Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon

Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon–DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.     

Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex

The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone (CAZ) present at conventional synapses. In mice deficient for the CAZ protein Bassoon, ribbons are not anchored to the presynaptic membrane but float freely in the cytoplasm. Exploiting this phenotype, we dissected the molecular structure of the photoreceptor ribbon complex. Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1. A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex. Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.     

Bassoon and Piccolo maintain synapse integrity by regulating protein ubiquitination and degradation

The presynaptic active zone (AZ) is a specialized microdomain designed for the efficient and repetitive release of neurotransmitter. Bassoon and Piccolo are two high molecular weight components of the AZ, with hypothesized roles in its assembly and structural maintenance. However, glutamatergic synapses lacking either protein exhibit relatively minor defects, presumably due to their significant functional redundancy. In the present study, we have used interference RNAs to eliminate both proteins from glutamatergic synapses, and find that they are essential for maintaining synaptic integrity. Loss of Bassoon and Piccolo leads to the aberrant degradation of multiple presynaptic proteins, culminating in synapse degeneration. This phenotype is mediated in part by the E3 ubiquitin ligase Siah1, an interacting partner of Bassoon and Piccolo whose activity is negatively regulated by their conserved zinc finger domains. Our findings demonstrate a novel role for Bassoon and Piccolo as critical regulators of presynaptic ubiquitination and proteostasis.     

Active zone proteins are dynamically associated with synaptic ribbons in rat pinealocytes

Synaptic ribbons (SRs) are prominent organelles that are abundant in the ribbon synapses of sensory neurons where they represent a specialization of the cytomatrix at the active zone (CAZ). SRs occur not only in neurons, but also in neuroendocrine pinealocytes where their function is still obscure. In this study, we report that pinealocyte SRs are associated with CAZ proteins such as Bassoon, Piccolo, CtBP1, Munc13-1, and the motorprotein KIF3A and, therefore, consist of a protein complex that resembles the ribbon complex of retinal and other sensory ribbon synapses. The pinealocyte ribbon complex is biochemically dynamic. Its protein composition changes in favor of Bassoon, Piccolo, and Munc13-1 at night and in favor of KIF3A during the day, whereas CtBP1 is equally present during the night and day. The diurnal dynamics of the ribbon complex persist under constant darkness and decrease after stimulus deprivation of the pineal gland by constant light. Our findings indicate that neuroendocrine pinealocytes possess a protein complex that resembles the CAZ of ribbon synapses in sensory organs and whose dynamics are under circadian regulation.     

Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon

Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in normal synaptic transmission, which can be attributed to the inactivation of a significant fraction of glutamatergic synapses. At these synapses, vesicles are clustered and docked in normal numbers but are unable to fuse. Phenotypically, the loss of Bassoon causes spontaneous epileptic seizures. These data show that Bassoon is not essential for synapse formation but plays an essential role in the regulated neurotransmitter release from a subset of glutamatergic synapses.     

The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina

The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.     

Bassoon speeds vesicle reloading at a central excitatory synapse

Sustained rate-coded signals encode many types of sensory modalities. Some sensory synapses possess specialized ribbon structures, which tether vesicles, to enable high-frequency signaling. However, central synapses lack these structures, yet some can maintain signaling over a wide bandwidth. To analyze the underlying molecular mechanisms, we investigated the function of the active zone core component Bassoon in cerebellar mossy fiber to granule cell synapses. We show that short-term synaptic depression is enhanced in Bassoon knockout mice during sustained high-frequency trains but basal synaptic transmission is unaffected. Fluctuation and quantal analysis as well as quantification with constrained short-term plasticity models revealed that the vesicle reloading rate was halved in the absence of Bassoon. Thus, our data show that the cytomatrix protein Bassoon speeds the reloading of vesicles to release sites at a central excitatory synapse.     

CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.     

Detection of heteromerization of more than two proteins by sequential BRET-FRET

Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.     

Assembly of active zone precursor vesicles: obligatory trafficking of presynaptic cytomatrix proteins Bassoon and Piccolo via a trans-Golgi compartment

Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.     

Imaging kinase--AKAP79--phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton-localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 A of each other, thus providing the first direct evidence of a ternary kinase-scaffold-phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.     

