The constant dynamic movement of synapses and their components has emerged in the last decades as a key feature of synaptic transmission and its plasticity. Intramolecular protein movements drive conformation changes important to transduce transmitter binding into signaling. Constant cytoskeletal rearrangements power synapse shape movements. Vesicular trafficking at the pre- and postsynapse underlies transmitter release and receptor traffic between the cell surface and intracellular compartments, respectively. Receptor movement in the plane of the plasma membrane by thermally powered Brownian diffusion movement and reversible trapping by receptor-scaffold interactions has emerged as the main mechanism to dynamically organize the synaptic membrane in nanoscale domains. We will discuss here the different conceptual and methodological advances that have led to a rethinking of the synapse as an organelle whose function is tightly linked to its dynamic organization.
In addition to guiding proteins to defined genomic loci, DNA can act as an allosteric ligand that influences protein structure and activity. Here we compared genome-wide binding, transcriptional regulation and, using NMR, the conformation of two glucocorticoid receptor (GR) isoforms that differ by a single amino acid insertion in the lever arm, a domain that adopts DNA sequence-specific conformations. We show that these isoforms differentially regulate gene expression levels through two mechanisms : differential DNA binding and altered communication between GR domains. Our studies suggest a versatile role for DNA in both modulating GR activity and also in directing the use of GR isoforms. We propose that the lever arm is "fulcrum" for bidirectional allosteric signaling, conferring conformational changes in the DNA reading head that influence DNA sequence selectivity, as well as changes in the dimerization domain that connect functionally with remote regulatory surfaces, thereby influencing which genes are regulated and the magnitude of their regulation.
Sustained synaptic transmission requires vesicle recycling and refilling with transmitter, two processes considered to proceed independently. Contrary to this assumption, we show here that depletion of cytosolic transmitter at GABAergic synapses reversibly reduces the number of recycling vesicles. Using paired recordings in hippocampal cultures, we show that repetitive activity causes two phases of reduction of the postsynaptic response. The first involves the classical depletion of the readily releasable and recycling pools, while the second reflects impairment of vesicle filling as GABA is consumed, since it can only be reversed by uptake of GABA or its precursors, glutamate or glutamine. Surprisingly, this second phase is associated with reduced quantal release, a faster depression rate and lower FM5-95 labeling, suggesting that the size of the cycling vesicular pool is regulated by cytosolic transmitter availability. Regulation of vesicular cycling may represent a general mechanism of presynaptic plasticity, matching synaptic release to transmitter supply.
RNA interference defends against viral infection in plant and animal cells. The nematode Caenorhabditis elegans and its natural pathogen, the positive-strand RNA virus Orsay,have recently emerged as a new animal model of host-virus interaction. Using a genome-wide association study in C. elegans wild populations and quantitative trait locus mapping, we identify a 159 base-pair deletion in the conserved drh-1 gene (encoding a RIG-I-like helicase) as a major determinant of viral sensitivity. We show that DRH-1 is required for the initiation of an antiviral RNAi pathway and the generation of virus-derived siRNAs (viRNAs). In mammals, RIG-I-domain containing proteins trigger an interferon-based innate immunity pathway in response to RNA virus infection. Our work in C. elegans demonstrates that the RIG-I domain has an ancient role in viral recognition. We propose that RIG-I acts as modular viral recognition factor that couples viral recognition to different effector pathways including RNAi and interferon responses.
receptors (NMDARs) are glutamate-gated ion channels and are crucial for neuronal communication. NMDARs form tetrameric complexes that consist of several homologous subunits. The subunit composition of NMDARs is plastic, resulting in a large number of receptor subtypes. As each receptor subtype has distinct biophysical, pharmacological and signalling properties, there is great interest in determining whether individual subtypes carry out specific functions in the CNS in both normal and pathological conditions. Here, we review the effects of subunit composition on NMDAR properties, synaptic plasticity and cellular mechanisms implicated in neuropsychiatric disorders. Understanding the rules and roles of NMDAR diversity could provide new therapeutic strategies against dysfunctions of glutamatergic transmission.
Although feedback loops are essential in development, their molecular implementation and precise functions remain elusive. Using enhancer knockout in mice, we demonstrate that a direct, positive autoregulatory loop amplifies and maintains the expression of Krox20, a transcription factor governing vertebrate hindbrain segmentation. By combining quantitative data collected in the zebrafish with biophysical modelling that accounts for the intrinsic stochastic molecular dynamics, we dissect the loop at the molecular level. We find that it underpins a bistable switch that turns a transient input signal into cell fate commitment, as we observe in single cell analyses. The stochasticity of the activation process leads to a graded input-output response until saturation is reached. Consequently, the duration and strength of the input signal controls the size of the hindbrain segments by modulating the distribution between the two cell fates. Moreover, segment formation is buffered from severe variations in input level. Finally, the progressive extinction of Krox20 expression involves a destabilization of the loop by repressor molecules. These mechanisms are of general significance for cell type specification and tissue patterning.
The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. However, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted three-dimensional and quantitative nanoscopic imaging based on single-molecule detections to characterize the ultra-structure of inhibitory synapses and to count scaffold proteins and receptor binding sites. We observed a close correspondence between the spatial organization of gephyrin scaffolds and glycine receptors at spinal cord synapses. Endogenous gephyrin was clustered at densities of 5,000–10,000 molecules/mm2. The stoichiometry between gephyrin molecules and receptor binding sites was approximately 1:1, consistent with a two-dimensional scaffold in which all gephyrin molecules can contribute to receptor binding. The competition of glycine and GABAA receptor complexes for synaptic binding sites highlights the potential of single-molecule imaging to quantify synaptic plasticity on the nanoscopic scale.
Microtubules are subject to a variety of posttranslational modifications that potentially regulate cytoskeletal functions. Two modifications, glutamylation and glycylation, are highly enriched in the axonemes of most eukaryotes, and might therefore play particularly important roles in cilia and flagella. Here we systematically analyze the dynamics of glutamylation and glycylation in developing mouse ependymal cilia and the expression of the corresponding enzymes in the brain. By systematically screening enzymes of the TTLL family for specific functions in ependymal cilia, we demonstrate that the glycylating enzymes TTLL3 and TTLL8 were required for stability and maintenance of ependymal cilia, whereas the polyglutamylase TTLL6 was necessary for coordinated beating behavior. Our work provides evidence for a functional separation of glutamylating and glycylating enzymes in mammalian ependymal cilia. It further advances the elucidation of the functions of tubulin posttranslational modifications in motile cilia of the mammalian brain and their potential importance in brain development and disease.
Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA Polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, refuting their notion as statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.