Search

Abstract The mechanisms that enable synapses to achieve temporally and spatially precise signaling at nano-scale while being fluid with the cytosol are poorly understood. Liquid-liquid phase separation (LLPS) is emerging as a key principle governing subcellular organization; however, the impact of synaptic LLPS on neurotransmission is unclear. Here, using rat primary hippocampal cultures, we show that robust disruption of neuronal LLPS with aliphatic alcohols severely dysregulates action potential-dependent neurotransmission, while spontaneous neurotransmission persists. Synaptic LLPS maintains synaptic vesicle pool clustering and recycling as well as the precise organization of active zone RIM1/2 and Munc13 nanoclusters, thus supporting fast evoked Ca2+-dependent release. These results indicate although LLPS is necessary within the nanodomain of the synapse, the disruption of this nano-organization largely spares spontaneous neurotransmission. Therefore, properties of in vitro micron sized liquid condensates translate to the nano-structure of the synapse in a functionally specific manner regulating action potential-evoked release.

Summary: Homeostatic plasticity mechanisms act in a negative feedback manner to stabilize neuronal firing around a set point. Classically, homeostatic synaptic plasticity is elicited via rather drastic manipulation of activity in a neuronal population. Here, we employed a chemogenetic approach to regulate activity via eliciting G protein-coupled receptor (GPCR) signaling in hippocampal neurons to trigger homeostatic synaptic plasticity. We demonstrate that chronic activation of hM4D(Gi) signaling induces mild and transient activity suppression, yet still triggers synaptic upscaling akin to tetrodotoxin (TTX)-induced complete activity suppression. Therefore, this homeostatic regulation was irrespective of Gi-signaling regulation of activity, but it was mimicked or occluded by direct manipulation of cyclic AMP (cAMP) signaling in a manner that intersected with the retinoic acid receptor alpha (RARα) signaling pathway. Our data suggest chemogenetic tools can uniquely be used to probe cell-autonomous mechanisms of synaptic scaling and operate via direct modulation of second messenger signaling bypassing activity regulation.

Summary: Generating stable human embryonic stem cells (hESCs) with targeted genetic mutations allows for the interrogation of protein function in numerous cellular contexts while maintaining a relatively high degree of isogenicity. We describe a step-by-step protocol for generating knockout hESC lines with mutations in genes involved in synaptic transmission using CRISPR-Cas9. We describe steps for gRNA design, cloning, stem cell transfection, and clone isolation. We then detail procedures for gene knockout validation and differentiation of stem cells into functional induced neurons. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Calcium (Ca2+) signaling is tightly regulated within a presynaptic bouton. Here, we visualize Ca2+ signals within hippocampal presynaptic boutons using GCaMP8s tagged to synaptobrevin, a synaptic vesicle protein. We identify evoked presynaptic Ca2+ transients (ePreCTs) that derive from synchronized voltage-gated Ca2+ channel openings, spontaneous presynaptic Ca2+ transients (sPreCTs) that originate from ryanodine sensitive Ca2+ stores, and a baseline Ca2+ signal that arises from stochastic voltage-gated Ca2+ channel openings. We find that baseline Ca2+, but not sPreCTs, contributes to spontaneous glutamate release. We employ photobleaching as a use-dependent tool to probe nano-organization of Ca2+ signals and observe that all three occur in non-overlapping domains within the synapse at near-resting conditions. However, increased depolarization induces intermixing of these Ca2+ domains via both local and non-local synaptic vesicle turnover. Our findings reveal nanosegregation of Ca2+ signals within a presynaptic terminal that derive from multiple sources and in turn drive specific modes of neurotransmission.

