SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpincpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpincpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpincpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpincpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

Mutation m.15923A>G in the MT-TT gene causes mild myopathy - case report of an adult-onset phenotype.

BACKGROUND: Only five patients have previously been reported to harbor mutations in the MT-TT gene encoding mitochondrial tRNA threonine. The m.15923A > G mutation has been found in three severely affected children. One of these patients died within days after birth and two had a phenotype of myoclonic epilepsy with ragged red fibers (MERRF) in early childhood. We have now found the mutation in an adult patient with mild myopathy. CASE PRESENTATION: The patient is a 64-year-old Finnish man, who developed bilateral ptosis, diplopia and exercise intolerance in his fifties. Family history was unremarkable. Muscle histology showed cytochrome c-oxidase (COX) negative and ragged red fibres. The m.15923A > G mutation heteroplasmy was 33% in the skeletal muscle and 2% in buccal epithelial cells. The mutation was undetectable in the blood. Single-fibre analysis was performed and COX-negative fibres had a substantially higher heteroplasmy of 92%, than the normal fibres in which it was 43%. CONCLUSIONS: We report the fourth patient with m. 15923A > G and with a remarkably milder phenotype than the previous three patients. Our findings and recent biochemical studies suggest that the mutation m.15923A > G is a definite disease-causing mutation. Our results also suggest that heteroplasmy of the m.15923A > G mutation correlates with the severity of the phenotype. This study expands the catalog of the phenotypes caused by mutations in mtDNA.

Case report: a novel frameshift mutation in the mitochondrial cytochrome c oxidase II gene causing mitochondrial disorder.

BACKGROUND: Mitochondrial cytochrome c oxidase 2, MT-CO2, encodes one of the three subunits, which form the catalytic core of cytochrome c oxidase (COX), complex IV. Mutations in MT-CO2 are rare and the associated phenotypes are variable including nonsyndromic and syndromic forms of mitochondrial diseases. CASE PRESENTATION: We describe a 30-year-old man with cognitive decline, epilepsy, psychosis, exercise intolerance, sensorineural hearing impairment, retinitis pigmentosa, cataract and lactic acidosis. COX-deficient fibers and ragged red fibers were abundant in the muscle. Sequencing of mitochondrial DNA (mtDNA) revealed a novel frameshift mutation m.8156delG that was predicted to cause altered C-terminal amino acid sequence and to lead to truncation of the COX subunit 2. The deletion was heteroplasmic being present in 26% of the mtDNA in blood, 33% in buccal mucosa and 95% in muscle. Deletion heteroplasmy correlated with COX-deficiency in muscle histochemistry. The mother and the siblings of the proband did not harbor the deletion. CONCLUSIONS: The clinical features and muscle histology of the proband suggested a mitochondrial disorder. The m.8156delG deletion is a new addition to the short list of pathogenic mutations in the mtDNA-encoded subunits of COX. This case illustrates the importance of mtDNA sequence analysis in patients with an evident mitochondrial disorder.

Dynamic Micropatterning Reveals Substrate-Dependent Differences in the Geometric Control of Cell Polarization and Migration.

Cells are highly dynamic and adopt variable shapes and sizes. These variations are biologically important but challenging to investigate in a spatiotemporally controlled manner. Micropatterning, confining cells on microfabricated substrates with defined geometries and molecular compositions, is a powerful tool for controlling cell shape and interactions. However, conventional binary micropatterns are static and fail to address dynamic changes in cell polarity, spreading, and migration. Here, a method for dynamic micropatterning is reported, where the non-adhesive surface surrounding adhesive micropatterns is rapidly converted to support specific cell-matrix interactions while allowing simultaneous imaging of the cells. The technique is based on ultraviolet photopatterning of biotinylated polyethylene glycol-grafted poly-L-lysine, and it is simple, inexpensive, and compatible with a wide range of streptavidin-conjugated ligands. Experiments using biotinylation-based dynamic micropatterns reveal that distinct extracellular matrix ligands and bivalent integrin-clustering antibodies support different degrees of front-rear polarity in human glioblastoma cells, which correlates to altered directionality and persistence upon release and migration on fibronectin. Unexpectedly, however, neither an asymmetric cell shape nor centrosome orientation can fully predict the future direction of migration. Taken together, biotinylation-based dynamic micropatterns allow easily accessible and highly customizable control over cell morphology and motility.

