ABSTRACT
T follicular helper (Tfh) cells are essential for the development of germinal center B cells and high-affinity antibody-producing B cells in humans and mice. Here, we identify the guanine nucleotide exchange factor (GEF) Rin-like (Rinl) as a negative regulator of Tfh generation. Loss of Rinl leads to an increase of Tfh in aging, upon in vivo immunization and acute LCMV Armstrong infection in mice, and in human CD4+ T cell in vitro cultures. Mechanistically, adoptive transfer experiments using WT and Rinl-KO naïve CD4+ T cells unraveled T cell-intrinsic GEF-dependent functions of Rinl. Further, Rinl regulates CD28 internalization and signaling, thereby shaping CD4+ T cell activation and differentiation. Thus, our results identify the GEF Rinl as a negative regulator of global Tfh differentiation in an immunological context and species-independent manner, and furthermore, connect Rinl with CD28 internalization and signaling pathways in CD4+ T cells, demonstrating for the first time the importance of endocytic processes for Tfh differentiation.
Subject(s)
CD28 Antigens , Guanine Nucleotide Exchange Factors , Humans , Animals , Mice , Signal Transduction , Cell Differentiation , Adoptive TransferABSTRACT
Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is the key player during the formation of the neuromuscular junction. Signal transduction events downstream of MuSK activation induce both pre- and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors at synaptic sites. More recently, regulated MuSK endocytosis and degradation have been implicated as crucial events for MuSK signalling activity, implicating a cross-talk between signalling and endocytosis. In the present study, we use a live imaging approach to study MuSK endocytosis. We find that MuSK is internalized via a clathrin-, dynamin-dependent pathway. MuSK is transported to Rab7-positive endosomes for degradation and recycled via Rab4- and Rab11-positive vesicles. MuSK activation by Dok7 mildly affects the localization of MuSK on the cell surface but has no effect on the rate of MuSK internalization. Interestingly, MuSK colocalizes with actin and Arf6 at the cell surface and during endosomal trafficking. Disruption of the actin cytoskeleton or the proper function of Arf6 concentrates MuSK in cell protrusions. Moreover, inhibition of Arf6 or cytoskeletal rearrangements impairs acetylcholine receptor clustering and phosphorylation. These results suggest that MuSK uses both classical and nonclassical endosomal pathways that involve a variety of different components of the endosomal machinery.
Subject(s)
ADP-Ribosylation Factors/metabolism , Actins/metabolism , Clathrin/metabolism , Endocytosis , Receptor Protein-Tyrosine Kinases/metabolism , ADP-Ribosylation Factor 6 , Agrin/metabolism , Animals , COS Cells , Chlorocebus aethiops , Cytoskeleton/metabolism , Dynamins/metabolism , Endosomes/enzymology , Mice , Muscle Proteins/metabolism , Protein Transport , Proteolysis , Receptors, Cholinergic/metabolism , rab GTP-Binding Proteins/metabolism , rab4 GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis.
