ABSTRACT By combining the osmotic stress technique with small-angle x-ray scattering measurements, we followed the
structural response of the casein micelle to an overall increase in concentration. When the aqueous phase that separates
the micelles is extracted, they behave as polydisperse repelling spheres and their internal structure is not affected. When
they are compressed, the micelles lose water and shrink to a smaller volume. Our results indicate that this compression is nonaffine,
i.e., some parts of the micelle collapse, whereas other parts resist deformation. We suggest that this behavior is consistent
with a spongelike casein micelle having a triple hierarchical structure. The lowest level of the structure consists of the CaP
nanoclusters that serve as anchors for the casein molecules. The intermediate level consists of 10- to 40-nm hard regions that
resist compression and contain the nanoclusters. Those regions are connected and/or partially merged with each other, thus
forming a continuous and porous material. The third level of structure is the casein micelle itself, with an average size of
100 nm. In our view, such a structure is consistent with the observation of 10- to 20-nm casein particles in the Golgi vesicles
of lactating cells: upon aggregation, those particles would rearrange, fuse, and/or swell to form the spongelike micelle.