Effects of xylitol on xylitol-sensitive versus xylitol-resistant Streptococcus mutans strains in a three-species in vitro biofilm.

We studied the effects of xylitol on biofilms containing xylitol-resistant (Xr) and xylitol-sensitive (Xs) Streptococcus mutans, Actinomyces naeslundii and S. sanguinis. The biofilms were grown for 8 and 24 h on hydroxyapatite discs. The viable microorganisms were determined by plate culturing techniques and fluorescence in situ hybridization (FISH) was performed using a S. mutans-specific probe. Extracellular cell-bound polysaccharides (EPS) were determined by spectrofluorometry from single-species S. mutans biofilms. In the presence of 5 % xylitol, the counts of the Xs S. mutans decreased tenfold in the young (8 h) biofilm (p < 0.05) but no effect was seen in the mature (24 h) biofilm. No decrease was observed for the Xr strains, and FISH confirmed these results. No differences were detected in the EPS production of the Xs S. mutans grown with or without xylitol, nor between Xr and Xs S. mutans strains. Thus, it seems that xylitol did not affect the EPS synthesis of the S. mutans strains. Since the Xr S. mutans strains, not inhibited by xylitol, showed no xylitol-induced decrease in the biofilms, we conclude that growth inhibition could be responsible for the decrease of the counts of the Xs S. mutans strains in the clinically relevant young biofilms.

Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells.

While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin ß5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.

A Strong Contractile Actin Fence and Large Adhesions Direct Human Pluripotent Colony Morphology and Adhesion.

Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure.

Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription.

Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal-not cancer-stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression.

Integrin endosomal signalling suppresses anoikis.

Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localizes with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage independence and metastasis.

Vimentin-ERK Signaling Uncouples Slug Gene Regulatory Function.

Epithelial-mesenchymal transition (EMT) in cells is a developmental process adopted during tumorigenesis that promotes metastatic capacity. In this study, we advance understanding of EMT control in cancer cells with the description of a novel vimentin-ERK axis that regulates the transcriptional activity of Slug (SNAI2). Vimentin, ERK, and Slug exhibited overlapping subcellular localization in clinical specimens of triple-negative breast carcinoma. RNAi-mediated ablation of these gene products inhibited cancer cell migration and cell invasion through a laminin-rich matrix. Biochemical analyses demonstrated direct interaction of vimentin and ERK, which promoted ERK activation and enhanced vimentin transcription. Consistent with its role as an intermediate filament, vimentin acted as a scaffold to recruit Slug to ERK and promote Slug phosphorylation at serine-87. Site-directed mutagenesis established a requirement for ERK-mediated Slug phosphorylation in EMT initiation. Together, these findings identified a pivotal step in controlling the ability of Slug to organize hallmarks of EMT.

A ZO-1/α5ß1-integrin complex regulates cytokinesis downstream of PKCε in NCI-H460 cells plated on fibronectin.

Recently, we demonstrated that integrin adhesion to the extracellular matrix at the cleavage furrow is essential for cytokinesis of adherent cells. Here, we report that tight junction protein ZO-1 (Zonula Occludens-1) is required for successful cytokinesis in NCI-H460 cells plated on fibronectin. This function of ZO-1 involves interaction with the cytoplasmic domain of α5-integrin to facilitate recruitment of active fibronectin-binding integrins to the base of the cleavage furrow. In the absence of ZO-1, or a functional ZO-1/α5ß1-integrin complex, proper actin-dependent constriction between daughter cells is impaired and cells fail cytokinesis. Super-resolution microscopy reveals that in ZO-1 depleted cells the furrow becomes delocalized from the matrix. We also show that PKCε-dependent phosphorylation at Serine168 is required for ZO-1 localization to the furrow and successful cell division. Altogether, our results identify a novel regulatory pathway involving the interplay between ZO-1, α5-integrin and PKCε in the late stages of mammalian cell division.