| PURPOSE: To study the role of synaptotagmin-controlled fusion of docked vesicles in invasion and metastasis induced by chemokines in lymphomas and potentially melanomas (part 1), and the role of myosin-driven vesicle transport in the activation of the integrin LFA-1, required for lymphoma invasion and metastasis (part 2). BACKGROUND: Part 1. We have shown that the CXCR4 receptor for the chemokine SDF-1 is required for invasion and metastasis of a lymphoma, and that signals via the Cdc42 GTPase are essential. Cdc42 induces formation of filopodia, but was recently also implicated in fusion of vesicles with the plasma membrane. Therefore, we have tested the role of vesicle fusion in invasion. Since responses to chemokines are rapid, the vesicles should be docked at the membrane. Fusion of docked vesicles is regulated by members of the synaptotagmin (Syt) family, not only in nerve cells but also in other cells. Desai et al. (J. Cell Biol. 150:1125, 2000) showed that synaptotagmin multimers, formed by interaction between C2B domains upon a calcium signal, trigger fusion. A truncated Syt-1 protein, consisting of only the C2B domain, inhibits multimer formation of all synaptotagmin family members and blocks exocytosis when added to permeabilized cells. The lymphoma cells express Syt-3 which has been implicated in regulated exocytosis in pancreatic cells. Its oligomerization is also blocked by Syt-1 C2B. We overexpressed Syt-1 C2B in lymphoma cells and found that this completely blocked chemotaxis towards SDF-1, and completely blocked invasion and metastasis. This result strongly supports the notion that regulated exocytosis is essential for invasion induced by SDF-1, and therefore probably also in invasion and metastasis, dependent on other chemokines and similar factors. PLAN OF INVESTIGATION: Part 1. We aim to obtain further evidence for this hypothesis and to study synaptotagmin involvement in metastasis of other tumor cell types, primarily hematopoietic malignancies and melanomas, in which similar signal pathways have been implicated. To show that Syt-3 is involved, wild-type Syt-3 will be overexpressed which should reverse inhibition by Syt-1 C2B. We will inhibit synaptotagmins by Syt-1 C2B in three hematopoietic tumor cell lines with different invasion mechanisms and metastatic behavior, and in B16 melanoma, and test effects on metastasis. Furthermore, we aim to demonstrate the existence and role of docked vesicles. Vesicle fusion depends on transient interaction between proteins called v- and t-SNAREs. On docked vesicles, the proteins remain bound. Co-immune precipitation of v- and t-SNAREs can be used as an assay for docked vesicles, and in preliminary results we have thus observed that fusion is induced by SDF-1. Using the available transfectants in which other involved signal molecules are blocked, we can assess whether they act upstream or downstream of the fusion event. We will also visualize the docked vesicles using cells transfected with GFP fused to vesicle components. For technical reasons, those experiments will be done mainly in macrophage cell lines. BACKGROUND: Part 2. SDF-1 induces motility, but in addition it activates the integrin adhesion molecule LFA-1, and this is also required for invasion. This activation is blocked by inhibitors of myosin. We observed that LFA-1 is highly concentrated on filopodia, in small discrete clusters. We hypothesize that these clusters are derived from vesicles containing recycled LFA-1, which are rapidly transported to the newly formed filopodia by myosin activity. LFA-1 'activation' (i.e. increased adhesion) would thus be due to clustering and concentration of LFA-1 on filopodia. Preliminary results supporting this hypothesis are that LFA-1 is recycled, accumulates within cells when exocytosis is blocked by primaquine, and that LFA-1-dependent migration and invasion are specifically blocked by primaquine. PLAN OF INVESTIGATION: Part 2. We aim to study the mechanism of LFA-1 activation and, in particular, to test the hypothesis that this involves myosin-driven vesicle transport. Recycling will be studied by reversible biotinylation, and effects of SDF-1 and myosin inhibitors assessed. We will selectively inhibit recycling pathways using dominant-negative inhibitors of Rab4, Rab 11 and vesicle-associated myosins, and by reducing their expression levels with newly developed retroviral siRNA technology. This project is expected to provide novel insights into the mechanisms of metastasis of hematopoietic malignancies and possibly melanomas. |
