Genetic activation of glucokinase in a minority of pancreatic beta cells causes hypoglycemia in mice.

AIMS: Glucokinase (GK) is expressed in the glucose-sensing cells of the islets of Langerhans and plays a critical role in glucose homeostasis. Here, we tested the hypothesis that genetic activation of GK in a small subset of ß-cells is sufficient to change the glucose set-point of the whole islet. MATERIAL AND METHODS: Mouse models of cell-type specific GK deficiency (GKKO) and genetic enzyme activation (GKKI) in a subset of ß-cells were obtained by crossing the αGSU (gonadotropin alpha subunit)-Cre transgene with the appropriate GK mutant alleles. Metabolic analyses consisted of glucose tolerance tests, perifusion of isolated islets and intracellular calcium measurements. KEY FINDINGS: The αGSU-Cre transgene produced genetically mosaic islets, as Cre was active in 15 ± 1.2 % of ß-cells. While mice deficient for GK in a subset of islet cells were normal, unexpectedly, GKKI mice were chronically hypoglycemic, glucose intolerant, and had a lower threshold for glucose stimulated insulin secretion. GKKI mice exhibited an average fasting blood glucose level of 3.5 mM. GKKI islets responded with intracellular calcium signals that spread through the whole islets at 1 mM and secreted insulin at 3 mM glucose. SIGNIFICANCE: Genetic activation of GK in a minority of ß-cells is sufficient to change the glucose threshold for insulin secretion in the entire islet and thereby glucose homeostasis in the whole animal. These data support the model in which ß-cells with higher GK activity function as 'hub' or 'trigger' cells and thus control insulin secretion by the ß-cell collective within the islet.

RhoA as a Signaling Hub Controlling Glucagon Secretion From Pancreatic α-Cells.

Glucagon hypersecretion from pancreatic islet α-cells exacerbates hyperglycemia in type 1 diabetes (T1D) and type 2 diabetes. Still, the underlying mechanistic pathways that regulate glucagon secretion remain controversial. Among the three complementary main mechanisms (intrinsic, paracrine, and juxtacrine) proposed to regulate glucagon release from α-cells, juxtacrine interactions are the least studied. It is known that tonic stimulation of α-cell EphA receptors by ephrin-A ligands (EphA forward signaling) inhibits glucagon secretion in mouse and human islets and restores glucose inhibition of glucagon secretion in sorted mouse α-cells, and these effects correlate with increased F-actin density. Here, we elucidate the downstream target of EphA signaling in α-cells. We demonstrate that RhoA, a Rho family GTPase, plays a key role in this pathway. Pharmacological inhibition of RhoA disrupts glucose inhibition of glucagon secretion in islets and decreases cortical F-actin density in dispersed α-cells and α-cells in intact islets. Quantitative FRET biosensor imaging shows that increased RhoA activity follows directly from EphA stimulation. We show that in addition to modulating F-actin density, EphA forward signaling and RhoA activity affect α-cell Ca2+ activity in a novel mechanistic pathway. Finally, we show that stimulating EphA forward signaling restores glucose inhibition of glucagon secretion from human T1D donor islets.

Screen for Small-Molecule Modulators of Circadian Rhythms Reveals Phenazine as a Redox-State Modifying Clockwork Tuner.

A high-throughput cell-based screen identified redox-active small molecules that produce a period lengthening of the circadian rhythm. The strongest period lengthening phenotype was induced by a phenazine carboxamide (VU661). Comparison to two isomeric benzquinoline carboxamides (VU673 and VU164) shows the activity is associated with the redox modulating phenazine functionality. Furthermore, ex vivo cell analysis using optical redox ratio measurements shows the period lengthening phenotype to be associated with a shift to the NAD/FAD oxidation state of nicotinamide and flavine coenzymes.

SGLT2 inhibitors therapy protects glucotoxicity-induced ß-cell failure in a mouse model of human KATP-induced diabetes through mitigation of oxidative and ER stress.

