Phosphorylation of the actin-binding protein profilin2a at S137 modulates bidirectional structural plasticity at dendritic spines.

Background: Synaptic plasticity requires constant adaptation of functional and structural features at individual synaptic connections. Rapid re-modulation of the synaptic actin cytoskeleton provides the scaffold orchestrating both morphological and functional modifications. A major regulator of actin polymerization not only in neurons but also in various other cell types is the actin-binding protein profilin. While profilin is known to mediate the ADP to ATP exchange at actin monomers through its direct interaction with G-actin, it additionally is able to influence actin dynamics by binding to membrane-bound phospholipids as phosphatidylinositol (4,5)-bisphosphate (PIP2) as well as several other proteins containing poly-L-proline motifs including actin modulators like Ena/VASP, WAVE/WASP or formins. Notably, these interactions are proposed to be mediated by a fine-tuned regulation of post-translational phosphorylation of profilin. However, while phosphorylation sites of the ubiquitously expressed isoform profilin1 have been described and analyzed previously, there is still only little known about the phosphorylation of the profilin2a isoform predominantly expressed in neurons. Methods: Here, utilizing a knock-down/knock-in approach, we replaced endogenously expressed profilin2a by (de)phospho-mutants of S137 known to alter actin-, PIP2 and PLP-binding properties of profilin2a and analyzed their effect on general actin dynamics as well as activity-dependent structural plasticity. Results and Discussion: Our findings suggest that a precisely timed regulation of profilin2a phosphorylation at S137 is needed to mediate actin dynamics and structural plasticity bidirectionally during long-term potentiation and long-term depression, respectively.

Metaplasticity mechanisms restore plasticity and associativity in an animal model of Alzheimer's disease.

Dynamic regulation of plasticity thresholds in a neuronal population is critical for the formation of long-term plasticity and memory and is achieved by mechanisms such as metaplasticity. Metaplasticity tunes the synapses to undergo changes that are necessary prerequisites for memory storage under physiological and pathological conditions. Here we discovered that, in amyloid precursor protein (APP)/presenilin-1 (PS1) mice (age 3-4 mo), a prominent mouse model of Alzheimer's disease (AD), late long-term potentiation (LTP; L-LTP) and its associative plasticity mechanisms such as synaptic tagging and capture (STC) were impaired already in presymptomatic mice. Interestingly, late long-term depression (LTD; L-LTD) was not compromised, but the positive associative interaction of LTP and LTD, cross-capture, was altered in these mice. Metaplastic activation of ryanodine receptors (RyRs) in these neurons reestablished L-LTP and STC. We propose that RyR-mediated metaplastic mechanisms can be considered as a possible therapeutic target for counteracting synaptic impairments in the neuronal networks during the early progression of AD.

The molybdenum cofactor biosynthesis complex interacts with actin filaments via molybdenum insertase Cnx1 as anchor protein in Arabidopsis thaliana.

The pterin based molybdenum cofactor (Moco) plays an essential role in almost all organisms. Its biosynthesis is catalysed by six enzymes in a conserved four step reaction pathway. The last three steps are located in the cytoplasm, where a multimeric protein complex is formed to protect the intermediates from degradation. Bimolecular fluorescence complementation was used to test for cytoskeleton association of the Moco biosynthesis enzymes with actin filaments and microtubules using known cytoskeleton associated proteins, thus permitting non-invasive in vivo studies. Coding sequences of binding proteins were cloned via the GATEWAY system. No Moco biosynthesis enzyme showed any interaction with microtubules. However, alone the two domain protein Cnx1 exhibited interaction with actin filaments mediated by both domains with the Cnx1G domain displaying a stronger interaction. Cnx6 showed actin association only if unlabelled Cnx1 was co-expressed in comparable amounts. So Cnx1 is likely to be the anchor protein for the whole biosynthesis complex on actin filaments. A stabilization of the whole Moco biosynthesis complex on the cytoskeleton might be crucial. In addition a micro-compartmentation might either allow a localisation near the mitochondrial ATM3 exporter providing the first Moco intermediate or near one of the three molybdate transporters enabling efficient molybdate incorporation.

