A targeted siRNA screen identifies regulators of Cdc42 activity at the natural killer cell immunological synapse.

Natural killer (NK) cells kill tumor cells and virally infected cells, and an effective NK cell response requires processes, such as motility, recognition, and directional secretion, that rely on cytoskeletal rearrangement. The Rho guanosine triphosphatase (GTPase) Cdc42 coordinates cytoskeletal reorganization downstream of many receptors. The Rho-related GTPase from plants 1 (ROP1) exhibits oscillatory activation behavior at the apical plasma membrane of growing pollen tubes; however, a similar oscillation in Rho GTPase activity has so far not been demonstrated in mammalian cells. We hypothesized that oscillations in Cdc42 activity might occur within NK cells as they interact with target cells. Through fluorescence lifetime imaging of a Cdc42 biosensor, we observed that in live NK cells forming immunological synapses with target cells, Cdc42 activity oscillated after exhibiting an initial increase. We used protein-protein interaction networks and structural databases to identify candidate proteins that controlled Cdc42 activity, leading to the design of a targeted short interfering RNA screen. The guanine nucleotide exchange factors RhoGEF6 and RhoGEF7 were necessary for Cdc42 activation within the NK cell immunological synapse. In addition, the kinase Akt and the p85α subunit of phosphoinositide 3-kinase (PI3K) were required for Cdc42 activation, the periodicity of the oscillation in Cdc42 activity, and the subsequent polarization of cytotoxic vesicles toward target cells. Given that PI3Ks are targets of tumor therapies, our findings suggest the need to monitor innate immune function during the course of targeted therapy against these enzymes.

Integrating receptor signal inputs that influence small Rho GTPase activation dynamics at the immunological synapse.

The Rho GTPase Cdc42 regulates cytoskeletal changes at the immunological synapse (IS) that are critical to T-cell activation. By imaging fluorescent activity biosensors (Raichu) using fluorescence lifetime imaging microscopy, Cdc42 activation was shown to display kinetics that are conditional on the specific receptor input (through two IS-associated receptors, CD3 and beta1 integrin). CD3-triggered Cdc42 activity is dependent on the cyto-2 (NPIY) motif of the beta1 integrin cytoplasmic domain. Perturbations of the ezrin-radixin-moesin (ERM) function blocked CD3- and beta1-dependent increases in Cdc42 activity. Both IS-associated receptors probably lie on a serial molecular pathway and transduce signals through the ERM-dependent machinery that is responsible for the remodeling and stabilization of the synapse. Cdc42 activity is impaired in beta1 integrin-deficient T cells that form conjugates with antigen-presenting cells but is partially restored in the context of an antigen-specific synapse. This restoration of Cdc42 activity is due, at least in part, to the recruitment and activation of beta2 integrin.

HS1-associated protein X-1 regulates carcinoma cell migration and invasion via clathrin-mediated endocytosis of integrin alphavbeta6.

Enhanced expression levels of integrin alphavbeta6 have been linked to more aggressive invasive carcinoma cell behavior and poorer clinical prognosis. However, how alphavbeta6 determines invasion and the dynamics of integrin alphavbeta6 regulation in tumor cells are poorly understood. We have identified the 35-kDa HS1-associated protein X-1 (HAX-1) protein as a novel binding partner of the beta6 cytoplasmic tail using a yeast two-hybrid screen. We show that alphavbeta6-dependent migration is blocked following small interfering RNA (siRNA)-mediated depletion of HAX-1 in oral squamous cell carcinoma cell lines. Using both siRNA and membrane-permeable peptides, we show that alphavbeta6-dependent migration and invasion require HAX-1 to bind directly to beta6 and thereby regulate clathrin-mediated endocytosis of alphavbeta6 integrins. Progression of oral cancer is associated with enhanced expression of alphavbeta6 and HAX-1 proteins in patient tissue. This report establishes that integrin endocytosis is required for alphavbeta6-dependent carcinoma cell motility and invasion and suggests that this process is an important mechanism in cancer progression.

