Inmaculada Ayala

Multiple regulatory inputs converge on cortactin to control invadopodia biogenesis and extracellular matrix degradation.

Invadopodia are proteolytically active protrusions formed by invasive tumoral cells when grown on an extracellular matrix (ECM) substratum. Although many molecular components have been defined, less is known of the formation and regulation of invadopodia. The multidomain protein cortactin, which is involved in the regulation of actin polymerisation, is one such component, but how cortactin is modulated to control the formation of invadopodia has not been elucidated. Here, a new invadopodia synchronization protocol is used to show that the cortactin N-terminal acidic and SH3 domains, involved in Arp2/3 complex and N-WASP binding and activation, respectively, are both required for invadopodia biogenesis. In addition, through a combination of RNA interference and a wide array of cortactin phosphorylation mutants, we were able to show that three convergent regulatory inputs based on the regulation of cortactin phosphorylation by Src-family kinases, Erk1/Erk2 and PAK are necessary for invadopodia formation and extracellular matrix degradation. These findings suggest that cortactin is a scaffold protein bringing together the different components necessary for the formation of the invadopodia, and that a fine balance between different phosphorylation events induces subtle changes in structure to calibrate cortactin function.

Invadopodia: specialized tumor cell structures for the focal degradation of the extracellular matrix

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.

ACTIN MICROFILAMENTS ARE ESSENTIAL FOR THE CYTOLOGICAL POSITIONING AND MORPHOLOGY OF THE GOLGI COMPLEX

The organization and function of the Golgi complex was studied in normal rat kidney cells following disruption of the actin cytoskeleton induced by cytochalasin D. In cells treated with these reagents, the reticular and perinuclear Golgi morphology acquired a cluster shape restricted to the centrosome region. Golgi complex alteration affected all Golgi subcompartments as revealed by double fluorescence staining with antibodies to the cis/middle Mannosidase II and the trans-Golgi network TGN38 proteins or vital staining with the lipid derivate C6-NBD-ceramide. The ultrastructural and stereological analysis showed that the Golgi cisternae remained attached in a stacked conformation, but they were swollen and contained electron-dense intra-cisternal bodies. The Golgi complex cluster remained linked to microtubules since it was fragmented and dispersed after treatment with nocodazole. Moreover, the reassembly of Golgi fragments after the disruption of the microtubuli with nocodazole does not utilize the actin microfilaments. The actin microfilament requirement for the disassembly and reassembly of the Golgi complex and for the ER-Golgi vesicular transport were also studied. The results show that actin microfilaments are not needed for either the retrograde fusion of the Golgi complex with the endoplasmic reticulum promoted by brefeldin A or the anterograde reassembly after the removal of the drug, or the ER-Golgi transport of VSV-G glycoprotein. However, actin microfilaments are directly involved in the subcellular localization and the morphology of the Golgi complex.

Faciogenital Dysplasia Protein Fgd1 Regulates Invadopodia Biogenesis and Extracellular Matrix Degradation and Is Up-regulated in Prostate and Breast Cancer

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.

Invadopodia biogenesis is regulated by caveolin‐mediated modulation of membrane cholesterol levels

Invadopodia are proteolytically active protrusions formed by invasive tumoural cells when grown on an extracellular matrix (ECM) substratum. Clearly, invadopodia are specialized membrane domains acting as sites of signal transduction and polarized delivery of components required for focalized ECM degradation. For these reasons, invadopodia are a model to study focal ECM degradation by tumour cells. We investigated the features of invadopodia membrane domains and how altering their composition would affect invadopodia biogenesis and function. This was achieved through multiple approaches including manipulation of the levels of cholesterol and other lipids at the plasma membrane, alteration of cholesterol trafficking by acting on caveolin 1 expression and phosphorylation. We show that cholesterol depletion impairs invadopodia formation and persistence, and that invadopodia themselves are cholesterol‐rich membranes. Furthermore, the inhibition of invadopodia formation and ECM degradation after caveolin 1 knock‐down was efficiently reverted by simple provision of cholesterol. In addition, the inhibitory effect of caveolin 3DGV expression, a mutant known to block cholesterol transport to the plasma membrane, was similarly reverted by provision of cholesterol. We suggest that invadopodia biogenesis, function and structural integrity rely on appropriate levels of plasma membrane cholesterol, and that invadopodia display the properties of cholesterol‐rich membranes. Also, caveolin 1 exerts its function in invadopodia formation by regulating cholesterol balance at the plasma membrane. These findings support the connection between cholesterol, cancer and caveolin 1, provide further understanding of the role of cholesterol in cancer progression and suggest a mechanistic framework for the proposed anti‐cancer activity of statins, tightly related to their blood cholesterol‐lowering properties.