Profilin isoforms modulate astrocytic morphology and the motility of astrocytic processes.

The morphology of astrocytic processes determines their close structural association with synapses referred to as the 'tripartite synapse'. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes. Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as isoform-specific functions.

Making synapses strong: metaplasticity prolongs associativity of long-term memory by switching synaptic tag mechanisms.

One conceptual mechanism for the induction of associative long-term memory is that a synaptic tag, set by a weak event, can capture plasticity-related proteins from a nearby strong input, thus enabling associativity between the 2 (synaptic tagging and capture, STC). So far, STC has been observed for only a limited time of 60 min. Nevertheless, association of weak memory forms can occur beyond this period and its mechanism is not well understood. Here we report that metaplasticity induced by ryanodine receptor activation or synaptic activation of metabotropic glutamate receptors prolongs the durability of the synaptic tag, thus extending the time window for associative interactions mediating storage of long-term memory. We provide evidence that such metaplasticity alters the mechanisms of STC from a CaMKII-mediated (in non-primed STC) to a protein kinase Mzeta (PKMζ)-mediated process (in primed STC). Thus the association of weak synapses with strong synapses in the "late" stage of associative memory formation occurs only through metaplasticity. The results also reveal that the short-lived, CaMKII-mediated tag may contribute to a mechanism for a fragile form of memory while metaplasticity enables a PKMζ-mediated synaptic tag capable of prolonged interactions that induce a more stable form of memory that is resistant to reversal.

Neuronal profilin isoforms are addressed by different signalling pathways.

Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.

Biochemical and spectroscopic characterization of the human mitochondrial amidoxime reducing components hmARC-1 and hmARC-2 suggests the existence of a new molybdenum enzyme family in eukaryotes.

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b(5), and NADH/FAD-dependent cytochrome b(5) reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.

Fine-tuning of neuronal architecture requires two profilin isoforms.

Two profilin isoforms (PFN1 and PFN2a) are expressed in the mammalian brain. Although profilins are essential for regulating actin dynamics in general, the specific role of these isoforms in neurons has remained elusive. We show that knockdown of the neuron-specific PFN2a results in a significant reduction in dendrite complexity and spine numbers of hippocampal neurons. Overexpression of PFN1 in PFN2a-deficient neurons prevents the loss of spines but does not restore dendritic complexity. Furthermore, we show that profilins are involved in differentially regulating actin dynamics downstream of the pan-neurotrophin receptor (p75(NTR)), a receptor engaged in modulating neuronal morphology. Overexpression of PFN2a restores the morphological changes in dendrites caused by p75(NTR) overexpression, whereas PFN1 restores the normal spine density. Our data assign specific functions to the two PFN isoforms, possibly attributable to different affinities for potent effectors also involved in actin dynamics, and suggest that they are important for the signal-dependent fine-tuning of neuronal architecture.

Testis-expressed profilins 3 and 4 show distinct functional characteristics and localize in the acroplaxome-manchette complex in spermatids.

BACKGROUND: Multiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. The testis-specific isoforms profilin-3 (PFN3) and profilin-4 (PFN4) may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the "somatic" profilins, profilin-1 (PFN1) and profilin-2 (PFN2). RESULTS: Ligand interactions and spatial distributions of PFN3 and PFN4 were compared by biochemical, molecular and immunological methods; PFN1 and PFN2 were employed as controls. beta-actin, phosphoinositides, poly-L-proline and mDia3, but not VASP, were confirmed as in vitro interaction partners of PFN3. In parallel experiments, PFN4 bound to selected phosphoinositides but not to poly-L-proline, proline-rich proteins, or actin. Immunofluorescence microscopy of PFN3 and PFN4 revealed distinct subcellular locations in differentiating spermatids. Both were associated first with the acroplaxome and later with the transient manchette. Predicted 3D structures indicated that PFN3 has the actin-binding site conserved, but retains only approximately half of the common poly-L-proline binding site. PFN4, in comparison, has lost both, polyproline and actin binding sites completely, which is well in line with the experimental data. CONCLUSION: The testis-specific isoform PFN3 showed major hallmarks of the well characterized "somatic" profilin isoforms, albeit with distinct binding affinities. PFN4, on the other hand, did not interact with actin or polyproline in vitro. Rather, it seemed to be specialized for phospholipid binding, possibly providing cellular functions which are distinct from actin dynamics regulation.

