Liangyi Chen

Functional stoichiometry of the unitary calcium-release-activated calcium channel

Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca2+ release-activated Ca2+ (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orai1 tandem multimers and a fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orai1 molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orai1 subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca2+ stores were depleted, the channels clustered in aggregates containing ≈1,300 channels, amplifying the local Ca2+ entry.

Mapping the Interacting Domains of STIM1 and Orai1 in Ca2+ Release-activated Ca2+ Channel Activation

STIM1 and Orai1 are essential components of Ca2+ release-activated Ca2+ channels (CRACs). After endoplasmic reticulum Ca2+ store depletion, STIM1 in the endoplasmic reticulum aggregates and migrates toward the cell periphery to co-localize with Orai1 on the opposing plasma membrane. Little is known about the roles of different domains of STIM1 and Orai1 in protein clustering, migration, interaction, and, ultimately, opening CRAC channels. Here we demonstrate that the coiled-coil domain in the C terminus of STIM1 is crucial for its aggregation. Amino acids 425–671 of STIM1, which contain a serine-proline-rich region, are important for the correct targeting of the STIM1 cluster to the cell periphery after calcium store depletion. The polycationic region in the C-terminal tail of STIM1 also helps STIM1 targeting but is not essential for CRAC channel activation. The cytoplasmic C terminus but not the N terminus of Orai1 is required for its interaction with STIM1. We further identify a highly conserved region in the N terminus of Orai1 (amino acids 74–90) that is necessary for CRAC channel opening. Finally, we show that the transmembrane domain of Orai1 participates in Orai1-Orai1 interactions.

Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits.

The Ca2+ release-activated Ca2+ (CRAC) channel pore is formed by Orai1 and gated by STIM1 after intracellular Ca2+ store depletion. To resolve how many STIM1 molecules are required to open a CRAC channel, we fused different numbers of Orai1 subunits with functional two-tandem cytoplasmic domains of STIM1 (residues 336-485, designated as S domain). Whole-cell patch clamp recordings of these chimeric molecules revealed that CRAC current reached maximum at a stoichiometry of four Orai1 and eight S domains. Further experiments indicate that two-tandem S domains specifically interact with the C-terminus of one Orai1 subunit, and CRAC current can be gradually increased as more Orai1 subunits can interact with S domains or STIM1 proteins. Our data suggest that maximal opening of one CRAC channel requires eight STIM1 molecules, and support a model that the CRAC channel activation is not in an “all-or-none” fashion but undergoes a graded process via binding of different numbers of STIM1.

Direct Quantification of Fusion Rate Reveals a Distal Role for AS160 in Insulin-stimulated Fusion of GLUT4 Storage Vesicles

Insulin-stimulated GLUT4 translocation to the plasma membrane constitutes a key process for blood glucose control. However, convenient and robust assays to monitor this dynamic process in real time are lacking, which hinders current progress toward elucidation of the underlying molecular events as well as screens for drugs targeting this particular pathway. Here, we have developed a novel dual colored probe to monitor the translocation process of GLUT4 based on dual color fluorescence measurement. We demonstrate that this probe is more than an order of magnitude more sensitive than the current technology for detecting fusion events from single GLUT4 storage vesicles (GSVs). A small fraction of fusion events were found to be of the “kiss-and-run” type. For the first time, we show that insulin stimulation evokes a ∼40-fold increase in the fusion of GSVs in 3T3-L1 adipocytes, compared with basal conditions. The probe can also be used to monitor the prefusion behavior of GSVs. By quantifying both the docking and fusion rates simultaneously, we demonstrate a proportional inhibition in both docking and fusion of GSVs by a dominant negative mutant of AS160, indicating a role for AS160 in the docking of GSVs but not in the regulation of GSV fusion after docking.

