The adult thymus provides a variety of specialized microenvironments that support and direct T cell differentiation and selection. In this review, we summarize recent advances in the understanding of the function of microenvironments in shaping a diverse T cell repertoire. In particular, we focus on how thymocytes move in and out of these specialized thymic compartments in response to homing signals, differential chemokine gradients and other factors that regulate T cell migration. In addition, we discuss the diverse developmental signals provided by these microenvironments that contribute to the generation of divergent T cell lineages.

Gap junctions are important in maintaining lens transparency and metabolic homeostasis. In this paper, we report that the gap junction-forming protein, connexin (Cx) 45.6, was specifically truncated during lens development and that the majority of the truncated fragments were located in the differentiated lens fibers. When isolated lens membranes were treated by caspase-3, the truncated fragments of Cx45.6 were reproduced, and this truncation occurred at the COOH terminus of Cx45.6. Moreover, when primary lens cells were treated with apoptosis-inducing reagents, Cx45.6 was cleaved similarly as the in vitro treatment by caspase-3, and this cleavage was blocked by a caspase-3 inhibitor. These results suggest that caspase-3 is responsible for the development-associated cleavage of Cx45.6. The cleavage site of Cx45.6 was identified between amino acid residues Glu 367 and Gly 368 . We have shown previously that Ser 363 is an in vivo phosphorylated site by casein kinase II, and this specific phosphorylation leads to a rapid turnover of Cx45.6. Interestingly, we found here that when Ser 363 was phosphorylated by casein kinase II, the cleavage of Cx45.6 catalyzed by caspase-3 was inhibited. This study, for the first time, demonstrates that a connexin can be a direct target of an apoptotic protease and that cleavage by caspase-3-like protease leads to the development-associated truncation of a lens connexin. Finally, caspase-3-mediated cleavage can be regulated by casein kinase II-mediated phosphorylation, suggesting that Cx45.6 turnover and specific cleavage by caspase-3-like protease is alternatively modulated.

Little is known about the dynamics of the interactions between thymocytes and other cell types, as well as the spatiotemporal distribution of thymocytes during positive selection in the microenvironment of the cortex. We used two-photon laser scanning microscopy of the mouse thymus to visualize thymocytes and dendritic cells (DCs) and to characterize their interactions in the cortex. We show that thymocytes make frequent contacts with DCs in the thymic cortex and that these associations increase when thymocytes express T cell receptors that mediate positive selection. We also show that cortical DCs and the chemokine CCL21 To what extent the cortex itself is divided into distinct functional compartments is not yet clear. At one end of the spectrum, it is possible that the cortex is functionally homogeneous. According to this view, cortical thymocytes would invariably be in contact with thymic epithelial cells capable of mediating positive selection, and positive selection could take place with equal probability throughout the cortex. On the other hand, there are indications that cortical thymic epithelial cells are heterogeneous (reviewed in Ref. 7), raising the possibility of functionally distinct regions of the cortex specialized for mediating selection events. The anatomical segregation of thymocytes in the cortex based on their TCR specificity provides an indication for such heterogeneity (8). However, distinct functional regions of the thymic cortex have not yet been identified. Two-photon laser scanning microscopy (TPLSM) provides an opportunity to examine the cellular migration and interaction events in tissues that underlie selection events within the thymus (reviewed in Ref. 9). Initial studies have revealed that thymocytes undergo a calcium-dependent stopping in response to positive selection signals in the thymus (10) and that interaction of thymocytes with MHC-bearing stromal cells can lead to prolonged but dynamic cell-cell contacts (11). Additionally, analysis of cell migration in the cortex of intact thymic lobes provided evidence that thymocytes move via a random walk in the cortex before positive selection and undergo rapid, directed migration to the medulla as a result of positive selection (12). These studies provide an early glimpse into the regulation of thymocyte migration by positive selection. However, the interactions of thymocytes with other cell types in the intact thymus and how these interactions change during the process of positive selection have not yet been addressed.Herein we examine the impact of the chemokine receptor CCR7 and TCR repertoire selection on thymocyte-DC interactions in the cortex of intact thymic lobes. We show that thymic DCs form intimate associations with cortical capillaries near sources of CCR7 ligands in the cortex. We find that thymocytes extensively

These results suggest that Cx45.6 plays an important role in stimulating lens cell differentiation and fiber formation, which is different from the other lens connexins, Cx43 and Cx56. This stimulatory effect is independent of gap junction-mediated intercellular communication and lens cell proliferation.

