The SRC substrate Tks5, podosomes (invadopodia), and cancer cell invasion.

Some years ago, we employed a screen of phage cDNA expression libraries to identify novel substrates of the protein tyrosine kinase Src. One of these, Tks5 (previously known as Fish), is a large scaffolding protein with an amino-terminal PX domain and five SH3 domains. In normal fibroblasts, Tks5 is cytoplasmic, but the protein is found in podosomes when the cells are transformed with Src. Using short interfering RNA technology, we have shown that Tks5 is required for podosome formation. Furthermore, cells with reduced Tks5 expression are poorly invasive through Matrigel. Tks5 is expressed and localized to podosomes in invasive human cancer cell lines and in tumor tissue, particularly breast cancers and melanomas. In these cells too, Tks5 is required for invasion. Our future work will focus on the identification of the binding partners of Tks5 that are responsible for podosome formation and invasion, and on determining the role of Tks5 in animal models of metastasis.

Dynamic localization of rop GTPases to the tonoplast during vacuole development.

Vacuoles are essential pleomorphic organelles that undergo dynamic changes during cell growth and differentiation in plants. How developmental signals are linked to vacuole biogenesis and development is poorly understood. In this report, we show that a Rop GTPase is localized to developing vacuoles in pea (Pisum sativum cv Extra Early Alaska). Rop belongs to the RHO family of Ras-related small GTP-binding proteins that are key molecular switches in a wide variety of eukaryotic signal transduction pathways. Using indirect immunofluorescence and an anti-Rop antibody, we showed that Rop proteins accumulate to high levels in rapidly growing tapetal cells of pea anthers. In these cells, Rop is localized to an endomembrane system that exists as dynamic pleomorphic networks: a perinuclear fine network decorated with punctate dots, a network composed of small spheres and tubules, and interconnected chambers. Colocalization with a tonoplast annexin VCaB42 shows that these dynamic networks represent the tonoplast. Our results suggest that the dynamic Rop-containing tonoplast networks represent a unique stage of vacuole development. The specific localization of Rop to developing vacuoles supports a role for Rop in signal transduction that mediates vacuole development in plants.

A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion.

Yeast vacuoles undergo priming, docking, and homotypic fusion, although little has been known of the connections between these reactions. Vacuole-associated Vam2p and Vam6p (Vam2/6p) are components of a 65S complex containing SNARE proteins. Upon priming by Sec18p/NSF and ATP, Vam2/6p is released as a 38S subcomplex that binds Ypt7p to initiate docking. We now report that the 38S complex consists of both Vam2/6p and the class C Vps proteins [Reider, S. E. and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307-2327]. This complex includes Vps33p, a member of the Sec1 family of proteins that bind t-SNAREs. We term this 38S complex HOPS, for homotypic fusion and vacuole protein sorting. This unexpected finding explains how Vam2/6p associates with SNAREs and provides a mechanistic link of the class C Vps proteins to Ypt/Rab action. HOPS initially associates with vacuole SNAREs in "cis" and, after release by priming, hops to Ypt7p, activating this Ypt/Rab switch to initiate docking.

A Vacuole-Associated Annexin Protein, VCaB42, Correlates with the Expansion of Tobacco Cells.

A Ca-dependent membrane-binding protein of the annexin family, VCaB42, has previously been shown to associate with vacuolar vesicles at physiological levels of Ca. In this study we used suspension-cultured cells of tobacco (Nicotiana tabacum BY-2) to show that VCaB42 is enriched 4.5-fold in intact vacuoles, whereas evacuolated protoplasts show a 12-fold reduction in VCaB42. VCaB42 distribution is thus comparable to that of the vacuole-associated H+-ATPase but is distinct from the endoplasmic reticulum-localized protein calnexin. Because VCaB42 is a vacuole-associated annexin, and given the putative function of annexins in vesicle fusion, we hypothesize a role for this protein in the vacuolation process of expanding cells. Consistent with this hypothesis, we show that VCaB42 levels correlate with age-associated and hormonally induced changes in cell volume in tobacco suspension cultures. The association of VCaB42 with vacuoles and its correlative pattern of expression relative to the expansion of cells is consistent with a possible role for VCaB42 in the early events of vacuole biogenesis.

A 42-kilodalton annexin-like protein is associated with plant vacuoles.

A 42-kD, calcium-dependent, membrane-binding protein (VCaB42) was associated with partially purified vacuole membrane. Membrane-dissociation assays indicated that VCaB42 binding to vacuole membranes was selective for calcium over other cations and that 50% of VCaB42 remained membrane bound at 61 +/- 11 nM free calcium. A 13-amino acid sequence obtained from VCaB42 showed 85% similarity with the endonexin fold, a sequence found in the annexin family of proteins that is thought to be essential for calcium and lipid binding. The greatest similarity in amino acid sequence was observed with annexin VIII (VAC-beta). The calcium-binding properties and sequence similarities suggest that VCaB42 is a member of the annexin family of calcium-dependent, membrane-binding proteins. Functional assays for VCaB42 on vacuole membrane transport processes indicated that it did not significantly affect the initial rate of calcium uptake into vacuole membrane vesicles. Because VCaB42 is vacuole localized (likely on the cytosolic surface of the vacuole) and is 50% dissociated within the physiological range of cytosolic free calcium, we hypothesize that this protein is a sensor that monitors cytosolic calcium levels and transmits that information to the vacuole.