Characterizing interactions of Synaptotagmin‐1 with the SNARE complex is crucial to understand the mechanism of neurotransmitter release. X‐ray crystallography revealed how the Synaptotagmin‐1 C2B domain binds to the SNARE complex through a so‐called primary interface and to a complexin‐1‐SNARE complex through a so‐called tripartite interface. Mutagenesis and electrophysiology supported the functional relevance of both interfaces, and extensive additional data validated the primary interface. However, ITC evidence suggesting that binding via the tripartite interface occurs in solution was called into question by subsequent NMR data. Here, we describe joint efforts to address this apparent contradiction. Using the same ITC approach with the same C2B domain mutant used previously (C2BKA‐Q) but including ion exchange chromatography to purify it, which is crucial to remove polyacidic contaminants, we were unable to observe the substantial endothermic ITC signal that was previously attributed to binding of this mutant to the complexin‐1‐SNARE complex through the tripartite interface. We were also unable to detect substantial populations of the tripartite interface in NMR analyses of the ITC samples or in measurements of paramagnetic relaxation effects, despite the high sensitivity of this method to detect weak protein complexes. However, these experiments do not rule out the possibility of very low affinity (KD > 1 mm) binding through this interface. These results emphasize the need to develop methods to characterize the structure of synaptotagmin‐1‐SNARE complexes between two membranes and to perform further structure–function analyses to establish the physiological relevance of the tripartite interface.

Synaptic neurotransmitter release is an evolutionarily conserved process that mediates rapid information transfer between neurons as well as several peripheral tissues. Release of neurotransmitters are ensured by successive events such as synaptic vesicle docking and priming that prepare synaptic vesicles for rapid fusion. These events are orchestrated by interaction of different presynaptic proteins and are regulated by presynaptic calcium. Recent studies have identified various mutations in different components of neurotransmitter release machinery resulting in aberrant neurotransmitter release, which underlie a wide spectrum of psychiatric and neurological symptoms. Here, we review how these genetic alterations in different components of the core neurotransmitter release machinery affect the information transfer between neurons and how aberrant synaptic release affects nervous system function.

Summary: Visualizing the nano-organization of the synapse is fundamental to elucidating the structure-function relationship of the nervous system. The advent of super-resolution microscopy provides a tool to assess and quantify the dynamic organization of numerous proteins at the synapse. Here we present a protocol assessing inhibitory synapse scaffold protein, gephyrin, in rat primary hippocampal cultures using dSTORM microscopy. We delineate the steps for artemisinin treatment, immunocytochemistry, dSTORM image acquisition, single-molecule localization, and the analysis of synaptic scaffold protein dynamics.For complete details on the use and execution of this protocol, please refer to Guzikowski and Kavalali (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Rapid release of neurotransmitters in synchrony with action potentials is considered a key hardwired property of synapses. Here, in glutamatergic synapses formed between induced human neurons, we show that action potential-dependent neurotransmitter release becomes progressively desynchronized as synapses mature and age. In this solely excitatory network, the emergence of NMDAR-mediated transmission elicits endoplasmic reticulum (ER) stress leading to downregulation of key presynaptic molecules, synaptotagmin-1 and cysteine string protein α, that synchronize neurotransmitter release. The emergence of asynchronous release with neuronal maturity and subsequent aging is maintained by the high-affinity Ca2+ sensor synaptotagmin-7 and suppressed by the introduction of GABAergic transmission into the network, inhibition of NMDARs, and ER stress. These results suggest that long-term disruption of excitation-inhibition balance affects the synchrony of excitatory neurotransmission in human synapses.

Summary: Immunolabeling of surface AMPA receptors (AMPARs) can be used for in vivo or ex vivo examination of synaptic scaling, a type of homeostatic plasticity. Here, we present a protocol to analyze changes in synaptic weights using immunohistochemistry for surface AMPARs coupled with optical imaging analysis. We detail immunostaining of AMPARs in mouse brain sections, followed by confocal imaging of surface AMPARs in dendritic region of hippocampal CA1. We then describe using Fiji/ImageJ and rank order plots for analyzing synaptic weight.For complete details on the use and execution of this protocol, please refer to Suzuki et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Synapses maintain both action potential-evoked and spontaneous neurotransmitter release; however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release in primary hippocampal cultures. In nonneuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of nonphotobleached probes with a time constant of ~10 s. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.

