Michael E. Werner

αB-Crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer

Recent gene profiling studies have identified a new breast cancer subtype, the basal-like group, which expresses genes characteristic of basal epithelial cells and is associated with poor clinical outcomes. However, the genes responsible for the aggressive behavior observed in this group are largely unknown. Here we report that the small heat shock protein alpha-basic-crystallin (alphaB-crystallin) was commonly expressed in basal-like tumors and predicted poor survival in breast cancer patients independently of other prognostic markers. We also demonstrate that overexpression of alphaB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, alphaB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing alphaB-crystallin by RNA interference inhibited these abnormalities. alphaB-Crystallin overexpression also induced EGF- and anchorage-independent growth, increased cell migration and invasion, and constitutively activated the MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by alphaB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing alphaB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that alphaB-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors.

Actin and microtubules drive differential aspects of planar cell polarity in multiciliated cells

Actin dynamics are required for proper cilia spacing, global coordination of cilia polarity, and coordination of metachronic cilia beating, whereas cytoplasmic microtubule dynamics are required for local coordination of polarity between neighboring cilia.

Understanding ciliated epithelia: The power of Xenopus

Ciliated epithelia are important in a wide variety of biological contexts where they generate directed fluid flow. Here we address the fundamental advances in understanding ciliated epithelia that have been achieved using Xenopus as a model system. Xenopus embryos are covered with a ciliated epithelium that propels fluid unidirectionally across their surface. The external nature of this tissue, coupled with the molecular tools available in Xenopus and the ease of microscopic analysis on intact animals has thrust Xenopus to the forefront of ciliated epithelia biology. We discuss advances in understanding the molecular regulators of ciliated epithelia cell fate as well as basic aspects of ciliated epithelia cell biology including ciliogenesis and cell polarity. genesis 50:176–185, 2012.

Crucial Role of the Specificity-determining Loop of the Integrin β4 Subunit in the Binding of Cells to Laminin-5 and Outside-in Signal Transduction

Within each hemidesmosome, α6β4 integrin plays a crucial role in hemidesmosome assembly by binding to laminin-5 in the basement membrane zone of epithelial tissue. Recent analyses have implicated “specificity-determining loops” (SDLs) in the I-like domain of β integrin in regulating ligand binding. Here, we investigated the function of an SDL-like motif within the extracellular I-like domain of β4 integrin. We generated point mutations within the SDL of β4 integrin tagged with green fluorescent protein (GFP-β4K150A and GFP-β4Q155L). We also generated a mutation within the I-like domain of the β4 integrin, lying outside the SDL region (GFP-β4V284E). We transfected constructs encoding the mutated β4 integrins and a GFP-conjugated wild type β4 integrin (GFP-β4WT) into 804G cells, which assemble hemidesmosomes, and human endothelial cells, which express little endogenous β4 integrin. In transfected 804G cells, GFP-β4WT and GFP-β4V284E colocalize with hemidesmosome proteins, whereas hemidesmosomal components in cells expressing GFP-β4K150A and GFP-β4Q155L are aberrantly localized. In endothelial cells, GFP-β4WT and mutant proteins are co-expressed at the cell surface with α6 integrin. When transfected endothelial cells are plated onto laminin-5 matrix, GFP-β4WT and GFP-β4V284E localize with laminin-5, whereas GFP-β4K150A and GFP-β4Q155L do not. GFP-β4WT and GFP-β4V284E expressed in endothelial cells associate with the adaptor protein Shc when the cells are stimulated with laminin-5. However, GFP-β4K150A and GFP-β4Q155L fail to associate with Shc even when laminin-5 is present, thus impacting downstream signaling. These results provide evidence that the SDL segment of the β4 integrin subunit is required for ligand binding and is involved in outside-in signaling.

