CE13 - Biologie Cellulaire, biologie du développement et de l’évolution 

MICROTUBULE INNER PROTEINS (MIPS): from molecular identification to structural properties and functions – MIP-MAP

«MICROTUBULAR INTERNAL PROTEINS (MIP): from identification to their structural and functional properties

Following the discovery of the presence of MAP6 inside neuronal microtubules, MIP-MAP aims to determine whether other members of the MAP6 family are also MIPs as well as their roles in the physiology of cilia and flagella, structures in which they are highly expressed. Regarding MAP6, the aim is to determine the ultrastructural basis of its intraluminal localization and to detect microtubules containing MAP6 inside neurons.

characterisation of MAP6 menbers

The objectives of the program are to i) characterize the molecular properties of the MAP6 family of proteins as MIPs ii) describe their localization in cytoplasmic MTs and/or cilia/flagellar MT doublets iii) analyze their roles in the physiology of sperm and airway cells.

This program will be carried out by two research groups from Grenoble with complementary expertise, on the study of the properties of MTs in vitro and in vivo (team A Andrieux/I Arnal) and the structure/function relationships of ciliary and flagellar proteins (team C Arnoult).

Premières images obtenues pour MAP6 en dehors des MTs autour de la résolution de 4.7Å
*Obtention de souris KO pour SAXO1 et SAXO2
*Validation de l'étiquette alfa comme outil pour les souris KI.

The originality of the MIP-MAP program is based on the combination of different model systems (from the molecule to the cells and animal models) and advanced optical and electronic imaging methods. This program will allow a better understanding of mammalian MIPs and the mechanisms of intra-luminal stabilization of MTs. In terms of applications, the results could help to understand ciliopathies and infertility syndromes.

to come

Microtubules (MTs) are hollow tubes composed of tubulin heterodimers that play crucial roles in cell division and migration, intracellular trafficking and ciliary/flagellar motility. Virtually all cells contain cytoplasmic singlet MTs. Doublet MTs organized in a nine-fold radial symmetry are found in conserved structures, the axonemes in cilia and flagella. Non-motile cilia act as cellular antennae, whereas motile cilia and flagella, found on specialized cells such as multiciliated epithelial cells and sperm, beat to drive fluid flow or cellular motility. Cilia dysfunctions cause a number of ciliopathies including central nervous system malformation, cystic kidney disorders, and diseases of the airways; flagellar defects induce male sterility.
The remarkable stability of doublet MTs supports a stable length of cilia/flagella and withstands their high-frequency beating. In unicellular organisms (Chlamydomonas, Tetrahymena), this stability was shown to be driven by MT Inner Proteins (MIPs) localized in the MT lumen; 33 of these MT-stabilizing MIPs were recently identified in Chlamydomonas flagella. The molecular identities of MIPs in mammals remained totally unknown until we discovered that the MT-associated protein, MAP6, localizes in the lumen of singlet neuronal MTs. MAP6 is thus the first mammalian MIP. We subsequently found that one Chlamydomonas flagella MIP, FAP363, is an ancestor of mammalian MAP6, and that the two proteins share similar MT-binding domains, Mn modules, anchoring FAP363 to the MT’s inner surface. Three other genes encoding proteins containing similar Mn modules exist in mammals, SAXO1, SAXO2 and MAP6d1. The corresponding proteins are expressed at high levels in sperm, ciliated cells and neurons, respectively. Their shared MT-binding Mn modules suggest that these proteins could be the first family of mammalian MIPs (MAP6-family), and that its members could play crucial roles in axonemal and neuronal MT stability. Study of MAP6-family proteins represents a unique opportunity to characterize mammalian MIPs and create knowledge in a truly virgin field of research.
The objectives of this program are to (i) characterize the molecular properties of MAP6 proteins as MIPs, (ii) describe their localization in various types of MT arrangements (cytoplasmic MTs, non-motile/motile cilia, flagella), and (iii) assess their roles in the physiology of sperm and airway cells. We will thus 1/determine whether MAP6d1, SAXO1 and SAXO2 localize in the MT lumen (cell-free systems/cellular extracts, cryo-electron microscopy) 2/solve the high-resolution structure of the canonical neuronal MAP6 inside MTs to establish the structural basis of singlet MT luminal stabilization (cell-free systems, cryo-electron microscopy) 3/detect and visualize MAP6, MAP6d1, SAXO1 and SAXO2 in various types of MTs including singlet neuronal MTs and ciliary/flagellar doublet MTs (expansion microscopy, super-resolution light imaging applied to various cell types) 4/determine how MAP6 and SAXO1 affect sperm flagella and fertility, and how SAXO2 influences ciliary function in airway cells (MAP6 and SAXO1/2 KO cells and mice).
To complete this ground-breaking program, we will combine the strengths of two Grenoble-based teams with complementary and world-recognized expertise relating to in vitro and in vivo studies of MTs (A Andrieux/I Arnal) and structural/functional studies of ciliary/flagellar proteins (C Arnoult). The originality of the MIPMAP project lies in the combined use of several model systems (molecules, multiple cell types, and animal models) and advanced imaging methods (light- and electron-microscopy, expansion microscopy). Overall, this program will substantially advance our knowledge of mammalian MIPs and provide molecular elements shedding new light on how MTs are stabilized by proteins located inside their lumen. In terms of potential applications and translation, results might help to understand ciliopathies and infertility syndromes.

Project coordination

ANNIE ANDRIEUX (GRENOBLE INSTITUT DES NEUROSCIENCES (GIN))

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.

Partner

U 1216 GRENOBLE INSTITUT DES NEUROSCIENCES (GIN)
IAB Institut pour l'Avancée des Biosciences

Help of the ANR 537,798 euros
Beginning and duration of the scientific project: December 2020 - 48 Months

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