Intrinsic heterogeneity of primary cilia revealed through spatial proteomics

Area of Science:

• Cell Biology
• Genetics
• Proteomics

Background:

• Primary cilia are essential organelles in most human cells, crucial for various cellular functions.
• Dysfunction of primary cilia leads to hereditary ciliopathies, a group of disorders with diverse clinical presentations.
• Understanding the precise roles of cilia in different cell types is limited by challenges in analyzing their protein composition.

Purpose of the Study:

• To expand the Human Protein Atlas by characterizing the spatial proteome of primary cilia using antibody-based spatial proteomics.
• To identify and map the subciliary locations of proteins within primary cilia across different cell types.
• To investigate cell-type specificity and heterogeneity in the ciliary proteome.

Main Methods:

• Utilized antibody-based spatial proteomics to analyze the protein composition of primary cilia.
• Examined 128,156 individual cilia across three distinct cell lines.
• Mapped the subciliary localization of 715 identified proteins.

Main Results:

• Identified the subciliary locations of 715 proteins, significantly expanding the knowledge of the ciliary proteome.
• Found that 69% of the ciliary proteome is cell-type specific, highlighting functional specialization.
• Observed significant heterogeneity, with 78% of cilia showing variations in protein composition within a single cell type.
• Discovered 91 novel cilia proteins and identified a potential genetic link (CREB3 variant) in a patient with ciliopathy-like features.

Conclusions:

• Primary cilia function as dynamic sensors, adapting their proteome to environmental cues and cellular demands.
• The developed spatial cilia atlas provides a valuable resource for advancing research into cilia biology and the mechanisms underlying ciliopathies.
• Revealed extensive cell-type specificity and intra-cilia heterogeneity, underscoring the complexity of ciliary function.