Type 1 diabetes studies consistently generate data showing islet β-cell dysfunction and T cell-mediated anti-β-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the β-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single β-cells. Consistent with immunohistological studies, β-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These β-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC trans-activator protein expressed by a subset of insulin+CD68- β-cells, specifically found in islets with lymphocytic infiltrates. β-Cell surface expression of HLA Class II was detected on a portion of CD45-insulin+ β-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic β-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. β-Cell expression of Class II molecules suggests that β-cells may interact directly with islet-infiltrating CD4+ T cells and may play an immunopathogenic role.

Low birth weight is a risk factor for gestational and type 2 diabetes (T2D). Since mammalian target of rapamycin (mTOR) controls pancreatic β-cell mass and hormone release, we hypothesized that nutritional insult in utero might permanently alter mTOR signaling. Mice were fed a low-protein (LP, 8%) or control (C, 20%) diet throughout pregnancy, and offspring examined until 130 days age. Mice receiving LP were born 12% smaller and β-cell mass was significantly reduced throughout life. Islet mTOR levels were lower in LP-exposed mice and localized predominantly to α-rather than β-cells. Incubation of isolated mouse islets with rapamycin significantly reduced cell proliferation while increasing apoptosis. mRNA levels for mTORC complex genes mTOR, Rictor and Raptor were elevated at 7 days in LP mice, as were the mTOR and Raptor proteins. Proglucagon gene expression was similarly increased, but not insulin or the immune/metabolic defense protein STING. In human and mouse pancreas STING was strongly associated with islet β-cells. Results support long-term changes in islet mTOR signaling in response to nutritional insult in utero, with altered expression of glucagon and insulin and a reduced β-cell mass. This may contribute to an increased risk of gestational or type 2 diabetes.

AIMS/HYPOTHESIS: in type 1 diabetes, selective beta cell loss occurs within the inflamed milieu of insulitic islets. This milieu is generated via the enhanced secretion of proinflammatory cytokines and by the loss of anti-inflammatory molecules such as IL-4 and IL-13. While the actions of proinflammatory cytokines have been well-studied in beta cells, the effects of their anti-inflammatory counterparts have received relatively little attention and we have addressed this. METHODS: Clonal beta cells, isolated human islets and pancreas sections from control individuals and those with type 1 diabetes were employed. Gene expression was measured using targeted gene arrays and by quantitative RT-PCR. Protein expression was monitored in cell extracts by western blotting and in tissue sections by immunocytochemistry. Target proteins were knocked down selectively with interference RNA. RESULTS: Cytoprotection achieved with IL-4 and IL-13 is mediated by the early activation of signal transducer and activator of transcription 6 (STAT6) in beta cells, leading to the upregulation of anti-apoptotic proteins, including myeloid leukaemia-1 (MCL-1) and B cell lymphoma-extra large (BCLXL). We also report the induction of signal regulatory protein-α (SIRPα), and find that knockdown of SIRPα is associated with reduced beta cell viability. These anti-apoptotic proteins and their attendant cytoprotective effects are lost following siRNA-mediated knockdown of STAT6 in beta cells. Importantly, analysis of human pancreas sections revealed that STAT6 is markedly depleted in the beta cells of individuals with type 1 diabetes, implying the loss of cytoprotective responses. CONCLUSIONS/INTERPRETATION: Selective loss of STAT6 may contribute to beta cell demise during the progression of type 1 diabetes.

