. Molecular proximity of seprase and the urokinase-type plasminogen activator receptor on malignant melanoma cell membranes: dependence on beta1 integrins and the cytoskeleton. Carcinogenesis 23, 1593-1601.

Invasive tumor-derived or transformed cells, cultured on a flat extracellular matrix substratum, extend specialized proteolytically active plasma membrane protrusions. These structures, termed invadopodia, are responsible for the focal degradation of the underlying substrate. Considerable progress has been made in recent years towards understanding the basic molecular components and regulatory circuits and the ultrastructural features of invadopodia. This has generated substantial interest in invadopodia as a paradigm to study the complex interactions between the intracellular trafficking, signal transduction and cytoskeleton regulation machineries; hopes are high that they may also represent valid biological targets to help advance the anti-cancer drug discovery process. Current knowledge will be reviewed here with an emphasis on the many open questions in invadopodia biology.

The integration of endocytic routes is critical to regulate receptor signaling. A non-clathrin endocytic pathway (NCE) of the epidermal growth factor receptor (EGFR) is activated at high ligand concentrations and targets receptors to degradation, attenuating signaling. Here we performed an unbiased molecular characterization of EGFR-NCE. We identified NCE-specific regulators, including the endoplasmic reticulum (ER)-resident protein reticulon-3 (RTN3), and a specific cargo, CD147. RTN3 was critical for EGFR/CD147-NCE, promoting the creation of plasma membrane (PM)-ER contact sites that were required for the formation/maturation of NCE invaginations. Ca 2+ release at these sites, triggered by IP3-dependent activation of ER Ca 2+ channels, was needed for the completion of EGFR internalization. Thus, we identified a mechanism of EGFR endocytosis that relies on ER-PM contact sites and local Ca 2+ signaling. This manuscript has been accepted for publication in Science and it is under embargo until the publication date. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS.Correspondence

Invadopodia are proteolytically active membrane protrusions that extend from the ventral surface of invasive tumoral cells grown on an extracellular matrix (ECM). The core machinery controlling invadopodia biogenesis is regulated by the Rho GTPase Cdc42. To understand the upstream events regulating invadopodia biogenesis, we investigated the role of Fgd1, a Cdc42-specific guanine nucleotide exchange factor. Loss of Fgd1 causes the rare inherited human developmental disease faciogenital dysplasia. Here, we show that Fgd1 is required for invadopodia biogenesis and ECM degradation in an invasive cell model and functions by modulation of Cdc42 activation. We also find that Fgd1 is expressed in human prostate and breast cancer as opposed to normal tissue and that expression levels matched tumor aggressiveness. Our findings suggest a central role for Fgd1 in the focal degradation of the ECM in vitro and, for the first time, show a connection between Fgd1 and cancer progression, proposing that it might function during tumorigenesis. [Cancer Res 2009;69(3):747-52]

Summary Adaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling.

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