Dendritic spines in hippocampal neurons mature from a filopodia-like precursor into a mushroom-shape with an enlarged post-synaptic density (PSD) and serve as the primary post-synaptic location of the excitatory neurotransmission that underlies learning and memory. Using myosin II regulatory mutants, inhibitors, and knockdowns, we show that non-muscle myosin IIB (MIIB) activity determines where spines form and whether they persist as filopodia-like spine precursors or mature into a mushroom-shape. MIIB also determines PSD size, morphology, and placement in the spine. Local inactivation of MIIB leads to the formation of filopodia-like spine protrusions from the dendritic shaft. However, di-phosphorylation of the regulatory light chain on residues Thr18 and Ser19 by Rho kinase is required for spine maturation. Inhibition of MIIB activity or a mono-phosphomimetic mutant of RLC similarly prevented maturation even in the presence of NMDA receptor activation. Expression of an actin cross-linking, non-contractile mutant, MIIB R709C, showed that maturation into a mushroom-shape requires contractile activity. Loss of MIIB also leads to an elongated PSD morphology that is no longer restricted to the spine tip; whereas increased MIIB activity, specifically through RLC-T18, S19 di-phosphorylation, increases PSD area. These observations support a model whereby myosin II inactivation forms filopodia-like protrusions that only mature once NMDA receptor activation increases RLC di-phosphorylation to stimulate MIIB contractility, resulting in mushroom-shaped spines with an enlarged PSD.

BACKGROUND Fragile X Syndrome (FXS) is the most common type of mental retardation attributable to a single-gene mutation. It is caused by FMR1 gene silencing and the consequent loss of its protein product, Fragile X Mental Retardation Protein (FMRP). Fmr1 global knock out (KO) mice recapitulate many behavioral and synaptic phenotypes associated with FXS. Abundant evidence suggests that astrocytes are important contributors to neurological diseases. This study investigates astrocytic contributions to the progression of synaptic abnormalities and learning impairments associated with FXS. METHODS Taking advantage of the Cre-lox system, we generated and characterized mice in which FMRP is selectively deleted or exclusively expressed in astrocytes. We performed in vivo two-photon imaging to track spine dynamics/morphology along dendrites of neurons in the motor cortex and examined associated behavioral defects. RESULTS We found that adult astrocyte-specific Fmr1 KO mice displayed an increased spine density in the motor cortex and impaired motor-skill learning. The learning defect coincided with a lack of enhanced spine dynamics in the motor cortex that normally occurs in response to motor skill acquisition. While spine density was normal at one month of age in astrocyte-specific Fmr1 KO mice, new spines formed at an elevated rate. Furthermore, expression of FMRP only in astrocytes was insufficient to rescue most spine or behavioral defects. CONCLUSIONS Our work suggests a joint astrocytic-neuronal contribution to FXS pathogenesis and reveals that heightened spine formation during adolescence precedes the overabundance of spines and behavioral defects found in adult Fmr1 KO mice.

A group of phosphorylatable serine residues within the nonhelical domain of NMII-B controls the ability of NMII-B to generate stable migratory front–rear polarity.

Dendritic spines are micron-sized protrusions that constitute the primary post-synaptic sites of excitatory neurotransmission in the brain. Spines mature from a filopodia-like protrusion into a mushroom-shaped morphology with a post-synaptic density (PSD) at its tip. Modulation of the actin cytoskeleton drives these morphological changes as well as the spine dynamics that underlie learning and memory. Several PSD molecules respond to glutamate receptor activation and relay signals to the underlying actin cytoskeleton to regulate the structural changes in spine and PSD morphology. α-Actinin-2 is an actin filament cross-linker, which localizes to dendritic spines, enriched within the post-synaptic density, and implicated in actin organization. We show that loss of α-actinin-2 in rat hippocampal neurons creates an increased density of immature, filopodia-like protrusions that fail to mature into a mushroom-shaped spine during development. α-Actinin-2 knockdown also prevents the recruitment and stabilization of the PSD in the spine, resulting in failure of synapse formation, and an inability to structurally respond to chemical stimulation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor. The Ca2+-insensitive EF-hand motif in α-actinin-2 is necessary for the molecule's function in regulating spine morphology and PSD assembly, since exchanging it for the similar but Ca2+-sensitive domain from α-actinin-4, another α-actinin isoform, inhibits its function. Furthermore, when the Ca2+-insensitive domain from α-actinin-2 is inserted into α-actinin-4 and expressed in neurons, it creates mature spines. These observations support a model whereby α-actinin-2, partially through its Ca2+-insensitive EF-hand motif, nucleates PSD formation via F-actin organization and modulates spine maturation to mediate synaptogenesis.

Nuclear import and export is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (referred to as importins and exportins). The yeast karyopherin Kap114p has previously been shown to import histones H2A and H2B, Nap1p, and a component of the preinitiation complex (PIC), TBP. Using a proteomic approach, we have identified several potentially new cargoes for Kap114p. These cargoes include another PIC component, the general transcription factor IIB or Sua7p, which interacted directly with Kap114p. Consistent with its role as a Sua7p import factor, deletion of KAP114 led to specific mislocalization of Sua7p to the cytoplasm. An interaction between Sua7p and TBP was also detected in cytosol, raising the possibility that both Sua7p and TBP can be coimported by Kap114p. We have also shown that Kap114p possesses multiple overlapping binding sites for its partners, Sua7p, Nap1p, and H2A and H2B, as well as RanGTP and nucleoporins. In addition, we have assembled an in vitro complex containing Sua7p, Nap1p, and histones H2A and H2B, suggesting that this Kap may import several proteins simultaneously. The import of more than one cargo at a time would increase the efficiency of each import cycle and may allow the regulation of coimported cargoes. INTRODUCTIONIn eukaryotic cells the nucleocytoplasmic transport of most proteins and some RNAs is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (also referred to as importins and exportins; reviewed in Weis, 2003;Harel and Forbes, 2004;Mosammaparast and Pemberton, 2004). After synthesis in the cytoplasm, most nuclear protein cargoes are bound by a member of the karyopherin family, through direct interaction with a nuclear localization sequence contained in the cargo protein. Transport through the nuclear pore complex occurs via transient interactions of the karyopherin with the NPC. Once in the nucleus, the karyopherin encounters a high concentration of RanGTP, which acts as a regulator of transport. Interaction of the karyopherin with RanGTP leads to dissociation of the karyopherin from its nuclear cargo (Weis, 2003;Harel and Forbes, 2004;Mosammaparast and Pemberton, 2004). In some circumstances, nuclear-binding partners of the cargo appear to also play a role in stimulating the dissociation of Kap and cargo (Senger et al., 1998;Lee and Aitchison, 1999;Pemberton et al., 1999). In yeast, there are 14 members of the karyopherin family, with Ͼ20 members in mammalian cells (Mosammaparast and Pemberton, 2004). Karyopherins appear to function in either nuclear import or nuclear export, with only two examples of a Kap that works in both directions (Weis, 2003;Harel and Forbes, 2004;Mosammaparast and Pemberton, 2004). In yeast, 11 members of the karyopherin family must import at least 1500 nuclear proteins, suggesting that each receptor must have many cargoes. To date specific transport receptor-cargo pairs have only been shown for ϳ30 cargoes; in addition the NLS sequences recognized by most Kaps is n...