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Updated: May 30, 2026
Single-Molecule Localization Microscopy of Membrane Proteins using Single-Antibody Labeling
Published on: March 20, 2026
The C-terminal tail of tetraspanin protein CD9 contributes to its function and molecular organization
Hong-Xing Wang1, Tatiana V Kolesnikova, Carilee Denison
1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
Tetraspanin protein CD9 supports sperm-egg fusion, and regulates cell adhesion, motility, metastasis, proliferation and signaling. The large extracellular loop and transmembrane domains of CD9 engage in functionally important interactions with partner proteins. However, neither functional nor biochemical roles have been shown for the CD9 C-terminal tail, despite it being highly conserved throughout vertebrate species. To gain new insight into the CD9 tail, three C-terminal amino acids (Glu-Met-Val) were replaced with residues corresponding to C-terminal amino acids from tetraspanin protein CD82 (Pro-Lys-Tyr). Wild-type and mutant CD9 were then stably expressed in MOLT-4, K562, U937, RD and HT1080 cells. Whereas wild-type CD9 inhibited cell adhesion and spreading on fibronectin, mutant CD9 did not. Wild-type CD9 also promoted homotypic cell-cell aggregation and microvilli formation, whereas mutant CD9 did not. Protein interactions of wild-type and mutant CD9 were compared quantitatively using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with liquid-chromatography-tandem mass spectrometry (LC-MS/MS) technology. SILAC results showed that, despite wild-type and mutant CD9 having identical expression levels, mutant CD9 and its major transmembrane interacting partners were recovered in substantially reduced amounts from 1% Brij 96 lysates. Immunoprecipitation experiments confirmed that mutant CD9 recovery was decreased in Brij 96, but not in more stringent Triton X-100 detergent. Additionally, compared with wild-type CD9 complexes, mutant CD9 complexes were larger and more oligomerized in Brij 96 detergent, consistent with decreased Brij 96 solubility, perhaps due to more membrane domains packing more tightly together. In conclusion, multiple CD9 functions depend on its C-terminal tail, which affects the molecular organization of CD9 complexes, as manifested by their altered solubilization in Brij 96 and organization on the cell surface.
Insights
The CD9 protein
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Tetraspanin CD9 is crucial for sperm-egg fusion and regulates cell adhesion, motility, metastasis, proliferation, and signaling.
- The functional and biochemical roles of the conserved CD9 C-terminal tail remain largely unknown.
- Understanding the CD9 tail's role is key to elucidating its diverse cellular functions.
Purpose of the Study:
- To investigate the functional and biochemical significance of the CD9 C-terminal tail.
- To determine how modifications to the CD9 C-terminal tail affect its interactions and cellular functions.
- To elucidate the role of the CD9 tail in protein complex organization and cell surface behavior.
Main Methods:
- Site-directed mutagenesis to alter the CD9 C-terminal tail (CD9-CoV-PKY mutant).
- Stable expression of wild-type and mutant CD9 in various cell lines (MOLT-4, K562, U937, RD, HT1080).
- Cell adhesion, spreading, homotypic aggregation, and microvilli formation assays.
- Quantitative proteomics using stable isotope labeling with amino acids in cell culture (SILAC) coupled with LC-MS/MS.
- Immunoprecipitation assays in different detergent conditions (Brij 96, Triton X-100).
Main Results:
- Mutant CD9 lacking the native C-terminal tail (CD9-CoV-PKY) failed to inhibit cell adhesion and spreading on fibronectin.
- Wild-type CD9-mediated homotypic cell-cell aggregation and microvilli formation were abolished in the mutant.
- SILAC and immunoprecipitation revealed reduced recovery of mutant CD9 and its interaction partners in Brij 96 lysates, indicating altered detergent solubility.
- Mutant CD9 complexes exhibited increased size and oligomerization in Brij 96, suggesting altered molecular organization and membrane packing.
Conclusions:
- The CD9 C-terminal tail is essential for multiple CD9 functions, including regulation of cell adhesion, aggregation, and microvilli formation.
- The CD9 tail critically influences the molecular organization and detergent solubility of CD9-protein complexes.
- Alterations in the CD9 tail affect its interactions and localization within the cell membrane, impacting its overall cellular roles.
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