Yu Itoh

Involvement of afadin in barrier function and homeostasis of mouse intestinal epithelia

Afadin interacts with the cytoplasmic region of nectins, which are immunoglobulin-like cell adhesion molecules at adherens junctions, and links them to the actin cytoskeleton. Afadin regulates activities of cells in culture such as directional motility, proliferation and survival. We used Cre-loxP technology to generate mice conditionally lacking afadin specifically in the intestinal epithelia after birth. The loss of afadin caused increased paracellular permeability in the intestinal mucosa and enhanced susceptibility to the tissue destruction induced by dextran sulfate sodium. The junctional architecture of the intestinal epithelia appeared to be preserved, whereas the deficiency of afadin caused the mislocalization of nectin-2 and nectin-3 from adherens junctions to basolateral membrane domains but not that of other components of apical junctions. By contrast, such phenotypic changes were undetected in mice lacking nectin-2, nectin-3 or both. These findings suggest that afadin plays crucial roles, independently of the role as the nectin–afadin module, in barrier function and homeostasis of the intestinal epithelia once the epithelial structure has been established.

Increased susceptibility to spontaneous lung cancer in mice lacking LIM-domain only 7

LIM‐domain only (LMO) 7 is a multifunctional protein that is predicted to regulate the actin cytoskeleton, assembly of adherens junctions in epithelial cells, and gene expression. LMO7 was highly expressed in the mouse lung and predominantly localized to the apical membrane domain of bronchiolar epithelial cells. Although mice lacking LMO7 were viable and fertile in specific pathogen‐free conditions, they developed protruding epithelial lesions in the terminal and respiratory bronchioles and alveolar ducts at 14–15 weeks of age. Furthermore, they tended to develop spontaneous adenocarcinoma in the lung at over 90 weeks of age. The cumulative incidence ratios of lung cancer were 22% in LMO7−/– mice and 13% in LMO7+/– mice whereas no primary lung cancer was observed in wild‐type mice. Ex vivo analyses of the cancer cells showed numerical chromosome abnormalities and tumorigenicity in nude mice. These results suggest that LMO7 can act as a tumor suppressor whose deficiency confers a genetic predisposition to naturally occurring lung cancer. (Cancer Sci 2009; 100: 608–616)

Multiple roles of afadin in the ultrastructural morphogenesis of mouse hippocampal mossy fiber synapses: SAI et al.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply‐branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin‐deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin‐deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin‐1, nectin‐3, and N‐cadherin were localized at the control typical PAJs, whereas nectin‐1 and nectin‐3 were localized at the afadin‐deficient atypical PAJs to extents lower than those in the control synapse and N‐cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin‐1 and nectin‐3 independently of afadin and N‐cadherin and that the typical PAJs are formed by afadin and N‐cadherin cooperatively with nectin‐1 and nectin‐3. Serial block face‐scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin‐deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.

Localization of nectin-2δ at perivascular astrocytic endfoot processes and degeneration of astrocytes and neurons in nectin-2 knockout mouse brain

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules. In the nervous system, among four members (nectin-1, -2, -3, and -4), nectin-1 and -3 are asymmetrically localized at puncta adherentia junctions formed between the mossy fiber terminals and the dendrites of CA3 pyramidal neurons in the mouse hippocampus and heterophilic trans-interactions between nectin-1 and nectin-3 are involved in the selective interaction of axons and dendrites of cultured neurons. By contrast, nectin-2, which has two splicing variants, nectin-2α and -2δ, has not been well characterized in the brain. We showed here that nectin-2α was expressed in both cultured mouse neurons and astrocytes whereas nectin-2δ was selectively expressed in the astrocytes. Nectin-2δ was localized at the adhesion sites between adjacent cultured astrocytes, but in the brain it was localized on the plasma membranes of astrocytic perivascular endfoot processes facing the basement membrane of blood vessels. Genetic ablation of nectin-2 caused degeneration of astrocytic perivascular endfoot processes and neurons in the cerebral cortex. These results uncovered for the first time the localization and critical functions of nectin-2 in the brain.

Nectin-1 spots regulate the branching of olfactory mitral cell dendrites

Olfactory mitral cells extend lateral secondary dendrites that contact the lateral secondary and apical primary dendrites of other mitral cells in the external plexiform layer (EPL) of the olfactory bulb. The lateral dendrites further contact granule cell dendrites, forming dendrodendritic reciprocal synapses in the EPL. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. We recently showed that the immunoglobulin-like cell adhesion molecule nectin-1 constitutes a novel adhesion apparatus at the contacts between mitral cell lateral dendrites, between mitral cell lateral and apical dendrites, and between mitral cell lateral dendrites and granule cell dendritic spine necks in the deep sub-lamina of the EPL of the developing mouse olfactory bulb and named them nectin-1 spots. We investigated here the role of the nectin-1 spots in the formation of dendritic structures in the EPL of the mouse olfactory bulb. We showed that in cultured nectin-1-knockout mitral cells, the number of branching points of mitral cell dendrites was reduced compared to that in the control cells. In the deep sub-lamina of the EPL in the nectin-1-knockout olfactory bulb, the number of branching points of mitral cell lateral dendrites and the number of dendrodendritic reciprocal synapses were reduced compared to those in the control olfactory bulb. These results indicate that the nectin-1 spots regulate the branching of mitral cell dendrites in the deep sub-lamina of the EPL and suggest that the nectin-1 spots are required for odor information processing in the olfactory bulb.

Dynamic Change of Polarity in Primary Cultured Spheroids of Human Colorectal Adenocarcinoma and Its Role in Metastasis

Intestinal epithelial cells possess apical-basal polarity, which governs the exchange of nutrients and waste. Perturbation of cell polarity appears to be a general feature of cancers, although most colorectal cancers are differentiated adenocarcinomas, in which polarity is maintained to some extent. Little is known about the role of dysregulated polarity in cancer. The cancer tissue-originated spheroid method was applied to the preparation and culture of spheroids. Spheroids were cultured in suspension or in type I collagen gel. Polarity was assessed by IHC of apical markers and electron microscopy. Two types of polarity status in spheroids were observed: apical-in, with apical membrane located at cavities inside the spheroids in type I collagen gel; and apical-out, with apical membrane located at the outermost layer of spheroids in suspension. These polarities were highly interchangeable. Inhibitors of Src and dynamin attenuated the polarity switch. In patients, clusters of cancer cells that invaded vessels had both apical-in and apical-out morphologic features, whereas primary and metastatic tumors had apical-in features. In a mouse liver metastasis model, apical-out spheroids injected into the portal vein became apical-in spheroids in the liver within a few days. Inhibitors of Src and dynamin significantly decreased liver metastasis. Polarity switching was observed in spheroids and human cancer. The polarity switch was critical in an experimental liver metastasis model.

Genetic Ablation of Afadin Causes Mislocalization and Deformation of Paneth Cells in the Mouse Small Intestinal Epithelium

Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.