Connexin (Cx) 45.6, an avian counterpart of rodent Cx50, is phosphorylated in vivo, but the sites and function of the phosphorylation have not been elucidated. Our peptide mapping experiments showed that the Ser 363 site in the carboxyl (COOH) terminus of Cx45.6 was phosphorylated and that this site is within casein kinase (CK) II consensus sequence, although showing some similarity to CKI sequence. The peptide containing Ser 363 could be phosphorylated in vitro by CKII, but not by CKI. Furthermore, CKII phosphorylated Cx45.6 in embryonic lens membrane and the fusion protein containing the COOH terminus of Cx45.6. Two-dimensional peptide mapping experiments showed that one of the Cx45.6 peptides phosphorylated in vivo migrated to the same spot as one of those phosphorylated by CKII in vitro. Furthermore, CKII activity could be detected in lens lysates. To assess the function of this phosphorylation event, exogenous wild type and mutant Cx45.6 (Ser 363 3 Ala) were expressed in lens primary cultures by retroviral infection. The mutant Cx45.6 was shown to be more stable having a longer half-life compared with wild type Cx45.6. Together, the evidence suggests that CKII is likely a kinase responsible for the Ser 363 phosphorylation, leading to the destablization and degradation of Cx45.6. The connexin degradation induced by phosphorylation has a broad functional significance in the regulation of gap junctions in vivo.Gap junctions are channels between two adjacent cells, which allow passage of small molecules (M r Յ 1000) such as small metabolites, ions, and second messengers. The gap junction-mediated cell-to-cell communications are important in maintaining cell and tissue functions (1). The structural components of gap junctions are members of a family of related membrane proteins called connexins, which consist of four conserved transmembrane domains and two extracellular loops, while their cytoplasmic regions are unique. The COOH terminus, the most variable region among connexins, contains several putative kinase consensus sequences. The phosphorylation of connexins has been indicated in the intracellular trafficking of connexins (2), channel assembly (3

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junctionforming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a K D(app) of 7.5 and 10.3 M, respectively. The K D(app) for the full-length loop region is 7.7 M. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.

During thymic development, T cell progenitors undergo positive selection based on the ability of their T cell Ag receptors (TCR) to bind MHC ligands on thymic epithelial cells. Positive selection determines T cell fate, in that thymocytes whose TCR bind MHC class I (MHC-I) develop as CD8-lineage T cells, whereas those that bind MHC class II (MHC-II) develop as CD4 T cells. Positive selection also induces migration from the cortex to the medulla driven by the chemokine receptor CCR7. In this study, we show that CCR7 is up-regulated in a larger proportion of CD4+CD8+ thymocytes undergoing positive selection on MHC-I compared with MHC-II. Mice bearing a mutation of Th-POK, a key CD4/CD8-lineage regulator, display increased expression of CCR7 among MHC-II-specific CD4+CD8+ thymocytes. In addition, overexpression of CCR7 results in increased development of CD8 T cells bearing MHC-II-specific TCR. These findings suggest that the timing of CCR7 expression relative to coreceptor down-regulation is regulated by lineage commitment signals.

Gap junctions are important in maintaining lens homeostasis. Here we report that connexin 45.6 (Cx45.6) was partially truncated to a 46 kDa fragment during chicken lens development. This specific truncation initiated during embryonic days and the truncated fragment accumulated towards the later developmental stages. When membranes of the embryonic lens were subjected to caspase-3 treatment, the 46 kDa fragment of Cx45.6 was reproduced, suggesting apoptotic protease caspase-3 is a potential protease involved. The COOH-terminus of Cx45.6 in GST-fusion protein was also cleaved by caspase-3, confirming that Cx45.6 is a direct substrate of caspase-3. Induction of apoptosis in lens primary cultures regenerated the 46 kDa fragment and this cleavage was blocked by a caspase-3 inhibitor. Alteration of amino acid residue Asp364 or Glu367 to Ala prevented Cx45.6 from cleavage by caspase-3, suggesting that the cleavage site of Cx45.6 is likely to be between Glu367 and Gly361. Phosphorylation of Ser363, a known substrate for casein kinase II (CKII) in vivo, inhibited the cleavage of Cx45.6 by caspase-3. Thus, this study demonstrates that a lens connexin can be a direct target of caspase-3 and the cleavage by caspase-3 leads to the development-associated truncation of Cx45.6. Finally, caspase-3 mediated truncation can be modulated by the specific connexin phosphorylation.