3D morphology of the human hepatic ferritin mineral core: new evidence for a subunit structure revealed by single particle analysis of HAADF-STEM images

Abstract

Ferritin, the major iron storage protein, has dual functions; it sequesters redox activity of intracellular iron and facilitates iron turn-over. Here we present high angle annular dark field (HAADF) images from individual hepatic ferritin cores within tissue sections, these images were obtained using spherical aberration corrected scanning transmission electron microscopy (STEM) under controlled electron fluence. HAADF images of the cores suggest a cubic morphology and a polycrystalline (ferrihydrite) subunit structure that is not evident in equivalent bright field images. By calibrating contrast levels in the HAADF images using quantitative electron energy loss spectroscopy, we have estimated the absolute iron content in any one core, and produced a three dimensional reconstruction of the average core morphology. The core is composed of up to eight subunits, consistent with the eight channels in the protein shell that deliver iron to the central cavity. We find no evidence of a crystallographic orientation relationship between core subunits. Our results confirm that the ferritin protein shell acts as a template for core morphology and within the core, small (approximately 2 nm), surface-disordered ferrihydrite subunits connect to leave a low density centre and a high surface area that would allow rapid turn-over of iron in biological systems.

Fig. 1

The effect of accumulated electron fluence on (a) the valence and (b) the oxygen co-ordination of iron in haemosiderin and ferritin mineral cores (HMC and FMC respectively) as measured by fitting reference spectra to Fe L_2,3-ionisation EELS edges recorded in the STEM. (c) The change in valence with progressively increasing electron fluence as measured by taking Fe L₃ to L₂- ratios from the same spectra used for a and b, following the method of van Aken and Liebscher (2002). (d) The highest fluence haemosiderin Fe L_2,3-ionisation EELS edge with the four fitting reference spectra shown underneath and their intensity scaled by the fitting coefficients such that when summed they produce the best fit to the mixed valence spectrum (inset is the residual after fitting).

Fig. 2

STEM-HAADF images of ferritin molecule cores within thin sections of fixed and unstained tissue. (a) Ferritin cores located in a hereditary haemochromatosis human liver biopsy. Strongly scattering iron-rich cores are visible as bright spots in the image and occur throughout the cytoplasm. Some cellular structures can be identified: A: endoplasmic reticulum; B: ribosome; C: organelle (probably mitochondrion). (b) Intermediate magnification HAADF image within the tissue section showing clear and regular subunit structure to the cytosolic ferritin cores with a line profile of the signal to background across a core inset. (c) Bright field STEM image of the same area as (b); note the significant decrease in signal to background of the inset line profile across a core compared to the HAADF image. (d) High magnification image of cytosolic ferritin cores from the same biopsy showing atomic lattice resolution of a subunit within a core. While other parts of the core may also be crystalline, only the bottom-right corner of the core is oriented along a crystallographic zone axis such that iron atom columns are resolvable/visible in the image. Note the non-facetted nature of the core edges. The lattice d-spacings (0.273 nm for spacing 1 and 0.272 nm for spacing 2, and 0.276 nm for spacing 3) and angles between the spacings (58.7° and 60.2°) are consistent with a ferrihydrite crystal structure (Drits et al., 1993). (e) Corresponding bright field STEM image of the core shown in (d) here the image is dominated by phase contrast effects such as the granularity in the embedded tissue surrounding the core and the lattice fringes in the subunit of the core that is lying on a crystallographic zone axis. i.e., the size and shape of the core is not clear here in comparison to the HAADF image because the parts of the core that are not lying on a crystallographic zone axis are partially obscured.

Fig. 3

Summary of the single particle reconstruction procedure for an average hepatic ferritin molecule core with an estimated iron loading of between 1100–1850 iron atoms. (a) An example of one of 133 HAADF images containing multiple ferritin cores, used for the reconstruction. (b) Absolute quantification of the iron atom content of a core using EELS correlates linearly with the normalised HAADF image intensity for a set of 16 ferritin cores with a range of different iron loadings. (c) Distribution of the iron content of 1241 ferritin cores in a tissue section showing a normal distribution with a mean of 1500 ± 300 iron atoms (upper and lower limits are 4,000 and 200 iron atoms). (d) Selected views of the initial models and final reconstructions (view 1–3) of a core developed from the 750 ferritin cores each with an iron atom content in the most populated range of between 1100–1850; despite different initial models a cubic-like subunit structure with a low density centre is evident in all of the final reconstructions. (e) The top row shows 750 core images all bandpass filtered, aligned and classified using the EMAN program into 9 of the 18 projection classes. The bottom row shows 9 re-projections, at 12° spacing, of the final reconstruction developed from initial model (a). The re-projections show strong correlation with the raw shape classes. In both cases the scale bar is 10 nm.

Fig. 4

Schematic cross-section (viewing direction: parallel to one of the four-fold symmetry channels in the protein shell) of a hepatic ferritin core depicting our proposed formation mechanism. This is a modification of a schematic of core formation by Lewin et al. (2005). (a) Early stage of iron deposition in the ferritin central cavity. The sites near the ends of the three-fold symmetry iron entry channels (where the protein shell subunits, shown as grey lobes, have specific oxidation sites) are favourable for the incoming Fe²⁺ to deposit and be oxidised. The yellow circles represent oxidised iron (Fe³⁺). (b) As the iron cellular concentration increases, more Fe²⁺ is shuffled into the molecule and may rapidly deposit and oxidise on the surface of any existing Fe³⁺ deposits near the entry channels; consequently, core subunits are formed. (c) With higher iron-filling, a cubic-like core structure with eight subunits (only four of which are shown) develops. Oxidation of further incoming Fe²⁺, results in the early deposited Fe³⁺ diffusing inwards forming closely packed crystalline structures of ferrihydrite (dark red circles in contrast to the loosely packed Fe³⁺ (yellow circles)), the atomic structure of such a subunit structure is seen experimentally in Fig. 2d. The surface of each core subunit is disordered facilitating dynamic load and release activities consistent with the ‘last-in first-out’ hypothesis (Hoy et al., 1974). (d) An example of a commonly observed HAADF image of a single ferritin core of similar iron loading and lying in a similar orientation to the schematic; the four-fold symmetry arrangement of the subunits and a low density central region are clearly evident.