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Ultrafast transient vibrational action spectra of cryogenically cooled Re(CO)3(CH3CN)3+ ions are presented. Nonlinear spectra were collected in the time domain by monitoring the photodissociation of a weakly bound N2 messenger tag as a function of delay times and phases between a set of three infrared pulses. Frequency-resolved spectra in the carbonyl stretch region show relatively strong bleaching signals that oscillate at the difference frequency between the two observed vibrational features as a function of the pump–probe waiting time. This observation is consistent with the presence of nonlinear pathways resulting from underlying cross-peak signals between the coupled symmetric–asymmetric C≡O stretch pair. The successful demonstration of frequency-resolved ultrafast transient vibrational action spectroscopy of dilute molecular ion ensembles provides an exciting, new framework for the study of molecular dynamics in isolated, complex molecular ion systems. [ABSTRACT FROM AUTHOR]

Phenol-benzimidazole and phenol-pyridine dyad complexes have served as popular model systems for the study of proton-coupled electron transfer (PCET) kinetics in solution-phase experiments. Interpretation of measured PCET rates in terms of key structural parameters, such as the H-bond donor–acceptor distance, however, remains challenging. Herein, we report vibrational spectra in the electronic ground state for a series of phenol-benzimidazole and phenol-pyridine complexes isolated and cryogenically cooled in an ion trap. The four models studied each display highly red-shifted and broadened OH stretching transitions that arise from strong H-bonding interactions between the phenol OH group and the basic N site on benzimidazole/pyridine rings. The OH stretch transition in each model displays relatively strong absorption onsets near 2500 cm−1 with broad shoulders that extend asymmetrically to higher frequencies over hundreds of wavenumbers. In contrast, the deuterated isotopologues yield much weaker OD stretch transitions that appear symmetrically broadened. The spectral breadth and shape of the OD stretch transitions are ascribed to variations in OD stretch frequencies that arise from zero-point distributions in the proton donor–acceptor low-frequency soft mode vibration. The asymmetric structure of the OH stretch transitions is attributed to a set of combination bands between the OH stretch and a series of low-frequency H-bond soft modes. The spectra and modeling highlight the importance of OH stretch-soft mode couplings, which are thought to play important roles in PCET and proton transfer dynamics. [ABSTRACT FROM AUTHOR]

Abstract: This article describes a perfusion system for biophysical single cell experiments at the physiological temperature. Our system regulates temperature of test solutions using a small heat exchanger that includes several capillaries. Water circulating inside the heat exchanger warms or cools test solutions flowing inside the capillaries. Temperature-controlled solutions are delivered directly to a single cell(s) through a multibarreled manifold that switches solutions bathing a cell in less than 1s. This solution exchange is optimal for patch clamp, single-cell microamperometry, and microfluorometry experiments. Using this system, we demonstrate that exocytosis from pancreatic β cells and activation of TRPV1 channels are temperature sensitive. We also discuss how to measure local temperature near a single cell under investigation. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Pancreatic beta-cells maintain glucose homeostasis by their regulated Ca(2+)-dependent secretion of insulin. Several cellular mechanisms control intracellular Ca(2+) levels, but their relative significance in mouse beta-cells is not fully known. We used photometry to measure the dynamics of cytosolic Ca(2+) ([Ca(2+)](i)) clearance after brief, depolarization-induced Ca(2+) entry. Treatment with thapsigargin or cyclopiazonic acid, inhibitors of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pumps, nearly doubled the peak and slowed the decay of the depolarization-induced Ca(2+) transients. The remaining thapsigargin-insensitive decay was slowed further by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) and plasma membrane Na(+)/Ca(2+) exchanger (NCX) via alkalization of the bath solution, by adding lanthanum, or by substitution of Na(+) with Li(+). Mitochondrial Ca(2+) uptake contributed little to clearance in thapsigargin-pretreated cells. Together, the SERCA, PMCA, and NCX transport mechanisms accounted for 89 to 97% of clearance in normal solutions. We developed a quantitative model for the dynamic role of removal mechanisms over a wide range of [Ca(2+)](i). According to our model, 50 to 64% of initial Ca(2+) removal is via the SERCA pump, whereas the NCX contributes 21-30% of the extrusion at high [Ca(2+)](i), and the PMCA contributes 21-27% at low [Ca(2+)](i). [ABSTRACT FROM AUTHOR]

