Specialeforsvar: Louise Halkjær-Knudsen

Regulation of TGFβ signaling at the primary cilium

Hovedvejledere: Søren Tvorup Christensen and Lotte Bang Pedersen (The Cilia Group, BIO)

Censor: Steen Gammeltoft

Abstract
Previous studies have shown that canonical TGFβ signaling through the activation of SMAD2/3 relies on clathrin-mediated endocytosis of TGFβ receptors at the pocket region of the primary cilium. However, little is known on sorting and trafficking of TGFβ receptors to the cilium and to which extent retrograde IFT is required for receptor internalization and activation of SMAD2/3. Here, we investigated the presence of ciliary targeting sequences in TGFβ receptor I (TGFβRI) by sequentially deleting parts of the intracellular domain of the receptor and analyzing the ciliary localization of deletion constructs by IFM analysis. We have defined two sequences of interest located in the juxtamembrane and C-terminal domains of the receptor but whether these areas are sufficient to mediate ciliary localization is not yet elucidated. Furthermore, we investigated the effect of impaired retrograde IFT on TGFβ signaling using MEF Dynll1GT/GT cells carrying a mutation in the gene encoding the cytoplasmic dynein light chain LC8-type1. We found that TGFβ stimulation in MEF Dynll1GT/GT is associated with a decrease in TGFβRI accumulation at the ciliary pocket as well as a decrease in SMAD2/3 activation. In contrast, activation of non-canonical signaling, i.e. via phosphorylation of ERK and AKT, was greatly increased in the mutant cells. Based on these findings, we suggest that impaired retrograde IFT disrupts the balancing between canonical and non-canonical TGFβ signaling. Finally, we investigated the role of the centrosomal distal appendage protein, Cep128, in regulating TGFβ signaling at the base of the primary cilium. We found that siRNA-mediated knockdown of Cep128 reduced TGFβ-mediated activation of SMAD2/3, and that this was associated with defects in localization of early and Rab11-positive endosomes at the ciliary pocket. These results provide novel information on how the primary cilium is set up for coordination of TGFβ signaling and how signal transduction relies on multiple regulatory pathways.