Structural and functional studies of cilia and Intraflagellar Transport by 3D cryo-EM
Previous and Current Research
Cryo-transmission electron microscopy (Cryo-EM) is a powerful technology that can be used to reveal the three-dimensional architecture and the assembly of macromolecular machines, organelles and cells. In the last years, I have been using this technology to study the 3D structure of eukaryotic cilia and flagella.
Cilia and flagella are organelles that have major motility and sensory functions in eukaryotic cells, ranging from protists to mammals. Defects in the assembly or function of these organelles can be linked to several human diseases, called ciliopathies. Their structure is based on a stereotyped assembly of microtubules, called the axoneme, which consists of more than 300 different polypeptides, organized in a variety of macromolecular complexes. Together they give rise to the complex and exciting dynamics of cilia and flagella.
Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules. It is required for the assembly, maintenance, and sensory function of cilia and flagella. IFT-particles, which are organized in IFT-trains, are assemblies of more than 20 polypeptides. The trains enter the flagellum at the basal body and bring axonemal components to and from the flagellar tip. The regulation mechanisms of IFT entry, cargo loading/unloading, motor switch, or the position and role of individual IFT-particle proteins are still unknown.
With Prof. Lupetti (University of Siena) and prof. Joel Rosenbaum (Yale University), I have applied room temperature dual-axis electron tomography and sub-tomogram averaging on plastic-embedded IFT-trains in Chlamydomonas flagella. For the first time we showed the 3D structure and spatial assembly of IFT particles in situ (Pigino et al. 2009)(Fig.1). Using wild type and mutant cells with defects in IFT, we identified structural differences between the anterograde and retrograde IFT-trains and built the first 3D model of anterograde IFT trains.
In the lab of Dr. Ishikawa (ETH Zurich/PSI), I have investigated the 3D structure of various axonemal components in flagella and cilia of Chlamydomonas, Tetrahymena, and sea urchin sperms. We have used cryo-electron tomography and single particle analysis to reveal the 3D architecture of radial spokes (RS) in Chlamydomonas and Tetrahymena (Pigino et al. 2011)(Fig.2). In some axonemes, RS are ubiquitous components, thought to be mechanochemical transducers that are involved in controlling dynein-driven microtubule sliding. The analysis of Chlamydomonas mutants enabled us to identify specific locations of subsets of the 23 RS proteins (RSPs). Our 3D reconstructions show a twofold rotational symmetry, suggesting that fully assembled RSs are produced by dimerization of cytoplasmic RS precursors. Based on our cryo-ET data, we where able to propose a model for subdomain organization within the RS and a model for the interactions between RS proteins with other axonemal components.
My research group is currently investigating the 3D structure of cilia and flagella components in various species and cell types with the ultimate goal of explaining the mechanisms of flagellar/ciliary assembly and function.
Future Projects and Goals
We will continue to study the relationship between the 3D structure and the function of macromolecular complexes using a combination of 3D Electron Microscopy, Cryo-Electron Tomography, single particle analysis, correlative fluorescence microscopy, image analysis, and needed biochemical techniques. Special emphasis will be given to:
- The IFT machinery (using cryo-electron tomographic analysis of IFT trains in situ and subtomogram averaging, as well as cryo-single particle analysis of isolated IFT-particles).
- The ciliogenesis and flagellar assembly.
- The 3D structure of primary cilia axonemes.
Methodological and Technical Expertise
- 3D electron microscopy and various techniques for TEM (plastic embedding, high-pressure freezing, freeze substitution, negative staining, ultramicrotomy, etc.)
- cryo-electron microscopy
- cryo-electron tomography, sub-tomogram averaging, and single particle analysis
- correlative light- and electron-microscopy
- Immunolabelling and immunogold labelling
- TIRF microscopy
- single molecule tracking
- cell culture
- flagella and cilia isolation
- purification of axonemal protein components
Pigino, G., & Ishikawa, T.
Axonemal radial spokes: 3D structure, function and assembly.
Bioarchitecture, 2(2), 50–58 (2012)
Pigino, G., Maheshwari, A., Bui, K. H., Shingyoji, C., Kamimura, S., & Ishikawa, T.
Comparative structural analysis of eukaryotic flagella and cilia from Chlamydomonas, Tetrahymena, and sea urchins.
J Struct Biol 178: 199–206 (2012)
Pigino G., Bui K.H., Maheshwari A., Lupetti P., Diener D., Ishikawa T.
Cryoelectron tomography of radial spokes in cilia and flagella.
J Cell Biol; 195(4):673–87 (2011)
Pigino G., Geimer S., Lanzavecchia S., Paccagnini E., Cantele F., Diener D.R., Rosenbaum J.L., Lupetti P.
Electron-tomographic Analysis of Intraflagellar Transport Particle Trains in Situ.
J Cell Biol 187: 135–148 (2009)
Cantele F., Paccagnini E., Pigino G., Lupetti P., Lanzavecchia S.
Simultaneous alignment of dual-axes tilt series.
Journal Structural Biology 169: 192–199 (2010)
Salvi, E., Cantele, F., Zampighi, L., Fain, N., Pigino, G., Zampighi, G., Lanzavecchia, S.
JUST (Java User Segmentation Tool) for semi-automatic segmentation of tomographic maps.
Journal Structural Biology 161: 287–297 (2008)