KIF13A drives AMPA receptor synaptic delivery for long-term potentiation via endosomal remodeling.

The regulated trafficking of AMPA-type glutamate receptors (AMPARs) from dendritic compartments to the synaptic membrane in response to neuronal activity is a core mechanism for long-term potentiation (LTP). However, the contribution of the microtubule cytoskeleton to this synaptic transport is still unknown. In this work, using electrophysiological, biochemical, and imaging techniques, we have found that one member of the kinesin-3 family of motor proteins, KIF13A, is specifically required for the delivery of AMPARs to the spine surface during LTP induction. Accordingly, KIF13A depletion from hippocampal slices abolishes LTP expression. We also identify the vesicular protein centaurin-α1 as part of a motor transport machinery that is engaged with KIF13A and AMPARs upon LTP induction. Finally, we determine that KIF13A is responsible for the remodeling of Rab11-FIP2 endosomal structures in the dendritic shaft during LTP. Overall, these results identify specific kinesin molecular motors and endosomal transport machinery that catalyzes the dendrite-to-synapse translocation of AMPA receptors during synaptic plasticity.

Unveiling the age and origin of biogenic aggregates produced by earthworm species with their NIRS fingerprint in a subalpine meadow of Central Pyrenees.

In this study the near-infrared reflectance (NIR) spectra signals (750-2,500 nm) of soil samples was compared with the NIR signals of the biogenic aggregates produced in the lab by three earthworm species, i.e., Aporrectodea rosea (Savigny 1826), Lumbricus friendi Cognetti, 1904 and Prosellodrilus pyrenaicus (Cognetti, 1904) from subalpine meadows in the Central Pyrenees. NIR spectral signatures of biogenic aggregates, root-aggregates, and non-aggregated soil were obtained together with soil carbon (C), nitrogen (N), [Formula: see text] and [Formula: see text] determinations. The concentrations of C, N and C:N ratio in the three types of soil aggregates identified were not statistically significant (ANOVA, p>0.05) although non-macroaggregated soil had slightly higher C concentrations (66.3 g kg-1 dry soil) than biogenic aggregates (earthworm- and root-aggregates, 64.9 and 63.5 g kg-1 dry soil, respectively), while concentrations of [Formula: see text] and [Formula: see text] were highest in the root-attached aggregates (3.3 and 0.31 mg kg dry soil-1). Total earthworm density and biomass in the sampled area was 137.6 ind. m-2, and 55.2 g fresh weight m-2, respectively. The biomass of aggregates attached to roots and non-macroaggregated soil was 122.3 and 134.8 g m-2, respectively, while biomass of free (particulate) organic matter and invertebrate biogenic aggregates was 62.9 and 41.7 g m-2, respectively. Multivariate analysis of NIR spectra signals of field aggregates separated root aggregates with high concentrations of [Formula: see text] and [Formula: see text] (41.5% of explained variance, axis I) from those biogenic aggregates, including root aggregates, with large concentrations of C and high C:N ratio (21.6% of total variability, axis II). Partial Least Square (PLS) regressions were used to compare NIR spectral signals of samples (casts and soil) and develop calibration equations relating these spectral data to those data obtained for chemical variables in the lab. After a derivatization process, the NIR spectra of field aggregates were projected onto the PLS factorial plane of the NIR spectra from the lab incubation. The projection of the NIR spectral signals onto the PLSR models for C, N, [Formula: see text] and [Formula: see text] from casts produced and incubated in the lab allowed us to identify the species and the age of the field biogenic aggregates. Our hypothesis was to test whether field aggregates would match or be in the vicinity of the NIR signals that corresponded to a certain species and the age of the depositions produced in the lab. A NIRS biogenic background noise (BBN) is present in the soil as a result of earthworm activity. This study provides insights on how to analyse the role of these organisms in important ecological processes of soil macro-aggregation and associated organic matter dynamics by means of analyzing the BBN in the soil matrix.

A retention-release mechanism based on RAB11FIP2 for AMPA receptor synaptic delivery during long-term potentiation.

It is well--established that Rab11-dependent recycling endosomes drive the activity-dependent delivery of AMPA receptors (AMPARs) into synapses during long-term potentiation (LTP). Nevertheless, the molecular basis for this specialized function of recycling endosomes is still unknown. Here, we have investigated RAB11FIP2 (FIP2 hereafter) as a potential effector of Rab11-dependent trafficking during LTP in rat hippocampal slices. Surprisingly, we found that FIP2 operates independently from Rab11 proteins, and acts as a negative regulator of AMPAR synaptic trafficking. Under basal conditions, FIP2 associates with AMPARs at immobile compartments, separately from recycling endosomes. Using shRNA-mediated knockdown, we found that FIP2 prevents GluA1 (encoded by the Gria1 gene) AMPARs from reaching the surface of dendritic spines in the absence of neuronal stimulation. Upon induction of LTP, FIP2 is rapidly mobilized, dissociates from AMPARs and undergoes dephosphorylation. Interestingly, this dissociation of the FIP2-AMPAR complex, together with FIP2 dephosphorylation, is required for LTP, but the interaction between FIP2 and Rab11 proteins is not. Based on these results, we propose a retention-release mechanism, where FIP2 acts as a gate that restricts the trafficking of AMPARs, until LTP induction triggers their release and allows synaptic delivery.