Caspase Proteolysis of the Integrin β4 Subunit Disrupts Hemidesmosome Assembly, Promotes Apoptosis, and Inhibits Cell Migration

Caspases are a conserved family of cell death proteases that cleave intracellular substrates at Asp residues to modify their function and promote apoptosis. In this report we identify the integrin β4 subunit as a novel caspase substrate using an expression cloning strategy. Together with its α6 partner, α6β4 integrin anchors epithelial cells to the basement membrane at specialized adhesive structures known as hemidesmosomes and plays a critical role in diverse epithelial cell functions including cell survival and migration. We show that integrin β4 is cleaved by caspase-3 and -7 at a conserved Asp residue (Asp1109) in vitro and in epithelial cells undergoing apoptosis, resulting in the removal of most of its cytoplasmic tail. Caspase cleavage of integrin β4 produces two products, 1) a carboxyl-terminal product that is unstable and rapidly degraded by the proteasome and 2) an amino-terminal cleavage product (amino acids 1–1109) that is unable to assemble into mature hemidesmosomes. We also demonstrate that caspase cleavage of integrin β4 sensitizes epithelial cells to apoptosis and inhibits cell migration. Taken together, we have identified a previously unrecognized proteolytic truncation of integrin β4 generated by caspases that disrupts key structural and functional properties of epithelial cells and promotes apoptosis.

c21orf59/kurly Controls Both Cilia Motility and Polarization

Cilia are microtubule-based projections that function in the movement of extracellular fluid. This requires cilia to be: (1) motile and driven by dynein complexes and (2) correctly polarized on the surface of cells, which requires planar cell polarity (PCP). Few factors that regulate both processes have been discovered. We reveal that C21orf59/Kurly (Kur), a cytoplasmic protein with some enrichment at the base of cilia, is needed for motility; zebrafish mutants exhibit characteristic developmental abnormalities and dynein arm defects. kur was also required for proper cilia polarization in the zebrafish kidney and the larval skin of Xenopus laevis. CRISPR/Cas9 coupled with homologous recombination to disrupt the endogenous kur locus in Xenopus resulted in the asymmetric localization of the PCP protein Prickle2 being lost in mutant multiciliated cells. Kur also makes interactions with other PCP components, including Disheveled. This supports a model wherein Kur plays a dual role in cilia motility and polarization.

Bbof1 is required to maintain cilia orientation

Multiciliate cells (MCCs) are highly specialized epithelial cells that employ hundreds of motile cilia to produce a vigorous directed flow in a variety of organ systems. The production of this flow requires the establishment of planar cell polarity (PCP) whereby MCCs align hundreds of beating cilia along a common planar axis. The planar axis of cilia in MCCs is known to be established via the PCP pathway and hydrodynamic cues, but the downstream steps required for cilia orientation remain poorly defined. Here, we describe a new component of cilia orientation, based on the phenotypic analysis of an uncharacterized coiled-coil protein, called bbof1. We show that the expression of bbof1 is induced during the early phases of MCC differentiation by the master regulator foxj1. MCC differentiation and ciliogenesis occurs normally in embryos where bbof1 activity is reduced, but cilia orientation is severely disrupted. We show that cilia in bbof1 mutants can still respond to patterning and hydrodynamic cues, but lack the ability to maintain their precise orientation. Misexpression of bbof1 promotes cilia alignment, even in the absence of flow or in embryos where microtubules and actin filaments are disrupted. Bbof1 appears to mediate cilia alignment by localizing to a polar structure adjacent to the basal body. Together, these results suggest that bbof1 is a basal body component required in MCCs to align and maintain cilia orientation in response to flow.

Using Xenopus Skin to Study Cilia Development and Function

Cilia are prevalent biological structures that are important for cell signaling and for generating fluid flow (or motility). Cilia are found throughout biology from single-celled organisms to vertebrates, and many model systems have been employed for their analysis. Here, we describe the use of Xenopus larval skin as a system for the study of ciliogenesis and ciliary function. In particular, we describe basic molecular and embryological manipulations and imaging techniques that have proven particularly useful for understanding the polarized beating of cilia and the generation of directed fluid flow (Werner & Mitchell, 2012). However, these same tools have the potential to benefit a large number of cilia-related biological questions.

Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1.

During radial intercalation of epithelial cells, Par3 and aPKC promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration.

Planar Cell Polarity: Microtubules Make the Connection with Cilia

Despite decades of research there are still basic aspects of planar cell polarity that are not well understood. Recent work in mouse tracheal epithelial cells links microtubules with both establishing asymmetry as well as responding to this asymmetry to coordinate cellular orientation.