Expansion microscopy for visualization of protein clusters in cultured cells and brain tissues

Abstract

Many biological studies necessitate high-resolution imaging and further analysis of cellular organelles and molecules. Expansion microscopy (ExM) enables achieving nanometer-level resolution with a standard fluorescence microscope by physically expanding the sample in the gel by multiple factors. In the present study, ExM was used to examine protein clusters of STIM1, a calcium-binding protein, and IP3R, a calcium-gated channel (inositol triphosphate receptor). The endoplasmic reticulum (ER) calcium sensor STIM1 translocates to the ER-plasma membrane junctions, forming clusters to activate store-operated calcium entry (SOCE) upon ER calcium decrease [1]. Expansion microscopy provides the advantage of expanding the sample in all three axes, including the Z-axis, enabling detection of premembrane proteins without relying on TIRF microscopy. STIM1 interacts with end-binding protein 1 (EB1) located at the plus ends of microtubules, which regulates SOCE. This study presents a quantitative approach for analyzing protein clusters using expansion microscopy. STIM1 and its non-EB-binding mutant, STIM1-TR/NN, were used as examples. In endothelial cells, Ca2+ is released into the cytoplasm from the ER through the main channels of Inositol-1,4,5-triphosphate receptors (IP3R). A previous study demonstrated that these receptors interact with the EB protein through the SxIP amino acid motif, similarly to STIM proteins, regulating clustering and calcium signaling [2]. In hippocampal neurons, the type 1 IP3 receptor forms clusters required for efficient calcium release through the channel in response to stimuli. To investigate the function of IP3 receptor isoform 1 in the brain, IP3R clusters were analyzed in wild-type mice and 5xFAD mice modeling Alzheimer’s disease (AD) since this receptor was observed to exhibit increased activity during this pathological condition [3]. HEK293T cells at 50–70% confluence underwent transfection using mCherry-STIM1 and mCherry-STIM1-TR/NN plasmids to assess the clustering of STIM1 proteins. Cells were fixed and stained with primary mCherry protein antibodies and secondary antibodies conjugated to the Alexa Fluor 594 fluorophore to enhance fluorescence. Next, the cells underwent isotropic expansion in the gel using expansion microscopy. The ExM method was executed following the protocol outlined by Asano et al. [4]. The sample was expanded through dual addition of sterile, distilled water for a period of 20 minutes. To examine the clustering of IP3R proteins, we obtained frontal slices of the brains of two groups of mice: control and 5xFAD (a mouse model for Alzheimer’s disease), which were 40–50 microns thick. Brain slices were immunohistochemically stained with IP3R1 primary antibody and Alexa Fluor 488 secondary antibody followed by expansion microscopy protocol [4]. The ImageJ and Icy software were used for processing images. Using ImageJ, the neurons’ intensity was determined, leading to the formation of three groups with different fluorescence intensity levels. A certain binarization threshold was implemented in Adaptive3DThreshold, depending on the level. The grouping approach enabled neutralizing the effect of potential disparities in IP3R protein expression levels on the analysis of differences in fluorescence intensity. According to the literature data, analysis of the results indicates that when the bond with tubulin microtubules is disrupted, STIM1 aggregates more when the calcium store is depleted in comparison to its standard STIM1 variant [5]. Upon evaluation of IP3R cluster morphometric parameters in transgenic mice compared to the control group, we observed an increase in the size and number of IP3R protein clusters in mice of the 5xFAD line within the groups exhibiting the highest neuronal intensity and the groups with the highest and average neuronal intensity. The Western blot method was used to validate the results and demonstrated overexpression of the IP3R protein in 5xFAD mice. This study represents the first time that IP3R protein clusters aggregate more in a mouse model of AD, which is congruent with the literature regarding high IP3R activity in AD [3].