Pancreatic β-cells maintain glucose homeostasis by their regulated Ca[sup 2+]-dependent secretion of insulin. Several cellular mechanisms control intracellular Ca[sup 2+] levels, but their relative significance in mouse β-cells is not fully known. We used photometry to measure the dynamics of cytosolic Ca[sup 2+]([Ca[sup 2+]][sub i]) clearance after brief, depolarization-induced Ca[sup 2+] entry. Treatment with thapsigargin or cyclopiazonic acid, inhibitors of the sarco-endoplasmic reticulum Ca[sup 2+]-ATPase (SERCA) pumps, nearly doubled the peak and slowed the decay of the depolarization-induced Ca[sup 2+] transients. The remaining thapsigargin-insensitive decay was slowed further by inhibition of the plasma membrane Ca[sup 2+]-ATPase (PMCA) and plasma membrane Na[sup +]/Ca[sup 2+] exchanger (NCX) via alkalization of the bath solution, by adding lanthanum, or by substitution of Na[sup +] with Li[sup +]. Mito- chondrial Ca[sup 2+] uptake contributed little to clearance in thapsigargin-pretreated cells. Together, the SERCA, PMCA, and NCX transport mechanisms accounted for 89 to 97% of clearance in normal solutions. We developed a quantitative model for the dynamic role of removal mechanisms over a wide range of [Ca[sup 2+]][sub i]. According to our model, 50 to 64% of initial Ca[sup 2+] removal is via the SERCA pump, whereas the NCX contributes 21-30% of the extrusion at high [Ca[sup 2+]][sub i], and the PMCA contributes 21-27% at low [Ca[sup 2+]][sub i]. [ABSTRACT FROM AUTHOR]

Mesenchymal migration usually happens on adhesive substrates, while cells adopt amoeboid migration on low/nonadhesive surfaces. Protein‐repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to these perceptions, this work discovers a unique locomotion of macrophages on adhesive–nonadhesive alternate substrates in vitro that they can overcome nonadhesive PEG gaps to reach adhesive regions in the mesenchymal mode. Adhering to extracellular matrix regions is a prerequisite for macrophages to perform further locomotion on the PEG regions. Podosomes are found highly enriched on the PEG region in macrophages and support their migration across the nonadhesive regions. Increasing podosome density through myosin IIA inhibition facilitates cell motility on adhesive–nonadhesive alternate substrates. Moreover, a developed cellular Potts model reproduces this mesenchymal migration. These findings together uncover a new migratory behavior on adhesive–nonadhesive alternate substrates in macrophages. [ABSTRACT FROM AUTHOR]

Over the past decade, novel optical imaging tools have been developed for imaging neuronal activities along with the evolution of fluorescence indicators with brighter expression and higher sensitivity. Miniature microscopes, as revolutionary approaches, enable the imaging of large populations of neuron ensembles in freely behaving rodents and mammals, which allows exploring the neural basis of behaviors. Recent progress in two‐photon miniature microscopes and mesoscale single‐photon miniature microscopes further expand those affordable methods to navigate neural activities during naturalistic behaviors. In this review article, two‐photon miniature microscopy techniques are summarized historically from the first documented attempt to the latest ones, and comparisons are made. The driving force behind and their potential for neuroscientific inquiries are also discussed. Current progress in terms of the mesoscale, i.e., the large field‐of‐view miniature microscopy technique, is addressed as well. Then, pipelines for registering single cells from the data of two‐photon and large field‐of‐view miniature microscopes are discussed. Finally, we present the potential evolution of the techniques. [ABSTRACT FROM AUTHOR]

Zinc biology, featuring intertwining signaling networks and critical importance to human health, witnesses exciting opportunities in the big data era of physiology. Here, we report a class of red‐ and far‐red‐emitting Zn2+ probes with Kd values ranging from 190 nM to 74 μM, which are particularly suitable for real‐time monitoring the high concentration of Zn2+ co‐released with insulin during vesicular secretory events. Compared to the prototypical rhodamine‐based Zn2+ probes, the new class exploits morpholino auxochromes which eliminates phototoxicity during long‐term live recording of isolated islets. A Si‐rhodamine‐based Zn2+ probe with high turn‐on ratio (>100), whose synthesis was enabled by a new route featuring late‐stage N‐alkylation, allowed simultaneous recording of Ca2+ influx, mitochondrial signal, and insulin secretion in isolated mouse islets. The time‐lapse multicolor fluorescence movies and their analysis, enabled by red‐shifted Zn2+ and other orthogonal physiological probes, highlight the potential impact of biocompatible fluorophores on the fields of islet endocrinology and system biology. [ABSTRACT FROM AUTHOR]

