Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.

Cancer cells exploit different strategies to escape from the primary tumor, gain access to the circulation, disseminate throughout the body, and form metastases, the leading cause of death by cancer. Invadopodia, proteolytically active plasma membrane extensions, are essential in this escape mechanism. Cortactin is involved in every phase of invadopodia formation, and its overexpression is associated with increased invadopodia formation, extracellular matrix degradation, and cancer cell invasion.

Fascin Rigidity and L-plastin Flexibility Cooperate in Cancer Cell Invadopodia and Filopodia.

Invadopodia and filopodia are dynamic, actin-based protrusions contributing to cancer cell migration, invasion, and metastasis. The force of actin bundles is essential for their protrusive activity. The bundling protein fascin is known to play a role in both invadopodia and filopodia.

Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP-1 macrophages and dendritic cells.

Podosomes are dynamic degrading devices present in myeloid cells among other cell types. They consist of an actin core with associated regulators, surrounded by an adhesive ring. Both fascin and cortactin are known constituents but the role of fascin actin bundling is still unclear and cortactin research rather focuses on its homologue hematopoietic lineage cell-specific protein-1 (HS1).

Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization.

Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion.

A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis.

Introduction: Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer.

L-plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages.

Podosomes are cellular structures acting as degradation 'hot-spots' in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein.

Functional complexes between YAP2 and ZO-2 are PDZ domain-dependent, and regulate YAP2 nuclear localization and signalling.

The Hippo pathway regulates the size of organs by controlling two opposing processes: proliferation and apoptosis. YAP2 (Yes kinase-associated protein 2), one of the three isoforms of YAP, is a WW domain-containing transcriptional co-activator that acts as the effector of the Hippo pathway in mammalian cells. In addition to WW domains, YAP2 has a PDZ-binding motif at its C-terminus.

TAZ interacts with zonula occludens-1 and -2 proteins in a PDZ-1 dependent manner.

The transcriptional coactivator TAZ recognizes L/PPxY motifs in transcription factors like Runx1/2 through its WW domain. We show that the first PDZ domain of zona occludens-1 (ZO-1) and 2 (ZO-2) interacts with the carboxy-terminal PDZ binding motif of TAZ. Deletion of this motif abrogates binding.

The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain.

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive.

The tandem PDZ protein Syntenin interacts with the aminoacyl tRNA synthetase complex in a lysyl-tRNA synthetase-dependent manner.

Syntenin-1 is a tandem PDZ protein that binds a diverse array of signaling molecules that are often associated with cell adhesion and intracellular trafficking. With the use of a MS-based functional proteomics approach, we identified several members of the aminoacyl-tRNA synthetase macromolecular (ARS) complex in a syntenin-1 pull down assay. Interaction of these proteins with syntenin-1 was confirmed by co-immunoprecipitation from cultured cells.

A new role for nuclear transport factor 2 and Ran: nuclear import of CapG.

The small GTPase Ran plays a central role in nucleocytoplasmic transport. Nuclear transport of Ran itself depends on nuclear transport factor 2 (NTF2). Here, we report that NTF2 and Ran control nuclear import of the filamentous actin capping protein CapG.

Downregulation of gelsolin family proteins counteracts cancer cell invasion in vitro.

Gelsolin and CapG are both actin binding proteins that modulate a variety of physiological processes by interacting differently with the actin cytoskeleton. Several studies suggest that overexpression of these proteins promotes invasion in vitro. In this study we explored the contribution of these proteins in human cancer cell invasion and motility.

The tandem PDZ domains of syntenin promote cell invasion.

Syntenin is a tandem PDZ protein that has recently been shown to be overexpressed in several cancer cells and tissues, and that might play an active role in tumor cell invasion and metastasis. Here we show that overexpression of the tandem PDZ domains of syntenin in non-invasive cells is necessary and sufficient to stimulate these cells to invade a collagen I matrix, and this effect can be regulated by ligand binding to the PDZ domains. Furthermore, we show that syntenin-induced invasion requires signaling through ras, rho and PI3K/MAPK signaling pathways and involves changes in cell-cell adhesion.

Molecular basis for dissimilar nuclear trafficking of the actin-bundling protein isoforms T- and L-plastin.

T- and L-plastin are highly similar actin-bundling proteins implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. We show that T-plastin localizes predominantly to the cytoplasm, whereas L-plastin distributes between nucleus and cytoplasm in HeLa or Cos cells. T-plastin shows nuclear accumulation upon incubation of cells with the CRM1 antagonist leptomycin B (LMB).

Increased importin-beta-dependent nuclear import of the actin modulating protein CapG promotes cell invasion.

CapG (gCap39) is a ubiquitous gelsolin-family actin modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis and motility. CapG is the only gelsolin-related actin binding protein that localizes constitutively to both nucleus and cytoplasm. Structurally related proteins like severin and fragmin are cytoplasmic because they contain a nuclear export sequence that is absent in CapG.

Gelsolin-induced epithelial cell invasion is dependent on Ras-Rac signaling.

Gelsolin is a widely distributed actin binding protein involved in controlling cell morphology, motility, signaling and apoptosis. The role of gelsolin in tumor progression, however, remains poorly understood. Here we show that expression of green fluorescent protein (GFP)-tagged gelsolin in MDCK-AZ, MDCKtsSrc or HEK293T cells promotes invasion into collagen type I.