In epithelial cell movements, which occur during wound healing or embryonic morphogenesis, sheets of cells move together as a unit. Molecular mechanisms that regulate this sheet movement have been largely unknown, although cell locomotion or movement mechanisms for individual cells, such as for fibroblastic cells, have been extensively studied. Here, we show that, during wound healing, sheets of MDCK epithelial cells migrate coordinately as a unit, and wound-induced activation of ERK MAP kinase (ERK1/2) propagates in cell sheets in accordance with the cell sheet movement. Inhibition of ERK1/2 activation by specific MEK inhibitors or by expressing dominant-negative ERK2 results in marked inhibition of the sheet movement during wound healing, and inhibition of the cell sheet movement by disrupting actin cytoskeleton suppresses propagation of ERK1/2 activation. These results indicate that cell movement and ERK1/2 activation form a positive feedback loop, which facilitates cell sheet migration. Moreover, we find that Src family kinase inhibitors suppress both cell migration and propagation of ERK1/2 activation, suggesting that Src family kinase may participate in this feedback loop. Interestingly, neither cell sheet migration as a unit nor migration-dependent propagation of ERK1/2 activation occurs during wound healing in fibroblastic 3Y1 cells. Thus, our results identify specific requirements of ERK1/2 MAP kinase for epithelial cell sheet movement.

The classical mitogen-activated protein kinase (MAPK, also known as ERK) pathway is widely involved in eukaryotic signal transductions. In response to extracellular stimuli, MAPK becomes activated and translocates from the cytoplasm to the nucleus. At least two pathways for the nuclear import of MAPK are shown to exist; passive diffusion of a monomer and Ran-dependent active transport of a dimer, the detailed molecular mechanism of which is unknown. In this study, we have reconstituted nuclear import of MAPK in vitro by using digitonin-permeabilized cells with GFP-fused MAPK protein (GFP-MAPK), which is too large to pass through the nuclear pore by passive diffusion. GFP-MAPK was able to accumulate in the nucleus irrespective of its phosphorylation state. This import of GFP-MAPK occurred even in the absence of any soluble cytosolic factors or ATP but was inhibited by wheat germ agglutinin or an excess amount of importin-␤ or at low temperatures. Moreover, MAPK directly bound to an FG repeat region of nucleoporin CAN/Nup214 in vitro. Taken together, these results suggest the third pathway for nuclear import of MAPK, in which MAPK passes through the nuclear pore by directly interacting with the nuclear pore complex.The classical mitogen-activated protein kinase (MAPK, 1 also known as ERK) cascade is among the key signaling pathways regulating many cellular events, such as cell proliferation, cell differentiation, and early embryonic development. Extracellular stimuli such as growth factors induce sequential activation of three protein kinases, MAP kinase kinase kinase, such as Raf, MAP kinase kinase (MAPKK, also known as MEK) and MAPK (i.e. ERK) (1-9). The activation of MAPK leads to its translocation from the cytoplasm to the nucleus (10 -12) where MAPK phosphorylates and activates several nuclear targets, including transcription factors (13). The nuclear translocation of MAPKs appears to be a prerequisite for proper cellular responses (14).In contrast to MAPKs, classical MAPKKs, MEK1 (i.e. MKK1) and MEK2 (i.e. MKK2), always localize to the cytoplasm (12,15,16). We have previously shown that the cytoplasmic localization of MAPKK is achieved by its nuclear export signal at the N terminus (17). Moreover, the cytoplasmic localization of MAPK is achieved by its specific binding to MAPKK, and nuclear translocation of MAPK accompanies the dissociation of the MAPKK⅐MAPK complex (18). Phosphorylation of MAPK by MAPKK is necessary and sufficient for the dissociation of MAPK from MAPKK (19).How does MAPK translocate to the nucleus? Khokhlatchev et al. (20) have demonstrated that phosphorylation of MAPK promotes its dimerization and nuclear translocation. There are at least two pathways for the nuclear import of MAPK, passive diffusion of a monomer and active transport of a dimer (19). RanQ69L, a dominant negative mutant of low molecular weight GTPase Ran, inhibits the active transport of MAPK, suggesting the involvement of an importin-␤ family protein(s) in the active transport of MAPK (19).In this study, we have analyze...

SummaryThe basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3, 4, 5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6, 7, 8, 9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.

