Protocol to generate fast-dissociating recombinant antibody fragments for multiplexed super-resolution microscopy.

Multiplexed high-density label super-resolution microscopy image reconstruction by integrating exchangeable single-molecule localization (IRIS) enables elucidating fine structures and molecular distribution in cells and tissues. However, fast-dissociating binders are required for individual targets. Here, we present a protocol for generating antibody-based IRIS probes from existing antibody sequences. We describe steps for retrieving antibody sequences from databases. We then detail the construction, purification, and evaluation of recombinant probes after site-directed mutagenesis at the base of complementarity-determining region loops. The protocol accelerates dissociation rates without compromising the binding specificity. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.

Engineered fast-dissociating antibody fragments for multiplexed super-resolution microscopy.

Image reconstruction by integrating exchangeable single-molecule localization (IRIS) achieves multiplexed super-resolution imaging by high-density labeling with fast exchangeable fluorescent probes. However, previous methods to develop probes for individual targets required a great amount of time and effort. Here, we introduce a method for generating recombinant IRIS probes with a new mutagenesis strategy that can be widely applied to existing antibody sequences. Several conserved tyrosine residues at the base of complementarity-determining regions were identified as candidate sites for site-directed mutagenesis. With a high probability, mutations at candidate sites accelerated the off rate of recombinant antibody-based probes without compromising specific binding. We were able to develop IRIS probes from five monoclonal antibodies and three single-domain antibodies. We demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections with high binding density. It is now practically feasible to generate fast-dissociating fluorescent probes for multitarget super-resolution imaging.

Tuberous Sclerosis Complex (TSC) Inactivation Increases Neuronal Network Activity by Enhancing Ca2+ Influx via L-Type Ca2+ Channels.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder characterized by hamartomas in various organs, such as the brain, lungs, and kidneys. Epilepsy, along with autism and intellectual disability, is one of the neurologic impairments associated with TSC that has an intimate relationship with developmental outcomes and quality of life. Sustained activation of the mammalian target of rapamycin (mTOR) via TSC1 or TSC2 mutations is known to be involved in the onset of epilepsy in TSC. However, the mechanism by which mTOR causes seizures remains unknown. In this study, we showed that, human induced pluripotent stem cell-derived TSC2-deficient (TSC2-/-) neurons exhibited elevated neuronal activity with highly synchronized Ca2+ spikes. Notably, TSC2-/- neurons presented enhanced Ca2+ influx via L-type Ca2+ channels (LTCCs), which contributed to the abnormal neurite extension and sustained activation of cAMP response element binding protein (CREB), a critical mediator of synaptic plasticity. Expression of Cav1.3, a subtype of LTCCs, was increased in TSC2-/- neurons, but long-term rapamycin treatment suppressed this increase and reversed the altered neuronal activity and neurite extensions. Thus, we identified Cav1.3 LTCC as a critical downstream component of TSC-mTOR signaling that would trigger enhanced neuronal network activity of TSC2-/- neurons. We suggest that LTCCs could be potential novel targets for the treatment of epilepsy in TSC.SIGNIFICANCE STATEMENT There is a close relationship between elevated mammalian target of rapamycin (mTOR) activity and epilepsy in tuberous sclerosis complex (TSC). However, the underlying mechanism by which mTOR causes epilepsy remains unknown. In this study, using human TSC2-/- neurons, we identified elevated Ca2+ influx via L-type Ca2+ channels as a critical downstream component of TSC-mTOR signaling and a potential cause of both elevated neuronal activity and neurite extension in TSC2-/- neurons. Our findings demonstrate a previously unrecognized connection between sustained mTOR activation and elevated Ca2+ signaling via L-type Ca2+ channels in human TSC neurons, which could cause epilepsy in TSC.

Dual-FRET imaging of IP3 and Ca2+ revealed Ca2+-induced IP3 production maintains long lasting Ca2+ oscillations in fertilized mouse eggs.

In most species, fertilization induces Ca2+ transients in the egg. In mammals, the Ca2+ rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP3 generated by PLCζ induces Ca2+ release from the intracellular Ca2+ store through IP3 receptor, termed IP3-induced Ca2+ release. Here, we developed new fluorescent IP3 sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 µM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca2+ and IP3, using IRIS-2s as the IP3 sensor, we developed a new single fluorophore Ca2+ sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP3 concentration ([IP3]) starts to increase before the onset of the first Ca2+ wave. [IP3] stayed at the elevated level with small peaks followed after Ca2+ spikes through Ca2+ oscillations. We detected delays in the peak of [IP3] compared to the peak of each Ca2+ spike, suggesting that Ca2+-induced regenerative IP3 production through PLC produces small [IP3] rises to maintain [IP3] over the basal level, which results in long lasting Ca2+ oscillations in fertilized eggs.

A novel multi lines analysis tool of Ca2+ dynamics reveals the nonuniformity of Ca2+ propagation.