AIMS/HYPOTHESIS: Sphingolipids play important roles in beta cell physiology, by regulating proinsulin folding and insulin secretion and in controlling apoptosis, as studied in animal models and cell cultures. Here we investigate whether sphingolipid metabolism may contribute to the pathogenesis of human type 1 diabetes and whether increasing the levels of the sphingolipid sulfatide would prevent models of diabetes in NOD mice. METHODS: We examined the amount and distribution of sulfatide in human pancreatic islets by immunohistochemistry, immunofluorescence and electron microscopy. Transcriptional analysis was used to evaluate expression of sphingolipid-related genes in isolated human islets. Genome-wide association studies (GWAS) and a T cell proliferation assay were used to identify type 1 diabetes related polymorphisms and test how these affect cellular islet autoimmunity. Finally, we treated NOD mice with fenofibrate, a known activator of sulfatide biosynthesis, to evaluate the effect on experimental autoimmune diabetes development. RESULTS: We found reduced amounts of sulfatide, 23% of the levels in control participants, in pancreatic islets of individuals with newly diagnosed type 1 diabetes, which were associated with reduced expression of enzymes involved in sphingolipid metabolism. Next, we discovered eight gene polymorphisms (ORMDL3, SPHK2, B4GALNT1, SLC1A5, GALC, PPARD, PPARG and B4GALT1) involved in sphingolipid metabolism that contribute to the genetic predisposition to type 1 diabetes. These gene polymorphisms correlated with the degree of cellular islet autoimmunity in a cohort of individuals with type 1 diabetes. Finally, using fenofibrate, which activates sulfatide biosynthesis, we completely prevented diabetes in NOD mice and even reversed the disease in half of otherwise diabetic animals. CONCLUSIONS/INTERPRETATION: These results indicate that islet sphingolipid metabolism is abnormal in type 1 diabetes and suggest that modulation may represent a novel therapeutic approach. DATA AVAILABILITY: the RNA expression data is available online at https://www.dropbox.com/s/93mk5tzl5fdyo6b/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%2C%20RNA%20expression.xlsx?dl=0. A list of SNPs identified is available at https://www.dropbox.com/s/yfojma9xanpp2ju/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%20SNP.xlsx?dl=0.

Objective: to identify the genetic cause of disease in 2 previously unreported families with forms of distal hereditary motor neuropathies (dHMNs). Methods: the first family comprises individuals affected by dHMN type V, which lacks the cardinal clinical feature of vocal cord paralysis characteristic of dHMN-VII observed in the second family. Next-generation sequencing was performed on the proband of each family. Variants were annotated and filtered, initially focusing on genes associated with neuropathy. Candidate variants were further investigated and confirmed by dideoxy sequence analysis and cosegregation studies. Thorough patient phenotyping was completed, comprising clinical history, examination, and neurologic investigation. Results: dHMNs are a heterogeneous group of peripheral motor neuron disorders characterized by length-dependent neuropathy and progressive distal limb muscle weakness and wasting. We previously reported a dominant-negative frameshift mutation located in the concluding exon of the SLC5A7 gene encoding the choline transporter (CHT), leading to protein truncation, as the likely cause of dominantly-inherited dHMN-VII in an extended UK family. In this study, our genetic studies identified distinct heterozygous frameshift mutations located in the last coding exon of SLC5A7, predicted to result in the truncation of the CHT C-terminus, as the likely cause of the condition in each family. Conclusions: This study corroborates C-terminal CHT truncation as a cause of autosomal dominant dHMN, confirming upper limb predominating over lower limb involvement, and broadening the clinical spectrum arising from CHT malfunction.

Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3K392R, on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3K392R and healthy-control cells, but in later stages, NEUROG3 expression was upregulated prematurely in STAT3K392R cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3K392R-activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3K392R. Collectively, our results demonstrate that the STAT3K392R mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression.

The presynaptic, high-affinity choline transporter is a critical determinant of signalling by the neurotransmitter acetylcholine at both central and peripheral cholinergic synapses, including the neuromuscular junction. Here we describe an autosomal recessive presynaptic congenital myasthenic syndrome presenting with a broad clinical phenotype due to homozygous choline transporter missense mutations. The clinical phenotype ranges from the classical presentation of a congenital myasthenic syndrome in one patient (p.Pro210Leu), to severe neurodevelopmental delay with brain atrophy (p.Ser94Arg) and extend the clinical outcomes to a more severe spectrum with infantile lethality (p.Val112Glu). Cells transfected with mutant transporter construct revealed a virtually complete loss of transport activity that was paralleled by a reduction in transporter cell surface expression. Consistent with these findings, studies to determine the impact of gene mutations on the trafficking of the Caenorhabditis elegans choline transporter orthologue revealed deficits in transporter export to axons and nerve terminals. These findings contrast with our previous findings in autosomal dominant distal hereditary motor neuropathy of a dominant-negative frameshift mutation at the C-terminus of choline transporter that was associated with significantly reduced, but not completely abrogated choline transporter function. Together our findings define divergent neuropathological outcomes arising from different classes of choline transporter mutation with distinct disease processes and modes of inheritance. These findings underscore the essential role played by the choline transporter in sustaining acetylcholine neurotransmission at both central and neuromuscular synapses, with important implications for treatment and drug selection.