Progressive loss of pancreatic ß-cell functional mass and anti-diabetic drug responsivity are classic findings in diabetes, frequently attributed to compensatory insulin hypersecretion and ß-cell exhaustion. However, loss of ß-cell mass and identity still occurs in mouse models of human KATP-gain-of-function induced Neonatal Diabetes Mellitus (NDM), in the absence of insulin secretion. Here we studied the temporal progression and mechanisms underlying glucotoxicity-induced loss of functional ß-cell mass in NDM mice, and the effects of sodium-glucose transporter 2 inhibitors (SGLT2i) therapy. Upon tamoxifen induction of transgene expression, NDM mice rapidly developed severe diabetes followed by an unexpected loss of insulin content, decreased proinsulin processing and increased proinsulin at 2-weeks of diabetes. These early events were accompanied by a marked increase in ß-cell oxidative and ER stress, without changes in islet cell identity. Strikingly, treatment with the SGLT2 inhibitor dapagliflozin restored insulin content, decreased proinsulin:insulin ratio and reduced oxidative and ER stress. However, despite reduction of blood glucose, dapagliflozin therapy was ineffective in restoring ß-cell function in NDM mice when it was initiated at >40 days of diabetes, when loss of ß-cell mass and identity had already occurred. Our data from mouse models demonstrate that: i) hyperglycemia per se, and not insulin hypersecretion, drives ß-cell failure in diabetes, ii) recovery of ß-cell function by SGLT2 inhibitors is potentially through reduction of oxidative and ER stress, iii) SGLT2 inhibitors revert/prevent ß-cell failure when used in early stages of diabetes, but not when loss of ß-cell mass/identity already occurred, iv) common execution pathways may underlie loss and recovery of ß-cell function in different forms of diabetes. These results may have important clinical implications for optimal therapeutic interventions in individuals with diabetes, particularly for those with long-standing diabetes.

Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins.

Fluorescence fluctuation spectroscopy can be used to measure the aggregation of fluorescently labeled molecules and is typically performed using time series data. Spatial intensity distribution analysis and fluorescence moment image analysis are established tools for measuring molecular brightnesses from single-color images collected with laser scanning microscopes. We have extended these tools for analysis of two-color images to resolve heteromeric interactions between molecules labeled with spectrally distinct chromophores. We call these new methods two-color spatial intensity distribution analysis and two-color spatial cumulant analysis (2c-SpCA). To implement these techniques on a hyperspectral imaging system, we developed a spectral shift filtering technique to remove artifacts due to intrinsic cross talk between detector bins. We determined that 2c-SpCA provides better resolution from samples containing multiple fluorescent species; hence, this technique was carried forward to study images of living cells. We used fluorescent heterodimers labeled with enhanced green fluorescent protein and mApple to quantify the effects of resonance energy transfer and incomplete maturation of mApple on brightness measurements. We show that 2c-SpCA can detect the interaction between two components of trimeric G-protein complexes. Thus, 2c-SpCA presents a robust and computationally expedient means of measuring heteromeric interactions in cellular environments.

Ca2+-Stimulated AMPK-Dependent Phosphorylation of Exo1 Protects Stressed Replication Forks from Aberrant Resection.

Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.

Regulation of islet glucagon secretion: Beyond calcium.

The islet of Langerhans plays a key role in glucose homeostasis through regulated secretion of the hormones insulin and glucagon. Islet research has focused on the insulin-secreting ß-cells, even though aberrant glucagon secretion from α-cells also contributes to the aetiology of diabetes. Despite its importance, the mechanisms controlling glucagon secretion remain controversial. Proper α-cell function requires the islet milieu, where ß- and δ-cells drive and constrain α-cell dynamics. The response of glucagon to glucose is similar between isolated islets and that measured in vivo, so it appears that the glucose dependence requires only islet-intrinsic factors and not input from blood flow or the nervous system. Elevated intracellular free Ca2+ is needed for α-cell exocytosis, but interpreting Ca2+ data is tricky since it is heterogeneous among α-cells at all physiological glucose levels. Total Ca2+ activity in α-cells increases slightly with glucose, so Ca2+ may serve a permissive, rather than regulatory, role in glucagon secretion. On the other hand, cAMP is a more promising candidate for controlling glucagon secretion and is itself driven by paracrine signalling from ß- and δ-cells. Another pathway, juxtacrine signalling through the α-cell EphA receptors, stimulated by ß-cell ephrin ligands, leads to a tonic inhibition of glucagon secretion. We discuss potential combinations of Ca2+ , cAMP, paracrine and juxtacrine factors in the regulation of glucagon secretion, focusing on recent data in the literature that might unify the field towards a quantitative understanding of α-cell function.