Synapses maintain synchronous, asynchronous, and spontaneous modes of neurotransmission through distinct molecular and biochemical pathways. Traditionally a single synapse was assumed to have a homogeneous organization of molecular components both at the active zone and post-synaptically. However, recent advancements in experimental tools and the further elucidation of the physiological significance of distinct forms of release have challenged this notion. In comparison to rapid evoked release, the physiological significance of both spontaneous and asynchronous neurotransmission has only recently been considered in parallel with synaptic structural organization. Active zone nanostructure aligns with postsynaptic nanostructure creating a precise trans-synaptic alignment of release sites and receptors shaping synaptic efficacy, determining neurotransmission reliability, and tuning plasticity. This review will discuss how studies delineating synaptic nanostructure create a picture of a molecularly heterogeneous active zone tuned to distinct forms of release that may dictate diverse synaptic functional outputs.

Summary: Ketamine is a noncompetitive glutamatergic N-methyl-d-aspartate receptor (NMDAR) antagonist that exerts rapid antidepressant effects. Preclinical studies identify eukaryotic elongation factor 2 kinase (eEF2K) signaling as essential for the rapid antidepressant action of ketamine. Here, we combine genetic, electrophysiological, and pharmacological strategies to investigate the role of eEF2K in synaptic function and find that acute, but not chronic, inhibition of eEF2K activity induces rapid synaptic scaling in the hippocampus. Retinoic acid (RA) signaling also elicits a similar form of rapid synaptic scaling in the hippocampus, which we observe is independent of eEF2K functioni. The RA signaling pathway is not required for ketamine-mediated antidepressant action; however, direct activation of the retinoic acid receptor α (RARα) evokes rapid antidepressant action resembling ketamine. Our findings show that ketamine and RARα activation independently elicit a similar form of multiplicative synaptic scaling that is causal for rapid antidepressant action.

Synaptic transmission is initiated via spontaneous or action-potential evoked fusion of synaptic vesicles. At excitatory synapses, glutamatergic receptors activated by spontaneous and evoked neurotransmission are segregated. Although inhibitory synapses also transmit signals spontaneously or in response to action potentials, they differ from excitatory synapses in both structure and function. Therefore, we hypothesized that inhibitory synapses may have different organizing principles. We report picrotoxin, a GABAAR antagonist, blocks neurotransmission in a use-dependent manner at rat hippocampal synapses and therefore can be used to interrogate synaptic properties. Using this tool, we uncovered partial segregation of inhibitory spontaneous and evoked neurotransmission. We found up to 40% of the evoked response is mediated through GABAARs which are only activated by evoked neurotransmission. These data indicate GABAergic spontaneous and evoked neurotransmission processes are partially non-overlapping, suggesting they may serve divergent roles in neuronal signaling.

Summary: Brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), regulate long-term potentiation (LTP) in the hippocampus, although the sites of BDNF-TrkB receptors in this process are controversial. We used a viral-mediated approach to delete BDNF or TrkB specifically in CA1 and CA3 regions of the Schaffer collateral pathway. Deletion of BDNF in CA3 or CA1 revealed that presynaptic BDNF is involved in LTP induction, while postsynaptic BDNF contributes to LTP maintenance. Similarly, loss of presynaptic or postsynaptic TrkB receptors leads to distinct LTP deficits, with presynaptic TrkB required to maintain LTP, while postsynaptic TrkB is essential for LTP formation. In addition, loss of TrkB in CA3 significantly diminishes release probability, uncovering a role for presynaptic TrkB receptors in basal neurotransmission. Taken together, this direct comparison of presynaptic and postsynaptic BDNF-TrkB reveals insight into BDNF release and TrkB activation sites in hippocampal LTP. : Lin et al. directly compare a role for presynaptic and postsynaptic BDNF and TrkB receptors in hippocampal LTP. They find that LTP induction is mediated by anterograde BDNF-TrkB signaling, while both anterograde and retrograde BDNF-TrkB signaling persists presynaptically and postsynaptically for LTP maintenance. Keywords: BDNF, TrkB, hippocampus, synaptic plasticity, LTP, presynaptic, postsynaptic

