Joseph L. Dynes

Dynamics of bidirectional transport of Arc mRNA in neuronal dendrites

The mRNA for Arc (activity‐regulated cytoskeletal protein) is delivered into dendrites and localizes selectively at active synapses. Here we use a green fluorescent protein‐based labeling system and confocal microscopy to define the transport kinetics of exogenously expressed mRNA from chimaeric Arc constructs (Arc/MS2 mRNA) in the dendrites of living rat neurons in culture. Arc/MS2 mRNA assembles into particles that move independently, bidirectionally, and intermittently in a fashion indicative of transport. Transport velocities range from below 6 to 65 μm/minute, which is consistent with actin‐based and microtubule‐based transport, respectively. In general, orthograde translocations are longer than retrograde translocations. Rapidly translocating Arc/MS2 mRNA particles sometimes reverse direction and decrease velocity just before stopping, suggesting that local signals regulate Arc mRNA targeting movements. These observations identify several phases of Arc mRNA movement that serve as potential points for regulating Arc mRNA localization. J. Comp. Neurol. 500:433–447, 2007.

Arc mRNA docks precisely at the base of individual dendritic spines indicating the existence of a specialized microdomain for synapse‐specific mRNA translation

Arc (aka Arg 3.1) is induced by neural activity and learning experience. Arc mRNA is rapidly exported into dendrites where it localizes near activated synapses. By imaging green fluorescent protein (GFP)‐tagged mRNA in living neurons in culture, we show that fusion transcripts containing the Arc 3′UTR (untranslated region) localize with remarkable precision in a microdomain at the base of dendritic spines. Transcripts with the Arc 3′UTR that encode a reporter protein rather than Arc show precise localization. Localization persists in the presence of translation inhibitors, indicating that localization does not require ongoing translation. Similarly, polyribosome complexes remained stably positioned at spine bases in brain tissue treated with the translation inhibitor (puromycin) that releases ribosomes from mRNA. Single particle tracking revealed that Arc mRNA particles positioned at spine bases exhibited highly constrained submicron movements. These observations imply the existence of a microdomain at the spine base where Arc mRNA docks in association with a previously unknown mRNA‐binding structural element. J. Comp. Neurol., 520:3105–3119, 2012.

Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry

Significance Almost all cell types rely on calcium signals to maintain homeostasis and trigger specific cell responses. In eukaryotic cells, store-operated Ca2+ entry (SOCE) is one of the mechanisms used to ensure control over cytosolic Ca2+ signaling and internal Ca2+ stores. Mutations in either Orai1 or stromal interaction molecule 1 (STIM1) lead to lethal severe combined immune deficiencies. Using a morphological approach with transmission and freeze–fracture electron microscopy, we describe STIM1-Orai1 interactions and visualize the distribution of individual Orai1 channels on the cell surface. This approach confirms STIM1-Orai1 interaction at specialized endoplasmic reticulum (ER)–plasma membrane junctions following ER depletion and provides new insight on STIM1-Orai1 stoichiometry. Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze–fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)–plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze–fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic “particles”; TG treatment led to aggregation of these particles into sharply delimited “puncta” positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.

Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx

Significance Calcium ions serve as intracellular signals controlling many aspects of cell behavior. Here, we create fusions of genetically encoded calcium indicators and cell surface Orai calcium channels that report calcium influx through the channel. These fluorescent fusion proteins will allow us to see in living cells Orai signals that underlie cell functions, especially those essential for mounting an adaptive immune response. Their output is bright enough to record the intermittent openings of a single Orai channel for the first time to our knowledge. Our recordings reveal several patterns of channel activity, including oscillations. These responses help us understand Orai channels as molecular machines, and our work enables Orai calcium signals to be identified, mapped, and related to the functional systems of the body. Orai1 comprises the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel. When bound and activated by stromal interacting molecule 1 (STIM1), an endoplasmic reticulum (ER)-resident calcium sensor, Orai1 channels possess high selectivity for calcium but extremely small conductance that has precluded direct recording of single-channel currents. We have developed an approach to visualize Orai1 activity by fusing Orai1 to fluorescent, genetically encoded calcium indicators (GECIs). The GECI–Orai1 probes reveal local Ca2+ influx at STIM1–Orai1 puncta. By whole cell recording, these fusions are fully functional as CRAC channels. When GECI–Orai1 and the CRAC-activating domain (CAD) of STIM1 were coexpressed at low levels and imaged using a total internal reflectance fluorescence microscope, cells exhibited sporadic fluorescence transients the size of diffraction-limited spots and the brightness of a few activated GECI proteins. Transients typically rose rapidly and fell into two classes according to duration: briefer “flickers” lasting only a few hundred milliseconds, and longer “pulses” lasting one to several seconds. The size, intensity, trace shape, frequency, distribution, physiological characteristics, and association with CAD binding together demonstrate that GECI–Orai1 fluorescence transients correspond to single-channel Orai1 responses. Single Orai1 channels gated by CAD, and small Orai1 puncta gated by STIM1, exhibit repetitive fluctuations in single-channel output. CAD binding supports a role in open state maintenance and reveals a second phase of CAD/STIM1 binding after channel opening. These first recordings of single-channel Orai1 currents reveal unexpected dynamics, and when paired with CAD association, support multiple single-channel states.

