Spatial Distribution of Intracellular Ion Concentrations in Aggregate-Forming HeLa Cells Analyzed by μ-XRF Imaging

Abstract

Protein aggregation is a hallmark of several severe neurodegenerative disorders such as Huntington's, Parkinson's, or Alzheimer's disease. Metal ions play a profound role in protein aggregation and altered metal-ion homeostasis is associated with disease progression. Here we utilize μ-X-ray fluorescence imaging in combination with rapid freezing to resolve the elemental distribution of phosphorus, sulfur, potassium, and zinc in huntingtin exon-1-mYFP expressing HeLa cells. Using quantitative XRF analysis, we find a threefold increase in zinc and a 10-fold enrichment of potassium that can be attributed to cellular stress response. While the averaged intracellular ion areal masses are significantly different in aggregate-containing cells, a local intracellular analysis shows no different ion content at the location of intracellular inclusion bodies. The results are compared to corresponding experiments on HeLa cells forming pseudoisocyanine chloride aggregates. As those show similar results, changes in ion concentrations are not exclusively linked to huntingtin exon-1 amyloid formation.

Keywords: X-ray fluorescence; huntingtin; potassium; pseudoisocyanine chloride; zinc.

Figure 1

a) Domain structure of the overexpressed Htt‐ex1‐mYFP protein. An N‐terminal domain (N17) and a polyproline domain (PolyP) flank the PolyQ domain that is native (Q25) or extended (Q72). mYFP is used for C‐terminal fluorescence detection; b) exemplary phase contrast microscopy (LM) and visible‐light fluorescence (LF) microscopy as well as X‐ray fluorescence images of individual HeLa cells. Cells were transfected with Htt‐ex1 proteins with different polyglutamine lengths (Q25 & Q72) and compared to untreated, non‐transfected cells (NT).

Figure 2

Concentrations determined within individual Htt‐transfected (Q25 & Q72) and non‐transfected HeLa cells for the elements a) phosphorus, b) sulfur, c) potassium, and d) zinc. The concentrations were determined for each pixel in the cells and the distribution is represented as a boxplot. The colored boxes represent values from 25 %–75 %, while the whiskers represent values from 5 % −95 %. A total of 171,660 XRF spectra recorded on 42 cells were analyzed.

Figure 3

Comparison of local intracellular area concentrations of IB‐containing Htt‐ex1 cells for a) phosphorus, b) sulfur, c) potassium, and d) zinc. The left data point of the data pairs represents the concentration in areas containing IBs, the right data point represents regions with the same area but diffuse (soluble) Htt‐ex1. The position of the inclusion bodies was located by visible light fluorescence images of the cells. Each box color represents one Htt‐ex1‐transfected HeLa cell. Cells with significant differences between the two regions (p<0.05) were marked with an asterisk. The difference in mYFP fluorescence intensity for the areas within the respective individual cells is shown in e). The mYFP fluorescence intensity was significantly different for all box plot pairs (p<0.05). The corresponding p‐values are summarized in Table S2.

Figure 4

a) Normalized absorption spectrum of monomeric PIC (black) and the J‐aggregated PIC (blue) and its normalized fluorescence emission of the aggregated state (red). b) Phase contrast microscopy (LM), visible‐light fluorescence microscopy (LF) and X‐ray fluorescence images of PIC‐aggregate‐containing cells, 10 min and 60 min after treatment. Distribution of phosphorous, sulfur, potassium, and zinc after different incubation times in a PIC‐containing medium were examined.

Figure 5

Intracellular element area concentrations in HeLa cells 10 min and 60 min after PIC treatment for the elements a) phosphorus, b) sulfur, c) potassium, and d) zinc. The concentrations were determined for each pixel in the cell and the distribution per cell is represented as a boxplot. The colored boxes represent values from 25 %–75 %, while the whiskers represent values from 5 %–95 %. A total of 303207 XRF spectra recorded on 114 cells were analyzed, only 134771 from 51 cells are shown as boxplots for the sake of clarity.