Abstract Neurotransmission and secretion of hormones involve a sequence of protein/lipid interactions with lipid turnover impacting on vesicle trafficking and ultimately fusion of secretory vesicles with the plasma membrane. We previously demonstrated that sphingosine, a sphingolipid metabolite, promotes formation of the SNARE complex required for membrane fusion and also increases the rate of exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and in hippocampal neurons. Recently a fungi-derived sphingosine homologue, FTY720, has been approved for treatment of multiple sclerosis. In its non-phosphorylated form FTY720 accumulates in the central nervous system, reaching high levels which could affect neuronal function. Considering close structural similarity of sphingosine and FTY720 we investigated whether FTY720 has an effect on regulated exocytosis. Our data demonstrate that FTY720 can activate vesicular synaptobrevin for SNARE complex formation and enhance exocytosis in neuroendocrine cells and neurons.

Emerging evidence suggests that spontaneous neurotransmitter release contributes to the maintenance of synaptic efficacy. Here the authors selectively reduce spontaneous glutamatergic transmission while leaving the stimulus-evoked responses intact and show that this leads to homeostatic scaling at the postsynaptic side in cultured neurons and alters synaptic plasticity in acute brain slices.

Homeostatic scaling allows neurons to maintain stable activity patterns by globally altering their synaptic strength in response to changing activity levels. Suppression of activity by the blocking of action potentials increases synaptic strength through an upregulation of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Although this synaptic upscaling was shown to require transcription, the molecular nature of the intrinsic transcription program underlying this process and its functional significance have been unclear. Using RNA-seq, we identified 73 genes that were specifically upregulated in response to activity suppression. In particular, Neuronal pentraxin-1 (Nptx1) increased within 6 hr of activity blockade, and knockdown of this gene blocked the increase in synaptic strength. Nptx1 induction is mediated by calcium influx through the T-type voltage-gated calcium channel, as well as two transcription factors, SRF and ELK1. Altogether, these results uncover a transcriptional program that specifically operates when neuronal activity is suppressed to globally coordinate the increase in synaptic strength.

Coupling of synaptic vesicle fusion and retrieval constitutes a core mechanism ensuring maintenance of presynaptic function. Recent studies using fast-freeze electron microscopy and capacitance measurements reported an ultrafast mode of endocytosis operating at physiological temperatures. Here, using rat hippocampal neurons, we optically monitored single synaptic vesicle endocytosis with high time resolution using the vesicular glutamate transporter, synaptophysin and the V0a1 subunit of the vacuolar ATPase as probes. In this setting, we could distinguish three components of retrieval operating at ultrafast (~150–250 ms, ~20% of events), fast (~5–12 s, ~40% of events) and ultraslow speeds (>20 s, ~40% of events). While increasing Ca2+ slowed the fast events, increasing temperature accelerated their time course. In contrast, the kinetics of ultrafast events were only mildly affected by these manipulations. These results suggest that synaptic vesicle proteins can be retrieved with ultrafast kinetics, although a majority of evoked fusion events are coupled to slower retrieval mechanisms.

Synaptic vesicle recycling is essential for sustained and reliable neurotransmission. A key component of synaptic vesicle recycling is the synaptic vesicle biogenesis process that is observed in synapses and that maintains the molecular identity of synaptic vesicles. However, the mechanisms by which synaptic vesicles are retrieved and reconstituted after fusion remain unclear. The complex molecular composition of synaptic vesicles renders their rapid biogenesis a daunting task. Therefore, in this context, kiss-and-run type transient fusion of synaptic vesicles with the plasma membrane without loss of their membrane composition and molecular identity remains a viable hypothesis that can account for the fidelity of the synaptic vesicle cycle. In this article, we discuss the biological implications of this problem as well as its possible molecular solutions.

