Live‐cell super‐resolution imaging of actin using LifeAct‐14 with a PAINT‐based approach

We present direct‐LIVE‐PAINT, an easy‐to‐implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct‐LIVE‐PAINT with an actin‐binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct‐LIVE‐PAINT can be used to image actin structures below the diffraction‐limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells.     

Single‐molecule imaging reveals Tau trapping at nanometer‐sized dynamic hot spots near the plasma membrane that persists after microtubule perturbation and cholesterol depletion

Accumulation of aggregates of the microtubule‐binding protein Tau is a pathological hallmark of Alzheimer''s disease. While Tau is thought to primarily associate with microtubules, it also interacts with and localizes to the plasma membrane. However, little is known about how Tau behaves and organizes at the plasma membrane of live cells. Using quantitative, single‐molecule imaging, we show that Tau exhibits spatial and kinetic heterogeneity near the plasma membrane of live cells, resulting in the formation of nanometer‐sized hot spots. The hot spots lasted tens of seconds, much longer than the short dwell time (∼ 40 ms) of Tau on microtubules. Pharmacological and biochemical disruption of Tau/microtubule interactions did not prevent hot spot formation, suggesting that these are different from the reported Tau condensation on microtubules. Although cholesterol removal has been shown to reduce Tau pathology, its acute depletion did not affect Tau hot spot dynamics. Our study identifies an intrinsic dynamic property of Tau near the plasma membrane that may facilitate the formation of assembly sites for Tau to assume its physiological and pathological functions.     

Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications

Serotonergic neurotransmission in prefrontal cortex (PFC) plays a key role in regulating emotion and cognition under normal and pathological conditios. Increasing evidence suggests that serotonin receptors are involved in the complex regulation of GABAergic inhibitory transmission in PFC. Activation of postsynaptic 5-HT₂ receptors in PFC pyramidal neurons inhibits GABA_A-receptor currents via phosphorylation of GABA_A receptor γ2 subunits by RACK1-anchored PKC. In contrast, activation of postsynaptic 5-HT₄ receptors produces an activity-dependent bi-directional regulation of GABA-evoked currents in PFC pyramidal neurons, which is mediated through phosphorylation of GABA_A-receptor β subunits by anchored PKA. On the presynaptic side, GABAergic inhibition is regulated by 5-HT through the activation of 5-HT₂, 5-HT₁, and 5-HT₃ receptors on GABAergic intereneurons. These data provide a molecular and cellular mechanism for serotonin to dynamically regulate synaptic transmission and neuronal excitability in the PFC network, which may underlie the actions of many antidepressant and antipsychotic drugs.     

GABAergic signaling in primary lens epithelial and lentoid cells and its involvement in intracellular Ca2+ modulation

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouse lenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on the intracellular Ca²⁺ ([Ca²⁺]_i) level. We have found that these cultures express specific cellular markers for lens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thus recapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-B receptors (GABA_AR and GABA_BR) with the specific agonists muscimol and baclofen, respectively induces [Ca²⁺]_i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and were dependent on extracellular Ca²⁺. Bicuculline did not change the GABA-evoked Ca²⁺ responses in Ca²-containing buffers, but suppressed them significantly in Ca²⁺-free buffers suggesting the two receptors couple to convergent Ca²⁺ mobilization mechanisms with different extracellular Ca²⁺ sensitivity. Prolonged activation of GABA_BR induced wave propagation of the Ca²⁺ signal and persistent oscillations. The number of cells reacting to GABA or GABA + bicuculline in P30 mouse LEC cultures expressing predominantly the synaptic type GABA_AR did not differ significantly from the number of reacting cells in P0 mouse LEC cultures. The GABA-induced Ca²⁺ transients in P30 (but not P0) mouse LEC could be entirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca²⁺ signaling may be involved in a variety of Ca²⁺-dependent cellular processes during lens growth and epithelial cell differentiation.     