Establishment of the early cilia preassembly protein complex during motile ciliogenesis.

Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Förster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.

Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic ß-cells.

Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS's capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic ß-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic ß-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.

FUCCI tracking shows cell-cycle-dependent Neurog3 variation in pancreatic progenitors.

During pancreas organogenesis, Neurog3HI endocrine-committing cells are generated from a population of Sox9+ mitotic progenitors with only a low level of Neurog3 transcriptional activity (Neurog3TA.LO ). Low-level Neurog3 protein, in Neurog3TA.LO cells, is required to maintain their mitotic endocrine-lineage-primed status. Herein, we describe a Neurog3-driven FUCCI cell-cycle reporter (Neurog3P2A.FUCCI ) derived from a Neurog3 BAC transgenic reporter that functions as a loxed cassette acceptor (LCA). In cycling Sox9+ Neurog3TA.LO progenitors, the majority of cells in S-G2 -M phases have undetectable levels of Neurog3 with increased expression of endocrine progenitor markers, while those in G1 have low Neurog3 levels with increased expression of endocrine differentiation markers. These findings support a model in which variations in Neurog3 protein levels are coordinated with cell-cycle phase progression in Neurog3TA.LO progenitors with entrance into G1 triggering a concerted effort, beyond increasing Neurog3 levels, to maintain an endocrine-lineage-primed state by initiating expression of the downstream endocrine differentiation program prior to endocrine-commitment.

Combined Deletion of Slc30a7 and Slc30a8 Unmasks a Critical Role for ZnT8 in Glucose-Stimulated Insulin Secretion.

Polymorphisms in the SLC30A8 gene, which encodes the ZnT8 zinc transporter, are associated with altered susceptibility to type 2 diabetes (T2D), and SLC30A8 haploinsufficiency is protective against the development of T2D in obese humans. SLC30A8 is predominantly expressed in pancreatic islet ß-cells, but surprisingly, multiple knockout mouse studies have shown little effect of Slc30a8 deletion on glucose tolerance or glucose-stimulated insulin secretion (GSIS). Multiple other Slc30a isoforms are expressed at low levels in pancreatic islets. We hypothesized that functional compensation by the Slc30a7 isoform, which encodes ZnT7, limits the impact of Slc30a8 deletion on islet function. We therefore analyzed the effect of Slc30a7 deletion alone or in combination with Slc30a8 on in vivo glucose metabolism and GSIS in isolated islets. Deletion of Slc30a7 alone had complex effects in vivo, impairing glucose tolerance and reducing the glucose-stimulated increase in plasma insulin levels, hepatic glycogen levels, and pancreatic insulin content. Slc30a7 deletion also affected islet morphology and increased the ratio of islet α- to ß-cells. However, deletion of Slc30a7 alone had no effect on GSIS in isolated islets, whereas combined deletion of Slc30a7 and Slc30a8 abolished GSIS. These data demonstrate that the function of ZnT8 in islets can be unmasked by removal of ZnT7 and imply that ZnT8 may affect T2D susceptibility through actions in other tissues where it is expressed at low levels rather than through effects on pancreatic islet function.

Precommitment low-level Neurog3 expression defines a long-lived mitotic endocrine-biased progenitor pool that drives production of endocrine-committed cells.

The current model for endocrine cell specification in the pancreas invokes high-level production of the transcription factor Neurogenin 3 (Neurog3) in Sox9(+) bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3(TA.LO) cell population, defined as Neurog3 transcriptionally active and Sox9(+) and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9(+) Neurog3(TA.LO) progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3(HI) cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9(+) Neurog3(TA.LO) progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9(+) Neurog3(TA.LO) endocrine-biased progenitors feed production of Neurog3(HI) endocrine-committed cells during pancreas organogenesis.

Dopamine Receptor Signaling in MIN6 ß-Cells Revealed by Fluorescence Fluctuation Spectroscopy.