Lithium is widely used as a treatment for Bipolar Disorder although the molecular mechanisms that underlie its therapeutic effects are under debate. In this study, we show brain-derived neurotrophic factor (BDNF) is required for the antimanic-like effects of lithium but not the antidepressant-like effects in mice. We performed whole cell patch clamp recordings of hippocampal neurons to determine the impact of lithium on synaptic transmission that may underlie the behavioral effects. Lithium produced a significant decrease in α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated miniature excitatory postsynaptic current (mEPSC) amplitudes due to postsynaptic homeostatic plasticity that was dependent on BDNF and its receptor tropomyosin receptor kinase B (TrkB). The decrease in AMPAR function was due to reduced surface expression of GluA1 subunits through dynamin-dependent endocytosis. Collectively, these findings demonstrate a requirement for BDNF in the antimanic action of lithium and identify enhanced dynamin-dependent endocytosis of AMPARs as a potential mechanism underlying the therapeutic effects of lithium.

Recent studies indicate that within individual synapses spontaneous and evoked release processes are segregated and regulated independently. In the hippocampus, earlier electrophysiological recordings suggested that spontaneous and evoked glutamate release can activate separate groups of postsynaptic NMDA receptors with limited overlap. However, it is still unclear how this separation of NMDA receptors is distributed across individual synapses. In a previous paper (Reese and Kavalali, 2015) we showed that NMDA receptor mediated spontaneous transmission signals to the postsynaptic protein translation machinery through Ca2+-induced Ca2+ release. Here, we show that in rat hippocampal neurons although spontaneous and evoked glutamate release driven NMDA receptor mediated Ca2+ transients often occur at the same synapse, these two signals do not show significant correlation or cross talk.

Spontaneous glutamate release-driven NMDA receptor activity exerts a strong influence on synaptic homeostasis. However, the properties of Ca2+ signals that mediate this effect remain unclear. Here, using hippocampal neurons labeled with the fluorescent Ca2+ probes Fluo-4 or GCAMP5, we visualized action potential-independent Ca2+ transients in dendritic regions adjacent to fluorescently labeled presynaptic boutons in physiological levels of extracellular Mg2+. These Ca2+ transients required NMDA receptor activity, and their propensity correlated with acute or genetically induced changes in spontaneous neurotransmitter release. In contrast, they were insensitive to blockers of AMPA receptors, L-type voltage-gated Ca2+ channels, or group I mGluRs. However, inhibition of Ca2+-induced Ca2+ release suppressed these transients and elicited synaptic scaling, a process which required protein translation and eukaryotic elongation factor-2 kinase activity. These results support a critical role for Ca2+-induced Ca2+ release in amplifying NMDA receptor-driven Ca2+ signals at rest for the maintenance of synaptic homeostasis.

The neuronal ceroid lipofuscinoses are a newly-recognized group of lysosomal storage disorders in which neurodegeneration predominates. The pathophysiological basis for this is unknown. In the current paper, we sought to determine whether neurons that lack the enzyme responsible for the infantile form of neuronal ceroid lipofuscinosis (INCL) display abnormalities in culture that could be related to the clinical disorder. Electrophysiological and fluorescent dye studies were performed using cortical neuronal cultures established from postnatal day 2 palmitoyl-protein thioesterase-1 (Ppt1) knockout mice. We found a 30% reduction in synaptic vesicle number per bouton that was progressive with time in culture as well as an elevation in lysosomal pH, whereas a number of passive and active membrane properties of the neurons were normal. The reduction in vesicle pool size was also reflected in a decrease in the frequency of miniature synaptic currents. The progressive and gradual decline in vesicle numbers and miniature event frequency we observed here may be an early indicator of synapse degeneration, in keeping with observations during competitive stimulation at the neuromuscular junction or age-related synapse elimination recently reported by others. PPT1 did not colocalize with synaptic vesicle or synapse markers, suggesting that lysosomal dysfunction leads indirectly to the synaptic abnormalities. We conclude that from an early age, neurons deficient in PPT1 enzyme activity display intrinsically abnormal properties that could potentially explain key features of the clinical disease, such as myoclonus and seizures.