In birds, profilin-2a is ubiquitously expressed and contributes to actin-based motility.

Profilins are small actin-binding proteins expressed in all eukaryotes. They are involved in the regulation of actin filament dynamics and various signalling pathways. The identification of a variety of profilin isoforms led to the assumption that there may be isoform-specific functions. In mammals, profilin-1 (PFN1) is ubiquitously expressed and engaged in the regulation of various motility processes in all cell types. By contrast, profilin-2a (PFN2a) is mainly restricted to neuronal cells and there is evidence that it is involved in neuronal plasticity and membrane trafficking. However, the PFN2a sequence is much better conserved than PFN1 throughout different phyla, indicating that its restricted expression and specialized function in mammals might be unique. Using isoform-specific antibodies, we show that the situation is different in birds. PFN2a is ubiquitously expressed in embryonic and adult chicken tissues at equal and frequently higher amounts than in mammals. Together with PFN1, it is present in cultivated chicken fibroblasts, but differentially localized. Knockdown experiments with miRNA reveal that PFN2a is involved in cell adhesion, spreading and locomotion, and silencing this isoform has pronounced consequences on these processes. Our results indicate profilin isoform expression is differentially regulated among vertebrates.

Profilin regulates the activity of p42POP, a novel Myb-related transcription factor.

Profilins, regulators of cytoplasmic actin dynamics, also bind to several nuclear proteins but the significance of these interactions is mostly unclear. Here, we describe a novel Myb-related transcription factor, p42POP, as a new ligand for profilin and show that profilin regulates its activity. p42POP comprises a unique combination of domains and is widely expressed in mouse tissues. In contrast to many other Myb proteins, it contains only one functional tryptophan-cluster motif. This is followed by an acidic domain, a leucine zipper that mediates dimerization and functional nuclear import and export signals that can direct p42POP to either the nuclear or the cytoplasmic compartment. Binding to profilins is mediated by a proline-rich cluster. p42POP-profilin complexes can be precipitated from cell lysates. In transfected cells displaying p42POP in the nucleus, nuclear profilin is markedly increased. When p42POP is anchored at mitochondrial membranes, profilin is targeted to this location. Hence, in a cellular environment, p42POP and profilin are found in the same protein complex. In luciferase assays, p42POP acts as repressor and this activity is substantially reduced by profilins, indicating that profilin can regulate p42POP activity and is therefore involved in gene regulation.

Linking the synapse to the cytoskeleton: a breath-taking role for microfilaments.

Cytoskeletal elements, in particular microtubules and microfilaments, are essential players in a large variety of phenomena requiring cellular and intracellular motility. To name but a few, they are intimately involved in determining cell shape and adhesion, establishment and maintenance of polarity, locomotion and organelle transport in all eukaryotic cells, including neurons. Here, we would like to focus on the synapse in the vertebrate central nervous system, proposing a model for a specific dialogue between neuronal microfilaments and other protein components in neurotransmission and synaptic plasticity.

Tumor suppressor activity of profilin requires a functional actin binding site.

Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1.

Complex formation between the postsynaptic scaffolding protein gephyrin, profilin, and Mena: a possible link to the microfilament system.

Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems, microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Here, we show that neuronal gephyrin interacts directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses.