The primary cilium is a several µm long microtubule-based protrusion that is present on almost all cells of our body. With only 1:10.000 of the cell’s volume it was long thought to be rudimentary, yet, the cilium is now recognized to play a major role in tissue homeostasis and patterning. Hence, ciliary defects contribute to neoplastic growth and lead to severe human disorders, termed ciliopathies with many different tissues affected. However, many cilia functions as well as the molecular causations of the underlying tissue-specific defects remain enigmatic.

Architecture of a primary cilium.
© Tommy Sroka




In addition to classic biochemical and fluorescence microscopy methods, our group employs novel biochemical proximity labeling methods using ascorbate peroxidase (APEX) to attach biotin-derivatives to nearby proteins, which allows subsequent isolation and identification by mass-spectrometry. Due to its tunable enzymatic activity APEX allowed us to generate snapshots of the ciliary proteome under different signaling states. We believe that further development and application of this powerful technology will allow us to decipher the protein content of cilia in different cell types and provide new clues on the unknown functions of cilia as well as molecular causations of ciliopathies.

Schematic cilia-APEX2 workflow.
© Tommy Sroka



A central signaling pathway impacting tissue patterning and embryonic development is Hedgehog signaling, which –when defective- can also cause severe forms of childhood cancers. Almost all known components of the Hedgehog signaling pathway in vertebrates can be found in the primary cilium, yet, not at the same time, since their localization depends on the signaling state. Moreover, we do not know the precise functional relationship between components and additional unknown factors are likely involved.

One goal of our studies is the precise description of the proteomic changes in cilia during cellular signaling events, such as Hedgehog signaling, by APEX-based time-resolved proteomic profiling using quantitative mass spectrometry methods. This will set the groundwork to reveal the molecular involvement of the cilium in specific signaling pathways and how cilia transform external signals into cellular responses.

Sonic hedgehog signaling in primary cilia. Left: unstimulated pathway. Right: stimulated with patched ligand and active state.

May, Sroka and Mick (2021). Front. Cell Dev. Biol. 9:664279.
doi: 10.3389/fcell.2021.664279 © Tommy Sroka


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