Insulin secretion defects are central to the development of type II diabetes mellitus. Glucose stimulation of insulin secretion has been extensively studied, but its regulation by other stimuli such as incretins and neurotransmitters is not as well understood. We investigated the mechanisms underlying the inhibition of insulin secretion by dopamine, which is synthesized in pancreatic ß-cells from circulating L-dopa. Previous research has shown that this inhibition is mediated primarily by activation of the dopamine receptor D3 subtype (DRD3), even though both DRD2 and DRD3 are expressed in ß-cells. To understand this dichotomy, we investigated the dynamic interactions between the dopamine receptor subtypes and their G-proteins using two-color fluorescence fluctuation spectroscopy (FFS) of mouse MIN6 ß-cells. We show that proper membrane localization of exogenous G-proteins depends on both the Gß and Gγ subunits being overexpressed in the cell. Triple transfections of the dopamine receptor subtype and Gß and Gγ subunits, each labeled with a different-colored fluorescent protein (FP), yielded plasma membrane expression of all three FPs and permitted an FFS evaluation of interactions between the dopamine receptors and the Gßγ complex. Upon dopamine stimulation, we measured a significant decrease in interactions between DRD3 and the Gßγ complex, which is consistent with receptor activation. In contrast, dopamine stimulation did not cause significant changes in the interactions between DRD2 and the Gßγ complex. These results demonstrate that two-color FFS is a powerful tool for measuring dynamic protein interactions in living cells, and show that preferential DRD3 signaling in ß-cells occurs at the level of G-protein release.

Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets.

The observation of ionic signaling dynamics in intact pancreatic islets has contributed greatly to our understanding of both α- and ß-cell function. Insulin secretion from ß-cells depends on the firing of action potentials and consequent rises of intracellular calcium activity ([Ca(2+)]i). Zinc (Zn(2+)) is cosecreted with insulin, and has been postulated to play a role in cell-to-cell cross talk within an islet, in particular inhibiting glucagon secretion from α-cells. Thus, measuring [Ca(2+)]i and Zn(2+) dynamics from both α- and ß-cells will elucidate mechanisms underlying islet hormone secretion. [Ca(2+)]i and intracellular Zn(2+) can be measured using fluorescent biosensors, but the most efficient sensors have overlapping spectra that complicate their discrimination. Hyperspectral imaging can be used to distinguish signals from multiple fluorophores, but available hyperspectral implementations are either too slow to measure the dynamics of ionic signals or not suitable for thick samples. We have developed a five-dimensional (x,y,z,t,λ) imaging system that leverages a snapshot hyperspectral imaging method, image mapping spectrometry, and light-sheet microscopy. This system provides subsecond temporal resolution from deep within multicellular structures. Using a single excitation wavelength (488 nm) we acquired images from triply labeled samples with two biosensors and a genetically expressing fluorescent protein (spectrally overlapping with one of the biosensors) with high temporal resolution. Measurements of [Ca(2+)]i and Zn(2+) within both α- and ß-cells as a function of glucose concentration show heterogeneous uptake of Zn(2+) into α-cells that correlates to the known heterogeneities in [Ca(2+)]i. These differences in intracellular Zn(2+) among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels.

Quantitative assessment of fluorescent proteins.

The advent of fluorescent proteins (FPs) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight ß-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.

Host and tumor derived MMP13 regulate extravasation and establishment of colorectal metastases in the liver.

BACKGROUND: Non alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the United States and worldwide. Our studies have previously shown an increase in metastatic burden in steatotic vs. normal livers using a mouse model of diet induced steatosis. In the present study we aim to identify and evaluate the molecular factors responsible for this increase in tumor burden. METHODS: We assessed changes in expression of a panel of matrix metalloproteinases (MMPs) using qRT-PCR between normal and steatotic livers and validated them with western blot analysis of protein levels. To evaluate the role of MMP13 on tumor development, we utilized a splenic injection model of liver metastasis in Wildtype and Mmp13 deficient mice, using either parental or stable Mmp13 knockdown cell lines. Further, to evaluate changes in the ability of tumor cells to extravasate we utilized whole organ confocal microscopy to identify individual tumor cells relative to the vasculature. MTT, migration and invasion assays were performed to evaluate the role of tumor derived MMP13 on hallmarks of cancer in vitro. RESULTS: We found that MMP13 was significantly upregulated in the steatotic liver both in mice as well as human patients with NAFLD. We showed a decrease in metastatic tumor burden in Mmp13-/- mice compared to wildtype mice, explained in part by a reduction in the number of tumor cells extravasating from the hepatic vasculature in the Mmp13-/- mice compared to wildtype mice. Additionally, loss of tumor derived MMP13 through stable knockdown in tumor cell lines lead to decreased migratory and invasive properties in vitro and metastatic burden in vivo. CONCLUSIONS: This study demonstrates that stromal as well as tumor derived MMP13 contribute to tumor cell extravasation and establishment of metastases in the liver microenvironment.

