Programs that direct cellular differentiation are dependent on the strict temporal expression of regulatory factors that can be provided by Rho GTPases. Ciliogenesis is a complex sequence of events involving the generation and docking of basal bodies at the apical membrane, followed by ciliary axoneme generation. Although a cilia proteome has been assembled, programs that direct ciliated cell differentiation are not well established, particularly in mammalian systems. Using mouse primary culture airway epithelial cells, we identified a critical stage of ciliogenesis requiring the temporal establishment of an apical web-like structure of actin for basal body docking and subsequent axoneme growth. Apical web formation and basal body docking were prevented by interruption of actin remodeling and were dependent on RhoA activation. Additional evidence for this program was provided by analysis of Foxj1-null mice that failed to dock basal bodies and lacked apical actin. Foxj1 expression coincided with actin web formation, activated RhoA and RhoB, and persisted despite RhoA inhibition, suggesting that Foxj1 promoted RhoA during ciliogenesis. Apical ezrin localization was also dependent on Foxj1, actin remodeling, and RhoA, but was not critical for ciliogenesis. Thus, temporal Foxj1 and RhoA activity are essential regulatory events for cytoskeletal remodeling during mammalian ciliogenesis.

During angiogenesis, microvascular endothelial cells (ECs) secrete proteinases that permit penetration of the vascular basement membrane as well as the interstitial extracellular matrix. This study tested the hypothesis that cathepsin S (Cat S) contributes to angiogenesis. Treatment of cultured ECs with inflammatory cytokines or angiogenic factors stimulated the expression of Cat S, whereas inhibition of Cat S activity reduced microtubule formation by impairing cell invasion. ECs from Cat S-deficient mice showed reduced collagenolytic activity and impaired invasion of collagens type I and IV. Cat S-deficient mice displayed defective microvessel development during wound repair. This abnormal angiogenesis occurred despite normal vascular endothelial growth factor and basic fibroblast growth factor levels, implying an essential role for extracellular matrix degradation by Cat S during microvessel formation. These results demonstrate a novel function of endothelium-derived Cat S in angiogenesis.

Chromosomal translocations in lymphoid tumours can involve antigen-receptor loci undergoing V(D)J recombination. Here, we show that translocations are recovered from the joining of RAG-generated double-strand breaks (DSBs) on one chromosome to an endonuclease-generated DSB on a second chromosome, providing evidence for the participation of non-RAG DSBs in some lymphoid translocations. Surprisingly, translocations are increased in cells deficient for the nonhomologous end-joining (NHEJ) protein Ku70, implicating non-canonical joining pathways in their etiology.

Cilia are traditionally classified as motile or primary. Motile cilia are restricted to specific populations of well-differentiated epithelial cells, including those in the airway, brain ventricles, and oviducts. Primary cilia are nonmotile, solitary structures that are present in many cell types, and often have sensory functions such as in the retina and renal tubules. Primary cilia were also implicated in the regulation of fundamental processes in development. Rare depictions of primary cilia in embryonic airways led us to hypothesize that primary cilia in airway cells are temporally related to motile ciliogenesis. We identified primary cilia in undifferentiated, cultured airway epithelial cells from mice and humans and in developing lungs. The solitary cilia in the airways express proteins considered unique to primary cilia, including polycystin-1 and polycystin-2. A temporal analysis of airway epithelial cell differentiation showed that cells with primary cilia acquire markers of motile ciliogenesis, suggesting that motile ciliated cells originate from primary ciliated cells. Whereas motile ciliogenesis requires Foxj1, primary ciliogenesis does not, and the expression of Foxj1 was associated with a loss of primary cilia, just before the appearance of motile cilia. Primary cilia were not found in well-differentiated airway epithelial cells. However, after injury, they appear in the luminal layer of epithelium and in basal cells. The transient nature of primary cilia, together with the temporal and spatial patterns of expression in the development and repair of airway epithelium, suggests a critical role of primary cilia in determining outcomes during airway epithelial cell differentiation.

Some cultured cell lines undergo typical apoptosis upon infection with influenza virus. However, the release of replicated virus into the culture medium does not change when apoptosis is inhibited. Since apoptotic cells are heterophagically eliminated at early stages of the apoptosis pathway, we anticipated that the coexistence of phagocytic cells with virus-infected cells affects the extent of virus growth. When influenza A virus-infected HeLa cells were mixed with activated mouse peritoneal macrophages, efficient phagocytosis, which was abrogated in the presence of a caspase inhibitor, occurred. At the same time, the release of virus into the culture medium was completely inhibited, and this required direct contact between virus-infected cells and macrophages. Furthermore, an immunoelectron microscopic analysis detected influenza virus particles associated with phagosome-like structures within macrophages. These results indicate that apoptosis-dependent phagocytosis of virus-infected cells may lead to direct elimination of the pathogen.