Integrins, extracellular matrix molecules, and cytoskeletal proteins 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? 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 normal 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 of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. We recently discovered that for the newly characterized lobopodial mode of 3D cell migration, myosin II-dependent contractility generates high-pressure lobopodial protrusions at the front of various primary human cells migrating in a 3D matrix. In these cells, the nucleus physically compartmentalizes the cytoplasm into forward and rear compartments to maintain differences in hydrostatic pressure; experimentally inducing loss of pressure results in rapid leading edge retraction. 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. Myosin II, actin, vimentin, and nesprin-3 form a molecular complex, and reducing the expression of nesprin-3 by RNAi decreases and equalizes the intracellular pressure. Thus, the nucleus can act as a piston that physically compartmentalizes the cytoplasm and increases the hydrostatic pressure between the nucleus and the leading edge of a motile cell to drive lamellipodia-independent 3D lobopodial cell migration. This work has added an additional class of 3D cell migration to the previously characterized lamellipodial/mesenchymal and amoeboid forms of migration. We previously demonstrated that myosin II could crosstalk to regulate levels of microtubule acetylation, though its biological significance was unclear. Our recent studies reveal that both fibroblasts and developing glands coordinate levels of cellular contractility and microtubule acetylation in a bi-directional, reciprocal fashion. Mechanistically, this balancing is achieved by competitive binding and dephosphorylation by myosin phosphatase of either myosin light chain or the key microtubule deacetylase HDAC6. This homeostatic balancing of contractility and microtubule acetylation controls the maturation of cell adhesions by regulating integrin recycling, the cell surface density of the alpha5-beta1 integrin, the matrix assembly of extracellular fibronectin to alter the efficiency of cell migration, and even the contractility of a 3D collagenous matrix. We conclude that this intracellular homeostatic balancing system between contractility and microtubule acetylation mediated by myosin phosphatase for controlled activation/deactivation of myosin II and HDAC6 can regulate integrins on the cell surface, fibronectin assembly in the extracellular matrix, and rates of cell migration. This combined knowledge regarding the regulation of cell migration and phenotype should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of the precise manner in which cells move and interact with their matrix environmentwill also facilitate tissue engineering studies.