6 research outputs found
A comparative analysis of the mobility of 45 proteins in the synaptic bouton
Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, we analyzed the motion of 45 GFP-tagged synaptic proteins expressed in cultured hippocampal neurons, using fluorescence recovery after photobleaching, particle tracking, and modeling. We compared synaptic vesicle proteins, endo- and exocytosis cofactors, cytoskeleton components, and trafficking proteins. We found that movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. We then analyzed protein movement thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology
Characterization of transient and progressive pulmonary fibrosis by spatially correlated phase contrast microCT, classical histopathology and atomic force microscopy
: Pulmonary fibrosis (PF) is a severe and progressive condition in which the lung becomes scarred over time resulting in pulmonary function impairment. Classical histopathology remains an important tool for micro-structural tissue assessment in the diagnosis of PF. A novel workflow based on spatial correlated propagation-based phase-contrast micro computed tomography (PBI-microCT), atomic force microscopy (AFM) and histopathology was developed and applied to two different preclinical mouse models of PF - the commonly used and well characterized Bleomycin-induced PF and a novel mouse model for progressive PF caused by conditional Nedd4-2 KO. The aim was to integrate structural and mechanical features from hallmarks of fibrotic lung tissue remodeling. PBI-microCT was used to assess structural alteration in whole fixed and paraffin embedded lungs, allowing for identification of fibrotic foci within the 3D context of the entire organ and facilitating targeted microtome sectioning of planes of interest for subsequent histopathology. Subsequently, these sections of interest were subjected to AFM to assess changes in the local tissue stiffness of previously identified structures of interest. 3D whole organ analysis showed clear morphological differences in 3D tissue porosity between transient and progressive PF and control lungs. By integrating the results obtained from targeted AFM analysis, it was possible to discriminate between the Bleomycin model and the novel conditional Nedd4-2 KO model using agglomerative cluster analysis. As our workflow for 3D spatial correlation of PBI, targeted histopathology and subsequent AFM is tailored around the standard procedure of formalin-fixed paraffin-embedded (FFPE) tissue specimens, it may be a powerful tool for the comprehensive tissue assessment beyond the scope of PF and preclinical research
Assembly of Simple Epithelial Keratin Filaments: Deciphering the Ion Dependence in Filament Organization
The intermediate filament proteins
keratin K8 and K18 constitute
an essential part of the cytoskeleton in simple epithelial cell layers,
structurally enforcing their mechanical resistance. K8/K18 heterodimers
form extended filaments and higher-order structures including bundles
and networks that bind to cell junctions. We study the assembly of
these proteins in the presence of monovalent or divalent ions by small-angle
X-ray scattering. We find that both ion species cause an increase
of the filament diameter when their concentration is increased; albeit,
much higher values are needed for the monovalent compared to the divalent
ions for the same effect. Bundling occurs also for monovalent ions
and at comparatively low concentrations of divalent ions, very different
from vimentin intermediate filaments, a fibroblast-specific cytoskeleton
component. We explain these differences by variations in charge and
hydrophobicity patterns of the proteins. These differences may reflect
the respective physiological situation in stationary cell layers versus
single migrating fibroblasts
The Structure of Gold-Nanoparticle Networks Cross-Linked by Di- and Multifunctional RAFT Oligomers
Gold nanoparticle (AuNP) network
structures featuring particles
from the two-phase BrustāSchiffrin synthesis and linear RAFT
oligomers of styrene with two and multiple trithiocarbonate (TTC)
groups along their backbone have been investigated in detail. Insights
into the internal structures of these particle networks could be obtained
from small-angle X-ray scattering experiments, showing that primary
AuNPs are cross-linked by the employed molecular linker. The extent
of AuNP network formation was investigated by means of dynamic light
scattering and UV/visible extinction spectroscopy, showing an abrupt
attenuation of network formation after a critical degree of polymerization
of the cross-linker is exceeded. Analysis of transmission electron
micrographs indicated a three-dimensional shape of the particle superstructures,
which is evenly filled with the primary AuNPs. From the results obtained
in this study, guidelines for the fabrication of nanoparticle networks
from the self-assembly with macromolecular cross-linkers are suggested
Xārays Reveal the Internal Structure of Keratin Bundles in Whole Cells
In recent years, X-ray imaging of
biological cells has emerged
as a complementary alternative to fluorescence and electron microscopy.
Different techniques were established and successfully applied to
macromolecular assemblies and structures in cells. However, while
the resolution is reaching the nanometer scale, the dose is increasing.
It is essential to develop strategies to overcome or reduce radiation
damage. Here we approach this intrinsic problem by combing two different
X-ray techniques, namely ptychography and nanodiffraction, in one
experiment and on the same sample. We acquire low dose ptychography
overview images of whole cells at a resolution of 65 nm. We subsequently
record high-resolution nanodiffraction data from regions of interest.
By comparing images from the two modalities, we can exclude strong
effects of radiation damage on the specimen. From the diffraction
data we retrieve quantitative structural information from intracellular
bundles of keratin intermediate filaments such as a filament radius
of 5 nm, hexagonal geometric arrangement with an interfilament distance
of 14 nm and bundle diameters on the order of 70 nm. Thus, we present
an appealing combined approach to answer a broad range of questions
in soft-matter physics, biophysics and biology