a whole stack of about 50 images of 30-70 m diameter can be acquired within a few seconds. MMM represents a significant advance towards high speed nonlinear optical tomography of fluorescent specimens.

Fluorescence resonance energy transfer analysis of protein–protein interactions in single living cells by multifocal multiphoton microscopy

Fluorescence resonance energy transfer (FRET) resolved by multifocal multiphoton microscopy (MMM) was successfully used to measure transport phenomena in living cells. We expressed different pairs of CFP-/YFP-fusion proteins involved in retrograde Golgi-to-ER transport to analyze sorting of the occupied KDEL-receptor into retrograde transport vesicles triggered by application of the external cholera toxin mutant CTXK63. FRET observed as a sensitized emission of the acceptor was confirmed by acceptor photobleaching and the dequenching of the donor was measured. FRET-MMM data obtained from single cells were compared with bulk cell experiments employing spectrofluorimetry. The importance of controlling the degree of overexpression of CFP-/YFP-fusion proteins for FRET analysis is stressed in this article. Using MMM we showed for the first time that FRET can be measured across the Golgi membrane. Finally, FRET-MMM records performed continuously over 2 h allowed to analyze intracellular retrograde transport and sorting events and to discuss these mechanisms on a single cell level.

New slant on photonic crystal fibers

We present the novel use of microstructured optical fibers not as light-pipes, but in a transverse geometry to manipulate the light propagating across the fiber. Fundamental and higher-order bandgaps were observed experimentally in this geometry using a number of techniques. The comparison of the measured spectra with photonic band structure and Finite-Difference Time-Domain simulations provide strong evidence that the spectral features are a result of the periodic nature of the fiber microstructure in the transverse direction.

Optical tuning of three-dimensional photonic crystals fabricated by femtosecond direct writing

In this letter, we report on an optically tunable three-dimensional photonic crystal that exhibits main gaps in the 3–4μm range. The photonic crystal is manufactured via a femtosecond direct writing technique. Optical tuning is achieved by a luminary polling technique with a low-power polarized laser beam. The refractive index variation resulting from liquid-crystal rotation causes a shift in the photonic band gap of up to 65 nm with an extinction of transmission of up to 70% in the stacking direction. Unlike other liquid-crystal tuning techniques where a pregenerated structure is infiltrated, this optical tuning method is a one-step process that allows arbitrary structures to be written into a solid liquid-crystal-polymer composite and leads to a high dielectric contrast.

Planar cavity modes in void channel polymer photonic crystals

Planar dielectric microcavities embedded in woodpile void channel photonic crystals with stop bands in the stacking direction ranging from 4.3 to 4.8 microm in wavelength were generated by femtosecond-laser direct writing in a solid polymer. Infrared transmission spectra revealed fundamental and second-order modes crossing the stop gap region with a free spectral range of 430 nm on varying the microcavity size from 0.3 to 2.25 microm. Supercell calculations confirmed the cavity size dependence of highly localized cavity modes, whereas the angle of incidence was accounted for using a simple Fabry-Perot model.

Nonlinear magneto-optical Kerr measurements on Fe(110)

For the clean Fe(110) surface Kerr angles close to 90° are observed in the frequency doubled light and for the oxygen covered surface asymmetries up to 100%. In the wavelength range from 740 to 840 nm a sizable amount of the second harmonic light is possibly generated by the bulk quadrupole like nonlinear polarization especially for p-polarized incident light. For s-polarized incident light a strong surface contribution is found.

Second harmonic generation from the Cu(001) surface

We report on measurements of the second harmonic (SH) yield from a Cu(001) surface in the wavelength range between 740 nm and 840 nm which is below the one photon excitation threshold of d-electrons. The SH-yield varies strongly with the azimuthal angle of the Cu crystal indicating a significant contribution of bulk anisotropic SH generation.

Multifocal Multiphoton Microscopy as an efficient 4-D imaging method

By splitting the beam of a mode-locked Ti:sapphire laser into an array of ~25 foci, multifocal multi-photon microscopy (MMM) is a fast and technically simple method for generating 3D-images (fig. 1) [1,2].