Extracellular stimuli evoke a robust increase in the concentration of intracellular Ca2+ ([Ca2+]c) throughout the cell to trigger various cellular responses, such as gene expression and apoptosis. This robust expansion of [Ca2+]c is called Ca2+ propagation. To date, it is thought that intracellular second messengers, such as inositol 1,4,5-trisphosphate (IP3) and intracellular Ca2+, and clusters of IP3 receptors (IP3Rs) regulate Ca2+ propagation. However, little is known about how the elevation in the [Ca2+]c spreads throughout the cell, especially in non-polar cell, including HeLa cell. In this study, we developed a novel multi lines analysis tool. This tool revealed that the velocity of Ca2+ propagation was inconstant throughout cell and local concentration of intracellular Ca2+ did not contribute to the velocity of Ca2+ propagation. Our results suggest that intracellular Ca2+ propagation is not merely the result of diffusion of intracellular Ca2+, and that, on the contrary, intracellular Ca2+ propagation seems to be regulated by more complicated processes.

A novel gain-of-function mutation in the ITPR1 suppressor domain causes spinocerebellar ataxia with altered Ca2+ signal patterns.

We report three affected members, a mother and her two children, of a non-consanguineous Irish family who presented with a suspected autosomal dominant spinocerebellar ataxia characterized by early motor delay, poor coordination, gait ataxia, and dysarthria. Whole exome sequencing identified a novel missense variant (c.106C>T; p.[Arg36Cys]) in the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor gene (ITPR1) as the cause of the disorder, resulting in a molecular diagnosis of spinocerebellar ataxia type 29. In the absence of grandparental DNA, microsatellite genotyping of healthy family members was used to confirm the de novo status of the ITPR1 variant in the affected mother, which supported pathogenicity. The Arg36Cys variant exhibited a significantly higher IP3-binding affinity than wild-type (WT) ITPR1 and drastically changed the property of the intracellular Ca2+ signal from a transient to a sigmoidal pattern, supporting a gain-of-function disease mechanism. To date, ITPR1 mutation has been associated with a loss-of-function effect, likely due to reduced Ca2+ release. This is the first gain-of-function mechanism to be associated with ITPR1-related SCA29, providing novel insights into how enhanced Ca2+ release can also contribute to the pathogenesis of this neurological disorder.

Development of a convenient and supersensitive high-throughput screening system for genetically encoded fluorescent probes of small molecules using a confocal microscope.

Monitoring the dynamic patterns of intracellular signaling molecules, such as inositol 1,4,5-trisphosphate (IP3) and Ca2+, that control many diverse cellular processes, provides us significant information to understand the regulatory mechanism of cellular functions. For searching more sensitive and higher dynamic range probes for signaling molecules, convenient and supersensitive high throughput screening systems are required. Here we show the optimal "in Escherichia coli (E. coli) colony" screening method based on the twin-arginine translocase (Tat) pathway and introduce a novel application of a confocal microscope as a supersensitive detection system to measure changes in the fluorescence intensity of fluorescent probes in E. coli grown on an agar plate. To verify the performance of the novel detection system, we compared the changes detected in the fluorescent intensity of genetically encoded Ca2+ indicator after Ca2+ exposure to two kinds of conventional fluorescence detection systems (luminescent image analyzer and fluorescence stereomicroscope). The rate of fluorescence change between Ca2+ binding and unbinding detected by novel supersensitive detection system was almost double than those measured by conventional detection systems. We also confirmed that the Tat pathway-based screening method is applicable to the development of genetically encoded probes for IP3. Our convenient and supersensitive screening system improves the speed of developing florescent probes for small molecules.

Probes for manipulating and monitoring IP3.

Inositol 1,4,5-trisphosphate (IP3) is an important second messenger produced via G-protein-coupled receptor- or receptor tyrosine kinase-mediated pathways. IP3 levels induce Ca2+ release from the endoplasmic reticulum (ER) via IP3 receptor (IP3R) located in the ER membrane. The resultant spatiotemporal pattern of Ca2+ signals regulates diverse cellular functions, including fertilization, gene expression, synaptic plasticity, and cell death. Therefore, monitoring and manipulating IP3 levels is important to elucidate not only the functions of IP3-mediated pathways but also the encoding mechanism of IP3R as a converter of intracellular signals from IP3 to Ca2+.

Redox-assisted regulation of Ca2+ homeostasis in the endoplasmic reticulum by disulfide reductase ERdj5.

Calcium ion (Ca2+) is an important second messenger that regulates numerous cellular functions. Intracellular Ca2+ concentration ([Ca2+]i) is strictly controlled by Ca2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca2+ from the cytosol into the ER in an ATPase activity-dependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond. Notably, ERdj5 activated SERCA2b at a lower ER luminal [Ca2+] ([Ca2+]ER), whereas a higher [Ca2+]ER induced ERdj5 to form oligomers that were no longer able to interact with the pump, suggesting [Ca2+]ER-dependent regulation. Binding Ig protein, an ER-resident molecular chaperone, exerted a regulatory role in the oligomerization by binding to the J domain of ERdj5. These results identify ERdj5 as one of the master regulators of ER calcium homeostasis and thus shed light on the importance of cross talk among redox, Ca2+, and protein homeostasis in the ER.

