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

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

Stimulation of insulin secretion increases the expression of the NKp46 ligand.

<p>(A-B) INS-1E cells were incubated for different periods of time (indicated in the Figure) in Krebs-Ringer bicarbonate HEPES–BSA buffer containing 3.3 mM and 16.7 mM glucose. Cells were harvested at each time point and NKp46 ligand expression was analyzed by cytospin (A) and by flow cytometry (B). Shown in (A) is the intensity (colors intensity is indicated in the right of the Figure) of the expression of NKp46 ligand in INS-1E cells using the NKp46-Ig. MagnificationX400, scale-50 µm. (B) Flow cytometry staining for the surface expression of the NKp46 ligand in INS-1E cells following stimulation at 16.7 mM glucose (green histograms-unstimulated cells, red-stimulated). The MFI values are indicated in the corresponding histograms. (C) INS-1E cells shown in A were incubated at 16.7 mM glucose for different periods of time and secreted insulin was analyzed by an ELISA assay. Results are expressed as microgram/liter (mic/l). Error bars (SD) are derived from triplicates. Data from one of three independent assays is shown.*p = 0.0125 (0/10min), **p = 0.014 (10/30min). (D) ELISA assays for the detection of the NKp46 ligand in the supernatants of the INS-1E cells shown in (A) before and during glucose stimulation. Data from one of two independent assays is shown.</p