Zinc biology, featuring intertwining signaling networks and critical importance to human health, witnesses exciting opportunities in the big data era of physiology. Here, we report a class of red‐ and far‐red‐emitting Zn2+ probes with Kd values ranging from 190 nM to 74 μM, which are particularly suitable for real‐time monitoring the high concentration of Zn2+ co‐released with insulin during vesicular secretory events. Compared to the prototypical rhodamine‐based Zn2+ probes, the new class exploits morpholino auxochromes which eliminates phototoxicity during long‐term live recording of isolated islets. A Si‐rhodamine‐based Zn2+ probe with high turn‐on ratio (>100), whose synthesis was enabled by a new route featuring late‐stage N‐alkylation, allowed simultaneous recording of Ca2+ influx, mitochondrial signal, and insulin secretion in isolated mouse islets. The time‐lapse multicolor fluorescence movies and their analysis, enabled by red‐shifted Zn2+ and other orthogonal physiological probes, highlight the potential impact of biocompatible fluorophores on the fields of islet endocrinology and system biology. [ABSTRACT FROM AUTHOR]

• More peripheral ER sheet structure in PLP1 duplication patients' cells. • Abnormal mitochondria morphology in PLP1 duplication patients' cells. • Reduced respiration level in PLP1 duplication patients' cells. • A higher level of ER-mitochondria interface was found near the ER sheet. • Closer contact of ER and mitochondria can alter mitochondria-associated membranes. Among the hypomyelinating leukodystrophies, Pelizaeus–Merzbacher disease (PMD) is a representative disorder. The disease is caused by different types of PLP1 mutations, among which PLP1 duplication accounts for ∼70% of the mutations. Previous studies have shown that PLP1 duplications lead to PLP1 retention in the endoplasmic reticulum (ER); in parallel, recent studies have demonstrated that PLP1 duplication can also lead to mitochondrial dysfunction. As such, the respective roles and interactions of the ER and mitochondria in the pathogenesis of PLP1 duplication are not clear. In both PLP1 patients' and healthy fibroblasts, we measured mitochondrial respiration with a Seahorse XF Extracellular Analyzer and examined the interactions between the ER and mitochondria with super-resolution microscopy (spinning-disc pinhole-based structured illumination microscopy, SD-SIM). For the first time, we demonstrated that PLP1 duplication mutants had closer ER-mitochondrion interfaces mediated through structural and morphological changes in both the ER and mitochondria-associated membranes (MAMs). These changes in both the ER and mitochondria then led to mitochondrial dysfunction, as reported previously. This work highlights the roles of MAMs in bridging PLP1 expression in the ER and pathogenic dysfunction in mitochondria, providing novel insight into the pathogenicity of mitochondrial dysfunction resulting from PLP1 duplication. These findings suggest that interactions between the ER and mitochondria may underlie pathogenic mechanisms of hypomyelinating leukodystrophies diseases at the organelle level. [ABSTRACT FROM AUTHOR]

Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination microscopy (SIM) suffers from aberrations caused by various sources. Although artefacts generated from inaccurate reconstruction parameter estimation and noise amplification can be minimized, aberrations due to the scattering of excitation light on samples have rarely been investigated. In this paper, by simulating multiple subcellular structure with the distinct refractive index from water, we study how different thicknesses of this subcellular structure scatter incident light on its optical path of SIM excitation. Because aberrant interference light aggravates with the increase in sample thickness, the reconstruction of the 2D-SIM SR image degraded with the change of focus along the axial axis. Therefore, this work may guide the future development of algorithms to suppress SIM artefacts caused by scattering in thick samples. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'. [ABSTRACT FROM AUTHOR]

Background and Aim: Transient global amnesia (TGA) was first described by Bender in 1956 and is characterized by sudden, temporary, and anterograde memory loss. This study aimed to explore the possible mechanisms of and lesions responsible for TGA. Methods: Retrospective data were collected from all patients with TGA admitted to Zhongshan Hospital, affiliated with Xiamen University, between October 1, 2011, and October 30, 2018. Information about the TGA condition, previous history, and clinical examination of the TGA and control groups was recorded. Functional magnetic resonance imaging was performed on the patients to explore the possible lesions responsible for TGA. Results: A total of 73 patients with TGA and 73 age- and gender-matched controls were included in the analysis. The differences in the migraine history (9/2, p = 0.038) were statistically significant in both groups, but no statistically significant difference was observed regarding the history of hypertension, diabetes, and other diseases. In addition, seven patients with TGA had lesions located in the hippocampal CA1 region; the dome column and hippocampal CA1 region exist in the same functional loop and play a synergistic role. The average follow-up period in the groups was 36 months. During the follow-up period, no significant differences in cerebral infarction, cerebral hemorrhage, CHD, or TGA attack between the groups were observed. Conclusion: Migraine may be a risk factor of TGA, and cerebral infarction may be one of the pathogeneses. The brain area responsible for TGA may involve a memory loop comprising the hippocampal CA1 region and the fornix column among other parts. [ABSTRACT FROM AUTHOR]

