Correlative light and immuno-electron microscopy of retinal tissue cryostat sections

<div><p>Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound. Utilising these approaches, we have (i) identified the same phagosomes by FM and EM in the retinal pigment epithelium (RPE) of retinal tissue (ii) shown the correct localisation of rhodopsin on photoreceptor outer segment disc like-structures in iPSC derived optic cups and (iii) identified a novel interaction between peroxisomes and melanosomes as well as phagosomes in the RPE. These data show that cryostat sections allow easy characterisation of target macromolecule localisation within tissue samples, thus providing a substantial improvement over many conventional methods that are limited to cultured cells. As OCT embedding is routinely used for FM this provides an easily accessible and robust method for further analysis of existing samples by high resolution EM.</p></div

Outline method for correlative light and electron microscopy of cryostat sections.

<p>Outline method for correlative light and electron microscopy of cryostat sections.</p

Mouse retinal tissue prepared conventionally and from OCT embedded sections for electron microscopy.

<p>(A & B) Conventional fixed tissue and (C & D) OCT sections of tissue prepared for EM show little difference in the preservation quality. Organelles including mitochondria (M), melanosomes (Me) and phagosomes (P), in addition to membranes such as basal infoldings (BI) and photoreceptor outer segment (OS) discs and structural features such as collagen in Bruchs membrane (Co) and fenestrae of choroidal endothelial cells (F) are well preserved. (A & C) the connecting cilium is indicated by CC and the photoreceptor inner segment IS. Scale bar = 500nm.</p

Correlative light electron and microscopy of cryostat sections can be used to identify rhodopsin enriched phagosomes in the retinal pigment epithelium (RPE) cell layer.

<p>The same region of RPE viewed by (A) fluorescence microscopy (FM) and (B) electron microscopy (EM), with an overlay of the two in (C). The boxed regions in (A-C) are shown at higher magnification in (D) highlighting regions that include FM rhodopsin staining (green) overlapping with phagosomes seen by EM. (E) Higher magnification of phagosomes (Ph) boxed in (D), surrounded by melanosomes (Me) and mitochondria (M). Scale bar = (A-C)– 10um (D)– 1um (E)– 250nm.</p

Correlative light and electron microscopy of cryostat sections of control iPSC optic cup using dual fluorescence microscopy (FM) and nano-gold rhodopsin labelling.

<p>(A) Diagram outlining the differentiation of inducible pluripotent stem cells (iPSCs) into optic cups with photoreceptors. (B) Confocal FM overlay on top of differential interference contrast (DIC) image (C) FM overlay on top of electron microscopy (EM) image. (D) EM with (E) a high magnification region showing concentrated rhodopsin localisation in the photoreceptor OS region of the optic cups. The connecting cilia in (D) is indicated by CC. Scale bar = (B & C) 100um, (D) 2um and (E) 200nm.</p

Immuno-electron microscopy (iEM) labelling of cryostat sections allows the identification of peroxisomes in the RPE, illustrating the close contact with phagosomes and melanosomes.

<p>(A-B) FM images showing the localisation of peroxisomes (red) around rhodopsin enriched phagosomes (green) in the RPE cell layer. (C) Low magnification electron microscopy image of RPE cell layer with gold labelled peroxisomes that are localised towards the basal cell surface close to the basal infolding (Bi) and Bruch’s membrane (Br) and away from the apical processes (Ap). The box highlights a region with a phagosome (Ph) that is shown at higher magnification in (D) with a gold labelled peroxisome in close proximity and other organelles nearby, including melanolipofuscin granules (MeLi) and mitochondria (M). (E) Gold labelled peroxisomes in contact with melanosomes (Me) with a high magnification image (F). Contacts between the peroxisomes and melanosomes (black arrows) and in the zoomed insert tethers between the organelles can be seen (white arrows) Scale = (A)– 10um (B)– 5um (C)– 1um (D-E)– 250nm (F)– 100nm.</p

AIPL1, FAT10 and NUB1 form a ternary complex.

