A Genome-wide Multidimensional RNAi Screen Reveals Pathways Controlling MHC Class II Antigen Presentation

Paul, Petra; Hoorn, Tineke van den; Jongsma, Marlieke L.M.; Bakker, Mark J.; Hengeveld, Rutger C C; Janssen, Lennert; Cresswell, Peter; Egan, David A.; Ham, Marieke van; Brinke, Anja ten; Ovaa, Huib; Beijersbergen, Roderick L.; Kuijl, Coenraad; Neefjes, Jacques

doi:10.1016/j.cell.2011.03.023

Cited by 141 publications

(125 citation statements)

References 83 publications

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“…Fusion to lysosomes is mediated by the recruitment of the tether complex homotypic fusion and protein sorting (HOPS) (16,17). The transport of MHC II to the late endolysosomal system seems to occur through endosomal maturation and seems to require dynein motors and ESCRT complexes (3,(18)(19)(20). Yet, the GTPase regulating the delivery of Ag to MHC II compartments for generating MHC II-peptide complexes remains unknown.…”

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confidence: 99%

MHC Class II Presentation Is Controlled by the Lysosomal Small GTPase, Arl8b

Michelet

Garg

Wolf

et al. 2015

The Journal of Immunology

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Dendritic cells (DCs) are specialized APCs with the ability to prime naive T cells. DCs first sample Ags from the environment and then orchestrate their processing and loading onto MHC class II (MHC II) Ag-presenting molecules in lysosomes. Once MHC II molecules have bound a peptide, the MHC II–peptide complex is delivered to the cell surface for presentation to CD4+ T cells. Regulation of Ag uptake via macropinocytosis and phagocytosis has been extensively studied, as well as trafficking in early endocytic vesicles notably regulated by the small GTPase Rab5 and its effectors. However, little is known about the regulators of Ag delivery from early endosomes to lysosomal compartments where the proper pH, proteases, MHC II, invariant chain, and HLA-DM reside, awaiting exogenous Ags for loading. In this article, we report the crucial role of the small GTPase ADP-ribosylation factor-like 8b (Arl8b) in MHC II presentation in DCs. We show for the first time, to our knowledge, that Arl8b localizes to MHC II compartments in DCs and regulates formation of MHC II–peptide complexes. Arl8b-silenced DCs display a defect in MHC II–Ag complex formation and its delivery to the cell surface during infection resulting in a defect in T cell recognition. Our results highlight the role of Arl8b as a trafficking regulator of the late stage of complex formation and MHC II presentation in DCs.

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confidence: 99%

MHC Class II Presentation Is Controlled by the Lysosomal Small GTPase, Arl8b

Michelet

Garg

Wolf

et al. 2015

The Journal of Immunology

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“…Ii fills the peptide loading groove of MHC-II and targets MHC-II to late endosomal compartments, generally named MIIC (Neefjes et al, 1990;Roche and Cresswell, 1990;Peters et al, 1991). The ESCRT machinery is important for MIIC sorting and function, as shown by an siRNA screen for MHC-II peptide loading and expression that revealed many components of the ESCRT 0, I, II and III system (Paul et al, 2011). In the MIIC, Ii is degraded by resident proteases, except for a short fragment called CLIP that is protected by the surrounding peptide-binding groove.…”

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confidence: 99%

“…We silenced four tetraspanin proteins located in the MVB (CD9, CD63, CD81 and CD82) and determined the effect on MHC-II expression and peptide loading, using flow cytometry as described before (Paul et al, 2011). All but the CD82 tetraspanin appeared to control MHC-II expression, although silencing the tetraspanin proteins did not prevent peptide loading.…”

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confidence: 99%

Dynamics within tetraspanin pairs affect MHC class II expression

Hoorn

Paul

Janssen

et al. 2012

Journal of Cell Science

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SummaryLate endosomal multivesicular bodies (MVBs) are complicated organelles with various subdomains located at the limiting membrane and the internal vesicles (ILVs). ILVs accumulate tetraspanins such as CD63 and CD82 that might form protein assemblies, including major histocompatibility complex class II (MHC-II) and its chaperone human leukocyte antigen (HLA)-DM. Here, we studied the effect of four late endosomal tetraspanin proteins on MHC-II expression. Silencing CD9, CD63 and CD81 enhanced MHC-II expression whereas silencing CD82 did not. No effect on peptide loading was observed. Using confocal FRET technology, we measured the dynamics of CD63 and CD82 interaction with MHC-II and its chaperone HLA-DM. CD63-CD82 interactions remained unaltered in the two MVB subdomains whereas the interactions between CD63 or CD82 homologous pairs changed. CD63 stably associated with MHC-II, and CD82 with HLA-DM, on both MVB subdomains whereas the CD82-MHC-II and CD63-HLA-DM interactions changed. These data visualize for the first time the protein dynamics of tetraspanin assemblies in MVB subdomains. CD63, unlike CD82, stably interacts with MHC-II at both MVB subdomains and controls MHC-II expression.

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“…Systematic analysis of kinesins has already been performed in the context of cell cycle regulation (Tanenbaum et al, 2009;Goshima and Vale, 2003;Neumann et al, 2010;Zhu et al, 2005a) and membrane trafficking (Collinet et al, 2010;Paul et al, 2011;Simpson et al, 2012), but information about their roles specifically in integrin trafficking is missing. We observed no effect on a2 integrin trafficking when downregulating 13 kinesins that had no effect in the primary screen (supplementary material Tables S2, S4), indicating high fidelity of our assays.…”

Section: Discussionmentioning

confidence: 99%

RNAi screen identifies KIF15 as a novel regulator of integrin endocytic trafficking

Eskova

Knapp

Matelska

et al. 2014

Journal of Cell Science

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ABSTRACTa2b1 integrin is one of the most important collagen-binding receptors, and it has been implicated in numerous thrombotic and immune diseases. a2b1 integrin is a potent tumour suppressor, and its downregulation is associated with increased metastasis and poor prognosis in breast cancer. Currently, very little is known about the mechanism that regulates the cell-surface expression and trafficking of a2b1 integrin. Here, using a quantitative fluorescence-microscopybased RNAi assay, we investigated the impact of 386 cytoskeletonassociated or -regulatory genes on a2 integrin endocytosis and found that 122 of these affected the intracellular accumulation of a2 integrin. Of these, 83 were found to be putative regulators of a2 integrin trafficking and/or expression, with no observed effect on the internalization of epidermal growth factor (EGF) or transferrin. Further interrogation and validation of the siRNA screen revealed a role for KIF15, a microtubule-based molecular motor, as a significant inhibitor of the endocytic trafficking of a2 integrin. Our data suggest a novel role for KIF15 in mediating plasma membrane localization of the alternative clathrin adaptor Dab2, thus impinging on pathways that regulate a2 integrin internalization.

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A Genome-wide Multidimensional RNAi Screen Reveals Pathways Controlling MHC Class II Antigen Presentation

Cited by 141 publications

References 83 publications

MHC Class II Presentation Is Controlled by the Lysosomal Small GTPase, Arl8b

MHC Class II Presentation Is Controlled by the Lysosomal Small GTPase, Arl8b

Dynamics within tetraspanin pairs affect MHC class II expression

RNAi screen identifies KIF15 as a novel regulator of integrin endocytic trafficking

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