Régulation des processus cellulaires par Huntingtin interacting protein 1-related (HIP1R)

HIP1R (Huntingtin interacting protein 1-related) a des rôles dans l’endocytose médiée par la clathrine, l’apoptose et la mitose. Elle induit l’assemblage de la clathrine, régule la polymérisation de l’actine, contribue au clivage de la caspase-9 et aide l’ancrage des chromosomes sur les microtubules lors de la métaphase. Les domaines protéiques responsables de ces contributions et les facteurs permettant à HIP1R de changer de rôle ne sont pas encore bien connus. Nous avons utilisé la technique du knock-down et nous avons réintroduit des versions mutées ou tronquées d’HIP1R pour caractériser ses rôles en modifiant ses liaisons avec ses partenaires. Les résultats indiqueraient qu’HIP1R participe principalement à l’internalisation des plaques de clathrine et qu’HIP1R n’induit pas l’activation de la caspase-3. Finalement, nous avons montré que la liaison d’HIP1R avec la clathrine lui confère son rôle lors de la mitose lui permettant ainsi de se localiser sur les microtubules pendant la métaphase

Régulation des processus cellulaires par Huntingtin interacting protein 1-related (HIP1R)

HIP1R (Huntingtin interacting protein 1-related) a des rôles dans l’endocytose médiée par la clathrine, l’apoptose et la mitose. Elle induit l’assemblage de la clathrine, régule la polymérisation de l’actine, contribue au clivage de la caspase-9 et aide l’ancrage des chromosomes sur les microtubules lors de la métaphase. Les domaines protéiques responsables de ces contributions et les facteurs permettant à HIP1R de changer de rôle ne sont pas encore bien connus. Nous avons utilisé la technique du knock-down et nous avons réintroduit des versions mutées ou tronquées d’HIP1R pour caractériser ses rôles en modifiant ses liaisons avec ses partenaires. Les résultats indiqueraient qu’HIP1R participe principalement à l’internalisation des plaques de clathrine et qu’HIP1R n’induit pas l’activation de la caspase-3. Finalement, nous avons montré que la liaison d’HIP1R avec la clathrine lui confère son rôle lors de la mitose lui permettant ainsi de se localiser sur les microtubules pendant la métaphase

Unintended perturbation of protein function using GFP nanobodies in human cells

Tagging a protein of interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed ‘dongles’, that could add, for example, an FKBP tag to a GFP-tagged protein of interest, enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2–GFP caused inhibition of dynamin function prior to knocksideways. The function of GFP-tagged tumor protein D54 (TPD54, also known as TPD52L2) in anterograde traffic was also perturbed by dongles. While these issues potentially limit the application of dongles, we discuss strategies for their deployment as cell biological tools

Multi‐modal adaptor‐clathrin contacts drive coated vesicle assembly

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of β2 hinge-appendage (β2HA). We find that the β2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, β2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of β2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the “sandwich site” on the appendage. We propose that β2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2

Tumor protein D54 defines a new class of intracellular transport vesicles

Transport of proteins and lipids from one membrane compartment to another is via intracellular vesicles. We investigated the function of tumor protein D54 (TPD54/TPD52L2) and found that TPD54 was involved in multiple membrane trafficking pathways: anterograde traffic, recycling, and Golgi integrity. To understand how TPD54 controls these diverse functions, we used an inducible method to reroute TPD54 to mitochondria. Surprisingly, this manipulation resulted in the capture of many small vesicles (30 nm diameter) at the mitochondrial surface. Super-resolution imaging confirmed the presence of similarly sized TPD54-positive structures under normal conditions. It appears that TPD54 defines a new class of transport vesicle, which we term intracellular nanovesicles (INVs). INVs meet three criteria for functionality. They contain specific cargo, they have certain R-SNAREs for fusion, and they are endowed with a variety of Rab GTPases (16 out of 43 tested). The molecular heterogeneity of INVs and the diverse functions of TPD54 suggest that INVs have various membrane origins and a number of destinations. We propose that INVs are a generic class of transport vesicle that transfer cargo between these varied locations

Cell biology of tumor protein D54 (TPD54)

The expression of Tumor protein D52 (TPD52) family members is deregulated in many types of cancer. When overexpressed, it is suggested that they increase cell proliferation and migration/invasion as well as avoid apoptosis. Deregulation in the expression of the TPDs is therefore linked to poor prognosis. Little characterisation has been carried out to date, but it is known that the TPDs are found in association with components of the membrane trafficking pathway. The aim of this work is to uncover how the least studied member of the family, TPD54, affects cellular processes involved in carcinogenesis, such as cell migration and invasion. By using the knocksideways method, we have been able to map the cellular localisation of TPD54 and have identified association partners. These associations have been confirmed by immunoprecipitation and mass spectrometry analysis. Amongst these was the small GTPase Rab14. We have also found that TPD54 is involved in the trafficking of receptors containing a dileucine motif in their cytosolic tail, but not a tyrosine-based or NPXY motif. With the mapping of the localisation of TPD54, we hypothesise that TPD54 is on the recycling route following the Golgi apparatus, and in association with Rab14, regulates the trafficking of receptors containing a dileucine motif. Integrins are receptors controlling cell migration. They can be trafficked through the Golgi apparatus before being recycled back to the plasma membrane. This recycling route is not well characterised. We therefore hypothesise that TPD54 regulates this route with Rab14, and that this is the reason why TPD54 is important for cell migration, and that a defect in its function can cause cancer

Du matériel à l’immatériel : réflexion sur une muséologie adaptée à la danse

La conservation et la mise en valeur du patrimoine vivant posent aujourd’hui un certain nombre de défis aux institutions muséales dès lors que leur mission de contribuer à la mémoire culturelle d’une nation justifie à elle seule l’ex­ploration de ces nouveaux patrimoines. Nous nous focaliserons ici sur le cas précis de la danse du fait de sa capacité à stimuler les pratiques muséales et, à travers l’invention de nouvelles modalités de conservation et de mise en valeur, à justifier des partena..