The role of CDC42, 1RSP53 and its binding partners in filopodia formation

Ph.DDOCTOR OF PHILOSOPH

Rho GTPase Cdc42 Is a Direct Interacting Partner of Adenomatous Polyposis Coli Protein and Can Alter Its Cellular Localization

Adenomatous Polyposis Coli (APC) is a tumor suppressor gene product involved in colon cancer. APC is a large multidomain molecule of 2843 amino acid residues and connects cell-cell adhesion, the F-actin/microtubule cytoskeleton and the nucleus. Here we show that Cdc42 interacts directly with the first three armadillo repeats of APC by yeast two-hybrid screens. We confirm the Cdc42-APC interaction using pulldown assays in vitro and FRET assays in vivo. Interestingly, Cdc42 interacts with APC at leading edge sites where F-actin is enriched. In contrast, Cdc42 interacts with the truncated mutant APC1–1638 in cellular puncta associated with the golgi-lysozome pathway in transfected CHO cells. In HCT116 and SW480 cells, Cdc42 induces the relocalization of endogenous APC and the mutant APC1–1338 to the plasma membrane and cellular puncta, respectively. Taken together, these data indicate that the Cdc42-APC interaction induces localization of both APC and mutant APC and may thus play a direct role in the functions of these proteins

Stimulation of synaptic vesicle exocytosis by the mental disease gene DISC1 is mediated by N-Type voltage-gated calcium channels

Lesions and mutations of the DISC1 (Disrupted-in-schizophrenia-1) gene have been linked to major depression, schizophrenia, bipolar disorder and autism, but the influence of DISC1 on synaptic transmission remains poorly understood. Using two independent genetic approaches-RNAi and a DISC1 KO mouse-we examined the impact of DISC1 on the synaptic vesicle (SV) cycle by population imaging of the synaptic tracer vGpH in hippocampal neurons. DISC1 loss-of-function resulted in a marked decrease in SV exocytic rates during neuronal stimulation and was associated with reduced Ca(2+) transients at nerve terminals. Impaired SV release was efficiently rescued by elevation of extracellular Ca(2+), hinting at a link between DISC1 and voltage-gated Ca(2+) channels. Accordingly, blockade of N-type Cav2.2 channels mimics and occludes the effect of DISC1 inactivation on SV exocytosis, and overexpression of DISC1 in a heterologous system increases Cav2.2 currents. Collectively, these results show that DISC1-dependent enhancement of SV exocytosis is mediated by Cav2.2 and point to aberrant glutamate release as a probable endophenotype of major psychiatric disorders

Death domain signaling by disulfide-linked dimers of the p75 neurotrophin receptor mediates neuronal death in the CNS

CITATION: Tanaka, K. et al. 2016. Death domain signaling by disulfide-linked dimers of the p75 neurotrophin receptor mediates neuronal death in the CNS. Journal of Neuroscience, 36(20):5587-5595, doi:10.1523/JNEUROSCI.4536-15.2016.The original publication is available at http://www.jneurosci.orgThe p75 neurotrophin receptor (p75NTR) mediates neuronal death in response to neural insults by activating a caspase apoptotic pathway. The oligomeric state and activation mechanism that enable p75NTR to mediate these effects have recently been called into question. Here, we have investigated mutant mice lacking the p75NTR death domain (DD) or a highly conserved transmembrane (TM) cysteine residue (Cys 259) implicated in receptor dimerization and activation. Neuronal death induced by proneurotrophins or epileptic seizures was assessed and compared with responses in p75NTR knock-out mice and wild-type animals. Proneurotrophins induced apoptosis of cultured hippocampal and cortical neurons from wild-type mice, but mutant neurons lacking p75NTR, only the p75NTRDD, or just Cys 259 were all equally resistant to proneurotrophin-induced neuronal death. Homo-FRET anisotropy experiments demonstrated that both NGF and proNGF induce conformational changes in p75NTR that are dependent on the TM cysteine. In vivo, neuronal death induced by pilocarpine-mediated seizures was significantly reduced in the hippocampus and somatosensory, piriform, and entorhinal cortices of all three strains of p75NTR mutant mice. Interestingly, the levels of protection observed in mice lacking the DD or only Cys 259were identical tothose of p75NTR knock-out mice eventhoughthe Cys 259mutant differedfromthe wild-type receptorin only one amino acid residue.We conclude that, both in vitro and in vivo, neuronal death induced by p75NTR requires the DD and TM Cys 259, supporting the physiological relevance of DD signaling by disulfide-linked dimers of p75NTR in the CNS.http://www.jneurosci.org/content/36/20/5587Publisher's versio

Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation and vesicle motility: implications for endocytosis.

Transducer of Cdc42-dependent actin assembly (Toca-1) consists of an F-BAR domain, a Cdc42 binding site and an SH3 domain. Toca-1 interacts with N-WASP, an activator of actin nucleation that binds Cdc42. Cdc42 may play an important role in regulating Toca-1 and N-WASP functions. We report here that the cellular expression of Toca-1 and N-WASP induces membrane tubulation and the formation of motile vesicles. Marker and uptake analysis suggests that the tubules and vesicles are associated with clathrin-mediated endocytosis. Forster resonance energy transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) analysis shows that Cdc42, N-WASP and Toca-1 form a trimer complex on the membrane tubules and vesicles and that Cdc42 interaction with N-WASP is critical for complex formation. Modulation of Cdc42 interaction with Toca-1 and/or N-WASP affects membrane tubulation, vesicle formation and vesicle motility. Thus Cdc42 may influence endocytic membrane trafficking by regulating the formation and activity of the Toca-1/N-WASP complex

Determination of in Vivo Dissociation Constant, KD, of Cdc42-Effector Complexes in Live Mammalian Cells Using Single Wavelength Fluorescence Cross-correlation Spectroscopy*S⃞

The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membrane-bound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (KD) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine KD of Cdc42-effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured KD for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nm, respectively. The determination of KD for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells

The Toca-1-N-WASP Complex Links Filopodial Formation to EndocytosisS⃞

The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essential cofactor for Cdc42-driven actin polymerization in vitro. Toca-1 consists of an N-terminal F-BAR domain, followed by a Cdc42 binding site (HR1 domain) and an SH3 domain, (the N-WASP interacting site). N-WASP is an activator of actin nucleation through the Arp2/3 complex. The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex. We report that Toca-1 induces filopodia and neurites as does N-WASP in N1E115 neuroblastoma cells. Toca-1 requires the F-BAR domain, Cdc42 binding site, and SH3 domain to induce filopodia. Toca-1 and N-WASP both require each other to induce filopodia. The expression of Toca-1 and N-WASP affects the distribution, size, and number of Rab5 positive membranes. Toca-1 interacts directly with N-WASP in filopodia and Rab5 membrane as seen by Forster resonance energy transfer. Thus the Toca-1-N-WASP complex localizes to and induces the formation of filopodia and endocytic vesicles. Last, three inhibitors of endocytosis, Dynamin-K44A, Eps15Δ95/295, and clathrin heavy chain RNA interference, block Toca-1-induced filopodial formation. Taken together, these data suggest that the Toca-1-N-WASP complex can link filopodial formation to endocytosis