Local and global dynamics of the basement membrane during branching morphogenesis require protease activity and actomyosin contractility.

Many epithelial tissues expand rapidly during embryonic development while remaining surrounded by a basement membrane. Remodeling of the basement membrane is assumed to occur during branching morphogenesis to accommodate epithelial growth, but how such remodeling occurs is not yet clear. We report that the basement membrane is highly dynamic during branching of the salivary gland, exhibiting both local and global remodeling. At the tip of the epithelial end bud, the basement membrane becomes perforated by hundreds of well-defined microscopic holes at regions of rapid expansion. Locally, this results in a distensible, mesh-like basement membrane for controlled epithelial expansion while maintaining tissue integrity. Globally, the basement membrane translocates rearward as a whole, accumulating around the forming secondary ducts, helping to stabilize them during branching. Both local and global dynamics of the basement membrane require protease and myosin II activity. Our findings suggest that the basement membrane is rendered distensible by proteolytic degradation to allow it to be moved and remodeled by cells through actomyosin contractility to support branching morphogenesis.

Region-specific epithelial cell dynamics during branching morphogenesis.

BACKGROUND: Epithelial cells of developing embryonic organs, such as salivary glands, can display substantial motility during branching morphogenesis. Their dynamic movements and molecules involved in their migration are not fully characterized. RESULTS: We generated transgenic mice expressing photo-convertible KikGR and tracked the movements of individual cells highlighted by red fluorescence in different regions of developing salivary glands. Motility was highest for outer bud epithelial cells adjacent to the basement membrane, lower in inner bud cells, and lowest in duct cells. The highly motile outer cells contacting the basement membrane were pleomorphic, whereas inner cells were rounded. Peripheral cell motility was disrupted by antibodies inhibiting α6+ß1 integrins and the nonmuscle myosin II inhibitor blebbistatin. Inner bud cell migration was unaffected by these inhibitors, but their rate of migration was stimulated by inhibiting E-cadherin. CONCLUSIONS: Cell motility in developing salivary glands was highest in cells in contact with the basement membrane. The basement membrane-associated motility of these outer bud cells depended on integrins and myosin II, but not E-cadherin. In contrast, motility of inner bud cells was restrained by E-cadherin. These findings identify the importance of integrin-dependent basement membrane association for the morphology, tissue organization, and lateral motility of morphogenetic epithelial cells.

EphB regulates L1 phosphorylation during retinocollicular mapping.

Interaction of the cell adhesion molecule L1 with the cytoskeletal adaptor ankyrin is essential for topographic mapping of retinal ganglion cell (RGC) axons to synaptic targets in the superior colliculus (SC). Mice mutated in the L1 ankyrin-binding motif (FIGQY(1229)H) display abnormal mapping of RGC axons along the mediolateral axis of the SC, resembling mouse mutant phenotypes in EphB receptor tyrosine kinases. To investigate whether L1 functionally interacts with EphBs, we investigated the role of EphB kinases in phosphorylating L1 using a phospho-specific antibody to the tyrosine phosphorylated FIGQY(1229) motif. EphB2, but not an EphB2 kinase dead mutant, induced tyrosine phosphorylation of L1 at FIGQY(1229) and perturbed ankyrin recruitment to the membrane in L1-transfected HEK293 cells. Src family kinases mediated L1 phosphorylation at FIGQY(1229) by EphB2. Other EphB receptors that regulate medial-lateral retinocollicular mapping, EphB1 and EphB3, also mediated phosphorylation of L1 at FIGQY(1229). Tyrosine(1176) in the cytoplasmic domain of L1, which regulates AP2/clathrin-mediated endocytosis and axonal trafficking, was not phosphorylated by EphB2. Accordingly mutation of Tyr(1176) to Ala in L1-Y(1176)A knock-in mice resulted in normal retinocollicular mapping of ventral RGC axons. Immunostaining of the mouse SC during retinotopic mapping showed that L1 colocalized with phospho-FIGQY in RGC axons in retinorecipient layers. Immunoblotting of SC lysates confirmed that L1 was phosphorylated at FIGQY(1229) in wild type but not L1-FIGQY(1229)H (L1Y(1229)H) mutant SC, and that L1 phosphorylation was decreased in the EphB2/B3 mutant SC. Inhibition of ankyrin binding in L1Y(1229)H mutant RGCs resulted in increased neurite outgrowth compared to WT RGCs in retinal explant cultures, suggesting that L1-ankyrin binding serves to constrain RGC axon growth. These findings are consistent with a model in which EphB kinases phosphorylate L1 at FIGQY(1229) in retinal axons to modulate L1-ankyrin binding important for mediolateral retinocollicular topography.

Cell-matrix adhesions in 3D.

Cells in a three-dimensional (3D) extracellular matrix environment often display different properties and behavior compared to cells cultured on a two-dimensional (2D) substrate. Recent studies characterizing the cell-matrix adhesions formed by cells within a 3D matrix have arrived at contradictory conclusions regarding the presence and composition of adhesions. Here we review this literature, and provide a comparative compilation of information found in published studies from the 3D cell-matrix adhesion field in order to identify shared and divergent conclusions and conceptually important areas that require further research. Although there is a general consensus that discrete cell-matrix adhesions exist in various 3D matrix environments, there are specific exceptions, particularly in cells undergoing amoeboid migration. There are also technical issues to consider when imaging adhesions in 3D matrix; for example, over-expression of a cytoskeletal cell adhesion component can potentially cloud the visualization of adhesions and even alter the mode of cell migration. Properties such as stiffness and local matrix topography may also affect the composition of cell-matrix adhesions. For example, even though cells contain integrin-based 3D adhesions, there can be substantial variability within these adhesions in the presence of force-dependent cytoskeletal components such as vinculin. These new findings and ideas provide promising new leads for understanding the regulation and function of cell-matrix adhesions in 3D matrix.

Direct comparisons of the morphology, migration, cell adhesions, and actin cytoskeleton of fibroblasts in four different three-dimensional extracellular matrices.

Interactions between cells and the extracellular matrix are at the core of tissue engineering and biology. However, most studies of these interactions have used traditional two-dimensional (2D) tissue culture, which is less physiological than three-dimensional (3D) tissue culture. In this study, we compared cell behavior in four types of commonly used extracellular matrix under 2D and 3D conditions. Specifically, we quantified parameters of cell adhesion and migration by human foreskin fibroblasts in cell-derived matrix or hydrogels of collagen type I, fibrin, or basement membrane extract (BME). Fibroblasts in 3D were more spindle shaped with fewer lateral protrusions and substantially reduced actin stress fibers than on 2D matrices; cells failed to spread in 3D BME. Cell-matrix adhesion structures were detected in all matrices. Although the shapes of these cell adhesions differed, the total area per cell occupied by cell-matrix adhesions in 2D and 3D was nearly identical. Fibroblasts migrated most rapidly in cell-derived 3D matrix and collagen and migrated minimally in BME, with highest migration directionality in cell-derived matrix. This identification of quantitative differences in cellular responses to different matrix composition and dimensionality should help guide the development of customized 3D tissue culture and matrix scaffolds for tissue engineering.