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2007 Scientific Report

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  • Tumor
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VARI | 2007 Research Interests Research in the Laboratory of Cell Structure and Signal Integration focuses on the molecular machinery responsible for the reorganization of the cell’s architecture during division and directed migration. Of particular interest is how defects in the machinery drive the progression to malignancy. The goal is to identify key control steps that are altered in disease states and exploit that knowledge to improve diagnostic and prognostic capabilities. We have been targeting key points in the cytoskeletal control system to devise novel targets for molecular therapy. The cytoskeleton comprises microfilaments, microtubules, and intermediate filaments. Each of these structures is a polymer whose assembly from individual monomer subunits is controlled by accessory proteins. While the term “cytoskeleton” implies a static or rigid structure within cells, the various filamentous structures are actually highly dynamic. Microfilaments, for example, are made of polymerized actin; these filaments rapidly polymerize, bundle, bend, depolymerize, or are severed so as to assume different shapes within the cell to fulfill a given function. In some cases, individual strands are woven into networks and contract against each other so that cells can attach to extracellular substrates and crawl along them. For example, actin/microfilament remodeling is crucial in the immune cells’ role to search for and destroy invading pathogens. Cancer cells use such remodeling to migrate from primary tumors (often located at an innocuous site) to a secondary site. At the secondary site, tumor cells grow and damage adjacent tissue, often leading to the eventual death of the patient. This process is called metastasis, and to date there are few, if any, effective anti-cancer therapies that block it. Thus, there is an important need to identify mechanisms that can be effectively targeted to block the spread of tumor cells throughout the body. The Rho family of small GTPases controls critical steps in cytoskeletal remodeling. The GTPases are triggered by signals dictated by activated growth or adhesion receptors and, in turn, bind to “effectors” that govern the machinery assembling the cytoskeleton. Some of these effector proteins directly participate in cytoskeletal remodeling. One fundamentally important set of GTPase effectors is the mammalian Diaphanous-related (mDia) formins. 7 Formins nucleate, processively elongate, and (in some cases) bundle filamentous actin (F-actin) through conserved formin homology-2 (FH2) domains. mDia proteins participate in many cytoskeletal remodeling events including cytokinesis, vesicle trafficking, and filopodia assembly while acting as effectors for Rho small GTPases. Rho proteins govern mDia proteins by regulating an intramolecular autoregulatory mechanism. GTPases binding to the mDia amino-terminal GTPase-binding domain (GBD) sterically hinder the adjacent Dia-inhibitory domain (DID) interaction with the carboxyl-terminal Dia-autoregulatory (DAD) domain (Fig. 1). The release of DAD allows the adjacent FH2 domain to then nucleate and elongate nonbranched actin filaments. Figure 1. Figure 1. mDia proteins are autoregulated nucleators of actin. Autoinhibition of mDia is mediated by interaction between the DID and DAD domains. Activated GTP-bound Rho proteins bind to the GBD where they interfere with DAD binding to DID. Then the free FH2 domains, which also function as dimerization interfaces, can nucleate actin monomers and processively elongate actin filaments. Tagged fusion proteins (CFP-Rho GTPase and YFP-mDia) are used in fluorescence resonance energy transfer (FRET) to monitor the sites of protein-protein interactions. Excitation of CFP by a specific wavelength of light results in emitted light at a wavelength that excites YFP, but YFP excitation occurs only if the proteins are close enough to approximate direct binding. This approach is used to generate the data shown in Fig. 2.

Van Andel Research Institute | Scientific Report The cytoskeleton not only provides the impetus for cell movement, but it also allows the internal architecture to be organized into different compartments having specific functions in the cellular responses to growth factors. Rho GTPases and the dynamic assembly and disassembly of actin filaments have been shown to have crucial roles in both the internalization and trafficking of growth factor receptors. While all three mammalian Diaphanous-related formins (mDia1, mDia2, and mDia3) have been localized on endosomes, their roles in actin nucleation, filament elongation, and/or bundling remains poorly understood in the context of intracellular trafficking. In a recent publication in Experimental Cell Research, we reported the functional relationship between RhoB, a GTPase known to associate with both early and late endosomes, and the formin mDia2. We were able to show that 1) RhoB and mDia2 interact on endosomes, as seen in Fig. 2 using the FRET approach; 2) GTPase activity—the ability to hydrolyze GTP to GDP—is required for the ability of RhoB to govern endosome dynamics; and 3) the actin dynamics controlled by RhoB and mDia2 is necessary for vesicle trafficking. These studies further suggested that Rho GTPases significantly influence the activity of mDia family formins in driving cellular membrane remodeling through the regulation of actin dynamics. 8 In another recent study, in the journal Current Biology, we reported how Diaphanous-interacting protein (DIP) binds to and regulates the activity of the formin mDia2 and its ability to assemble filopodia. Filopodia are small finger-like projections comprising several bundled nonbranched actin filaments emanating from the leading edge of migrating cells and essentially acting as sensors for directed cell movement. We investigated an interaction occurring between a conserved leucine-rich region (LRR) in DIP and the mDia FH2 domain. While DIP has been shown to interact with and stimulate N-WASp-dependent branched filament assembly via Arp2/3, it interfered with mDia2-dependent filament assembly and bundling. Figure 2. Figure 2. RhoB and mDia2 interact on a subset of vesicles bearing internalized EGF. CFP-RhoB and YFP-mDia2 interact on vesicles bearing internalized Texas Red–labeled epidermal growth factor. Cells expressing the two FRET probes (4 h after injection) were incubated with fluorescent EGF for 5 min prior to fixation. RhoB-mDia2 FRET occurs on a subset of vesicles (FRET is false-colored green, with Texas Red–EGF shown in red).

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