Unscrambling exit site patterns on the endoplasmic reticulum as a quenched demixing process.

The endoplasmic reticulum (ER) is a vital organelle in mammalian cells with a complex morphology. Consisting of sheet-like cisternae in the cell center, the peripheral ER forms a vast tubular network on which a dispersed pattern of a few hundred specialized domains (ER exit sites (ERESs)) is maintained. Molecular details of cargo sorting and vesicle formation at individual ERESs, fueling the early secretory pathway, have been studied in some detail. The emergence of spatially extended ERES patterns, however, has remained poorly understood. Here, we show that these patterns are determined by the underlying ER morphology, suggesting ERESs to emerge from a demixing process that is quenched by the ER network topology. In particular, we observed fewer but larger ERESs when transforming the ER network to more sheet-like morphologies. In contrast, little to no changes with respect to native ERES patterns were observed when fragmenting the ER, indicating that hampering the diffusion-mediated coarse graining of domains is key for native ERES patterns. Model simulations support the notion of effective diffusion barriers impeding the coarse graining and maturation of ERES patterns. We speculate that tuning a simple demixing mechanism by the ER topology allows for a robust but flexible adaption of ERES patterns, ensuring a properly working early secretory pathway in a variety of conditions.

Diffusion of Exit Sites on the Endoplasmic Reticulum: A Random Walk on a Shivering Backbone.

Major parts of the endoplasmic reticulum (ER) in eukaryotic cells are organized as a dynamic network of membrane tubules connected by three-way junctions. On this network, self-assembled membrane domains, called ER exit sites (ERES), provide platforms at which nascent cargo proteins are packaged into vesicular carriers for subsequent transport along the secretory pathway. Although ERES appear stationary and spatially confined on long timescales, we show here via single-particle tracking that they exhibit a microtubule-dependent and heterogeneous anomalous diffusion behavior on short and intermediate timescales. By quantifying key parameters of their random walk, we show that the subdiffusive motion of ERES is distinct from that of ER junctions, i.e., ERES are not tied to junctions but rather are mobile on ER tubules. We complement and corroborate our experimental findings with model simulations that also indicate that ERES are not actively moved by microtubules. Altogether, our study shows that ERES perform a random walk on the shivering ER backbone, indirectly powered by microtubular activity. Similar phenomena can be expected for other domains on subcellular structures, setting a caveat for the interpretation of domain-tracking data.

Atomic diffusion studied with coherent X-rays.

Knowledge of atomic diffusion is a fundamental issue in synthesis and stability of materials. Direct studies of the elementary diffusion event, that is, how the individual atoms 'jump', are scarce, as the available techniques are limited to selected systems. Here we show how by monitoring the spatial and temporal variations of the scattered coherent X-ray intensity the diffusion of single atoms can be studied. This is demonstrated for the intermetallic alloy Cu(90)Au(10). By measuring along several directions in reciprocal space, we can elucidate the dynamical behaviour of single atoms as a function of their neighbourhood. This method, usually referred to as X-ray photon correlation spectroscopy (XPCS), does not rely on specific atomic species or isotopes and can thus be applied to almost any system. Thus, given the advent of the next-generation X-ray sources, XPCS has the potential to become the main method for quantitatively understanding diffusion on the atomic scale.

Digital in-line holography with femtosecond VUV radiation provided by the free-electron laser FLASH.

Femtosecond vacuum ultraviolet (VUV) radiation provided by the free-electron laser FLASH was used for digital in-line holographic microscopy and applied to image particles, diatoms and critical point dried fibroblast cells. To realize the classical in-line Gabor geometry, a 1 microm pinhole was used as spatial filter to generate a divergent light cone with excellent pointing stability. At a fundamental wavelength of 8 nm test objects such as particles and diatoms were imaged at a spatial resolution of 620 nm. In order to demonstrate the applicability to biologically relevant systems, critical point dried rat embryonic fibroblast cells were for the first time imaged with free-electron laser radiation.

Anomalous dynamics of the endoplasmic reticulum network.

Large portions of the endoplasmic reticulum (ER) in eukaryotic cells are organized as dynamic networks whose segments are connected by three-way junctions. Here we show that ER junctions move subdiffusively with signatures of fractional Brownian motion and a strong dependence on the cytoskeleton's integrity: The time-averaged mean square displacement scales as 〈r^{2}(τ)〉_{t}∼τ^{α} with α≈0.5 in untreated cells and α≈0.3 when disrupting microtubules, with successive steps being anticorrelated in both cases. We explain our observations by considering ER junctions to move like monomers in (semi)flexible polymer segments immersed in a viscoelastic environment. We also report that ER networks have a nontrivial fractal dimension d_{f}≈1.6 on mesoscopic scales and we provide evidence that the organelle's dynamics is governed by fractons.

Detrended fluctuation analysis in x-ray photon correlation spectroscopy for determining coarsening dynamics in alloys.

We study the dynamics of precipitate coarsening in phase-separating alloys at late stages of phase separation by x-ray photon correlation spectroscopy (XPCS). For analyzing time series of fluctuating speckle intensities from small-angle scattering of coherent x rays, the method of detrended fluctuation analysis (DFA), which is ideal for determining power-law correlations, is applied. We discuss the application of DFA with respect to XPCS data by means of simulated time series. In particular, the effects of different signal-to-noise ratios are examined. Results from measurements of the two model systems Al-6 at. % Ag at 140 degrees C and Al-9 at. % Zn at 0 degrees C are presented. Since the DFA effectively removes adulterating trends in the data, quantitative agreement with Monte Carlo simulations is obtained. It is verified that two different coarsening mechanisms are predominant in the two systems--coarsening either by diffusion of single atoms or by movement of whole precipitates.

Hard X ray holographic diffraction imaging.

We determine the absolute electron density of a lithographically grown nanostructure with 25 nm resolution by combining hard x-ray Fourier transform holography with iterative phase retrieval methods. While holography immediately reveals an unambiguous image of the object, we deploy in addition iterative phase retrieval algorithms for pushing the resolution close to the diffraction limit. The use of hard (8 keV) x rays eliminates practically all constraints on sample environment and enables a destruction-free investigation of relatively thick or buried samples, making holographic diffraction imaging a very attractive tool for materials science. We note that the technique is ideally suited for subpicosecond imaging that will become possible with the emerging hard x-ray free-electron lasers.

Transient interface sharpening in miscible alloys.

We observed that diffuse interfaces sharpen rather than broaden in completely miscible ideal binary systems. This is shown in situ during heat treatments at gradually increasing temperatures by scattering of synchrotron radiation in coherent Mo/V multilayers containing initially diffuse interfaces. This effect provides a useful tool for the improvement of interfaces and offers a way to fabricate better x-ray or neutron mirrors, microelectronic devices, or multilayers with giant magnetic resistance.