Modern cellular pathology relies on conventional microscopes to study the etiology and pathogenesis of diseases, which suffer from limited spatial resolution, little dynamic information due to sample fixation, and the lack of molecular information. Different PLP1 mutants exhibited distinct localizations; for example, while PLP1 C353G-EGFP exhibited patterns similar to WT PLP1 (Fig. Overall, we envision that live-cell SR pathology enabled by Hessian deconvolution confers a much-needed tool, which can be applied to PMD-like diseases and other rare diseases caused by dominant gene mutations. [Extracted from the article]

Cytoskeletal‐based molecular motors produce force perpendicular to their direction of movement. However, it remains unknown whether and why motor proteins generate sidesteps movement along their filamentous tracks in vivo. Using Hessian structured illumination microscopy, we located green fluorescent protein (GFP)‐labeled intraflagellar transport (IFT) particles inside sensory cilia of live Caenorhabditis elegans with 3–6‐nanometer accuracy and 3.4‐ms resolution. We found that IFT particles took sidesteps along axoneme microtubules, demonstrating that IFT motors generate torque in a living animal. Kinesin‐II and OSM‐3‐kinesin collaboratively drive anterograde IFT. We showed that the deletion of kinesin‐II, a torque‐generating motor protein, reduced sidesteps, whereas the increase of neck flexibility of OSM‐3‐kinesin upregulated sidesteps. Either increase or decrease of sidesteps of IFT kinesins allowed ciliogenesis to the regular length, but changed IFT speeds, disrupted axonemal ninefold symmetry, and inhibited sensory cilia‐dependent animal behaviors. Thus, an optimum level of IFT kinesin sidestepping is associated with the structural and functional fidelity of cilia. Synopsis: Kinesin sidestepping has been observed in vitro, but its physiological relevance has not been investigated in vivo. In this study, optimal sidestepping of intraflagellar transport (IFT) kinesins in Caenorhabditis elegans sensory neurons is shown to regulate proper axoneme symmetry and animal chemosensation. IFT particles take sidesteps along axoneme microtubules, demonstrating torque generation of IFT motors in a living animal.Coordinated movement of kinesin‐II and OSM‐3 kinesin generates an intermediate level of sidestepping.Aberrant sidestepping allows ciliogenesis, but disrupts axonemal nine‐fold symmetryAberrant sidestepping inhibits sensory cilia‐dependent chemosensation. [ABSTRACT FROM AUTHOR]

[Display omitted] • A practical pipeline for in situ structural analysis of multicellular samples is presented. • Significantly increases sample throughput. • Subtomogram averaging of ribosomes from tissue samples provides comparable quality averages to cellular samples. • The subcellular structure of the islet shows a dramatic difference compared with that of wildly used β-cell line. The structural studies of macromolecules in their physiological context, particularly in tissue, is constrained by the bottleneck of sample preparation. In this study, we present a practical pipeline for preparing multicellular samples for cryo-electron tomography. The pipeline comprises sample isolation, vitrification, and lift-out-based lamella preparation using commercially available instruments. We demonstrate the efficacy of our pipeline by visualizing pancreatic β cells from mouse islets at the molecular level. This pipeline enables the determination of the properties of insulin crystals in situ for the first time, using unperturbed samples. [ABSTRACT FROM AUTHOR]

Mesenchymal Migration on Adhesive-Nonadhesive Alternate Surfaces in Macrophages (Adv. Sci. 23/2023) B Mesenchymal Migration b In article number 2301337, Fulin Xing, Leiting Pan, and co-workers uncover an incomparable mobility of macrophages that they could migrate across non-adhesive substrates in a mesenchymal mode on the adhesive-nonadhesive alternate substrates in vitro. Podosomes are found highly enriched on the non-adhesive region and supported this unique migratory behavior. [Extracted from the article]

This article presents an approach for space-variant Shack–Hartmann wavefront reconstruction based on image moment theory. We derive the relation between the moment of a pair of subimages and the local transformation coefficients. A special solution is obtained from this relation using the square guide 'star'. Our moment-based wavefront reconstruction has reduced computational complexity compared to the iteration-based algorithm. We execute image restorations using both the tiling strategy with 5 × 5 PSFs and the conventional strategy with a global average PSF. Our approach is supported by visual and quantitative evaluations. [ABSTRACT FROM AUTHOR]

