We have investigated the effects of inhibiting the expression of cofilin to understand its role in protrusion dynamics in metastatic tumor cells, in particular. We show that the suppression of cofilin expression in MTLn3 cells (an apolar randomly moving amoeboid metastatic tumor cell) caused them to extend protrusions from only one pole, elongate, and move rectilinearly. This remarkable transformation was correlated with slower extension of fewer, more stable lamellipodia leading to a reduced turning frequency. Hence, the loss of cofilin caused an amoeboid tumor cell to assume a mesenchymal-type mode of movement. These phenotypes were correlated with the loss of uniform chemotactic sensitivity of the cell surface to EGF stimulation, demonstrating that to chemotax efficiently, a cell must be able to respond to chemotactic stimulation at any region on its surface. The changes in cell shape, directional migration, and turning frequency were related to the re-localization of Arp2/3 complex to one pole of the cell upon suppression of cofilin expression.

It has been suggested that the phosphatydylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] phosphatase and tensin homolog PTEN plays a fundamental role in Dictyostelium discoideum chemotaxis. To identify that role, the behavior of a pten– mutant was quantitatively analyzed using two-dimensional and three-dimensional computer-assisted methods. pten– cells were capable of polarizing and translocating in the absence of attractant, and sensing and responding to spatial gradients, temporal gradients and natural waves of attractant. However, all of these responses were compromised (i.e. less efficient) because of the fundamental incapacity of pten– cells to suppress lateral pseudopod formation and turning. This defect was equally manifested in the absence, as well as presence, of attractant. PTEN, which is constitutively localized in the cortex of polarized cells, was found essential for the attractant-stimulated increase in cortical myosin II and F-actin that is responsible for the increased suppression of pseudopods during chemotaxis. PTEN, therefore, plays a fundamental role in the suppression of lateral pseudopod formation, a process essential for the efficiency of locomotion and chemotaxis, but not in directional sensing.

Interferon regulatory factor 6 (Irf6) regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post-conception failed to close their wound compared with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell-cell and cell-matrix adhesions were investigated. We show that wild-type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPaseactivating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration.

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named “facilitators” and “probes.” A third cell type, the “dervish”, is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.