Ndel1 palmitoylation: a new mean to regulate cytoplasmic dynein activity.

Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities, and failure to regulate this activity can result in neuronal migration retardation or neuronal degeneration. The activity of dynein is controlled by the LIS1-Ndel1-Nde1 protein complex that participates in intracellular transport, mitosis, and neuronal migration. These biological processes are subject to tight multilevel modes of regulation. Palmitoylation is a reversible posttranslational lipid modification, which can dynamically regulate protein trafficking. We found that both Ndel1 and Nde1 undergo palmitoylation in vivo and in transfected cells by specific palmitoylation enzymes. Unpalmitoylated Ndel1 interacts better with dynein, whereas the interaction between Nde1 and cytoplasmic dynein is unaffected by palmitoylation. Furthermore, palmitoylated Ndel1 reduced cytoplasmic dynein activity as judged by Golgi distribution, VSVG and short microtubule trafficking, transport of endogenous Ndel1 and LIS1 from neurite tips to the cell body, retrograde trafficking of dynein puncta, and neuronal migration. Our findings indicate, to the best of our knowledge, for the first time that Ndel1 palmitoylation is a new mean for fine-tuning the activity of the retrograde motor cytoplasmic dynein.

Differential activity-dependent secretion of brain-derived neurotrophic factor from axon and dendrite.

Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival and differentiation during development and for synaptic function and plasticity in the mature brain. BDNF-containing vesicles are widely distributed and bidirectionally transported in neurons, and secreted BDNF can act on both presynaptic and postsynaptic cells. Activity-dependent BDNF secretion from neuronal cultures has been reported, but it remains unknown where the primary site of BDNF secretion is and whether neuronal activity can trigger BDNF secretion from axons and dendrites with equal efficacy. Using BDNF fused with pH-sensitive green fluorescent protein to visualize BDNF secretion, we found that BDNF-containing vesicles exhibited markedly different properties of activity-dependent exocytic fusion at the axon and dendrite of cultured hippocampal neurons. Brief spiking activity triggered a transient fusion pore opening, followed by immediate retrieval of vesicles without dilation of the fusion pore, resulting in very little BDNF secretion at the axon. On the contrary, the same brief spiking activity induced "full-collapse" vesicle fusion and substantial BDNF secretion at the dendrite. However, full vesicular fusion with BDNF secretion could occur at the axon when the neuron was stimulated by prolonged high-frequency activity, a condition neurons may encounter during epileptic discharge. Thus, activity-dependent axonal secretion of BDNF is highly restricted as a result of incomplete fusion of BDNF-containing vesicles, and normal neural activity induces BDNF secretion from dendrites, consistent with the BDNF function as a retrograde factor. Our study also revealed a novel mechanism by which differential exocytosis of BDNF-containing vesicles may regulate BDNF-TrkB signaling between connected neurons.

Phosphorylation of CLASP2 by GSK-3beta regulates its interaction with IQGAP1, EB1 and microtubules.

Polarised cell migration is required for various cell behaviours and functions. Actin and microtubules are coupled structurally and distributed asymmetrically along the front-rear axis of migrating cells. CLIP-associating proteins (CLASPs) accumulate near the ends of microtubules at the front of migrating cells to control microtubule dynamics and cytoskeletal coupling. Regional inhibition of GSK-3beta is responsible for this asymmetric distribution of CLASPs. However, it is not known how GSK-3beta regulates the activity of CLASPs for linkage between actin and microtubules. Here we identified IQGAP1, an actin-binding protein, as a novel CLASP-binding protein. GSK-3beta directly phosphorylates CLASP2 at Ser533 and Ser537 within the region responsible for the IQGAP1 binding. Phosphorylation of CLASP2 results in the dissociation of CLASP2 from IQGAP1, EB1 and microtubules. At the leading edges of migrating fibroblasts, CLASP2 near microtubule ends partially colocalises with IQGAP1. Expression of active GSK-3beta abrogates the distribution of CLASP2 on microtubules, but not that of a nonphosphorylatable CLASP2 mutant. The phosphorylated CLASP2 does not accumulate near the ends of microtubules at the leading edges. Thus, phosphorylation of CLASP2 by GSK-3beta appears to control the regional linkage of microtubules to actin filaments through IQGAP1 for cell migration.

Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95.

Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

Dynamic protein palmitoylation in cellular signaling.

Protein S-palmitoylation, the most common lipid modification with the 16-carbon fatty acid palmitate, provides an important mechanism for regulating protein trafficking and function. The unique reversibility of protein palmitoylation allows proteins to rapidly shuttle between intracellular membrane compartments. Importantly, this palmitate cycling can be regulated by some physiological stimuli, contributing to cellular homeostasis and plasticity. Although the enzyme responsible for protein palmitoylation had been long elusive, DHHC family proteins, conserved from plants to mammals, have recently emerged as palmitoyl acyl transferases. Integrated approaches including advanced proteomics, live-cell imaging, and molecular genetics are beginning to clarify the molecular machinery for palmitoylation reaction in diverse aspects of cellular functions.

Identification of G protein alpha subunit-palmitoylating enzyme.

The heterotrimeric G protein alpha subunit (Galpha) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as Galpha palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated Galpha(q), Galpha(s), and Galpha(i2) in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased Galpha(q/11) palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that Galpha(q) rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous Galpha(q) shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the alpha(1A)-adrenergic receptor/Galpha(q/11)-mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling Galpha localization to the plasma membrane.

IQGAP3, a novel effector of Rac1 and Cdc42, regulates neurite outgrowth.

Rac1 and Cdc42, members of the Rho family GTPases, control diverse cellular processes such as cell migration and morphogenesis through their effectors. Among the effectors, IQGAP1 plays pivotal roles in the establishment of cytoskeletal architecture and intercellular adhesions in various cells. However, its roles remain to be clarified, especially in neuronal cells. We have identified IQGAP3 as a novel member of the IQGAP family, which is highly expressed in brain. We found that IQGAP3, an effector of Rac1 and Cdc42, associates directly with actin filaments and accumulates asymmetrically at the distal region of axons in hippocampal neurons. The depletion of IQGAP3 impairs neurite or axon outgrowth in neuronal cells with the disorganized cytoskeleton, but depletion of IQGAP1 does not. Furthermore, IQGAP3 is indispensable for Rac1/Cdc42-promoted neurite outgrowth in PC12 cells. Taken together, these results indicate that IQGAP3 can link the activation of Rac1 and Cdc42 with the cytoskeletal architectures during neuronal morphogenesis.

Roles of IQGAP1 in cell polarization and migration.

Cell polarization and migration are fundamental processes in all organisms and are stringently regulated during tissue development, chemotaxis and wound healing. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and the cytoskeleton. Linkage of microtubule plus ends to the cortical region is essential for polarized migration. +TIPs, including CLIP-170 and APC (adenomatous polyposis coli) are thought to function as capturing devices at specialized cortical regions. Rho family GTPases, particularly Rac1 and Cdc42, play pivotal roles in cell polarization and migration acting through their effectors. We found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. Activated Rac1 and Cdc42 enhance the binding of IQGAP1 to CLIP-170, and capture GFP-CLIP-170 at the base of leading edges and filopodia, respectively. Recently, we found that IQGAP1 directly binds to APC in addition to CLIP-170. IQGAP1 and APC interdependently localize to leading edges in migrating cells. IQGAP1 can link APC to actin filaments in vitro. Thus, activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and migration.

IQGAP1: a key regulator of adhesion and migration.

The dynamic rearrangement of cell-cell adhesion is one of the major physiological events in tissue development and tumor metastasis. Polarized cell migration, another key event, is a tightly regulated process that occurs during tissue development, chemotaxis and wound healing. Rho-family small GTPases, especially Rac1 and Cdc42, play pivotal roles in these processes through one of their effectors, IQGAP1. Recent studies reveal that IQGAP1 regulates cadherin-mediated cell-cell adhesion both positively and negatively. It captures and stabilizes microtubules through the microtubule-binding protein CLIP-170 near the cell cortex, leading to establishment of polarized cell morphology and directional cell migration. Furthermore, Rac1 and Cdc42 link the adenomatous polyposis coli (APC) protein to actin filaments through IQGAP1 at the leading edge and thereby regulate polarization and directional migration.

Regulation of microtubules in cell migration.

Directional cell migration is a fundamental process in all organisms that is stringently regulated during tissue development, chemotaxis and wound healing. Migrating cells have a polarized morphology with an asymmetrical distribution of signaling molecules and the cytoskeleton. Microtubules are indispensable for the directional migration of certain cells. Recent studies have shown that Rho family GTPases, which are key regulators of cell migration, affect microtubules, in addition to the actin cytoskeleton and adhesion. Rho family GTPases capture and stabilize microtubules through their effectors at the cell cortex, leading to a polarized microtubule array; in turn, microtubules modulate the activities of Rho family GTPases. In this article, we discuss how a polarized microtubule array is established and how microtubules facilitate cell migration.

Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration.

Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration.

Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact.

The small guanosine triphosphatase Rac1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and beta-catenin at sites of cell-cell contact. However, the modes of activation and action of Rac1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of Rac1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and beta-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of Rac1 reduced them. Knockdown of Rac1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in Rac1/Cdc42 binding. Knockdown of either Rac1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal Rac1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the Rac1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that Rac1 enhances the accumulation of actin filaments, E-cadherin, and beta-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of "E-cadherin-mediated cell-cell adhesion --> Rac1 activation --> actin-meshwork formation by IQGAP1 --> increasing E-cadherin-mediated cell-cell adhesion."

Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170.

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.