<p>Cells were transfected with constructs as indicated followed by immunoprecipitation and immunoblot analysis. (A) AIPL1 and FAT10 both co-precipitate with NUB1 (top panel); NUB1 and FAT10 both co-precipitate with AIPL1 (bottom panel). MG132 increased FAT10 steady-state levels and the amount of co-precipitated AIPL1 (top panel) or NUB1 (bottom panel). (B) NUB1 and AIPL1 both co-precipitate with FAT10. The AIPL1 C239R pathogenic mutant immunoprecipitated FAT10, but did not affect its NUB1-mediated degradation profile. (C) The AIPL1 C239R mutant did not immunoprecipitate NUB1 but FAT10 expression promoted their interaction. Heavy (h) and light (l) immunoglobulin chains are indicated. The position of molecular weight markers is indicated in kilodalton (kDa).</p

AIPL1 binds to FAT10 and FAT10-modified proteins.

<p>(A) Monomeric FAT10 co-precipitates with AIPL1 from transfected SK-N-SH cell lysates. Cells were transfected with the indicated constructs and immunoprecipitation was performed followed by immunoblot analysis. (B) Recombinant purified GST-AIPL1 but not GST can pull down free FAT10 and FAT10-conjugated proteins from HA-FAT10-transfected cell lysates. (C) Recombinant purified GST-AIPL1, but not GST, pulls down recombinant purified His6-FAT10. The position of molecular weight markers is indicated in kilodalton (kDa).</p

AIPL1 interacts with NUB1 to block the degradation of FAT10-DHFR.

<p>Cells were transfected with the indicated constructs, then treated 24 hours later with cycloheximide (CHX) to block protein synthesis and assess degradation of FAT10-DHFR over the indicated times. (A) AIPL1 blocked the NUB1-mediated degradation of FAT10-DHFR, and the effect was stronger in the presence of MG132. (B) AIPL1 delayed the degradation of FAT10-DHFR in the presence of NUB1. The percentage of FAT10 remaining was measured from 3 independent experiments (n = 3), and the level of significance calculated using the Wilcoxon signed-rank test. (C) Pathogenic AIPL1 mutants A197P and C239R were defective in blocking FAT10-DHFR degradation, while the G262S mutant was able to block degradation. (D) NUB1 co-precipitated with both WT and G262S AIPL1, but not with the A197P or C239R mutants. (E) FAT10-DHFR co-precipitated with WT AIPL1, and the mutants A197P, C239R and G262S.</p

AIPL1 alters the NUB1-mediated degradation of FAT10.

<p>SK-N-SH Cells were transfected with NUB1-FLAG, HA-FAT10 and Myc-AIPL1 vectors in the presence and absence of the proteasome inhibitor MG132, as indicated. Cell lysates were harvested 24 hours post-transfection and immunoprecipitates were analyzed by immunoblotting to detect the protein indicated. (A) NUB1 interacts with FAT10 and accelerates the degradation of free FAT10 and FAT10-modified proteins. The change in levels of FAT10 was measured from 3 independent experiments (n = 3) of duplicate samples. Heavy (h) and light (l) immunoglobulin chains are indicated. (B) NUB1 co-precipitates with AIPL1. (C) AIPL1 enhances the steady-state levels of free FAT10 and FAT10 modified proteins, both alone and in the presence of NUB1. The change in levels of FAT10 was measured from 5 independent experiments (n = 5) of duplicate samples. (D) HA-FAT10 was visualised by immunocytochemical analysis with anti-HA and Cy2-conjugated secondary antibody. NUB1-mediated degradation of FAT10 is altered by the presence of AIPL1. Scale bar is 20 µM. (C) and (E) A small proportion of AIPL1 is itself covalently modified with FAT10, as detected by anti-HA, anti-AIPL1 and anti-FAT10 (rabbit polyclonal) antibodies. The percentage of AIPL1 modified by FAT10 was measured from 3 independent experiments (n = 3). Conjugation of FAT10 to AIPL1 is prevented using a FAT10 diglycine deletion mutant. The position of molecular weight markers is indicated in kilodalton (kDa).</p