CELL AND MOLECULAR BIOLOGY OF INVADOPODIA

The controlled degradation of the extracellular matrix is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the extracellular matrix via specialized plasma membrane protrusions termed invadopodia. Considerable progress has been made in recent years toward understanding the basic molecular components and their ultrastructural features; generating substantial interest in invadopodia as a paradigm to study the complex interactions between the intracellular trafficking, signal transduction, and cytoskeleton regulation machineries. The next level will be to understand whether they may also represent valid biological targets to help advance the anticancer drug discovery process. Current knowledge will be reviewed here together with some of the most important open questions in invadopodia biology.

Morphological and biochemical analysis of the secretory pathway in melanoma cells with distinct metastatic potential

In this report, we have investigated whether alterations of the morphological and functional aspects of the biosecretory membrane system are associated with the metastatic potential of tumor cells. To this end, we have analyzed the morphology of the Golgi complex, the cytoskeleton organization and membrane trafficking steps of the secretory pathway in two human melanoma A375 cell line variants with low (A375‐P) and high metastatic (A375‐MM) potential. Immunofluorescence analysis showed that in A375‐P cells, the Golgi complex showed a collapsed morphology. Conversely, in A375‐MM cells, the Golgi complex presented a reticular and extended morphology. At the ultrastructural level, the Golgi complex of A375‐P cells was fragmented and cisternae were swollen. When the cytoskeleton was analyzed, the microtubular network appeared normal in both cell variants, whereas actin stress fibers were largely absent in A375‐P, but not in A375‐MM cells. In addition, the F‐actin content in A375‐P cells was significantly lower than in A375‐MM cells. These morphological differences in A375‐P cells were accompanied by acceleration and an increase in the endoplasmic reticulum to Golgi and the trans‐Golgi network to cell surface membrane transport, respectively. Our results indicate that in human A375 melanoma cells, metastatic potential correlates with a well‐structured morphofunctional organization of the Golgi complex and actin cytoskeleton.

Immunofluorescence and Immunoelectron Microscopy of Microinjected and Transfected Cultured Cells

Immunofluorescence microscopy is the most common method to analyze expression and localization of a given protein in cells that have been microinjected or transfected previously with the appropriate DNA constructs. It offers the advantage that it is quick, easy to perform, and allows examination of a large number of cells within a short time. However, illumination with UV light is often damaging for the cells, and the fluorescence tends to bleach as a result of the excitation of the fluorochrome by the UV light. Nowadays, these disadvantages have been overcome by sophisticated systems such as video intensification cameras and confocal microscopy. In addition, the information that can be obtained by immunofluorescence microscopy is also restricted by the limited resolution of the optical lens. Moreover, some fixation conditions can induce artifactual changes in the intracellular localization of a significant number of molecules (Melan and Sluder 1992; Griffiths et al. 1993). Therefore, if the precise localization of a protein is being studied, it will always be necessary to look at the fine structure of the cell, using immunoelectron microscopy techniques. Nevertheless, whenever possible, the utilization of immunofluorescence in combination with immunoelectron microscopy is preferable. In this chapter, we describe in detail protocols for immunofluorescence and immunoelectron microscopy analyses which are particularly suited for cells in culture.