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Cellular protrusions generated by the actin cytoskeleton are central to the process of building the body of the embryo. Problems with cellular protrusions underlie human diseases and syndromes, including implantation defects and pregnancy loss, congenital birth defects, and cancer. Cells use protrusive activity together with actin-myosin contractility to create an ordered body shape of the embryo. Here, I review how actin-rich protrusions are used by two major morphological cell types, epithelial and mesenchymal cells, during collective cell migration to sculpt the mouse embryo body. Pre-gastrulation epithelial collective migration of the anterior visceral endoderm is essential for establishing the anterior-posterior body axis. Gastrulation mesenchymal collective migration of the mesoderm wings is crucial for body elongation, and somite and heart formation. Analysis of mouse mutants with disrupted cellular protrusions revealed the key role of protrusions in embryonic morphogenesis and embryo survival. Recent technical approaches have allowed examination of the mechanisms that control cell and tissue movements in vivo in the complex 3D microenvironment of living mouse embryos. Advancing our understanding of protrusion-driven morphogenesis should provide novel insights into human developmental disorders and cancer metastasis. [ABSTRACT FROM AUTHOR]

Abstract Cell migration is part of many important in vivo biological processes and is influenced by chemical and physical factors such as substrate topography. Although the migratory behavior of different cell types on structured substrates has already been investigated, up to date it is largely unknown if specimen's age affects cell migration on structures. In this work, we investigated age-dependent migratory behavior of human endothelial cells from young (≤ 31 years old) and old (≥ 60 years old) donors on poly(dimethylsiloxane) microstructured substrates consisting of well-defined parallel grooves. We observed a decrease in cell migration velocity in all substrate conditions and in persistence length perpendicular to the grooves in cells from old donors. Nevertheless, in comparison to young cells, old cells exhibited a higher cell directionality along grooves of certain depths and a higher persistence time. We also found a systematic decrease of donor age-dependent responses of cell protrusions in orientation, velocity and length, all of them decreased in old cells. These observations lead us to hypothesize a possible impairment of actin cytoskeleton network and affected actin polymerization and steering systems, caused by aging. [ABSTRACT FROM AUTHOR]

Abstract: In the present study an in vitro bilayer model system of the pulmonary alveolocapillary barrier was established to investigate the role of the microvascular endothelium on re-epithelialization. The model system, confluent monolayer cultures on opposing sides of a porous membrane, consisted of a human microvascular endothelial cell line (HPMEC-ST1.6R) and an alveolar type II like cell line (A549), stably expressing EGFP and mCherry, respectively. These fluorescent proteins allowed the real time assessment of the integrity of the monolayers and the automated analysis of the wound healing process after a scratch injury. The HPMECs significantly attenuated the speed of re-epithelialization, which was associated with the proximity to the A549 layer. Examination of cross-sectional transmission electron micrographs of the model system revealed protrusions through the membrane pores and close contact between the A549 cells and the HPMECs. Immunohistochemical analysis showed that these close contacts consisted of heterocellular gap-, tight- and adherens-junctions. Additional analysis, using a fluorescent probe to assess gap-junctional communication, revealed that the HPMECs and A549 cells were able to exchange the fluorophore, which could be abrogated by disrupting the gap junctions using connexin mimetic peptides. These data suggest that the pulmonary microvascular endothelium may impact the re-epithelialization process. [Copyright &y& Elsevier]

Abstract: Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process. [Copyright &y& Elsevier]

Abstract: Breast tumor cells enter the bloodstream long before the development of clinically evident metastasis. However, the early presence of such bloodborne cells predicts poor patient outcome. Nearly 90% of human breast tumors arise as carcinomas from mammary epithelial cells, so it is important to study how these cells respond to the detached conditions that they would experience in the bloodstream. We report here that mammary epithelial cell lines produce long and dynamic protrusions of the plasma membrane when detached. Although human and mouse mammary epithelial cell lines die by apoptosis within 16 h of detachment, this protrusive response persists for days in cells overexpressing either Bcl-2 or Bcl-xL. Unlike actin-dependent invadopodia and podosomes, these protrusions are actually enhanced by actin depolymerization with Cytochalasin-D or Latrunculin-A. Immunofluorescence and Western blotting demonstrate that the protrusions are enriched in detyrosinated Glu-tubulin, a post-translationally modified form of α-tubulin that is found in stabilized microtubules. Video microscopy indicates that these protrusions promote cell–cell attachment, and inhibiting microtubule-based protrusions correlates with reduced extracellular matrix attachment. Since bloodborne metastasis depends on both cell–cell and cell–matrix attachment, microtubule-based protrusions in detached mammary epithelial cells provide a novel mechanism that could influence the metastatic spread of breast tumors. [Copyright &y& Elsevier]

Abstract: G protein-coupled receptor kinase-interacting protein (GIT)1 is a multidomain, adaptor protein that regulates cellular processes, such as migration and protrusive activity, by bringing together various signaling molecules, including PIX, PAK, and paxillin. Mutants of GIT1, which lack the C-terminal paxillin binding domain, fail to mediate its effects on migration and protrusions, suggesting that sites within this domain are critical to GIT1 function. In this study, we show that serine 709, which is located within the paxillin binding domain, regulates GIT1 function. Phosphorylation of serine 709 is necessary for GIT1-induced effects on protrusions. Phosphorylation of this site also regulates GIT1 interaction with paxillin, which could serve to target GIT1 to the leading edge of cells. As shown by an in vitro kinase assay, PAK phosphorylates GIT1 on serine 709. Taken together, our results indicate that GIT1 phosphorylation on serine 709 increases its binding to paxillin and regulates protrusive activity in cells. [Copyright &y& Elsevier]

Interlocking membrane domains are specialized membrane interdigitations in the form of ball-and-sockets and protrusions between lens fibre cells of all species. They are believed to play a key role in maintaining fibre–fibre stability and are therefore, important for normal lens function. Here we report the specific association of the clathrin/AP-2 adaptor complex and the branching F-actin network with the development of interlocking domains in rats and several other species. By thin-section electron microscopy we consistently observed a layer of distinct coating (∼25-nm thick) on the concave membrane surface of small and intermediate-sized developing interlocking domains. These membrane coats remarkably resembled the clathrin-coat of endocytic vesicles in which clathrin and the AP-2 adaptor are involved in the induction of coated pit formation during receptor-mediated endocytosis. We hypothesize that the clathrin/AP-2 complex is directly involved in the induction of interlocking domains in fibre cells. By immunoconfocal microscopy, co-labelling of a dotted-pattern of clathrin and AP-2 adaptor antibodies was seen along the cortical fibre cells. Immunoblot analysis further confirmed that clathrin and AP-2 adaptor antibodies specifically stained a polypeptide band of 180 and 106 kD, respectively, in the membrane fractions prepared separately from the outer and inner cortical fibres where interlocking domains are abundant but endocytic vesicles are absent. Immunoelectron microscopy showed that the clathrin antibody was localized along the interlocking membrane. In addition, branching actin filament networks were frequently observed within the cytoplasmic compartment of developing interlocking domains by TEM, in consistent with the findings by fluorescence and immunogold labelling of the F-actin antibody in the domains. These results demonstrate for the first time that the clathrin/AP-2 complex plays a new role for the formation of interlocking domains in lens fibre cells. Branching actin networks and possibly other cytoskeletal components are also associated with the development and maintenance of these interlocking domains. The coordinated ‘pulling and pushing’ actions generated by the clathrin/AP-2 complex and branching actin networks during interlocking domain formation are discussed. [Copyright &y& Elsevier]