Integrins, extracellular matrix molecules, cytoskeletal proteins, and regulators such as Btbd7 contribute to cell migration and signaling by complex, integrated mechanisms. We are addressing the following specific questions: 1. What subcellular structures and signaling pathways are important for efficient cell migration in two-dimensional and three-dimensional environments? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce effective cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, live-cell phase-contrast and confocal time-lapse microscopy, and methods for direct measurement of intracellular hydrostatic pressure in a single migrating mammalian cell. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions and regulation of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial governance of cell migration. We had previously discovered a new lobopodial mode of 3D cell migration in which myosin II-dependent contractility generates lobopodial protrusions at the front of various primary human cells migrating in a 3D cell-derived matrix by elevating intracellular hydrostatic pressure. In these cells, which include primary human fibroblasts, the nucleus physically compartmentalizes the cytoplasm into forward and rear compartments to maintain differences in hydrostatic pressure, which is high at the cell anterior. Actomyosin contractility acting via vimentin and the nucleoskeleton-intermediate filament linker protein nesprin-3 pulls the nucleus forward and pressurizes the front of the cell for cell protrusion. Consequently, the nucleus acts as a piston that physically compartmentalizes the cytoplasm and increases the hydrostatic pressure between the nucleus and the leading edge of motile cells to drive lamellipodia-independent 3D lobopodial cell migration. This nuclear piston mode of migration allows cells to switch from low-pressure lamellipodial migration to high-pressure protrusion-driven lobopodial migration in response to physical characteristics, such as linear elasticity, of the surrounding three-dimensional (3D) matrix. Although migrating tumor cells were known to change how they migrate in response to the 3D matrix, it was not clear whether they could switch between low- and high-pressure protrusions in analogy to primary human fibroblasts. An ongoing collaborative study with Ryan Petrie (now at Drexel University) has demonstrated that the nuclear piston is defective in human fibrosarcoma cells (malignant fibroblastic cells). Protease inhibition by inhibitors of matrix metalloproteases rescued the nuclear piston mechanism in polarized HT1080 and SW684 fibrosarcoma cells and re-established compartmentalized pressure. Rescuing this compartmentalized pressure required nesprin 3, actomyosin contractility, and integrin-mediated adhesion, fully consistent with normal lobopodia-based fibroblast migration. Interestingly, this activation of the nuclear piston mechanism slowed 3D migration of fibrosarcoma cells displaying mesenchymal but not amoeboid morphologies. Thus, inhibiting protease activity during polarized tumor cell 3D migration is sufficient to restore the nuclear piston migration mechanism and the compartmentalized anterior pressure characteristic of non-malignant cells. The major developmentally crucial migration of neural crest cells to form craniofacial structures depends on the extracellular matrix protein anosmin. Genetic defects in anosmin result in human Kallmann syndrome. We previously established that anosmin functions in neural crest formation, cell adhesion, and neuronal migration. We recently established cell adhesion-and-spreading assays for anosmin and used them for antibody inhibition analyses to search for hypothesized integrin adhesion receptors. We find that alpha5-beta1, alpha4-beta1, and alpha9-beta1 integrins are needed for effective human cell adhesive receptor function in cell adhesion and cell spreading on anosmin. In addition, cell adhesion to anosmin can be inhibited by certain peptides: RGD peptides known to affect interactions with alpha5-beta1, and CS1-based peptides known to disrupt adhesion to alpha4-beta1. This identification of the integrin adhesion receptors used by anosmin should facilitate further studies of this matrix protein altered in Kallmann syndrome. Cell migration is also a prominent feature of cellular behavior during mammalian branching morphogenesis, e.g., of salivary glands and lungs. Using cell migration analyses based on computer-assisted individual cell tracking in vitro and in vivo, we find that the newly characterized regulator Btbd7 is essential for the two-fold increased rate of migration of peripheral epithelial cells in developing mouse salivary glands. This enhanced Btbd7-dependent cell motility is accompanied by increased cell-cell separation in both in vitro and in vivo model systems. A significant portion of these effects of Btbd7 on cell motility are due to its non-transcriptional down-regulation of E-cadherin protein levels. These enhanced dynamic cell movements appear to be crucial for normal in vivo cleft formation and branching morphogenesis of several mammalian organs. This combined approach involving characterization of the regulation of cell migration and phenotype in various microenvironments should provide novel approaches to understanding, preventing, or ameliorating migratory processes used by cells during abnormal embryonic development and particularly in cancer. An in-depth understanding of the precise manner in which cells move and interact with their matrix environment will also facilitate tissue engineering studies.
| Endo, Yukinori; Ishiwata-Endo, Hiroko; Yamada, Kenneth M (2018) Cell adhesion to anosmin via ?5?1, ?4?1, and ?9?1 integrins. Cell Adh Migr 12:93-100 |
| DuChez, Brian J (2017) Automated Tracking of Cell Migration with Rapid Data Analysis. Curr Protoc Cell Biol 76:12.12.1-12.12.16 |
| Daley, William P; Matsumoto, Kazue; Doyle, Andrew D et al. (2017) Btbd7 is essential for region-specific epithelial cell dynamics and branching morphogenesis in vivo. Development 144:2200-2211 |
| Wang, Shaohe; Sekiguchi, Rei; Daley, William P et al. (2017) Patterned cell and matrix dynamics in branching morphogenesis. J Cell Biol 216:559-570 |
| Petrie, Ryan J; Harlin, Heather M; Korsak, Lulu I T et al. (2017) Activating the nuclear piston mechanism of 3D migration in tumor cells. J Cell Biol 216:93-100 |
| Huang, Hanxia; Konduru, Krishnamurthy; Solovena, Veronica et al. (2016) Therapeutic potential of the heme oxygenase-1 inducer hemin against Ebola virus infection. Curr Trends Immunol 17:117-123 |
| Doyle, Andrew D; Yamada, Kenneth M (2016) Mechanosensing via cell-matrix adhesions in 3D microenvironments. Exp Cell Res 343:60-66 |
| Joo, E Emily; Yamada, Kenneth M (2016) Post-polymerization crosstalk between the actin cytoskeleton and microtubule network. Bioarchitecture 6:53-9 |
| Petrie, Ryan J; Yamada, Kenneth M (2016) Multiple mechanisms of 3D migration: the origins of plasticity. Curr Opin Cell Biol 42:7-12 |
| Petrie, Ryan J; Yamada, Kenneth M (2015) Fibroblasts Lead the Way: A Unified View of 3D Cell Motility. Trends Cell Biol 25:666-674 |
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