Anja Mai

Competitive binding of Rab21 and p120RasGAP to integrins regulates receptor traffic and migration

P120RasGAP competes with Rab21 for binding to the cytoplasmic domain of integrin α-subunits, thereby promoting receptor escape from early endosomes and recycling to the plasma membrane.

PKCe Regulation of an a 5 Integrin-ZO-1 Complex Controls Lamellae Formation in Migrating Cancer Cells

Phosphorylated ZO-1 relocalizes from tight junctions to lamellae to associate with α5 integrin and control migration. From Adhesions to Lamellae The tight junction protein ZO-1 (zonula occludens-1) is involved in cell-cell adhesion, whereas integrins containing the α5 integrin subunit mediate both cell-cell and cell-matrix adhesion. Alterations in the abundance of ZO-1 and α5 are correlated with increased invasiveness in lung cancer cells. Through biochemical, RNA interference, and imaging techniques, Tuomi et al. found that ZO-1 and α5 interacted at the leading edge of migrating lung cancer cells, where the complex promoted lamellae formation and motility. Protein kinase Cɛ (PKCɛ) promoted the phosphorylation of ZO-1 at Ser168, which triggered the relocalization of ZO-1 from tight junctions to lamellae. The ZO-1–α5 complex was detected only in migrating cells in vitro and, intriguingly, was also present only in a subset of cells in metastatic lung tumors, suggesting that ZO-1 and α5 integrin may be involved in motility and invasion in vivo. Disruption of intercellular adhesions, increased abundance of α5β1 integrin, and activation of protein kinase Cɛ (PKCɛ) correlate with invasion and unfavorable prognosis in lung cancer. However, it remains elusive how these distinct factors contribute to the invasive behavior of cancer cells. Persistent cell motility requires the formation of stable lamellae at the leading edge of a migrating cell. Here, we report that the tight junction protein zonula occludens-1 (ZO-1) preferentially interacts with α5β1 integrin at the lamellae of migrating cells. Disruption of ZO-1 binding to an internal PDZ-binding motif in the α5 cytoplasmic tail prevented the polarized localization of ZO-1 and α5 at the leading edge. Furthermore, silencing of α5 integrin inhibited migration and invasion of lung cancer cells, and silencing of ZO-1 resulted in increased Rac activity and reduced directional cell motility. The formation of the α5–ZO-1 complex was dependent on PKCɛ: Phosphorylation of ZO-1 at serine-168 regulated the subcellular localization of ZO-1 and thus controlled its association with α5 integrin. In conclusion, PKCɛ activation drives the formation of a spatially restricted, promigratory α5–ZO-1 complex at the leading edge of lung cancer cells.

Integrin endosomal signalling suppresses anoikis

Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell–ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin–ECM downstream signalling, localizes with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage independence and metastasis.

Vimentin–ERK Signaling Uncouples Slug Gene Regulatory Function

Epithelial-mesenchymal transition (EMT) in cells is a developmental process adopted during tumorigenesis that promotes metastatic capacity. In this study, we advance understanding of EMT control in cancer cells with the description of a novel vimentin-ERK axis that regulates the transcriptional activity of Slug (SNAI2). Vimentin, ERK, and Slug exhibited overlapping subcellular localization in clinical specimens of triple-negative breast carcinoma. RNAi-mediated ablation of these gene products inhibited cancer cell migration and cell invasion through a laminin-rich matrix. Biochemical analyses demonstrated direct interaction of vimentin and ERK, which promoted ERK activation and enhanced vimentin transcription. Consistent with its role as an intermediate filament, vimentin acted as a scaffold to recruit Slug to ERK and promote Slug phosphorylation at serine-87. Site-directed mutagenesis established a requirement for ERK-mediated Slug phosphorylation in EMT initiation. Together, these findings identified a pivotal step in controlling the ability of Slug to organize hallmarks of EMT.

Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription

Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal—not cancer—stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression.

Distinct c-Met activation mechanisms induce cell rounding or invasion through pathways involving integrins, RhoA and HIP1

ABSTRACT Many carcinomas have acquired oncogenic mechanisms for activating c-Met, including c-Met overexpression and excessive autocrine or paracrine stimulation with hepatocyte growth factor (HGF). However, the biological outcome of c-Met activation through these distinct modes remains ambiguous. Here, we report that HGF-mediated c-Met stimulation triggers a mesenchymal-type collective cell invasion. By contrast, the overexpression of c-Met promotes cell rounding. Moreover, in a high-throughput siRNA screen that was performed using a library of siRNAs against putative regulators of integrin activity, we identified RhoA and the clathrin-adapter protein HIP1 as crucial c-Met effectors in these morphological changes. Transient RhoA activation was necessary for the HGF-induced invasion, whereas sustained RhoA activity regulated c-Met-induced cell rounding. In addition, c-Met-induced cell rounding correlated with the phosphorylation of filamin A and the downregulation of active cell-surface integrins. By contrast, a HIP1-mediated increase in &bgr;1-integrin turnover was required for the invasion triggered by HGF. Taken together, our results indicate that c-Met induces distinct cell morphology alterations depending on the stimulus that activates c-Met.

