Super-resolution microscopy reveals the number and distribution of topoisomerase IIα and CENH3 molecules within barley metaphase chromosomes.

Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination, respectively. Consequently, they are required for proper chromosome segregation during cell divisions. We combined two super-resolution techniques, structured illumination microscopy (SIM) to co-localize Topo IIα and CENH3, and photoactivated localization microscopy (PALM) to determine their molecule numbers in barley metaphase chromosomes.

Breakthrough instruments and products: Lattice Lightsheet 7-Non-invasive imaging of the subcellular dynamics of life.

Light sheet fluorescence microscopy has become an established method for fast and sensitive imaging of living specimens with minimum phototoxicity and photobleaching. By adding lattice structures to the light sheet, the ZEISS Lattice Lightsheet 7 makes this technique available for live cell imaging at subcellular resolution while also allowing microscopists to use their standard sample carriers.

A protocol to expand plant nuclei.

The resolution achieved by conventional light microscopy is limited by light diffraction. This obstacle can be overcome either by optical super-resolution techniques or by the recently developed method to physically expand specimens, called expansion microscopy (ExM). The method utilizes polymer chemistry and the ability of a swellable polyelectrolyte hydrogel to absorb water, and thus to expand its size.

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination.

Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices.

Label-Free Four-Dimensional Visualization of Anaerobically Growing Electroactive Biofilms.

Light sheet fluorescence microscopy (LSFM) allows nondestructive, label-free and in vivo imaging of large specimen, even at nontransparent surfaces. We show that LSFM can be applied for label-free analyses of prokaryotes on the example of electroactive biofilms. Biofilm growth is linked to the production of current serving as measure of metabolic activity in vivo by monitoring with high spatial and temporal resolution.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies.

The () gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly.

Abundance and distribution of RNA polymerase II in Arabidopsis interphase nuclei.

RNA polymerase II (RNAPII) is responsible for the transcription of most eukaryotic protein-coding genes. Analysing the topological distribution and quantification of RNAPII can contribute to understanding its function in interphase nuclei. Previously it was shown that RNAPII molecules in plant nuclei form reticulate structures within euchromatin of differentiated Arabidopsis thaliana nuclei rather than being organized in distinct 'transcription factories' as observed in mammalian nuclei.

The tumor suppressor PML specifically accumulates at RPA/Rad51-containing DNA damage repair foci but is nonessential for DNA damage-induced fibroblast senescence.

The PML tumor suppressor has been functionally implicated in DNA damage response and cellular senescence. Direct evidence for such a role based on PML knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking PML.

Molecular logic with a saccharide probe on the few-molecules level.

In this Communication we describe a two-component saccharide probe with logic capability. The combination of a boronic acid-appended viologen and perylene diimide was able to perform a complementary implication/not implication logic function. Fluorescence quenching and recovery with fructose was analyzed with fluorescence correlation spectroscopy on the level of a few molecules of the reporting dye.

FLIM and FCS detection in laser-scanning microscopes: increased efficiency by GaAsP hybrid detectors.

Photon counting detectors currently used in fluorescence lifetime microscopy have a number of deficiencies that result in less-than-ideal signal-to-noise ratio of the lifetimes obtained: either the quantum efficiency is unsatisfactory or the active area is too small, and afterpulsing or tails in the temporal response contribute to overall timing inaccuracy. We have therefore developed a new FLIM detector based on a GaAsP hybrid photomultiplier. Compared with conventional PMTs and SPADs, GaAsP hybrid detectors have a number of advantages: The detection quantum efficiency reaches or surpasses the efficiency of fast SPADs, and the active area is on the order of 5 mm², compared with 2.

Photon counting histogram analysis for two-dimensional systems.

Photon counting statistics in 3D photon counting histogram analysis for one-photon excitation is a function of the number of molecules of particular brightness in the excitation-detection volume of a confocal microscope. In mathematical form that volume is approximated by a three-dimensional Gaussian function which is embedded in the PCH theoretical equations. PCH theory assumes that a molecule can be found anywhere inside the excitation-detection volume with equal probability.

Note: An easy way to enable total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) by combining commercial devices for FCS and TIR microscopy.

Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) is a powerful method for studying dynamic processes at liquid-solid interfaces that may have numerous applications in biology, physics, and material science. Despite of its power and versatility, however, the use of TIR-FCS is still rather limited. The main reason for this is the need of a complex, in-house constructed optical setup whose assembly and adjustment is a quite difficult task.

Fluorescence fluctuation microscopy to reveal 3D architecture and function in the cell nucleus.

The three-dimensional (3D) architecture of the cell nucleus is determined not only by the presence of subnuclear domains, such as the nuclear envelope, chromosome territories, and nuclear bodies, but also by smaller domains which form in response to specific functions, such as RNA transcription, DNA replication, and DNA repair. Since both stable and dynamic structures contribute to nuclear morphology, it is important to study the biophysical principles of the formation of macromolecular assemblies within the nucleus. For this purpose, a variety of fluorescence fluctuation microscopy techniques can be applied.

Containment of extended length polymorphisms in silk proteins.

The spider silk gene family to the current date has been developed by gene duplication and homogenization events as well as conservation of crucial sequence parts. These evolutionary processes have created an amazing diversity of silk types each associated with specific properties and functions. In addition, they have led to allelic and gene variants within a species as exemplified by the major ampullate spidroin 1 gene of Nephila clavipes.

Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus.

We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA(r)) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity.

Fluorescence correlation spectroscopy to assess the mobility of nuclear proteins.

Recent developments in cell biology and microscopy techniques enable us to observe macromolecular assemblies in their natural setting: the living cell. These emerging technologies have revealed novel concepts in nuclear cell biology. In order to further elucidate the biochemistry of gene expression, replication, and genome maintenance, the major challenge is now to precisely determine the dynamics of nuclear proteins in the context of the structural organization of the nucleus.

Use of Kaede fusions to visualize recycling of G protein-coupled receptors.

The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein.

Environmental conditions impinge on dragline silk protein composition.

The silk formed in the major ampullate (MA) gland of the orb weaving spider Nephila clavipes is composed of two silk fibroins, which are called major ampullate spidroins 1 (MaSp1) and 2 (MaSp2). Analysis of proteolytic peptides and reactivity to spidroin type specific antibodies indicated that MaSp2 constituted only a minor part in the spinning dope as well as in the spun filaments. Upon starvation, a change in the silk's characteristic features was observed that was concomitant of a decrease in the contribution of MaSp2.

Composition and hierarchical organisation of a spider silk.

Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished.

Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication.

Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase alpha-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process.

Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication.

The initiation of SV40 (simian virus 40) DNA replication requires the co-operative interactions between the viral Tag (large T-antigen), RPA (replication protein A) and Pol (DNA polymerase alpha-primase) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with the mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex.

The conserved C-termini contribute to the properties of spider silk fibroins.

Spider silk fibroins can adopt different structural states at high protein concentrations. They are soluble within the spinning dope of the glands, but readily converted into insoluble polymers upon extrusion. A contribution of the C-termini to the maintenance and conversion of these states is suggested by their predicted secondary structures and biochemical behavior in vitro.

Differential polymerization of the two main protein components of dragline silk during fibre spinning.

Spider silks are some of the strongest materials found in nature. Achieving the high tensile strength and elasticity of the dragline of orb-weaving spiders, such as Nephila clavipes, is a principal goal in biomimetics research. The dragline has a composite nature and is predominantly made up by two proteins, the major ampullate spidroins 1 and 2 (refs 3, 6, 7), which can be considered natural block copolymers.

Characterization of the protein components of Nephila clavipes dragline silk.

Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads.