Basal body anchoring in ciliogenesis: structure-function analysis – ANCHOR

1) to identify interacting protein partners involved in the anchoring process using the proximity dependent biotin identification (BioID) technique and to dissect their functional interactions
2) to use cutting edge electron microscopy technologies in order to gain insights into the structural mechanisms underlying the transition zone formation at nanometric resolution

Setting up in the lab the BioID technic. This technic allows us to identify new partners at proximity of the bait. Those partners are cuurently under investigation.
We show that depletion of the two MKS module proteins induces spontaneous cilia autotomy, while the depletion of either NPHP4, Cep290 or Rpgrip1l inhibits the process. Our results constitute the first evidence for a role of conserved TZ proteins in deciliation and open new directions for understanding motile cilia physiology.
Tomograms allow to observe already described features such as: C-tubules terminate above the transition zone terminal plate, transition fibers emerge from the B tubule. However the obtained volumes allow a clarification of the structures and the twist adopted by them. In addition some cherry tails are occasionally observed starting from both the A and B tubule and might be the so-called Y-links;

The combination of transcriptomic, biochemical and ultrastructural methods should decipher the interaction network
required for basal body anchorage and transition zone assembly, and clarify the underlying mechanisms. The large
number of basal bodies in Paramecium, together with simple and reliable protocols for RNAi and cortex purification,
should give rise to original results, namely a projection into the 4D space of the nanometric-resolution data acquired in 3D.
These results would open the way to further studies linking atomic structure with transition zone assembly. Since, in
Paramecium, the structure of the transition zone matures along with the growth of a cilium, our study will provide a basis
for understanding the structural changes bringing its functionality to the transition zone.

Bengueddach, H., Lemullois, M., Aubusson-Fleury, A., and Koll, F. (2017). Basal body positioning and anchoring in the multiciliated cell Paramecium tetraurelia: roles of OFD1 and VFL3. Cilia 6, 6. (P1)

Tassin, A.-M., Lemullois, M., and Aubusson-Fleury, A. (2016). Paramecium tetraurelia basal body structure. Cilia 5, 6. (P1)

The cilium is a highly evolutionarily conserved cell appendage endowed with motility and sensory functions. This “cell antennae”, known as the primary cilium in most cells of multicellular organisms, has emerged as a key organelle in numerous developmental and physiological processes. For example, in human it is involved in the regulation of embryogenesis, tumorigenesis, kidney function, vision and smell. Consequently, inborn defects in human cilia result in patho-physiological conditions associated with diverse clinical manifestations called ciliopathies. Ciliogenesis is a multi-step process, including centriole/basal body duplication and maturation, followed by its migration, anchoring and docking to the cell surface where it templates the growth of the cilium. The molecular events implicated in centriole/basal body duplication are now well characterized. By contrast, the further steps are still under active investigation. Basal body anchoring involves multiple subcellular partners: the basal body itself, a membrane (either a vesicle or the plasma membrane) and cytoskeletal elements that guide the basal body and coordinate the operations. Docking triggers the formation of the transition zone, which mediates the interaction of the basal body with the membrane. This structural junction, located between the basal body and the cilium acts as a filter between the cellular and the cilium compartment and houses many proteins involved in human ciliopathies.
The major objective of our project is to understand the general mechanisms underlying the basal body anchoring process and transition zone formation. Paramecium, a large unicellular organism covered with cilia and expressing a whole set of proteins also present in metazoan, will be used as a stepping-stone for comparative analyses in mammalian cells. To duplicate its cell pattern at each cell division, Paramecium assembles thousands of basal bodies close to the cell surface in a rigorously ordered spatio-temporal sequence. The mechanisms of basal body anchoring can thus be easily investigated by biochemical and ultrastructural approaches, making of this simple organism an outstanding model. Hence, through a combination of GFP-fusion proteins, RNAi and low-resolution electron microscopy, we recently demonstrated that in Paramecium, centrin2, the ciliopathy protein OFD1, FOR20, and centrin3 are sequentially required for building of the transition zone and basal body anchoring. These findings further indicated that the anchoring process parallels the structural assembly of the transition zone. In mammals, regulation of anchoring by post-translational modifications such as ubiquitination, has recently been reported by our group, revealing a further level of complexity in this process.
We first aim at dissecting the spatio-temporal recruitment of proteins in Paramecium, as well as the physiological mechanisms involved in the basal body anchoring process and transition zone formation. In a second step, data derived from this approach will be applied to mammalian cells.
We propose multi-disciplinary approaches, combining biochemical, molecular and cell biology techniques with cryo-electron and STEM tomography. We plan: 1) to identify interacting protein partners involved in the anchoring process using the proximity dependent biotin identification (BioID) technique and to dissect their functional interactions, 2) to perform a transcriptomic screen to track additional members of the process, and 3) to use cutting edge electron microscopy technologies in order to gain insights into the structural mechanisms underlying the transition zone formation at nanometric resolution.
Together, the information should give rise to a 4D (space-time) analysis of the basal body anchoring process. Likewise, data from this study should be helpful for a better understanding of human ciliary defects.