Calcium Transport Mechanisms of PC12 Cells

Many studies of Ca2+ signaling use PC12 cells, yet the balance of Ca2+ clearance mechanisms in these cells is unknown. We used pharmacological inhibition of Ca2+ transporters to characterize Ca2+ clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), plasma membrane Ca2+ ATPase (PMCA), and Na+/Ca2+ exchanger (NCX) account for almost all Ca2+ clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of Ca2+ clearance possessing pharmacological characteristics consistent with secretory pathway Ca2+ ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall Ca2+ transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released Ca2+ instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed Ca2+ clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous Ca2+ binding ratio of PC12 cell cytoplasm, which we measured to be 268 ± 85. Our results indicate that Ca2+ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different Ca2+ transport mechanisms.

Contributions of Intracellular Compartments to Calcium Dynamics: Implicating an Acidic Store

Many cells show a plateau of elevated cytosolic Ca2+ after a long depolarization, suggesting delayed Ca2+ release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic β‐cells show a thapsigargin‐sensitive plateau (‘hump’) of Ca2+ after a 30 s depolarization but not after a 10 s depolarization. Surprisingly, this hump depends primarily on compartments other than the mitochondria or ER. It is reduced by only 22% upon blocking mitochondrial Na+–Ca2+ exchange and by only 18% upon blocking ryanodine or IP3 receptors together. Further, the time course of ER Ca2+ measured by a targeted cameleon does not depend on the duration of depolarizations. Instead, the hump is reduced 35% by treatments with the dipeptide glycylphenylalanine β‐napthylamide, a tool often used to lyse lysosomes. We show that this dipeptide does not disturb ER functions, but it lyses acidic compartments and releases Ca2+ into the cytosol. Moreover, it induces leaks in and possibly lyses insulin granules and stops mobilization of secretory granules to the readily releasable pool in β‐cells. We conclude that the dipeptide compromises dense‐core secretory granules and that these granules comprise an acidic calcium store in β‐cells whose loading and/or release is sensitive to thapsigargin and which releases Ca2+ after cytosolic Ca2+ elevation.

Orai-STIM–mediated Ca2+ release from secretory granules revealed by a targeted Ca2+ and pH probe

Significance Elevated cytoplasmic calcium modulates many cellular responses. Two Ca2+ sources feed most cytoplasmic Ca2+ elevations, the extracellular space and the endoplasmic reticulum. We found that secretory granules represent another Ca2+ source that releases Ca2+ in a physiologically interesting way. We developed a targeted probe for monitoring Ca2+ in secretory granules. It revealed a unique receptor-stimulated mechanism of Ca2+ release from secretory granules apparently involving “store-operated” Orai channels in the secretory granule membrane. The channels open when reticular stores are depleted. Such Ca2+ release from granules might elevate local Ca2+ concentrations and aid in the refilling of other cytoplasmic Ca2+ stores. Secretory granules (SGs) sequester significant calcium. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty of measuring SG calcium directly in living cells. We adapted the Förster resonance energy transfer-based D1-endoplasmic reticulum (ER) probe to develop a unique probe (D1-SG) to measure calcium and pH in secretory granules. It significantly localizes to SGs and reports resting free Ca2+ of 69 ± 15 μM and a pH of 5.8. Application of extracellular ATP to activate P2Y receptors resulted in a slow monotonic decrease in SG Ca2+ temporally correlated with the occurrence of store-operated calcium entry (SOCE). Further investigation revealed a unique receptor-mediated mechanism of calcium release from SGs that involves SG store-operated Orai channels activated by their regulator stromal interaction molecule 1 (STIM1) on the ER. SG Ca2+ release is completely antagonized by a SOCE antagonist, by switching to Ca2+-free medium, and by overexpression of a dominant-negative Orai1(E106A). Overexpression of the CRAC activation domain (CAD) of STIM1 resulted in a decrease of resting SG Ca2+ by ∼75% and completely abolished the ATP-mediated release of Ca2+ from SGs. Overexpression of a dominant-negative CAD construct (CAD-A376K) induced no significant changes in SG Ca2+. Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels and that during SG Ca2+ release, colocalization between SGs and STIM1 increases. We propose Orai channel opening on SG membranes as a potential mode of calcium release from SGs that may serve to raise local cytoplasmic calcium concentrations and aid in refilling intracellular calcium stores of the ER and exocytosis.

