The IntraFlagellar Transport machinery: regulation of motors and microtubules dynamics in vitro. – IRMM
The IntraFlagellar Transport machinery: regulation of motors and microtubules dynamics in vitro.
Intraflagellar transport proteins (IFTs) are essential for microtubules (MTs) organization both inside and outside of the cilium, through their interactions with MTs and/or MT associated motors. However, due to the complexity of the IFT machinery a lot of work remains to be done to precisely characterize those molecular mechanisms. Here, to circumvent this complexity, we propose to use simplified in vitro systems to reduce the molecular complexity of the cellular context.
Fine characterization of IFT proteins/Microtubules and motors intercations using a simplififed in vitro system
In this project, we propose to understand how IntraFlagellar Transport (IFT) proteins, that recently emerged as having both ciliary and non-ciliary functions, at different stages of the cell cycle, could regulate the activity and organization of microtubules and their associated motors. Indeed, despite tremendous progress to understand the cellular functions of IFTs, little is known on the precise molecular mechanisms of these complexes as regulators of molecular motors and MT structures. However, due to the complexity of the IFT machinery and its numerous potential molecular interactions at different stages of the cell cycle, a precise understanding of IFTs regulatory functions at the molecular level remains a challenge. Important work remains to be done to precisely characterize their direct role on the regulation of MT dynamics and organization, their specific interaction with cytoplasmic MT associated motors and the impact of these IFT/motor complexes on MTs dynamic and organization. Precisely addressing these questions at the molecular level can only be achieved using simplified in vitro systems to reduce the molecular complexity of the cellular context. Therefore, the general objective of the proposed project is to use purified proteins in an in vitro setup primarily based on TIRF microscopy in order to characterize, at the molecular level, the IFT-MTs-motors interactions and their influence on MT dynamics and organization .
AIM1. Characterization of IFTs-MTs interactions and their impact MT dynamics and organization.
1a- How do IFTs interact with MTs?
Previous works indicate direct IFT/tubulin-MTs interactions. We propose here to take advantage of IFTs subcomplexes with variable composition to validate those interactions by pull down experiments using stabilized MTs. Then, using TIRF microscopy we will precisely characterize the ability of these IFT subcomplexes to interact with either taxol stabilized or dynamic MTs (Figure1).
1b- Do IFTs affect MT dynamics and organization? To answer this question, we will first assess the ability of IFT subcomplexes to modulate MT dynamic instability. Then, we will look for their effects on the self-organization of MTs superstructures such as bundles, antiparallel arrays, MTs branching and MTs crossings (Figure1).
AIM2- Characterization of IFTs-motors interactions and their impact MT organization.
The kinesins superfamily perform a great variety of function both in cilia and cytoplasm. Here we are interested in two kinesins that interact with IFTs and are involved in cellular processes studied in the laboratory: Mklp2 and HSET/KifC1.
2a- How do IFTs interact with MTs bound motors and do they impact on motor activity? We will first validate IFTs-motors interaction using purified proteins in pull down assays. Then, IFTs complexes with variable composition will be used to map more finely the interacting partners. To assess the impact of IFTs on motor activity we will monitor by TIRF microscopy motor behavior on stabilized MTs in the presence of various, fluorescently labeled, subcomplexes (Figure1).
2b- How IFT/Motors complexes impact MTs organization? To address this question, we will add IFTs complexes to dynamic MTs nucleated from MT seeds and assess, first, their ability to modulate MT dynamics and, second, their effects an MTs sliding, bundling, branching and crossing (Figure1).
We have demonstrated a direct interaction between the Kinesin HSET and a subcomplex made of IFT88/70/52/46 using purified recombinant IFT originating from the organism Chlamydomonas Reinhardtii. This result is part of a recently published study which show the importance of IFT proteins for extra centrosome clustering in cancer cells, together with the kinesin HSET: Vitre, B., Taulet, N., Guesdon, A., Douanier, A., Dosdane, A., Cisneros, M., et al. (2020). IFT proteins interact with HSET to promote supernumerary centrosome clustering in mitosis . EMBO Reports Vol. 59, pp.9-15. doi:10.15252/embr.201949234.
