E-cadherin suppression directs cytoskeletal rearrangement and intraepithelial tumor cell migration in 3D human skin equivalents.

The link between loss of cell-cell adhesion, the activation of cell migration, and the behavior of intraepithelial (IE) tumor cells during the early stages of skin cancer progression is not well understood. The current study characterized the migratory behavior of a squamous cell carcinoma cell line (HaCaT-II-4) upon E-cadherin suppression in both 2D, monolayer cultures and within human skin equivalents that mimic premalignant disease. The migratory behavior of tumor cells was first analyzed in 3D tissue context by developing a model that mimics transepithelial tumor cell migration.

EspFU, a type III-translocated effector of actin assembly, fosters epithelial association and late-stage intestinal colonization by E. coli O157:H7.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 induces filamentous actin-rich 'pedestals' on intestinal epithelial cells. Pedestal formation in vitro requires translocation of bacterial effectors into the host cell, including Tir, an EHEC receptor, and EspF(U), which increases the efficiency of actin assembly initiated by Tir. While inactivation of espF(U) does not alter colonization in two reservoir hosts, we utilized two disease models to explore the significance of EspF(U)-promoted actin pedestal formation.

Collagenase promotes the cellular responses to injury and wound healing in vivo.

Objective: This study focuses on the growth-promoting and migration-enhancing role that Clostridial collagenase plays in vitro and in vivo.

Methods: For in vitro studies, biosynthesized extracellular matrices were treated with purified Clostridial collagenase, nonspecific proteases, or buffer controls. Keratinocytes were subsequently plated upon these matrices in the presence or absence of Clostridial collagenase and/or heparin-binding epidermal-like growth factor, and cell proliferation and migration were quantified.

Arf6 modulates the beta-actin specific capping protein, betacap73.

Recent work from our laboratory has revealed that isoactin cytoskeletal and membrane dynamics are coordinately regulated. In this chapter, we review some of the recent and relevant scientific literature focusing on key aspects of cytoskeletal and membrane-mediated signal transduction. Additionally, we highlight some of the strategic molecular, biochemical, and cell-based methodologies that we have either developed or implemented in our efforts aimed at revealing the pivotal role(s) that the actin isoforms play in controlling cell shape and motility during developmental and/or disease-associated events.

FGF-2 antagonizes the TGF-beta1-mediated induction of pericyte alpha-smooth muscle actin expression: a role for myf-5 and Smad-mediated signaling pathways.

Purpose: Although the FGF and TGF-beta families are known to play an important role in regulating vascular endothelial and smooth muscle cell behavior, the influence of these matrix-binding growth factors on microvascular pericyte morphogenesis is not well understood. The current study was undertaken to examine the molecular mechanisms that mediate the effects of the endothelium-produced growth regulators FGF-2 and TGF-beta1 on retinal pericyte proliferation and contractile phenotype.

Methods: Using purified retinal pericytes, a series of assays were implemented, including RT-PCR, DNA binding, immunoprecipitation, electrophoretic mobility shift, and indirect immunofluorescence, in an attempt to elucidate the FGF/TGF-beta1 signaling cascades that mediate retinal microvascular cell growth and contractile phenotype.

Rho GTPase signaling modulates cell shape and contractile phenotype in an isoactin-specific manner.

Rho family small GTPases (Rho, Rac, and Cdc42) play an important role in cell motility, adhesion, and cell division by signaling reorganization of the actin cytoskeleton. Here, we report an isoactin-specific, Rho GTPase-dependent signaling cascade in cells simultaneously expressing smooth muscle and nonmuscle actin isoforms. We transfected primary cultures of microvascular pericytes, cells related to vascular smooth muscle cells, with various Rho-related and Rho-specific expression plasmids.

Betacap73-ARF6 interactions modulate cell shape and motility after injury in vitro.

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, beta-actin, or beta-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates.