No abstract

Three of seven recently identified genes mutated in nonsyndromic mental retardation are involved in Rho family signaling. Two of the gene products, ␣-p-21-activated kinase (PAK) interacting exchange factor (␣PIX) and PAK3, form a complex with the synaptic adaptor protein G-protein-coupled receptor kinase-interacting protein 1 (GIT1). Using an RNA interference approach, we show that GIT1 is critical for spine and synapse formation. We also show that Rac is locally activated in dendritic spines using fluorescence resonance energy transfer. This local activation of Rac is regulated by PIX, a Rac guanine nucleotide exchange factor. PAK1 and PAK3 serve as downstream effectors of Rac in regulating spine and synapse formation. Active PAK promotes the formation of spines and dendritic protrusions, which correlates with an increase in the number of excitatory synapses. These effects are dependent on the kinase activity of PAK, and PAK functions through phosphorylating myosin II regulatory light chain (MLC). Activated MLC causes an increase in dendritic spine and synapse formation, whereas inhibiting myosin ATPase activity results in decreased spine and synapse formation. Finally, both activated PAK and activated MLC can rescue the defects of GIT1 knockdown, suggesting that PAK and MLC are downstream of GIT1 in regulating spine and synapse formation. Our results point to a signaling complex, consisting of GIT1, PIX, Rac, and PAK, that plays an essential role in the regulation of dendritic spine and synapse formation and provides a potential mechanism by which ␣PIX and PAK3 mutations affect cognitive functions in mental retardation.

Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein–coupled receptor kinase–interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

PAR-3 (partitioning-defective gene 3) is essential for cell polarization in many contexts, including axon specification. However, polarity proteins have not been implicated in later steps of neuronal differentiation, such as dendritic spine morphogenesis. Here, we show that PAR-3 is necessary for normal spine development in primary hippocampal neurons. Depletion of PAR-3 causes the formation of multiple filopodia- and lamellipodia-like dendritic protrusions - a phenotype similar to neurons expressing activated Rac. PAR-3 regulates spine formation by binding the Rac guanine nucleotide-exchange factor (GEF) TIAM1, and spatially restricting it to dendritic spines. Thus, a balance of PAR-3 and TIAM1 is essential to modulate Rac-GTP levels and to allow spine morphogenesis.

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinΔLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin–PKL interaction. In cells overexpressing paxillinΔLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLΔPBS2) induced phenotypic changes reminiscent of paxillinΔLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

Fus regulates translation of APC-associated RNAs at cell protrusions and misdirects their translation in cytoplasmic granules upon overexpression or expression of ALS–linked Fus mutants.

The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. Here we show that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, we further show that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity.

How signaling molecules achieve signal diversity and specificity is a long-standing cell biology question. Here we report the development of a targeted delivery method that permits specific expression of homologous Ras-family small GTPases (i.e., Ras, Rap2, and Rap1) in different subcellular microdomains, including the endoplasmic reticulum, lipid rafts, bulk membrane, lysosomes, and Golgi complex, in rodent hippocampal CA1 neurons. The microdomain-targeted delivery, combined with multicolor fluorescence protein tagging and high-resolution dual-quintuple simultaneous patch-clamp recordings, allows systematic analysis of microdomain-specific signaling. The analysis shows that Ras signals long-term potentiation via endoplasmic reticulum PI3K and lipid raft ERK, whereas Rap2 and Rap1 signal depotentiation and long-term depression via bulk membrane JNK and lysosome p38MAPK, respectively. These results establish an effective subcellular microdomain-specific targeted delivery method and unveil subcellular microdomain-specific signaling as the mechanism for homologous Ras and Rap to achieve signal diversity and specificity to control multiple forms of synaptic plasticity.