Spinning-Spot Shadowless TIRF Microscopy.

Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing near-membrane cellular structures and processes, including imaging of local Ca2+ transients with single-channel resolution. TIRF is most commonly implemented in epi-fluorescence mode, whereby laser excitation light is introduced at a spot near the periphery of the back focal plane of a high numerical aperture objective lens. However, this approach results in an irregular illumination field, owing to interference fringes and scattering and shadowing by cellular structures. We describe a simple system to circumvent these limitations, utilizing a pair of galvanometer-driven mirrors to rapidly spin the laser spot in a circle at the back focal plane of the objective lens, so that irregularities average out during each camera exposure to produce an effectively uniform field. Computer control of the mirrors enables precise scanning at 200 Hz (5ms camera exposure times) or faster, and the scan radius can be altered on a frame-by-frame basis to achieve near-simultaneous imaging in TIRF, widefield and ‘skimming plane’ imaging modes. We demonstrate the utility of the system for dynamic recording of local inositol trisphosphate-mediated Ca2+ signals and for imaging the redistribution of STIM and Orai proteins during store-operated Ca2+ entry. We further anticipate that it will be readily applicable for numerous other near-membrane studies, especially those involving fast dynamic processes.

T cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of ‘Salsa6f’, a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca2+ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca2+ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca2+ transients (‘sparkles’) as cells migrate.

Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility

Ca2+ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here, we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca2+ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca2+ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca2+ transients: short-duration ‘sparkles’ and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance.

Local Protein Synthesis at Synapses

Abstract This chapter provides an overview of mechanisms and machinery underlying local protein synthesis at synapses. Topics include the machinery localized at synapses that mediates local protein synthesis and processing, the messenger RNAs (mRNAs) that are present in dendrites and available for translation, RNA transport in dendrites, and aspects of mRNA targeting to synapses as revealed by studies of the immediate early gene Arc.

Cell-intrinsic activation of Orai1 regulates human T cell motility

Ca2+ signaling through the store-operated Ca2+ channel, Orai1, is crucial for T cell function, but a role in regulating T cell motility in lymph nodes has not been previously reported. Tracking human T cells in immunodeficient mouse lymph nodes and in microfabricated PDMS channels, we show that inhibition of Orai1 channel activity with a dominant-negative Orai1-E106A construct increases average T cell velocities by reducing the frequency of pauses in motile T cells. Orai1-dependent motility arrest occurs spontaneously during confined motility in vitro, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, we show these spontaneous pauses during T cell motility in vitro coincide with episodes of spontaneous cytosolic Ca2+ signaling. Our results demonstrate that Orai1, activated in a cell-intrinsic manner, regulates T cell motility patterns that accompany immune surveillance.

mRNA Trafficking to Synapses and Memory Formation

This chapter reviews evidence for the theory that local translation of mRNAs at synapses plays a critical role in synaptic modifications underlying memory consolidation. We consider mechanisms of neuronal cell biology that underlie mRNA transport into dendrites, localization at synapses, and control of local mRNA translation during synaptic plasticity. The final section reviews studies of the immediate early gene Arc, which provide insights into mechanisms linking local translation and memory consolidation.