Cilia are remarkable cellular devices that power cell motility and transduce extracellular signals. To assemble a cilium, a cylindrical array of 9 doublet microtubules push out an extension of the plasma membrane. Membrane tension regulates cilium formation; however, molecular pathways that link mechanical stimuli to ciliogenesis are unclear. Using genome editing, we introduced hereditary elliptocytosis (HE)- and spinocerebellar ataxia (SCA)-associated mutations into the Caenorhabditis elegans membrane skeletal protein spectrin. We show that these mutations impair mechanical support for the plasma membrane and change cell shape. RNA sequencing (RNA-seq) analyses of spectrin-mutant animals uncovered a global down-regulation of ciliary gene expression, prompting us to investigate whether spectrin participates in ciliogenesis. Spectrin mutations affect intraflagellar transport (IFT), disrupt axonemal microtubules, and inhibit cilium formation, and the endogenous spectrin periodically distributes along cilia. Mammalian spectrin also localizes in cilia and regulates ciliogenesis. These results define a previously unrecognized yet conserved role of spectrin-based mechanical support for cilium biogenesis. [ABSTRACT FROM AUTHOR]

Aims: To investigate the progression of obesity‐related type 2 diabetes mellitus (T2DM) in rhesus monkeys, especially dynamic changes in insulin and glucagon. Materials and methods: We followed a cohort of 52 rhesus monkeys for 7 years throughout the progression of obesity‐related T2DM. Intravenous glucose tolerance tests were performed every 6 months to evaluate dynamic changes in glucose, insulin and glucagon levels. Results: Obesity in rhesus monkeys increased the overall mortality and T2DM morbidity. During the progression of T2DM, glucagon remained consistently elevated, while insulin initially increased in compensation but then dropped to below normal levels when the monkeys developed overt T2DM. After a glucose challenge, both the first and second phases of insulin secretion increased during the early stage of T2DM; in later stages the first phase was delayed and the second phase was diminished. Conclusion: Our findings showed that, beside the decreased insulin level, hyperglucagonaemia also plays an important role in the development of T2DM. [ABSTRACT FROM AUTHOR]

Background: To assess whether intensive statin therapy reduces the occurrence of microemboli in patients with acute ischemic stroke. Methods: Patients with acute ischemic stroke within 72 h of onset were randomized to the intensive statin (atorvastatin 60 mg/day, adjusted to 20 mg/day after 7 days) and control (atorvastatin 20 mg/day) groups. Combined aspirin and clopidogrel were used for antiplatelet therapy. Microemboli were monitored by transcranial Doppler on days 1 (pre-treatment), 3, and 7. Metalloproteinase-9 (MMP-9), high-sensitivity C-reactive protein (hs-CRP), and National Institutes of Health Stroke Scale (NIHSS) score were assessed on days 1 and 7. The modified Rankin scale (mRS) was used on day 90. The primary outcome was the proportion of patients with microemboli on day 3. Results: There were 35 (58.3%) and 30 (52.6%) patients with microemboli in the intensive statin (n = 60) and control (n = 57) groups, respectively, on day 1 (p = 0.342). On day 3, there were significantly less microemboli in the intensive statin group (n = 9; 15.0%) compared with controls (n = 16; 28.1%; p = 0.002). No difference was observed in MMP-9 and hs-CRP levels on day 1, but on day 7, MMP-9 (median 79.3 vs. 95.9 μg/L; p = 0.004) and hs-CRP (median 2.01 vs. 3.60 mg/L; p = 0.020) levels were lower in the intensive statin group compared with controls. There were no differences in NIHSS scores on days 1 and 7. There was no difference in mRS on day 90. Conclusion: Intensive atorvastatin therapy in patients with acute ischemic stroke reduces the occurrence of microemboli and inflammation, with no overt adverse events. [ABSTRACT FROM AUTHOR]

To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis. [ABSTRACT FROM AUTHOR]

Syntaxin (Syn)-1A mediates exocytosis of predocked insulin-containing secretory granules (SGs) during first-phase glucose-stimulated insulin secretion (GSIS) in part via its interaction with plasma membrane (PM)-bound L-type voltage-gated calcium channels (Cav). In contrast, Syn-3 mediates exocytosis of newcomer SGs that accounts for second-phase GSIS. We now hypothesize that the newcomer SG Syn-3 preferentially binds and modulates R-type Cav opening, which was postulated to mediate second-phase GSIS. Indeed, glucose-stimulation of pancreatic islet β-cell line INS-1 induced a predominant increase in interaction between Syn-3 and Cavα1 pore-forming subunits of R-type Cav2.3 and to lesser extent L-type Cavs, while confirming the preferential interactions between Syn-1A with L-type (Cav1.2, Cav1.3) Cavs. Consistently, direct binding studies employing heterologous HEK cells confirmed that Syn-3 preferentially binds Cav2.3, whereas Syn-1A prefers L-type Cavs. We then used siRNA knockdown (KD) of Syn-3 in INS-1 to study the endogenous modulatory actions of Syn-3 on Cav channels. Syn-3 KD enhanced Ca2+ currents by 46% attributed mostly to R- and L-type Cavs. Interestingly, while the transmembrane domain of Syn-1A is the putative functional domain modulating Cav activity, it is the cytoplasmic domain of Syn-3 that appears to modulate Cav activity. We conclude that Syn-3 may mimic Syn-1A in the ability to bind and modulate Cavs, but preferring Cav2.3 to perhaps participate in triggering fusion of newcomer insulin SGs during second-phase GSIS. [ABSTRACT FROM AUTHOR]

