Arf6 and Rab22 mediate T cell conjugate formation by regulating clathrin-independent endosomal membrane trafficking.

Endosomal trafficking can influence the composition of the plasma membrane and the ability of cells to polarize their membranes. Here, we examined whether trafficking through clathrin-independent endocytosis (CIE) affects the ability of T cells to form a cell-cell conjugate with antigen-presenting cells (APCs). We show that CIE occurs in both the Jurkat T cell line and primary human T cells. In Jurkat cells, the activities of two guanine nucleotide binding proteins, Arf6 and Rab22 (also known as Rab22a), influence CIE and conjugate formation. Expression of the constitutively active form of Arf6, Arf6Q67L, inhibits CIE and conjugate formation, and results in the accumulation of vacuoles containing lymphocyte function-associated antigen 1 (LFA-1) and CD4, molecules important for T cell interaction with the APC. Moreover, expression of the GTP-binding defective mutant of Rab22, Rab22S19N, inhibits CIE and conjugate formation, suggesting that Rab22 function is required for these activities. Furthermore, Jurkat cells expressing Rab22S19N were impaired in spreading onto coverslips coated with T cell receptor-activating antibodies. These observations support a role for CIE, Arf6 and Rab22 in conjugate formation between T cells and APCs.

Rab and Arf G proteins in endosomal trafficking and cell surface homeostasis.

The dynamics of membrane fusion, fission, cargo sorting and organelle maturation in endosomal membrane systems is regulated by Rab and Arf small G proteins. Defining the regulators, effectors and sites of action for each Rab and Arf will enhance our understanding of how endocytic membrane traffic is orchestrated and functions in differentiated cell types. Recent work has also shown how Rab35 and Arf6 might serve as input sensors for 2 forms of endocytosis to balance membrane trafficking to preserve cell surface homeostasis.

Endosomal regulation of contact inhibition through the AMOT:YAP pathway.

Contact-mediated inhibition of cell proliferation is an essential part of organ growth control; the transcription coactivator Yes-associated protein (YAP) plays a pivotal role in this process. In addition to phosphorylation-dependent regulation of YAP, the integral membrane protein angiomotin (AMOT) and AMOT family members control YAP through direct binding. Here we report that regulation of YAP activity occurs at the endosomal membrane through a dynamic interaction of AMOT with an endosomal integral membrane protein, endotubin (EDTB). EDTB interacts with both AMOT and occludin and preferentially associates with occludin in confluent cells but with AMOT family members in subconfluent cells. EDTB competes with YAP for binding to AMOT proteins in subconfluent cells. Overexpression of the cytoplasmic domain or full-length EDTB induces translocation of YAP to the nucleus, an overgrowth phenotype, and growth in soft agar. This increase in proliferation is dependent upon YAP activity and is complemented by overexpression of p130-AMOT. Furthermore, overexpression of EDTB inhibits the AMOT:YAP interaction. EDTB and AMOT have a greater association in subconfluent cells compared with confluent cells, and this association is regulated at the endosomal membrane. These data provide a link between the trafficking of tight junction proteins through endosomes and contact-inhibition-regulated cell growth.

Rab14 regulation of claudin-2 trafficking modulates epithelial permeability and lumen morphogenesis.

Regulation of epithelial barrier function requires targeted insertion of tight junction proteins that have distinct selectively permeable characteristics. The insertion of newly synthesized proteins and recycling of internalized tight junction components control both polarity and junction function. Here we show that the small GTPase Rab14 regulates tight junction structure. In Madin-Darby canine kidney (MDCK) II cells, Rab14 colocalizes with junctional proteins, and knockdown of Rab14 results in increased transepithelial resistance. In cells without Rab14, there are small changes in the trafficking of claudin-1 and occludin. In addition, there is substantial depletion of the leaky claudin, claudin-2, but not other tight junction components. The loss of claudin-2 is complemented by inhibition of lysosomal function, suggesting that Rab14 sorts claudin-2 out of the lysosome-directed pathway. MDCK I cells lack claudin-2 endogenously, and knockdown of Rab14 in these cells does not result in a change in transepithelial resistance, suggesting that the effect is specific to claudin-2 trafficking. Furthermore, leaky claudins have been shown to be required for epithelial morphogenesis, and knockdown of Rab14 results in failure to form normal single-lumen cysts in three-dimensional culture. These results implicate Rab14 in specialized trafficking of claudin-2 from the recycling endosome.

Regulation of tight junction assembly and epithelial polarity by a resident protein of apical endosomes.

The establishment of tight junctions and cell polarity is an essential process in all epithelia. Endotubin is an integral membrane protein found in apical endosomes of developing epithelia when tight junctions and epithelial polarity first arise. We found that the disruption of endotubin function in cells in culture by siRNA or overexpression of the C-terminal cytoplasmic domain of endotubin causes defects in organization and function of tight junctions. We observe defects in localization of tight junction proteins, reduced transepithelial resistance, increased lanthanum penetration between cells and reduced ability of cells to form cysts in three-dimensional culture. In addition, in cells overexpressing the C-terminal domain of endotubin, we observe a delay in re-establishing the normal distribution of endosomes after calcium switch. These results suggest that endotubin regulates trafficking of polarity proteins and tight junction components out of the endosomal compartment, thereby providing a critical link between a resident protein of apical endosomes and tight junctions.

Correlation between extraversion and regional cerebral blood flow in response to olfactory stimuli.

OBJECTIVE: Extraversion, a trait associated with individual differences in approach motivation and the experience of positive emotional states, is negatively correlated with certain psychiatric disorders, including depression and social phobia. The authors examined the correlation between extraversion and regional cerebral blood flow (rCBF) while participants were exposed to olfactory stimuli in order to further characterize individual differences in hedonic processing associated with this trait. METHOD: Twelve healthy participants were exposed to pleasant and unpleasant odors while rCBF was measured using [(15)O] water PET. The NEO Five-Factor Inventory was used to assess extraversion. Associations between extraversion scores and rCBF in each olfactory stimulus condition were assessed by correlational analysis. RESULTS: During the pleasant smell condition, extraversion was correlated with rCBF in the amygdala and occipital cortex. During the unpleasant smell condition, extraversion was correlated with rCBF in the occipital cortex and inferior temporal gyrus. CONCLUSIONS: These results provide important evidence for the biological basis of extraversion and indicate that there are systematic individual differences in patterns of brain activation in response to affective stimuli.