Bassoon and the synaptic ribbon organize Ca²+ channels and vesicles to add release sites and promote refilling

At the presynaptic active zone, Ca2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca2+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca2+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca2+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca2+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca2+ channels and vesicles, and (2) promote vesicle replenishment.     

Bassoon's part in two presynaptic orchestras

The protein Bassoon is found in the cytoskeletal matrix at the active zone of conventional synapses and in presynaptic ribbons of photoreceptor synapses. Two new studies in Neuron show that Bassoon's most prominent role at conventional synapses is to enable vesicle cycling, whereas, in photoreceptors it attaches the ribbon to the presynaptic membrane.     

Regulation of Presynaptic Anchoring of the Scaffold Protein Bassoon by Phosphorylation-Dependent Interaction with 14-3-3 Adaptor Proteins

The proper organization of the presynaptic cytomatrix at the active zone is essential for reliable neurotransmitter release from neurons. Despite of the virtual stability of this tightly interconnected proteinaceous network it becomes increasingly clear that regulated dynamic changes of its composition play an important role in the processes of synaptic plasticity. Bassoon, a core component of the presynaptic cytomatrix, is a key player in structural organization and functional regulation of presynaptic release sites. It is one of the most highly phosphorylated synaptic proteins. Nevertheless, to date our knowledge about functions mediated by any one of the identified phosphorylation sites of Bassoon is sparse. In this study, we have identified an interaction of Bassoon with the small adaptor protein 14-3-3, which depends on phosphorylation of the 14-3-3 binding motif of Bassoon. In vitro phosphorylation assays indicate that phosphorylation of the critical Ser-2845 residue of Bassoon can be mediated by a member of the 90-kDa ribosomal S6 protein kinase family. Elimination of Ser-2845 from the 14-3-3 binding motif results in a significant decrease of Bassoon''s molecular exchange rates at synapses of living rat neurons. We propose that the phosphorylation-induced 14-3-3 binding to Bassoon modulates its anchoring to the presynaptic cytomatrix. This regulation mechanism might participate in molecular and structural presynaptic remodeling during synaptic plasticity.     

Single molecule detection of DNA looping by NgoMIV restriction endonuclease

Single molecule fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy were used to investigate DNA looping by NgoMIV restriction endonuclease. Using a linear double-stranded DNA (dsDNA) molecule labeled with a fluorescence donor molecule, Cy3, and fluorescence acceptor molecule, Cy5, and by varying the concentration of NgoMIV endonuclease from 0 to 3 x 10(-6) M, it was possible to detect and determine diffusion properties of looped DNA/protein complexes. FRET efficiency distributions revealed a subpopulation of complexes with an energy transfer efficiency of 30%, which appeared upon addition of enzyme in the picomolar to nanomolar concentration range (using 10(-11) M dsDNA). The concentration dependence, fluorescence burst size analysis, and fluorescence correlation analysis were all consistent with this subpopulation arising from a sequence specific interaction between an individual enzyme and a DNA molecule. A 30% FRET efficiency corresponds to a distance of approximately 65 A, which correlates well with the distance between the ends of the dsDNA molecule when bound to NgoMIV according to the crystal structure of this complex. Formation of the looped complexes was also evident in measurements of the diffusion times of freely diffusing DNA molecules with and without NgoMIV. At very high protein concentrations compared to the DNA concentration, FRET and fluorescence correlation spectroscopy results revealed the formation of larger DNA/protein complexes.     