SJ23B, a jatrophane diterpene activates classical PKCs and displays strong activity against HIV in vitro.

Existence of virus reservoirs makes the eradication of HIV infection extremely difficult. Current drug therapies neither eliminate these viral reservoirs nor prevent their formation. Consequently, new strategies are needed to target these reservoirs with the aim of decreasing their size. We analysed a series of jatrophane diterpenes isolated from Euphorbia hyberna and we found that one of them, SJ23B, induces the internalization of the HIV-1 receptors CD4, CXCR4 and CCR5 and prevents R5 and X4 viral infection in human primary T cells at the nanomolar range. Moreover, SJ23B is a potent antagonist of HIV-1 latency. Using Jurkat-LAT-GFP cells, a model for HIV-1 latency, we found that prostratin and SJ23B activate HIV-1 gene expression, with SJ23B being at least 10-fold more potent than prostratin. SJ23B did not elicit transforming foci activity in NIH 3T3 cells but is a potent activator of PKCalpha and delta as measured by in vitro kinase assays and by cellular translocation experiments. By using isoform-specific PKC inhibitors we found that cPKCs are critical for SJ23B-induced HIV-1 reactivation. We also showed that both SJ23B-induced IkappaBalpha degradation and NF-kappaB activation were inhibited by the classical PKC inhibitor, Gö6976. Accordingly, SJ23B synergizes with ionomycin to translocate PKCalpha to the plasma membrane and to activate the NF-kappaB pathway. Moreover, SJ23B activates both NF-kappaB and Sp1-dependent transcriptional activities in primary T cells. We have shown that diterpene jatrophanes represent a new member of anti-AIDS agents that could be developed for mitigating HIV reactivation.

Sprouty2 binds Grb2 at two different proline-rich regions, and the mechanism of ERK inhibition is independent of this interaction.

Sprouty2 has been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. Sprouty2 directly interacts with the adapter protein Grb2, member of the receptor tyrosine kinase-induced signaling pathways. In considering the functional role of Grb2, we investigated whether the interaction with this protein was responsible for ERK pathway inhibition. We found that the binding between Sprouty2 and Grb2 is constitutive, independent of Sprouty2 tyrosine phosphorylation, although it is increased when fibroblast growth factor receptor is activated. This connection is mediated by the N-terminal SH3 domain of Grb2 and two Sprouty2 proline-rich stretches (residues 59-64 and 303-307). Most importantly, a double Sprouty2 mutant (hSpry2 P59AP304A), which is unable to bind Grb2, developed at a similar inhibition level of fibroblast growth factor receptor-ERK pathway than that which originated from Sprouty2 wt. These results are evidence that the Sprouty2 mechanism of ERK inhibition is independent of Grb2 binding.

Grb2 is a negative modulator of the intrinsic Ras-GEF activity of hSos1.

hSos1 is a Ras guanine-nucleotide exchange factor. It was suggested that the carboxyl-terminal region of hSos1 down-regulates hSos1 functionality and that the intrinsic guanine-nucleotide exchange activity of this protein may be different before and after stimulation of tyrosine kinase receptors. Using different myristoylated hSos1 full-length and carboxyl-terminal truncated mutants, we show that Grb2 function accounts not only for recruitment of hSos1 to the plasma membrane but also for modulation of hSos1 activity. Our results demonstrate that the first two canonical Grb2 binding sites, inside the carboxyl-terminal region of hSos1, are responsible for this regulation. Following different approaches, such as displacement of Grb2 from the hSos1-Grb2 complex or depletion of Grb2 levels by small interfering RNA, we found that the full-length Grb2 proteins mediate negative regulation of the intrinsic Ras guanine-nucleotide exchange activity of hSos1.

The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras.

Ras proteins (H-, N-, and K-Ras) operate as molecular switches in signal transduction cascades controlling cell proliferation, differentiation, or apoptosis. The interaction of Ras with its effectors is mediated by the effector-binding loop, but different data about Ras location to plasma membrane subdomains and new roles for some docking/scaffold proteins point to signaling specificities of the different Ras proteins. To investigate the molecular mechanisms for these specificities, we compared an effector loop mutation (P34G) of three Ras isoforms (H-, N-, and K-Ras4B) for their biological and biochemical properties. Although this mutation diminished the capacity of Ras proteins to activate the Raf/ERK and the phosphatidylinositol 3-kinase/AKT pathways, the H-Ras V12G34 mutant retained the ability to cause morphological transformation of NIH 3T3 fibroblasts, whereas both the N-Ras V12G34 and the K-Ras4B V12G34 mutants were defective in this biological activity. On the other hand, although both the N-Ras V12G34 and the K-Ras4B V12G34 mutants failed to promote activation of the Ral-GDS/Ral A/PLD and the Ras/Rac pathways, the H-Ras V12G34 mutant retained the ability to activate these signaling pathways. Interestingly, the P34G mutation reduced specifically the N-Ras and K-Ras4B in vitro binding affinity to Ral-GDS, but not in the case of H-Ras. Thus, independently of Ras location to membrane subdomains, there are marked differences among Ras proteins in the sensitivity to an identical mutation (P34G) affecting the highly conserved effector-binding loop.