Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1

In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon–glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane

Anomalous Features of EMT during Keratinocyte Transformation

During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike “conventional EMT”, these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated

Ermin, a myelinating oligodendrocyte-specific protein that regulates cell morphology

Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, thereby allowing rapid propagation of action potentials. Despite the considerable clinical importance of myelination, little is known about the molecular mechanisms that enable oligodendrocytes to generate their specialized membrane wrapping. Here, we used microarray expression profiling of oligodendrocyte-ablated mutant mice to identify new glial molecules that are involved in CNS myelination. This effort resulted in the identification of Ermin, a novel cytoskeletal molecule that is exclusively expressed by oligodendrocytes. Ermin appears at a late stage during myelination, and in the mature nerves, it is localized to the outer cytoplasmic lip of the myelin sheath and the paranodal loops. In cultured oligodendrocytes, Ermin becomes visible in well differentiated MBP-positive cells, where it is concentrated at the tip of F-actin-rich processes (termed "Ermin spikes"). Ectopic expression of Ermin, but not of a mutant protein lacking its actin-binding domain, induced the formation of numerous cell protrusions and a pronounced change in cell morphology. Our results demonstrate that Ermin is a novel marker of myelinating oligodendroglia and suggest that it plays a role in cytoskeletal rearrangements during the late wrapping and/or compaction phases of myelinogenesis

Involvement of the Rho-mDia1 pathway in the regulation of Golgi complex architecture and dynamics

10.1091/mbc.E11-01-0007Molecular Biology of the Cell22162900-2911MBCE

The cytoskeletal adapter protein 4.1G organizes the internodes in peripheral myelinated nerves

Myelinating Schwann cells regulate the localization of ion channels on the surface of the axons they ensheath. This function depends on adhesion complexes that are positioned at specific membrane domains along the myelin unit. Here we show that the precise localization of internodal proteins depends on the expression of the cytoskeletal adapter protein 4.1G in Schwann cells. Deletion of 4.1G in mice resulted in aberrant distribution of both glial adhesion molecules and axonal proteins that were present along the internodes. In wild-type nerves, juxtaparanodal proteins (i.e., Kv1 channels, Caspr2, and TAG-1) were concentrated throughout the internodes in a double strand that flanked paranodal junction components (i.e., Caspr, contactin, and NF155), and apposes the inner mesaxon of the myelin sheath. In contrast, in 4.1G(−/−) mice, these proteins “piled up” at the juxtaparanodal region or aggregated along the internodes. These findings suggest that protein 4.1G contributes to the organization of the internodal axolemma by targeting and/or maintaining glial transmembrane proteins along the axoglial interface

The expression of the beta cell-derived autoimmune ligand for the killer receptor nkp46 is attenuated in type 2 diabetes.

NK cells rapidly kill tumor cells, virus infected cells and even self cells. This is mediated via killer receptors, among which NKp46 (NCR1 in mice) is prominent. We have recently demonstrated that in type 1 diabetes (T1D) NK cells accumulate in the diseased pancreas and that they manifest a hyporesponsive phenotype. In addition, we found that NKp46 recognizes an unknown ligand expressed by beta cells derived from humans and mice and that blocking of NKp46 activity prevented diabetes development. Here we investigated the properties of the unknown NKp46 ligand. We show that the NKp46 ligand is mainly located in insulin granules and that it is constitutively secreted. Following glucose stimulation the NKp46 ligand translocates to the cell membrane and its secretion decreases. We further demonstrate by using several modalities that the unknown NKp46 ligand is not insulin. Finally, we studied the expression of the NKp46 ligand in type 2 diabetes (T2D) using 3 different in vivo models and 2 species; mice and gerbils. We demonstrate that the expression of the NKp46 ligand is decreased in all models of T2D studied, suggesting that NKp46 is not involved in T2D

Expression of insulin and NKp46 ligand in T2D models.

<p>(A) Immunohistochemical staining of pancreatic tissues derived from 8–10 weeks old control C57BL/6 mice (top), 8–10 weeks old leptin deficient (OB/OB) mice (middle) and 5–6 weeks old leptin receptor deficient (DB/DB) mice (bottom). Staining was performed with NKp46-Ig. Magnificationx400. Representative of three independent staining is shown. Tissues were obtained from 3–4 mice in each group. (B) Immunofluorescence staining of pancreatic tissues derived from 8–10 weeks old control C57BL/6 mice (top), 8–10 weeks old OB/OB mice (middle) and 5–6 weeks old DB/DB mice (Bottom). Left images represent staining with NKp46-Ig (NKp46 ligand, green), middle images represent staining with anti-insulin (Insulin, red) and the right images represent the merge signal (Merge, yellow). Magnificationx400. Scale bars-50 µm. Representative of three independent staining is shown, 3–4 mice in each group.</p

Recovery of NKp46 ligand after reconstitution of leptin in ob/ob mice.

<p>Mice (ob/ob) were injected I.V with an adeno-vector encoding for GFP (Adeno GFP, A-C) or with an adeno-vector encoding for murine leptin (B-C). (B-C) Immunohistochemical (B) and Immunofluorescence (C) NKp46-Ig staining of pancreatic tissue derived from 8–12 weeks male ob/ob mice treated for 14 days with adeno-vector encoding for leptin (B, right and C lower) or with adeno-vector encoding for GFP (B, left and C upper). (C) Left images represent staining with NKp46-Ig (NKp46 ligand, green), middle images represent staining with anti-insulin antibody (Insulin, red) and the right images represent merge signal (Merge, yellow). (A and C) Magnificationx400, scale bar-50 µm, (B) Magnificationx200, scale bar-25 µm. Representative of two independent experiments, 3–4 mice in each group.</p

Insulinoma staining.

<p>(A-B) Immunofluorescence images of INS-1E (A) and MIN6 (B) beta cell lines. Left images represent staining with anti-insulin antibody (Insulin, red), middle images represent staining with the NKp46-Ig (NKp46 ligand, green) and the right images represent the merge signal (Merge, yellow). Arrows indicate areas positive for NKp46-Ig staining and negative for insulin. Representative of three independent staining is shown. Magnificationx1800, scale bar-10 µm.</p

Electron microscopy images of NKp46 ligand and of insulin.

<p>INS-1E cells were co-stained with NKp46-Ig (upper, large black dots, 15 nm) and KIR2DS4-Ig (lower) together with anti-insulin (small black dots, 6 nm).</p