Differential occurrence of reluctant openings in G-protein-inhibited N- and P/Q-type calcium channels

Voltage-dependent inhibition of N- and P/Q-type calcium channels by G proteins is crucial for presynaptic inhibition of neurotransmitter release, and may contribute importantly to short-term synaptic plasticity. Such calcium-channel modulation could thereby impact significantly the neuro-computational repertoire of neural networks. The differential modulation of N and P/Q channels could even further enrich their impact upon synaptic tuning. Here, we performed in-depth comparison of the G-protein inhibition of recombinant N and P/Q channels, expressed in HEK 293 cells with the m2 muscarinic receptor. While both channel types display classic features of G-protein modulation (kinetic slowing of activation, prepulse facilitation, and voltage dependence of inhibition), we confirmed previously reported quantitative differences, with N channels displaying stronger inhibition and greater relief of inhibition by prepulses. A more fundamental, qualitative difference in the modulation of these two channels was revealed by a modified tail-activation paradigm, as well as by a novel "slope" analysis method comparing time courses of slow activation and prepulse facilitation. The stark contrast in modulatory behavior can be understood within the context of the "willing-reluctant" model, in which binding of G-protein betagamma subunits to channels induces a reluctant mode of gating, where stronger depolarization is required for opening. Our experiments suggest that only N channels could be opened in the reluctant mode, at voltages normally spanned by neuronal action potentials. By contrast, P/Q channels appear to remain closed, especially over these physiological voltages. Further, the differential occurrence of reluctant openings is not explained by differences in the rate of G-protein unbinding from the two channels. These two scenarios predict very different effects of G-protein inhibition on the waveform of Ca(2+) entry during action potentials, with potentially important consequences for the timing and efficacy of synaptic transmission.     

Complex Role of Collybistin and Gephyrin in GABAA Receptor Clustering

Gephyrin and collybistin are key components of GABA_A receptor (GABA_AR) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA_AR subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA_AR α2 and α3 subunit intracellular M3–M4 domain (but not α1, α4, α5, α6, β1–3, or γ1–3) with gephyrin. Curiously, GABA_AR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA_AR α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA_AR α3 interacts with gephyrin, GABA_AR α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA_AR α2 and collybistin or GABA_AR α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA_AR α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA_AR α2 is capable of “activating ” collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA_AR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA_AR and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA_ARs, but not GlyRs or other GABA_AR subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA_ARs containing the α2 subunit.     

Calcium accumulation in neurites and cell bodies of rat cerebellar granule cells in culture: effects on GABAA receptor function

Summary. Accumulation of calcium in rat cerebellar granule cells in culture was studied by two photon laser scanning microscopy. Depolarizations by high extracellular potassium induced short-lived increases in calcium in both cell bodies and neurites. However, although the increase in neurites subsided completely after the initial peak, in cell bodies there was a persistent plateau until the high potassium stimulus was removed. On the contrary, the calcium signal due to NMDA receptors activation was persistent in both cell bodies and neurites and remained until the agonist was present.The nature of these calcium signals provides an interpretation key for the effects of NMDA receptors activation on GABA_A receptors. In particular, the persistent calcium increase in neurites may explain the decrease in GABA activated chloride currents which are related to activation of dendritic/synaptic GABA_A receptors.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.     

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent.In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i     

Novel self-associative and multiphasic nanostructured soft carriers based on amphiphilic hyaluronic acid derivatives

The purpose of the present study was to investigate the physicochemical properties in aqueous media of amphiphilic hyaluronic acid (HA) derivatives obtained by reaction of HA's hydroxyl groups with octenyl succinic anhydride (OSA). The self-associative properties of the resulting octenyl succinic anhydride-modified hyaluronic acid (OSA-HA) derivatives were studied by fluorescence spectroscopy using Nile Red as fluorophore. The morphology, size and surface charge of the OSA-HA assemblies were determined by transmission electron microscopy, dynamic light scattering and by measuring their electrophoretic mobility, respectively. OSA-HA was shown to spontaneously self-associate in aqueous media into negatively charged spherical and multiphasic nanostructures with a hydrodynamic diameter between 170 and 230 nm and to solubilize hydrophobic compounds such as Nile Red. This was a good indication that OSA-HA could be used as building block for the formulation of soft nanocarriers towards the encapsulation and controlled delivery of hydrophobic active ingredients or drugs.