Summary Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60–100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (∼10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of pre-formed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5β/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations. [ABSTRACT FROM AUTHOR]

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca2+ signalling. Store-operated Ca2+ entry mediated by dynamic STIM1-ORAI1 coupling represents a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially restricted biotin labelling in situ coupled with mass spectrometry and a secondary screen based on bimolecular fluorescence complementation, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches led to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca2+ influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the Ca2+-NFAT signalling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER-PM junctions. [ABSTRACT FROM AUTHOR]

The large dense-core vesicle (LDCV), a type of lysosome-related organelle, is involved in the secretion of hormones and neuropeptides in specialized secretory cells. The granin family is a driving force in LDCV biogenesis, but the machinery for granin sorting to this biogenesis pathway is largely unknown. The mu mutant mouse, which carries a spontaneous null mutation on the Muted gene (also known as Bloc1s5), which encodes a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), is a mouse model of Hermansky-Pudlak syndrome. Here, we found that LDCVs were enlarged in mu adrenal chromaffin cells. Chromogranin A (CgA, also known as CHGA) was increased in mu adrenals and muted- knockdown cells. The increased CgA in mu mice was likely due a failure to export this molecule out of immature LDCVs, which impairs LDCV maturation and docking. In mu chromaffin cells, the size of readily releasable pool and the vesicle release frequency were reduced. Our studies suggest that the muted protein is involved in the selective export of CgA during the biogenesis of LDCVs. [ABSTRACT FROM AUTHOR]

Glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells is critical to the maintenance of blood glucose homeostasis in animals. Both decrease in pancreatic beta-cell mass and defects in beta-cell function contribute to the onset of diabetes, although the underlying mechanisms remain largely unknown. Molecular imaging techniques can help beta-cell study in a number of ways. High-resolution fluorescence imaging techniques provide novel insights into the fundamental mechanisms underlying GSIS in isolated beta-cells or in situ in pancreatic islets, and dynamic changes of beta-cell mass and function can be noninvasively monitored in vivo by imaging techniques such as positron emission tomography and single-photon emission computed tomography. All these techniques will contribute to the better understanding of the progression of diabetes and the search for the optimized therapeutic measures that reverse deficits in beta-cell mass and function. [ABSTRACT FROM AUTHOR]

Inside Cover: Red- and Far-Red-Emitting Zinc Probes with Minimal Phototoxicity for Multiplexed Recording of Orchestrated Insulin Secretion (Angew. Keywords: 4D islet physiology; biocompatibility; fluorescent probe; insulin secretion; zinc EN 4D islet physiology biocompatibility fluorescent probe insulin secretion zinc 25586 25586 1 11/26/21 20211201 NES 211201 B Insulin secretion b from -cells in intact islets can now be recorded together with calcium signal and organelles. Tailored for 4D, long-term, and multiple-color recording of Zn SP 2+ sp /insulin co-secretion in -cells and islets, the new probes promise to unveil the spatial-temporal regulation of islet endocrinology. [Extracted from the article]

Innentitelbild: Red- and Far-Red-Emitting Zinc Probes with Minimal Phototoxicity for Multiplexed Recording of Orchestrated Insulin Secretion (Angew. 4D islet physiology, biocompatibility, fluorescent probe, insulin secretion, zinc Keywords: 4D islet physiology; biocompatibility; fluorescent probe; insulin secretion; zinc EN 4D islet physiology biocompatibility fluorescent probe insulin secretion zinc 25790 25790 1 11/26/21 20211201 NES 211201 B Die Insulinsekretion b aus -Zellen kann nun zusammen mit dem Kalziumsignal und den Organellen aufgezeichnet werden. [Extracted from the article]