We have expressed and purified, from insect cells, individual and subcomplexes of recombinant human IFT proteins (IFT88/70/52/46). We labelled those proteins with fluorescent markers and we started to study their interactions in vitro with the kinesin HSET using TIRF microscopy.
We have identified a direct intercation between IFT88/70/52/46 subcomplex and HSET and its importance for proper extra centrosome clustering in cancer cells.
We will now characterize the intercations between human recombinant IFT proteins with tubulin and microtubules as well as their intercations with the kinesin HSET using TIRF microscopy. This characterization which correspond to AIM1 and part of AIM2 of the project should be achieved within the next 12 months of the project.
We published one original article in EMBO Reports:
Vitre, B., Taulet, N., Guesdon, A., Douanier, A., Dosdane, A., Cisneros, M., et al. (2020). IFT proteins interact with HSET to promote supernumerary centrosome clustering in mitosis . EMBO Reports Vol. 59, pp.9-15. doi:10.15252/embr.201949234.
During cell cycle progression, the MT cytoskeleton is constantly remodelled to ensure that cellular processes occur properly. MTs reorganization is modulated by a variety of molecules including MTs associated proteins (MAPs) and/or molecular motors such as dyneins and kinesins. Our overall objective is to understand how IntraFlagellar Transport (IFT) proteins, that recently emerged as having both ciliary and non-ciliary functions at different stages of the cell cycle, could regulate the activity and organization of microtubules (MTs) and their associated motors. Indeed, despite tremendous progress to understand the cellular functions of IFTs, little is known on the precise molecular mechanisms of these complexes as regulators of molecular motors and MT structures.
Proteins of the IFT machinery are polarized cargo transport complexes that function in non-dividing and dividing cells in association with MTs and motors. They were initially described for their role in cilia and their link to polycystic kidney disease. However, several works aiming at characterizing the functions of IFT proteins in cellular systems and in vivo, including studies from the laboratory, indicate that IFTs also have extra ciliary functions. Indeed, those studies show that IFTs interact with MTs and cytoplasmic molecular motors, such as cytoplasmic dynein and the kinesin MKLP2, and can therefore regulate MT dynamics and the organization of the MT cytoskeleton. Those results reveal new regulatory functions of IFTs on MT cytoskeleton dynamic and organization. It is therefore important to understand in details those regulatory mechanisms. Due to the complexity of the IFT machinery and its numerous potential molecular interactions at different stages of the cell cycle, a precise understanding of IFTs functions at the molecular level remains a challenge.
In order to circumvent the difficulties arising from the complex nature of the cellular environment, we are proposing to address those questions by developing simplified in vitro systems. using purified proteins in an in vitro setup primarily based on TIRF microscopy, we propose here to decipher, at the molecular level, IFT-MTs-motors interactions and their influence on MT dynamics and organization. More specifically, we propose (AIM1) to characterize the interaction between IFT particles constituted of variable number of IFT proteins and stabilized or dynamic MTs. We will also assess how IFTs affect the dynamics and organization of MTs. We will focus primarily our study on sub complexes previously purified by our collaborator containing IFT88 for which non-ciliary roles have been characterized. We will also (AIM2) study how variable IFTs complexes interact and regulate the kinesins Mklp2 and HSET, as we know from recently published and on-going work that they interact with IFTs in vitro and in the cellular context.
Overall, this in vitro work will allow to reduce the molecular complexity of the IFT machinery and thus to better understand, at the molecular level the complex interactions occurring at different stages of the cell cycle and in the cellular environment. This in vitro approach will therefore be essential to complement work currently done in the team to understand IFT functions in cells and in vivo. Given that MKLP2 and HSET are required for cancer cell survival and have been suggested to be promising targets for cancer treatments, this project could, in the long term, results in the development of molecules that would specifically target their activities.
Monsieur Benjamin VITRE (Centre de Recherche en Biologie cellulaire de Montpellier)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CRBM Centre de Recherche en Biologie cellulaire de Montpellier
Help of the ANR 215,719 euros
Beginning and duration of the scientific project: January 2019 - 36 Months