Highlights d Labeled ECM in fly embryos can be examined from initiation to homeostasis d Quantifying ECM levels to homeostasis allows for modeling of basal turnover rate d Embryonic ECM has a half-life of 10 h, which was confirmed by pulse-chase analysis d Inhibiting MMPs or ECM interactions alters the basal turnover rate

Elucidating neuronal circuits and their plasticity in the cerebral cortex is one of the important questions in neuroscience research. Here we report novel axonal trajectories and their plasticity in the mouse somatosensory barrel cortex. We selectively visualized layer 2/3 neurons using in utero electroporation and examined the axonal trajectories of layer 2/3 neurons. We found that the axons of layer 2/3 neurons preferentially run in the septal regions of layer 4 and named this axonal pattern "barrel nets." The intensity of green fluorescent protein in the septal regions was markedly higher compared with that in barrel hollows. Focal in utero electroporation revealed that the axons in barrel nets were indeed derived from layer 2/3 neurons in the barrel cortex. During development, barrel nets became visible at postnatal day 10, which was well after the initial appearance of barrels. When whisker follicles were cauterized within 3 d after birth, the whisker-related pattern of barrel nets was altered, suggesting that cauterization of whisker follicles results in developmental plasticity of barrel nets. Our results uncover the novel axonal trajectories of layer 2/3 neurons with whisker-related patterns and their developmental plasticity in the mouse somatosensory cortex. Barrel nets should be useful for investigating the pattern formation and axonal reorganization of intracortical neuronal circuits.

Drosophila melanogaster plasmatocytes, the phagocytic cells among hemocytes, are essential for immune responses, but also play key roles from early development to death through their interactions with other cell types. They regulate homeostasis and signaling during development, stem cell proliferation, metabolism, cancer, wound responses, and aging, displaying intriguing molecular and functional conservation with vertebrate macrophages. Given the relative ease of genetics in Drosophila compared to vertebrates, tools permitting visualization and genetic manipulation of plasmatocytes and surrounding tissues independently at all stages would greatly aid a fuller understanding of these processes, but are lacking. Here, we describe a comprehensive set of transgenic lines that allow this. These include extremely brightly fluorescing mCherry-based lines that allow GAL4-independent visualization of plasmatocyte nuclei, the cytoplasm, or the actin cytoskeleton from embryonic stage 8 through adulthood in both live and fixed samples even as heterozygotes, greatly facilitating screening. These lines allow live visualization and tracking of embryonic plasmatocytes, as well as larval plasmatocytes residing at the body wall or flowing with the surrounding hemolymph. With confocal imaging, interactions of plasmatocytes and inner tissues can be seen in live or fixed embryos, larvae, and adults. They permit efficient GAL4-independent Fluorescence-Activated Cell Sorting (FACS) analysis/sorting of plasmatocytes throughout life. To facilitate genetic studies of reciprocal signaling, we have also made a plasmatocyte-expressing QF2 line that, in combination with extant GAL4 drivers, allows independent genetic manipulation of both plasmatocytes and surrounding tissues, and GAL80 lines that block GAL4 drivers from affecting plasmatocytes, all of which function from the early embryo to the adult.

No abstract

Argonaute 2 bound mature microRNA (Ago2‐miRNA) complexes are key regulators of the wound inflammatory response and function in the translational processing of target mRNAs. In this study, we identified four wound inflammation‐related Ago2‐miRNAs (miR‐139‐5p, miR‐142‐3p, miR‐142‐5p, and miR‐223) and show that miR‐223 is critical for infection control. miR‐223 Y/− mice exhibited delayed sterile healing with prolonged neutrophil activation and interleukin‐6 expression, and markedly improved repair of Staphylococcus aureus‐infected wounds. We also showed that the expression of miR‐223 was regulated by CCAAT/enhancer binding protein alpha in human neutrophils after exposure to S. aureus peptides. Treatment with miR‐223 Y/−‐derived neutrophils, or miR‐223 antisense oligodeoxynucleotides in S. aureus‐infected wild‐type wounds markedly improved the healing of these otherwise chronic, slow healing wounds. This study reveals how miR‐223 regulates the bactericidal capacity of neutrophils at wound sites and indicates that targeting miR‐223 might be of therapeutic benefit for infected wounds in the clinic.