Epithelial vimentin plays a functional role in mammary gland development

In the mammary gland, vimentin intermediate filaments are expressed in stromal cells and in basal epithelial cell populations, including gland-reconstituting mammary stem cells, with largely undefined functions. Here, we have studied how vimentin deficiency affects mouse mammary gland development. We find that, in adult vimentin knockout mice (Vim−/−), mammary ductal outgrowth is delayed. The adult Vim−/− glands display dilated ducts and a reduced basal-to-luminal mouse mammary epithelial cell (MMEC) ratio indicative of altered progenitor cell activity. Accordingly, isolated Vim−/− MMECs form fewer mammospheres and basal-like organoids in vitro than their wild-type counterparts. Importantly, reduced basal MMEC number translates into defects in Vim−/− mammary gland regeneration in vivo. Global gene expression profiling of basal MMECs reveals that lack of vimentin alters multiple pathways, including adhesion, cancer and Wnt signalling. Furthermore, vimentin contributes to stem-like cell properties in MDA-MB-231 breast cancer cells, wherein vimentin depletion reduces tumoursphere formation and attenuates expression of breast cancer stem cell-associated surface markers. Together, our findings identify vimentin as a positive regulator of stemness in the developing mouse mammary gland and in breast cancer cells. Summary: Loss of vimentin leads to altered gene expression and impedes the growth of basal mammary epithelium, which might be of importance in developing therapeutic strategies against breast cancer.

Vimentin Plays A Functional Role In Mammary Gland Regeneration

In the mammary gland, vimentin intermediate filaments are expressed in stromal cells and in basal epithelial cell populations including gland-reconstituting mammary stem cells (MaSC), with largely undefined functions. Here, we studied how vimentin deficiency affects mouse mammary gland development. Our results demonstrate that in adult vimentin knockout mice (Vim-/-) mammary ductal outgrowth is delayed. The adult Vim-/- glands are characterised by dilated ducts, an imbalance in the proportion of basal to luminal mammary epithelial cells and a reduction in cells expressing Slug (Snai2), an established MaSC regulator. All of these features are indicative of reduced progenitor cell activity. Accordingly, isolated Vim-/- mammary epithelial cells display reduced capacity to form mammospheres, and altered organoid structure, compared to wt counterparts, when plated in a 3D matrix in vitro. Importantly, altered basal epithelial cell number translates into defects in Vim-/- mammary gland regeneration in vivo in cleared fat pad transplantation studies. Furthermore, we show that vimentin contributes to stem-like cell properties in triple negative MDA-MB-231 breast cancer cells, wherein vimentin depletion reduces tumorsphere formation and alters expression of breast cancer stem cell-associated surface markers. Together, our findings identify vimentin as a positive regulator of stemness in the developing mouse mammary gland and in breast cancer cells.

ProLIF: quantitative integrin protein-protein interactions and synergistic membrane effects on proteoliposomes

ABSTRACT Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised cell lysates. However, quantitative methods to probe integrin–protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here, we describe ‘protein–liposome interactions by flow cytometry’ (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αβ heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P2 incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein–protein interactions with the potential for high-throughput/multiplex analyses. This article has associated First Person interviews with the first authors of the paper (see doi: 10.1242/jcs.223644 and doi: 10.1242/jcs.223719). Summary: This paper outlines a simple protocol to reconstitute integrin chimeras within liposomes and use flow cytometry to quantify the impact of lipid composition on integrin–talin interactions.

ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes

Integrin transmembrane heterodimeric receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. A diverse set of methods have been used to investigate cytoplasmic interactions between integrins and intracellular proteins. These predominantly consist of peptide-based pull-downs and biochemical immuno-isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe a technique called ProLIF (Protein-Liposome Iinenteractions by Flow cytometry) to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments on liposomes as individual α or β subunits or as αβ heterodimers and, using flow cytometry, to rapidly and quantitatively measure protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. By combining integrins with membrane lipids to generate proteoliposomes, the effects of membrane composition such as PI(4,5)P2 presence on protein recruitment to the integrin CTs can be analyzed. ProLIF requires no specific instrumentation, apart from a standard flow cytometer and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.