Abstract: Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage renal failure in children and young adults. NPHP8/RPGRIP1L is a novel ciliary gene that, when mutated, in addition to causing NPHP, also causes Joubert syndrome (JBTS) and Meckel syndrome (MKS). The exact function of NPHP8 and how defects in NPHP8 lead to human diseases are poorly understood. Here, we studied the Caenorhabditis elegans homolog nphp-8 (C09G5.8) and explored the possible function of NPHP-8 in ciliated sensory neurons. We determined the gene structure of nphp-8 through rapid amplification of cDNA ends (RACE) analysis and discovered an X-box motif that had been previously overlooked. Moreover, NPHP-8 co-localized with NPHP-4 at the transition zone at the base of cilia. Mutation of nphp-8 led to abnormal dye filling (Dyf) and shorter cilia lengths in a subset of ciliary neurons. In addition, chemotaxis to several volatile attractants was significantly impaired in nphp-8 mutants. Our data suggest that NPHP-8/RPGRIP1L plays an important role in cilia formation and cilia-mediated chemosensation in a cell type-specific manner. [Copyright &y& Elsevier]

Incretin-potentiated glucose-stimulated insulin secretion (GSIS) is critical to maintaining euglycemia, of which GLP-1 receptor (GLP-1R) on β-cells plays an indispensable role. Recently, α-cell-derived glucagon but not intestine-derived GLP-1 has been proposed as the critical hormone that potentiates GSIS via GLP-1R. However, the function of glucagon receptors (GCGR) on β-cells remains elusive. Here, using GCGR or GLP-1R antagonists, in combination with glucagon, to treat single β-cells, α-β cell clusters and isolated islets, we found that glucagon potentiates insulin secretion via β-cell GCGR at physiological but not high concentrations of glucose. Furthermore, we transfected primary mouse β-cells with RAB-ICUE (a genetically encoded cAMP fluorescence indicator) to monitor cAMP level after glucose stimulation and GCGR activation. Using specific inhibitors of different adenylyl cyclase (AC) family members, we revealed that high glucose concentration or GCGR activation independently evoked cAMP elevation via AC5 in β-cells, thus high glucose stimulation bypassed GCGR in promoting insulin secretion. Additionally, we generated β-cell-specific GCGR knockout mice which glucose intolerance was more severe when fed a high-fat diet (HFD). We further found that β-cell GCGR activation promoted GSIS more than GLP-1R in HFD, indicating the critical role of GCGR in maintaining glucose homeostasis during nutrient overload. [ABSTRACT FROM AUTHOR]

Abstract: Previously, we identified a clathrin-dependent slow endocytosis and a clathrin-independent fast endocytosis in pancreatic β cells, both triggered by elevated cytoplasmic Ca2+ concentration. In the current study, we attempted to explore the roles of different dynamin isoforms in these endocytotic processes. We first confirmed the existence of both neuron-specific dynamin 1 and ubiquitous dynamin 2 in INS-1 cells using both quantitative RT-PCR and Western blot experiments. By specifically knocking down the endogenous level of either dynamin isoform from INS-1 cells, we showed that dynamin 1 and dynamin 2 simultaneously participate in the clathrin-independent and -dependent membrane retrieval in pancreatic β cells. Transferrin internalization was also inhibited in cells with knock down of both dynamin 1 and dynamin 2. Based on these results, we argue that different dynamin isoforms play overlapping roles in different types of endocytosis. [Copyright &y& Elsevier]

Abstract: Membrane syntaxin plays essential roles in exocytosis in eukaryotic cells. The conservative Habc domain in plasma membrane syntaxins implies important roles for syntaxin targeting and function. Our previous study showed Habc domain was necessary for the trafficking and cluster distribution of syntaxin 1A on the plasma membrane. Here we identified which of the three domains (Ha, Hb and Hc) was essential for Stx1A trafficking and clustering. We found that, in INS-1 cells, the mutant truncated with either Ha, Hb or Hc domain could be sorted to the cell surface by a different mechanism compared to that of whole Habc truncated mutant. In contrast to wild type Stx1A, none of the mutants showed cluster distribution at the functional sites, suggesting that the physiological localization of Stx1A relies on intact Habc domain. Furthermore Munc18-1 is found not to be essential for Stx1A cluster distribution, despite important role in stabilizing membrane delivery of Stx1A. [Copyright &y& Elsevier]

Abstract: STIM1 and Orai1 have recently been identified to be crucial in the regulation of store-operated Ca2+ entry. However, it remains to be established how STIM1 couples store depletion to the functioning of Orai1 in the plasma membrane. Using quantitative measurement, we find little STIM1 on the surface membrane which is not increased by store depletion. We further demonstrate that Orai1 assembles into clusters that co-localize with STIM1 aggregations upon store depletion. The clustering of Orai1 is only seen when Oari1 are co-expressed with STIM1, but not when expressed alone. Moreover, ER retreat from cell periphery leads to mismatching of Orai1 and STIM1 puncta. Therefore, we propose that store depletion causes aggregation and translocation of STIM1 in close apposition to the plasma membrane, which in turn recruits Orai1 in the plasma membrane to the sites of STIM1 aggregates to assemble functional units of CRAC channels in a stoichiometric manner. [Copyright &y& Elsevier]

