Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid

Cell density is a key factor in tendon development, but the molecular mechanism remains unclear. Prior research has shown that a diffusible factor called SNZR is involved in cell density sensing. SNZR was purified through multiple chromatography steps and identified as a 16 kD protein band. N-terminal sequencing revealed a conserved gene across species, but its function is unknown. The gene encodes a large protein that may be cleaved into a smaller version of 94 amino acids. This smaller protein is hypothesized to bind a cofactor to appear larger on SDS gels. The protein lacks hydrophobic regions, raising questions about its secretion and signaling. This gap motivated the investigation into the gene's expression and the role of its cofactor.

The study aims to determine if the predicted 94 amino acid protein is the active form of SNZR. It explores whether this protein can be cleaved and how it binds to a cofactor. The research also investigates the protein's secretion and its role in cell density signaling. Expression of the gene in a human cell line is tested to observe morphological changes. Western blotting is used to detect intracellular and extracellular forms of the protein. The study examines how cofactor binding affects the protein's apparent size on gels. It seeks to identify the cofactor's composition and its tissue-specific role in signaling. The goal is to confirm the protein-lipid complex as the cell density sensor in tendon development.

The gene was cloned and tagged with myc and his for expression in U2OS cells. Western blotting was used to detect intracellular and extracellular protein forms. Mass spectrometry identified the cofactor as a ceramide phosphate-based lipid. Salt concentration and ultrafiltration experiments were conducted to separate cofactor and protein. Degradative enzymes helped confirm the lipid's structure and interaction with the protein. Frozen mouse tissue sections were stained with an antibody to the protein for localization. Cell culture experiments tested the effect of the cofactor on osteosarcoma cell growth. These methods combined molecular biology, biochemistry, and microscopy to validate the protein's role.

Expression of the gene in U2OS cells led to differentiation into bone-like structures. Western blotting showed two intracellular bands: full-length and a 26 kD cleaved form. Extracellularly, a 28 kD band was detected, larger than expected for the cleaved protein. Changing salt concentrations and ultrafiltration released the cofactor into the filtrate. Mass spectrometry identified the cofactor as a ceramide phosphate-based lipid complex. Tendon uses a different cofactor with two fatty acid chains linked to the phosphate. Adding the tendon cofactor to osteosarcoma cells inhibited growth, unlike in tendon cells. Antibody staining in mouse tissue confirmed the protein's presence at growing tendon ends.

The study confirms that a small protein binds to a tissue-specific phospholipid to form a signaling complex. This complex is membrane-associated but diffusible, enabling cell density sensing in tendon development. The protein's apparent size on gels is due to cofactor binding, not protein modification. The cofactor's composition and effect are tissue-specific, influencing cell behavior differently. Signal transduction is proposed to involve membrane ordering as protein/lipid concentration increases. These findings align with the authors' hypothesis of a molecular mechanism for cell density signaling. The protein-lipid complex is the missing link in understanding tendon morphogenesis. The results support the role of this complex in regulating proliferation and collagen production.