Activated ezrin promotes cell migration through recruitment of the GEF Dbl to lipid rafts and preferential downstream activation of Cdc42.

Establishment of polarized cell morphology is a critical factor for migration and requires precise spatial and temporal activation of the Rho GTPases. Here, we describe a novel role of the actin-binding ezrin/radixin/moesin (ERM)-protein ezrin to be involved in recruiting Cdc42, but not Rac1, to lipid raft microdomains, as well as the subsequent activation of this Rho GTPase and the downstream effector p21-activated kinase (PAK)1, as shown by fluorescence lifetime imaging microscopy. The establishment of a leading plasma membrane and the polarized morphology necessary for random migration are also dependent on ERM function and Cdc42 in motile breast carcinoma cells. Mechanistically, we show that the recruitment of the ERM-interacting Rho/Cdc42-specific guanine nucleotide exchange factor Dbl to the plasma membrane and to lipid raft microdomains requires the phosphorylated, active conformer of ezrin, which serves to tether the plasma membrane or its subdomains to the cytoskeleton. Together these data suggest a mechanism whereby precise spatial guanine nucleotide exchange of Cdc42 by Dbl is dependent on functional ERM proteins and is important for directional cell migration.

Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells.

While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor- and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.

Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions.

We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM). The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) alpha in carcinoma cells for both live- and fixed-cell experiments. The CXCR4-EGFP: PKCalpha-mRFP1 complex was found to be localized precisely to intracellular vesicles and cell protrusions when imaged by multiphoton fluorescence-FLIM. A comparison of the FRET efficiencies obtained using mRFP1-tagged regulatory domain or full-length PKCalpha as the acceptor revealed that PKCalpha, in the closed (inactive) form, is restrained from associating with the cytoplasmic portion of CXCR4. Live-cell FLIM experiments show that the assembly of this receptor:kinase complex is concomitant with the endocytosis process. This is confirmed by experimental evidence suggesting that the recycling of the CXCR4 receptor is increased on stimulation with phorbol ester and blocked on inhibition of PKC by bisindolylmaleimide. The EGFP-mRFP1 couple should be widely applicable, particularly to live-cell quantitative FRET assays.

DNA sequence specificity of triplex-binding ligands.

We have examined the ability of naphthylquinoline, a 2,7-disubstituted anthraquinone and BePI, a benzo[e]pyridoindole derivative, to stabilize parallel DNA triplexes of different base composition. Fluorescence melting studies, with both inter- and intramolecular triplexes, show that all three ligands stabilize triplexes that contain blocks of TAT triplets. Naphthylquinoline has no effect on triplexes formed with third strands composed of (TC)n or (CCT)n, but stabilizes triplexes that contain (TTC)n. In contrast, BePI slightly destabilizes the triplexes that are formed at (TC)n (CCT)n and (TTC)n. 2,7-Anthraquinone stabilizes (TC)n (CCT)n and (TTC)n, although it has the greatest effect on the latter. DNase I footprinting studies confirm that triplexes formed with (CCT)n are stabilized by the 2,7-disubstituted amidoanthraquinone but not by naphthylquinoline. Both ligands stabilize the triplex formed with (CCTT)n and neither affects the complex with (CT)n. We suggest that BePI and naphthylquinoline can only bind between adjacent TAT triplets, while the anthraquinone has a broader sequence of selectivity. These differences may be attributed to the presence (naphthylquinoline and BePI) or absence (anthraquinone) of a positive charge on the aromatic portion of the ligand, which prevents intercalation adjacent to C+GC triplets. The most stable structures are formed when the stacked rings (bases or ligand) alternate between charged and uncharged species. Triplexes containing alternating C+GC and TAT triplets are not stabilized by ligands as they would interrupt the alternating pattern of charged and uncharged residues.

Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis.

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.