To satisfy environment requirement of infrared search and trace optical system, an infrared diffractive/refractive hybrid optical system in 3.7–4.8μm with 11.42° of field of view for passive athermalization is presented. The system is consisted of three lenses, including two aspheric surfaces and a diffraction surface, which has only two materials Ge and Si. The optical system has compact structure, small volume and light weight. The image quality of the system approaches the diffraction limit in the temperature range −80°C to 160°C. It is compatible with staring focal plane array which has a format of 320×240 and the pixel pitch of 30μm. The system need not move the compensated lens repeatedly to obtain the best images from −80°C to 160°C and enhances the performance of target tracking and recognition. [ABSTRACT FROM AUTHOR]

Based on the most advanced staring focal plane array which had a format of 640×480 and the pixel pitch of 15μm, a set of all-sphere midwave infrared ahermalization optical system was designed. The working wavelength was in 3–5μm, the full field of view was 8.58°, the relative aperture was 1/2, the efficient focal length (EFL) was 80m. The opticalsystem consisted of four lenses with three kinds of material – Ge, ZnSe and Si. All surfaces were sphere, which was easier to process test, making the cost inexpensive, and it could avoid using diffractive surface and aspheric surface. The image quality of the system approaches the diffraction limit in the temperature range −60°C-180°C. The design results proved that, the high resolution midwave infrared optical system had compact structure, small volume, high resolution and excellent image quality, meeting the design requirements, so that it could be used for photoelectric detection and tracking system. [ABSTRACT FROM AUTHOR]

• A data-driven microscopy image enhancement network is developed to achieve quality enhancement of fluorescence microscopy images without requiring any manual intervention • Our method increases image quality and overcomes the restricts of photo-bleaching and photo-toxicity during imaging procedure • Experimental results demonstrate that our approach can achieve high quality enhancement for various types of fluorescence microscopy images that contain different object contents and are collected from different fluorescence microscopes platforms. Fluorescence microscopy imaging has become an essential technique in the biology and biomedical science which can provide comprehensive visualization of many biological processes, and the exposure time is one of the most critical parameters for fluorescence microscopy imaging. Short-exposure-time (SET) imaging overcomes the limitations of photo-bleaching and photo-toxicity, allowing comprehensive visualization of the biological processes. Unfortunately, SET images deteriorate the signal to noise ratio and the image quality when compared with the long-exposure-time (LET) images. Therefore, we introduce a data-driven microscopy image enhancement network (MIEN) to improve the quality of SET microscopy images without requiring any manual intervention, facilitating the production of high-resolution and high contrast images. The universal property and accuracy of the proposed network are validated on 38,500 real fluorescence microscopy images, which contain different object contents and are collected from various exposure time ratios and fluorescence microscopes platforms. Experimental results demonstrate that the proposed MIEN model is effective to enhance the quality of SET fluorescence microscopy images, and can be used to observe delicate changes in cells, tissues and organs with low photo-bleaching and photo-toxicity. [ABSTRACT FROM AUTHOR]

In many non-excitable cells, the depletion of endoplasmic reticulum (ER) Ca2+ stores leads to the dynamic formation of membrane contact sites (MCSs) between the ER and the plasma membrane (PM), which activates the store-operated Ca2+ entry (SOCE) to refill the ER store. Two different Ca2+-sensitive proteins, STIM1 and extended synaptotagmin-1 (E-syt1), are activated during this process. Due to the lack of live cell super-resolution imaging, how MCSs are dynamically regulated by STIM1 and E-syt1 coordinately during ER Ca2+ store depletion and replenishment remain unknown. With home-built super-resolution microscopes that provide superior axial and lateral resolution in live cells, we revealed that extracellular Ca2+ influx via SOCE activated E-syt1s to move towards the PM by ~12 nm. Unexpectedly, activated E-syt1s did not constitute the MCSs per se, but re-arranged neighboring ER structures into ring-shaped MCSs (230~280 nm in diameter) enclosing E-syt1 puncta, which helped to stabilize MCSs and accelerate local ER Ca2+ replenishment. Overall, we have demonstrated different roles of STIM1 and E-syt1 in MCS formation regulation, SOCE activation and ER Ca2+ store replenishment. [ABSTRACT FROM AUTHOR]