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Multiplex tissue profiling - Ciliated cellsCilia are small hair-like cell membrane-coated microtubule structures projecting out from the surface of virtually all vertebrate cells, either as a single non-motile sensory monocilia, as motile multicilia on specialized ciliated cells or as the sperm tail flagellum. Motile multicilia of ciliated cells, like the ones lining the inside of the fallopian tube och bronchi, facilitate the transportation of mucus. As part of the Tissue resource, we used multiplex immunohistochemistry-based fluorescence (mIHC/IF) for in-depth profiling of protein expression in motile ciliated cells. In the current version, 178 proteins have been analyzed in five tissues using 1 antibody panel.
The ciliated cells panelThe mIHC/IF panel for ciliated cells was developed to study the spatial protein expression of cilia proteins within the multiciliated cell. To address this, a panel of five markers was developed targeting the axoneme, basal body, transition zone, cytoplasm, and nucleus of motile ciliated cells. The panel was used on five different tissues expressing ciliated cells: bronchus, nasopharynx, cervix, endometrium, and fallopian tube. The mIHC/IF technique is based on the overlap between the location of the candidate protein and location of the marker proteins targeted by the panel antibodies. Figure 1 shows the different cell types/states/structures, and the corresponding marker proteins included in each panel. For more information about the multiplex panels, please visit Assays & annotations.
Figure 1. A schematic depiction of the ciliated cells panel on the left with a list of cell types, representing colors and marker proteins on the right. Protein expression in ciliated cellsCandidate proteins for in-depth profiling were selected from the Tissue resource based on cell type specific staining pattern using conventional immunohistochemistry. Multiplex immunohistochemistry has allowed us to analyze and visualize protein expression in ciliated cells at a higher resolution than ever before. For ciliated cells, the protein expression was analyzed in the cilia axoneme, cilia basal body, cilia transition zone, cytoplasm and nucleus.
Protein expression in cilia axonemeAs shown in Table 1, 128 genes have main subcellular location in the cilia axoneme. The axoneme of motile cilia is the main and beating part, stretching out of the epithelium to move fluid. An example of a protein found in cilia axoneme is CABCOCO1 with a function as a calcium-binding protein expressed in ciliated cells in female and male reproductive tissues as well as respiratory tissues. Two more proteins found in cilia axoneme are CFAP157 and DNAH2 with have functions related to cilia structure and function. Cilia and flagella-associated protein 157 (CFAP157) may be required to ensure a correct ultrastructure of the cilia whereas DNAH2 plays a central role in the ciliary beating by being part of the inner dynein arm complex.
Table 2. The number of proteins that have the cilia axoneme as main subcellular location for the studied tissues.
Protein expression in cilia basal bodyAs shown in Table 1, 7 genes have main protein location in the cilia basal body. The basal body is the structure under the cell membrane which holds the ciliary structure in place and decides its orientation. GAS2L2 is found in the cilia basal body where it is involved in cross-linking of microtubules and microfilaments, as well as regulating the orientation and performance of cilia in the airway. Other examples found were AGPS, a transferase involved in the beginning of lipid biosynthesis, as well as LRRC73 with a so far unknown protein function but it is enriched in respiratory ciliated cells according to scRNA-seq data.
Table 3. The number of proteins that have the cilia basal body as main subcellular location for the studied tissues.
Protein expression in cilia transition zoneAs shown in Table 1, 32 genes have main protein location in the cilia transition zone The transition zone is the structure at the base of the cilium, above the cell membrane, and acts as a gate controlling the entry and exit of ciliary proteins. Anoctamin 2 (ANO2) and sperm associated antigen 6 (SPAG6) are two examples of proteins found in the cilia transition zone. ANO2 is a calcium-activated chloride channel and SPAG6 is both important for the structure and function of sperm flagella and part of cilia biogenesis and degradation. A protein with a unknown function that was found in the cilia transition zone is ciliary rootlet coiled-coil (CROCC2), scRNAseq data showed it to be enriched in respiratory ciliated cells.
Table 4. The number of proteins that have the cilia transition zone as main subcellular location for the studied tissues.
Protein expression in cytoplasmAs shown in Table 1, 66 genes have main protein location in the cytoplasm in ciliated cells.Two examples of proteins found in the cytoplasm of ciliated cells are CDS1, an integral membrane protein in the inner mitochondrial membrane and endoplasmic reticulum and cilia and flagella associated protein 54 (CFAP54), required for the assembly and function of both cilia and flagella. Cathepsin S (CTSS) is a last example that is a lysosomal cysteine proteinase involved in the degradation of antigenic proteins for presentation on MHC class II molecules.
Table 5. The number of proteins that have the cytoplasm as main subcellular location in ciliated cells for the studied tissues.
Protein expression in nucleusAs shown in Table 1, 30 genes have main protein location in the nucleus in ciliated cells. An example of a protein found in the nucleus in ciliated cells is SOX17 which acts as a transcription regulator regulating for example embryonic development. Two other examples of proteins found in the nucleus of ciliated cells are WRAP53 and IK that respectively are a RNA chaperone, playing a key role in telomere maintenance, and a part of the spliceosome, involved in pre-mRNA splicing.
Table 6. The number of proteins that have the nucleus as main subcellular location in ciliated cells for the studied tissues.
Relevant links and publications Uhlén M et al., Tissue-based map of the human proteome. Science (2015) |
The Human Protein Atlas project is funded
