Protein subcellular localization is fundamental to the establishment of the body axis, cell migration, synaptic plasticity, and a vast range of other biological processes. Protein localization occurs through three mechanisms: protein transport, mRNA localization, and local translation. However, the relative contribution of each process to neuronal polarity remains unknown. Using neurons differentiated from mouse embryonic stem cells, we analyze protein and RNA expression and translation rates in isolated cell bodies and neurites genome-wide. We quantify 7323 proteins and the entire transcriptome, and identify hundreds of neurite-localized proteins and locally translated mRNAs. Our results demonstrate that mRNA localization is the primary mechanism for protein localization in neurites that may account for half of the neurite-localized proteome. Moreover, we identify multiple neurite-targeted non-coding RNAs and RNA-binding proteins with potential regulatory roles. These results provide further insight into the mechanisms underlying the establishment of neuronal polarity.

A method for spectral analysis of Förster resonance energy transfer (FRET) signals is presented, taking into consideration both the contributions of unpaired donor and acceptor fluorophores and the influence of incomplete labeling of the interacting partners. It is shown that spectral analysis of intermolecular FRET cannot yield accurate values of the Förster energy transfer efficiency E, unless one of the interactors is in large excess and perfectly labeled. Instead, analysis of donor quenching yields a product of the form Ef(d)p(a), where f(d) is the fraction of donor-type molecules participating in donor-acceptor complexes and p(a) is the labeling probability of the acceptor. Similarly, analysis of sensitized emission yields a product involving Ef(a). The analysis of intramolecular FRET (e.g., of tandem constructs) yields the product Ep(a). We use our method to determine these values for a tandem construct of cyan fluorescent protein and yellow fluorescent protein and compare them with those obtained by standard acceptor photobleaching and fluorescence lifetime measurements. We call the method lux-FRET, since it relies on linear unmixing of spectral components.

SummarySerotonin receptors 5-HT 1A and 5-HT 7 are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT 1A and 5-HT 7 receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5-HT 1A or 5-HT 7 receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT 7 -5-HT 7 homodimers, followed by the 5-HT 7 -5-HT 1A heterodimers and 5-HT 1A -5-HT 1A homodimers. Functionally, heterodimerization decreases 5-HT 1A -receptor-mediated activation of G i protein without affecting 5-HT 7 -receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT 1A receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT 1A receptor internalization and also enhances the ability of the 5-HT 1A receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT 7 receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT 1A -5-HT 7 heterodimers and, consequently, their functional importance undergoes pronounced developmental changes.

Glutamatergic synapses show large variations in strength and shortterm plasticity (STP). We show here that synapses displaying an increased strength either after posttetanic potentiation (PTP) or through activation of the phospholipase-C-diacylglycerol pathway share characteristic properties with intrinsically strong synapses, such as (i) pronounced short-term depression (STD) during high-frequency stimulation; (ii) a conversion of that STD into a sequence of facilitation followed by STD after a few conditioning stimuli at low frequency; (iii) an equalizing effect of such conditioning stimulation, which reduces differences among synapses and abolishes potentiation; and (iv) a requirement of long periods of rest for reconstitution of the original STP pattern. These phenomena are quantitatively described by assuming that a small fraction of "superprimed" synaptic vesicles are in a state of elevated release probability (p ∼ 0.5). This fraction is variable in size among synapses (typically about 30%), but increases after application of phorbol ester or during PTP. The majority of vesicles, released during repetitive stimulation, have low release probability (p ∼ 0.1), are relatively uniform in number across synapses, and are rapidly recruited. In contrast, superprimed vesicles need several seconds to be regenerated. They mediate enhanced synaptic strength at the onset of burst-like activity, the impact of which is subject to modulation by slow modulatory transmitter systems.posttetanic potentiation | short-term plasticity | calyx of Held | Munc13 | phorbol ester G lutamatergic synapses display a variety of dynamic changes in response to stimulation with action potential (AP) trains, ranging from immediate short-term depression to facilitation followed by depression (1). Both pharmacological (2-6) and molecular (7-9) perturbations have been described, which change such patterns from one to the other in a given synapse. Short-term plasticity (STP) has been shown to underlie many basic signal processing tasks of circuits in the central nervous system (10-13) and rapid changes of STP have been considered "... to be an almost necessary condition for the existence of (short-lived) activity states in the central nervous system" (ref. 14, p. 247). The balance between facilitation and depression is shifted during posttetanic potentiation (PTP) (15) and behavioral states are dynamically regulated by STP (16). Regulation occurs through slow, modulatory transmitter systems (17, 18). However, many open questions regarding the mechanisms underlying such changes remain. Modulation of presynaptic voltage-gated Ca 2+ channels by slow transmitter systems is probably the most powerful mechanism of changing release probability (p) of synaptic vesicles (SVs) (19)(20)(21). Changes in intrinsic [Ca 2+ ] i sensitivity of the release apparatus also contribute and have been investigated in the context of the phospholipase-C-diacylglycerol (PLC-DAG) signaling pathway (22-26) and posttetanic potentiation (15,(27)(28)(29)(30), but the infl...

Förster resonance energy transfer (FRET) has become an important tool for analyzing different aspects of interactions among biological macromolecules in their native environments. FRET analysis has also been successfully applied to study the spatiotemporal regulation of various cellular processes using genetically encoded FRET-based biosensors. A variety of procedures have been described for measuring FRET efficiency or the relative abundance of donor-acceptor complexes, based on analysis of the donor fluorescence lifetime or the spectrally resolved fluorescence intensity. The latter methods are preferable if one wants to not only quantify the apparent FRET efficiencies but also calculate donor-acceptor stoichiometry and observe fast dynamic changes in the interactions among donor and acceptor molecules in live cells. This review focuses on a comparison of the available intensity-based approaches used to measure FRET. We discuss their strengths and weaknesses in terms of FRET quantification, and provide several examples of biological applications.

Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.

It is commonly thought that clathrin-mediated endocytosis is the rate-limiting step of synaptic transmission in small CNS boutons with limited capacity for synaptic vesicles, causing short-term depression during high rates of synaptic transmission. Here, we show by analyzing synaptopHluorin fluorescence that 200 action potentials evoke the same cumulative amount of vesicle fusion, irrespective of the frequency of stimulation (5-40 Hz), implying the absence of vesicle reuse, since the method used (alkaline-trapping) measures only first-round exocytosis. After blocking all slow or specifically clathrin-mediated endocytosis, however, the same stimulation patterns cause a rapid stimulation-frequency-dependent release depression. This form of depression does not reflect insufficient vesicle supply, but appears to be the result of slow clearance of vesicular components from the release site. Our findings uncover an important yet overlooked role of endocytic proteins for release site clearance in addition to their well-characterized role in endocytosis itself.

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used single-vesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and γ-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters.