dc.description.abstracteng | The advances and technical improvements of X-ray imaging techniques, taking
advantage of X-ray focussing optics and high intensity synchrotron sources,
nowadays allow for the use of X-rays to probe the cellular nanoscale. Importantly,
X-rays permit thick samples to be imaged without sectioning or slicing. In this
work, two macromolecules, namely keratin intermediate filament (IF) proteins
and DNA, both essential components of cells, were studied by X-ray techniques.
Keratin IF proteins make up an integral part of the cytoskeleton of epithelial
cells and form a dense intracellular network of bundles. This network is built
from monomers in a hierarchical fashion. Thus, the keratin structure formation
spans a large range of length scales from a few nanometres (monomers) to
micrometres (networks). Here, keratin was studied at three different scales: i) filaments, ii) bundles and iii) networks. Solution small-angle X-ray scattering revealed distinct structural and organisational characteristics of these highly
charged polyelectrolyte filaments, such as increasing radius with increasing
salt concentration and spatial accumulation of ions depending on the salt
concentration. The results are quantified by employing advanced modelling of
keratin IFs by a core cylinder fl anked with Gaussian chains. Scanning micro-
diffraction was used to study keratin at the bundle scale. Very different
morphologies of keratin bundles were observed at different salt conditions. At
the network scale, new imaging approaches and analyses were applied to the
study of whole cells. Ptychography and scanning X-ray nano-diffraction imaging
were performed on the same cells, allowing for high resolution in real and
reciprocal space, thereby revealing the internal structure of these networks. By
using a fitting routine based on simulations of IFs packed on a hexagonal lattice,
the radius of each fi lament and distance between fi laments were retrieved.
In mammalian cells, each nucleus contains 2 nm-thick DNA double helices with
a total length of about 2 m. The DNA strands are packed in a highly hierarchical
manner into individual chromosomes. DNA was studied in intact cells by visible
light microscopy and scanning X-ray nano-diffraction, unveiling the compaction
und decompaction of DNA during the cell cycle. Thus, we obtained information on
the aggregation state of the nuclear DNA at a real space resolution on the order
of few hundreds nm. To exploit to the reciprocal space information, individual
diffraction patterns were analysed according to a generalised Porod’s law at a
resolution down to 10 nm. We were able to distinguish nucleoli, heterochromatin
and euchromatin in the nuclei and follow the compaction and decompaction
during the cell division cycle. | |