Fluorescent probes of the apoptolidins and their utility in cellular localization studies.

Apoptolidin A has been described among the top 0.1% most-cell-selective cytotoxic agents to be evaluated in the NCI 60 cell line panel. The molecular structure of apoptolidin A consists of a 20-membered macrolide with mono- and disaccharide moieties. In contrast to apoptolidin A, the aglycone (apoptolidinone) shows no cytotoxicity (>10 µM) when evaluated against several tumor cell lines. Apoptolidin H, the C27 deglycosylated analogue of apoptolidin A, displayed sub-micromolar activity against H292 lung carcinoma cells. Selective esterification of apoptolidins A and H with 5-azidopentanoic acid afforded azido-functionalized derivatives of potency equal to that of the parent macrolide. They also underwent strain-promoted alkyne-azido cycloaddition reactions to provide access to fluorescent and biotin-functionalized probes. Microscopy studies demonstrate apoptolidins A and H localize in the mitochondria of H292 human lung carcinoma cells.

Somatostatin and insulin mediate glucose-inhibited glucagon secretion in the pancreatic α-cell by lowering cAMP.

The dysregulation of glucose-inhibited glucagon secretion from the pancreatic islet α-cell is a critical component of diabetes pathology and metabolic disease. We show a previously uncharacterized [Ca(2+)]i-independent mechanism of glucagon suppression in human and murine pancreatic islets whereby cAMP and PKA signaling are decreased. This decrease is driven by the combination of somatostatin, which inhibits adenylyl cyclase production of cAMP via the Gαi subunit of the SSTR2, and insulin, which acts via its receptor to activate phosphodiesterase 3B and degrade cytosolic cAMP. Our data indicate that both somatostatin and insulin signaling are required to suppress cAMP/PKA and glucagon secretion from both human and murine α-cells, and the combination of these two signaling mechanisms is sufficient to reduce glucagon secretion from isolated α-cells as well as islets. Thus, we conclude that somatostatin and insulin together are critical paracrine mediators of glucose-inhibited glucagon secretion and function by lowering cAMP/PKA signaling with increasing glucose.

Inhibition of pancreatic ß-cell Ca2+/calmodulin-dependent protein kinase II reduces glucose-stimulated calcium influx and insulin secretion, impairing glucose tolerance.

Glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells is caused by Ca(2+) entry via voltage-dependent Ca(2+) channels. CaMKII is a key mediator and feedback regulator of Ca(2+) signaling in many tissues, but its role in ß-cells is poorly understood, especially in vivo. Here, we report that mice with conditional inhibition of CaMKII in ß-cells show significantly impaired glucose tolerance due to decreased GSIS. Moreover, ß-cell CaMKII inhibition dramatically exacerbates glucose intolerance following exposure to a high fat diet. The impairment of islet GSIS by ß-cell CaMKII inhibition is not accompanied by changes in either glucose metabolism or the activities of KATP and voltage-gated potassium channels. However, glucose-stimulated Ca(2+) entry via voltage-dependent Ca(2+) channels is reduced in islet ß-cells with CaMKII inhibition, as well as in primary wild-type ß-cells treated with a peptide inhibitor of CaMKII. The levels of basal ß-cell cytoplasmic Ca(2+) and of endoplasmic reticulum Ca(2+) stores are also decreased by CaMKII inhibition. In addition, CaMKII inhibition suppresses glucose-stimulated action potential firing frequency. These results reveal that CaMKII is a Ca(2+) sensor with a key role as a feed-forward stimulator of ß-cell Ca(2+) signals that enhance GSIS under physiological and pathological conditions.

A microfluidic-enabled mechanical microcompressor for the immobilization of live single- and multi-cellular specimens.

A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.