Ca2+ Triggers a Novel Clathrin-Independent but Actin-Dependent Fast Endocytosis in Pancreatic Beta Cells

The existence of clathrin‐independent recycling of secretory vesicles has been controversial. By combining patch‐clamp capacitance recording, optical methods and specific molecular interventions, we dissect two types of mechanistically different endocytosis in pancreatic β cells, both of which require GTP and dynamin. The fast one is a novel clathrin‐independent but actin‐dependent endocytosis that is triggered by high cytoplasmic Ca2+ concentration ([Ca2+]i). Large fluorescent dextran (10 nm in diameter) was able to be internalized by this pathway, indicating that it was not likely to be ‘kiss and run’. The slow endocytosis is a clathrin‐dependent process in which actin plays a complementary role. For the first time, we show that the rate constants for both types of endocytosis exhibit supralinear dependence on increase in [Ca2+]i. Compared with the slow endocytosis, higher [Ca2+]i level was required to fully accelerate the fast one, indicative of distinct Ca2+ sensors for different endocytosis. In the end, we show that physiologically relevant stimulation induces clathrin‐independent endocytosis in intact β cells, implying that it may contribute to the normal recycling of secretory vesicles in vivo.

Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice

Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscopes robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

Large-field high-resolution two-photon digital scanned light-sheet microscopy.

Recent advent of light-sheet fluorescent microscopy (LSFM) has revolutionized three-dimensional biological imaging with high temporal resolution and minimal photodamage, enabling long-term fluorescence imaging of tissues and small organisms [1-2]. By combining two-photon fluorescence excitation with LSFM, Truong et al. [3] have created a two-photon digital scanned light-sheet microscope (2P-DSLM), allowing for deep-tissue imaging of highly scattering Drosophila embryos and fast beating hearts of zebrafish. Similar to classical LSFM configurations, a 2P-DSLM uses a low numerical aperture (NA < 0.1) to achieve a long and homogenous illumination. This leads to a thick light sheet and thus reduces axial resolution and image contrast. On the other hand, Betzig and colleagues have used a Bessel beam to generate thin single-photon light sheet that yields superb axial resolution [4]. However, the field of view and the penetration depth are limited in such system. To achieve high axial resolution (thin light sheet) and large field of view simultaneously, here we have developed a novel two-photon three-axis digital scanned light-sheet microscope (2P3A-DSLM) based on ultrafast axial scanning of illumination focal spot with a tunable acoustic gradient (TAG) index device. This new technique provides a sub-micron axial resolution, a large field of view of 170 × 170 μm 2 in deep tissues, and a high temporal resolution limited only by the detection camera. In vivo imaging with 2P3A-DSLM resolved subcellular structures and dynamic processes in small organisms. A schematic illustration and configuration of the 2P3A-DSLM is shown in Figure 1A. A TAG lens uses acoustic wave to radially excite a fluid-filled cylindrical cavity and produce continuous changes in refrac-tive power that enables rapid change of its axial focal plane within 10 μs [5-6]. Here we used the TAG lens to rapidly translate the focal spot of a femtosecond laser (140 fs, repetition rate, 80 MHz, Chameleon Version II, Coherent) along the optics axis (x-direction) in front of the illumination objective (40× NA 0.8, Nikon). A gal-vo scanning mirror (GSM) was used to scan the laser beam vertically to the axis of the illumination objective (y-direction). By driving the TAG lens to refocus at ~450 kHz and the GSM at 1 kHz, we were able to create an ultra-thin scanned 2P light sheet within one millisecond. The size of the scanned light sheet can be modulated by adjusting the modulation intensity of the TAG (x-direction) and scanning amplitude of the galvo mirrors (y-direction) (Figure 1B-1D). During the fast …