Stable association between G alpha(q) and phospholipase C beta 1 in living cells

Signal transduction through G alpha(q) involves stimulation of phospholipase C beta (PLC beta) that results in increased intracellular Ca2+ and activation of protein kinase C. We have measured complex formation between G alpha(q) and PLC beta1 in vitro and in living PC12 and HEK293 cells by fluorescence resonance energy transfer. In vitro measurements show that PLC beta1 will bind to G alpha(q)(guanosine 5'-3-O-(thio)triphosphate) and also to G alpha(q)(GDP), and the latter association has a different protein-protein orientation. In cells, image analysis of fluorescent-tagged proteins shows that G alpha(q) is localized almost entirely to the plasma membrane, whereas PLC beta1 has a significant cytosolic population. By using fluorescence resonance energy transfer, we found that these proteins are pre-associated in the unstimulated state in PC12 and HEK293 cells. By determining the cellular levels of the two proteins in transfected versus nontransfected cells, we found that under our conditions overexpression should not significantly promote complex formation. G alpha(q)-PLC beta1 complexes are observed in both single cell measurements and measurements of a large (i.e. 10(6)) cell suspension. The high level (approximately 40% maximum) of FRET is surprising considering that G alpha(q) is more highly expressed than PLC beta1 and that not all PLC beta1 is plasma membrane-localized. Our measurements suggest a model in which G proteins and effectors can exist in stable complexes prior to activation and that activation is achieved through changes in intermolecular interactions rather than diffusion and association. These pre-formed complexes in turn give rise to rapid, localized signals.     

Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells

We investigate the potential of dual-color photon counting histogram (PCH) analysis to resolve fluorescent protein mixtures directly inside cells. Because of their small spectral overlap, we have chosen to look at the fluorescent proteins EGFP and mRFP1. We experimentally demonstrate that dual-color PCH quantitatively resolves a mixture of EGFP and mRFP1 in cells from a single measurement. To mimic the effect of protein association, we constructed a fusion protein of EGFP and mRFP1 (denoted EGFP-mRFP1). Fluorescence resonant energy transfer within the fusion protein alters the dual-channel brightness of the fluorophores. We describe a model for fluorescence resonant energy transfer effects on the brightness and incorporate it into dual-color PCH analysis. The model is verified using fluorescence lifetime measurements. Dual-color PCH analysis demonstrated that not all of the expressed EGFP-mRFP1 fusion proteins contained a fluorescent mRFP1 molecule. Fluorescence lifetime and emission spectra measurements confirmed this surprising result. Additional experiments show that the missing fluorescent fraction of mRFP1 is consistent with a dark state population of mRFP1. We successfully resolved this mixture of fusion proteins with a single dual-color PCH measurement. These results highlight the potential of dual-color PCH to directly detect and quantify protein mixtures in living cells.     

Excitation energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus: A specific effect of 1-hexanol on the optical properties of baseplate and energy transfer processes

The effect of 1-hexanol on spectral properties and the processes of energy transfer of the green gliding photosynthetic bacterium Chloroflexus aurantiacus was investigated with reference to the baseplate region. On addition of 1-hexanol to a cell suspension in a concentration of one-fourth saturation, a specific change in the baseplate region was induced: that is, a bleach of the 793-nm component, and an increase in absorption of the 813-nm component. This result was also confirmed by fluorescence spectra of whole cells and isolated chlorosomes. The processes of energy transfer were affected in the overall transfer efficiency but not kinetically, indicating that 1-hexanol suppressed the flux of energy flow from the baseplate to the B806-866 complexes in the cytoplasmic membranes. The fluorescence excitation spectrum suggests a specific site of interaction between bacteriochlorophyll (BChl) c with a maximum at 771 nm in the rod elements and BChl a with a maximum at 793 nm in the baseplate, which is a funnel for a fast transfer of energy to the B806-866 complexes in the membranes. The absorption spectrum of chlorosomes was resolved to components consistently on the basis, including circular dichroism and magnetic circular dichroism spectra; besides two major BChl c forms, bands corresponding to tetramer, dimer, and monomer were also discernible, which are supposed to be intermediary components for a higher order structure. A tentative model for the antenna system of C. aurantiacus is proposed.Abbreviations A670 a component whose absorption maximum is located at 670 nm - (B)Chl (bacterio)chlorophyll - CD circular dichroism - F675 a component whose emission maximum is located at 675 nm - FMO protein Fenna-Mathews-Olson protein - LD linear dichroism - LH light-harvesting - McD magnetic circular dichroism - PS photosystem - RC reaction center     

Bassoon,a Novel Zinc-finger CAG/Glutamine-repeat Protein Selectively Localized at the Active Zone of Presynaptic Nerve Terminals

The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 M _r, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.