Bidirectional Control of Synaptic GABAAR Clustering by Glutamate and Calcium.

GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABAA receptor (GABAAR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABAAR clustering and GABAergic transmission. This GABAAR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses.

Apoptosis induction-related cytosolic calcium responses revealed by the dual FRET imaging of calcium signals and caspase-3 activation in a single cell.

Stimulus-induced changes in the intracellular Ca(2+) concentration control cell fate decision, including apoptosis. However, the precise patterns of the cytosolic Ca(2+) signals that are associated with apoptotic induction remain unknown. We have developed a novel genetically encoded sensor of activated caspase-3 that can be applied in combination with a genetically encoded sensor of the Ca(2+) concentration and have established a dual imaging system that enables the imaging of both cytosolic Ca(2+) signals and caspase-3 activation, which is an indicator of apoptosis, in the same cell. Using this system, we identified differences in the cytosolic Ca(2+) signals of apoptotic and surviving DT40 B lymphocytes after B cell receptor (BCR) stimulation. In surviving cells, BCR stimulation evoked larger initial Ca(2+) spikes followed by a larger sustained elevation of the Ca(2+) concentration than those in apoptotic cells; BCR stimulation also resulted in repetitive transient Ca(2+) spikes, which were mediated by the influx of Ca(2+) from the extracellular space. Our results indicate that the observation of both Ca(2+) signals and cells fate in same cell is crucial to gain an accurate understanding of the function of intracellular Ca(2+) signals in apoptotic induction.

Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator.

Monitoring the pattern of intracellular Ca(2+) signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca(2+) indicators (GECIs) are used for monitoring intracellular Ca(2+) changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca(2+) signals using GECIs. We here compared the Ca(2+) signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca(2+) responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca(2+) signals.

Receptor-selective diffusion barrier enhances sensitivity of astrocytic processes to metabotropic glutamate receptor stimulation.

Metabotropic glutamate receptor (mGluR)-dependent calcium ion (Ca²+) signaling in astrocytic processes regulates synaptic transmission and local blood flow essential for brain function. However, because of difficulties in imaging astrocytic processes, the subcellular spatial organization of mGluR-dependent Ca²+ signaling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca²+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca²+ responses. Immunocytochemistry indicated that these polarized Ca²+ responses could be attributed to increased density of surface mGluR5 on processes relative to the soma. Single-particle tracking of surface mGluR5 dynamics revealed a membrane barrier that blocked the movement of mGluR5 between the processes and the soma. Overexpression of mGluR or expression of its carboxyl terminus enabled diffusion of mGluR5 between the soma and the processes, disrupting the polarization of mGluR5 and of mGluR-dependent Ca²+ signaling. Together, our results demonstrate an mGluR5-selective diffusion barrier between processes and soma that compartmentalized mGluR Ca²+ signaling in astrocytes and may allow control of synaptic and vascular activity in specific subcellular domains.

Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular Ca(2+) release channel, and its opening is controlled by IP(3) and Ca(2+). A single IP(3) binding site and multiple Ca(2+) binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca(2+) is unknown. In this study, we visualized conformational changes in IP(3)R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP(3) and Ca(2+) have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca(2+) dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca(2+) alone from the FRET changes evoked by both ligands together. A mutant IP(3)R containing a single amino acid substitution at K508, which is critical for IP(3) binding, did not exhibit this bell-shaped Ca(2+) dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca(2+) sensor, resulted in ∼10-fold reduction in the Ca(2+) dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP(3)R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca(2+) binding affinity, as the mechanism underlying the IP(3)-dependent regulation of the bell-shaped relationship between the IP(3)R activity and cytosolic Ca(2+).

[Detection of micro mutation in dystrophin gene of DMD female carrier].

We attempted to identify a mutation in dystrophin gene in a female patient who was suspected a Duchenne muscular dystrophy (DMD) carrier with muscle weakness of upper limbs and congestive heart failure. We examined the mutation hot spots in DMD gene, exon 3, 6, 8, 13, 17, 19, 43, 44, 45, 47, 48, 49, 50, 52, 60 by multiplex PCR which had been a diagnostic screening strategy, and detected an extra band in exon 43 product. We also detected an extra band in exon 43 products by SSCP analysis for detection of small mutations which could not be detected by multiplex PCR. As a result of sequencing a PCR product of an exon 43, we confirmed an allele having the insertion of a 2 base of AT in Intron42, which is described for the first time. Although we can not conclude that this insertion is responsible for DMD, but it may cause abnormal splicing. In carrier detection of DMD without genetic information of proband, it is difficult to detect mutations by multiplex PCR solely. Therefore, SSCP of PCR products are recommended to detect mutation in DMD carrier.