Presynaptic terminals release neurotransmitters spontaneously in a manner that can be regulated by Ca2+. However, the mechanisms underlying this regulation are poorly understood because the inherent stochasticity and low probability of spontaneous fusion events has curtailed their visualization at individual release sites. Here, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single spontaneous fusion events and found that they are retrieved extremely rapidly with faster re-acidification kinetics than their action potential-evoked counterparts. These fusion events were coupled to postsynaptic NMDA receptor-driven Ca2+ signals, and at elevated Ca2+ concentrations there was an increase in the number of vesicles that would undergo fusion. Furthermore, spontaneous vesicle fusion propensity in a synapse was Ca2+-dependent but regulated autonomously: independent of evoked fusion probability at the same synapse. Taken together, these results expand classical quantal analysis to incorporate endocytic and exocytic phases of single fusion events and uncover autonomous regulation of spontaneous fusion.

MEF2 (A-D) transcription factors govern development, differentiation and maintenance of various cell types including neurons. The role of MEF2 isoforms in the brain has been studied using in vitro manipulations with only MEF2C examined in vivo. In order to understand specific as well as redundant roles of the MEF2 isoforms, we generated brain-specific deletion of MEF2A and found that Mef2aKO mice show normal behavior in a range of paradigms including learning and memory. We next generated Mef2a and Mef2d brain-specific double KO (Mef2a/dDKO) mice and observed deficits in motor coordination and enhanced hippocampal short-term synaptic plasticity, however there were no alterations in learning and memory, Schaffer collateral pathway long-term potentiation, or the number of dendritic spines. Since previous work has established a critical role for MEF2C in hippocampal plasticity, we generated a Mef2a, Mef2c and Mef2d brain-specific triple KO (Mef2a/c/dTKO). Mef2a/c/d TKO mice have early postnatal lethality with increased neuronal apoptosis, indicative of a redundant role for the MEF2 factors in neuronal survival. We examined synaptic plasticity in the intact neurons in the Mef2a/c/d TKO mice and found significant impairments in short-term synaptic plasticity suggesting that MEF2C is the major isoform involved in hippocampal synaptic function. Collectively, these data highlight the key in vivo role of MEF2C isoform in the brain and suggest that MEF2A and MEF2D have only subtle roles in regulating hippocampal synaptic function.

Muscle contraction depends on release of Ca(2+) from the sarcoplasmic reticulum (SR) and reuptake by the Ca(2+)adenosine triphosphatase SERCA. We discovered a putative muscle-specific long noncoding RNA that encodes a peptide of 34 amino acids and that we named dwarf open reading frame (DWORF). DWORF localizes to the SR membrane, where it enhances SERCA activity by displacing the SERCA inhibitors, phospholamban, sarcolipin, and myoregulin. In mice, overexpression of DWORF in cardiomyocytes increases peak Ca(2+) transient amplitude and SR Ca(2+) load while reducing the time constant of cytosolic Ca(2+) decay during each cycle of contraction-relaxation. Conversely, slow skeletal muscle lacking DWORF exhibits delayed Ca(2+) clearance and relaxation and reduced SERCA activity. DWORF is the only endogenous peptide known to activate the SERCA pump by physical interaction and provides a means for enhancing muscle contractility.

Recent studies suggest an exquisite structural nano-organization within single synapses, where sites of evoked fusion - marked by clustering of synaptic vesicles, active zone proteins and voltage-gated calcium channels - are directly juxtaposed to postsynaptic receptor clusters within nanocolumns. This direct nanometer scale alignment between presynaptic fusion apparatus and postsynaptic receptors is thought to ensure the fidelity of synaptic signaling and possibly allow multiple distinct signals to occur without interference from each other within a single active zone. The functional specificity of this organization is made possible by the inherent nano-organization of calcium signals, where all the different calcium sources such as voltage-gated calcium channels, intracellular stores and store-operated calcium entry have dedicated local targets within their nanodomain to ensure precision of action. Here, we discuss synaptic nano-organization from the perspective of calcium signals, where some of the principal findings from early work in the 1980s continue to inspire current studies that exploit new genetic tools and super-resolution imaging technologies. [ABSTRACT FROM AUTHOR]