Nanoscale organization of tetraspanins during HIV-1 budding by correlative dSTORM/AFM

Dahmane, Selma; Doucet, Christine; Gall, Antoine Le; Chamontin, Célia; Dosset, Patrice; Murcy, Florent; Fernandez, Laurent; Salas, Desireé; Rubinstein, Eric; Mougel, Marylène; Nöllmann, Marcelo; Milhiet, Pierre-Emmanuel

doi:10.1039/c8nr07269h

Cited by 40 publications

(47 citation statements)

References 55 publications

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“…We also used FCS to assess the molecular brightness of ADAM10/Tspan15 BiFC dimers, to estimate the degree of clustering. The existence of some traces containing a brighter Tspan15/ADAM10 component was indicative of larger cluster formation, as has been observed for TspanC8s and other tetraspanins by super-resolution microscopy (35)(36)(37). We propose that FCS imaging of BiFC dimers will be useful for the study of other tetraspanin/partner protein complexes.…”

Section: Discussionsupporting

confidence: 59%

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

Koo

Harrison

Noy

et al. 2020

Journal of Biological Chemistry

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A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer’s disease and inflammation. ADAM10 is a “molecular scissor” that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigating this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

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Section: Discussionsupporting

confidence: 59%

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

Koo

Harrison

Noy

et al. 2020

Journal of Biological Chemistry

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“…We also used FCS to assess the molecular brightness of ADAM10/Tspan15 BiFC dimers, to estimate the degree of clustering. The existence of some traces containing a brighter Tspan15/ADAM10 component was indicative of larger cluster formation, as has been observed for TspanC8s and other tetraspanins by super-resolution microscopy (Zuidscherwoude et al 2015; Marjon et al 2016; Dahmane et al 2019). We propose that FCS imaging of BiFC dimers will be useful for the study of other tetraspanin/partner protein complexes.…”

Section: Discussionsupporting

confidence: 59%

Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

Koo

Harrison

Noy

et al. 2019

Preprint

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1A disintegrin and metalloprotease 10 (ADAM10) is essential for embryonic development and 2 impacts on diseases such as cancer, Alzheimer's and inflammatory diseases. ADAM10 is a 3 'molecular scissor' that proteolytically cleaves the extracellular region from over 100 substrates, 4 including Notch, amyloid precursor protein, cadherins, growth factors and chemokines. 5 ADAM10 was recently proposed to function as six distinct scissors with different substrates, 6 depending on its association with one of six regulatory tetraspanins, termed TspanC8s. 7 However, it remains unclear to what degree ADAM10 function is critically dependent on a 8 TspanC8 partner. To address this, we generated the first monoclonal antibodies to Tspan15 as a 9 model TspanC8. These were used to show that ADAM10 is the principal Tspan15-interacting 10 protein, that Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a 11 synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Together these findings 12 suggest that ADAM10 exists as an intimate ADAM10/TspanC8 scissor complex. 13 14 15A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitously-expressed transmembrane 16 protein which acts as a 'molecular scissor' by cleaving the extracellular region from over 100 17 substrates, a process termed ectodomain shedding (Lichtenthaler et al. 2018). ADAM10 is 18 essential for embryonic development by activating Notch proteins which determine cell fate. 19 Other substrates include cadherin adhesion molecules, amyloid precursor protein, and 20 transmembrane growth factors and chemokines. As such, ADAM10 is important for health and 21 in diseases such as cancer, Alzheimer's and inflammatory diseases (Wetzel et al. 2017). 22 The tetraspanins are a superfamily of 33 transmembrane proteins in mammals which interact 23 with specific transmembrane partner proteins and regulate their intracellular trafficking, lateral 24 mobility and clustering at the cell surface (Termini and Gillette 2017; van Deventer et al. 2017). 25 42 Jouannet et al. 2016; Noy et al. 2016; Seipold et al. 2018). Tspan15 is also upregulated, and is a 43 marker of poor prognosis, in certain cancers (Zhang et al. 2018; Hiroshima et al. 2019; 44 Sidahmed-Adrar et al. 2019) and promotes cancer progression in a mouse model (Zhang et al. 45 2018). The aims of this study were to generate the first Tspan15 mAbs and to test three 46 hypotheses that would support the theory that Tspan15 and ADAM10 exist together as a scissor 47 complex: first, that ADAM10 is the principal Tspan15-interacting protein; second, that Tspan15 48 expression requires ADAM10; and third, that covalently linking Tspan15 and ADAM10 together 49 as a single fusion protein yields a functional scissor. 50 51 Results 52Generation of Tspan15 mAbs 53 The majority of anti-tetraspanin mAbs have epitopes within the large extracellular loop (LEL). 54 However, it has been traditionally difficult to make mAbs to many tetraspanins due to lack of 55 efficacy of recombinant LELs a...

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“…Several studies have shown the localization of tetraspanin proteins in specific regions of curved cell membranes. For instance, CD9 and its related tetraspanin, CD81, are localized in the growing tips of virus buds and involved in the infection cycle, and are directly implicated in generating membrane curvature 13,14 . Molecular shapes of membrane proteins are highly associated with the protein localization as well as membrane shaping and remodeling [15][16][17][18] .…”

Section: Resultsmentioning

confidence: 99%

Structural insights into tetraspanin CD9 function

et al. 2020

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Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membranespanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.

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Nanoscale organization of tetraspanins during HIV-1 budding by correlative dSTORM/AFM

Abstract: Membrane partition and remodeling play a key role in numerous cell mechanisms, especially in viral replication cycles where viruses subvert the plasma membrane to enter and escape from the host cell.

Cited by 40 publications

References 55 publications

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

Structural insights into tetraspanin CD9 function

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