© 2016, BMJ Publishing Group. All rights reserved. Objectives: One of the theories connecting enterovirus (EV) infection of human islets with type 1 diabetes (T1D) is the development of a fertile field in the islets. This implies induction of appropriate proteins for the viral replication such as the coxsackie- adenovirus receptor (CAR). The aim of this study was to investigate to what extent CAR is expressed in human islets of Langerhans, and what conditions that would change the expression. Design: Immunohistochemistry for CAR was performed on paraffin-embedded pancreatic tissue from patients with T1D (n=9 recent onset T1D, n=4 long-standing T1D), islet autoantibody-positive individuals (n=14) and non-diabetic controls (n=24) individuals. The expression of CAR was also examined by reverse transcription PCR on microdissected islets (n=5), exocrine tissue (n=5) and on explanted islets infected with EV or exposed to chemokines produced by EV-infected islet cells. Results: an increased frequency of patients with T1D and autoantibody-positive individuals expressed CAR in the pancreas (p

AIMS/HYPOTHESIS: Human pancreatic beta cells may be complicit in their own demise in type 1 diabetes, but how this occurs remains unclear. One potentially contributing factor is hyperexpression of HLA class I antigens. This was first described approximately 30 years ago, but has never been fully characterised and was recently challenged as artefactual. Therefore, we investigated HLA class I expression at the protein and RNA levels in pancreases from three cohorts of patients with type 1 diabetes. The principal aims were to consider whether HLA class I hyperexpression is artefactual and, if not, to determine the factors driving it. METHODS: Pancreas samples from type 1 diabetes patients with residual insulin-containing islets (n = 26) from the Network for Pancreatic Organ donors with Diabetes (nPOD), Diabetes Virus Detection study (DiViD) and UK recent-onset type 1 diabetes collections were immunostained for HLA class I isoforms, signal transducer and activator of transcription 1 (STAT1), NLR family CARD domain containing 5 (NLRC5) and islet hormones. RNA was extracted from islets isolated by laser-capture microdissection from nPOD and DiViD samples and analysed using gene-expression arrays. RESULTS: Hyperexpression of HLA class I was observed in the insulin-containing islets of type 1 diabetes patients from all three tissue collections, and was confirmed at both the RNA and protein levels. The expression of β2-microglobulin (a second component required for the generation of functional HLA class I complexes) was also elevated. Both 'classical' HLA class I isoforms (i.e. HLA-ABC) as well as a 'non-classical' HLA molecule, HLA-F, were hyperexpressed in insulin-containing islets. This hyperexpression did not correlate with detectable upregulation of the transcriptional regulator NLRC5. However, it was strongly associated with increased STAT1 expression in all three cohorts. Islet hyperexpression of HLA class I molecules occurred in the insulin-containing islets of patients with recent-onset type 1 diabetes and was also detectable in many patients with disease duration of up to 11 years, declining thereafter. CONCLUSIONS/INTERPRETATION: Islet cell HLA class I hyperexpression is not an artefact, but is a hallmark in the immunopathogenesis of type 1 diabetes. The response is closely associated with elevated expression of STAT1 and, together, these occur uniquely in patients with type 1 diabetes, thereby contributing to their selective susceptibility to autoimmune-mediated destruction.

© 2015, Springer-Verlag Berlin Heidelberg. Aims/hypothesis: in humans, the rate of beta cell proliferation declines rapidly during the postnatal period and remains low throughout adult life. Recent studies suggest that this may reflect the distribution of cell cycle regulators which, unusually, are located in the cytosolic compartment of beta cells in islets isolated from adults. In the present work, we examined whether the localisation of cyclin-D molecules is also cytosolic in the islet cells of pancreatic samples studied in situ. Methods: Immunohistochemical approaches were employed to examine the subcellular localisation of cyclin-D1, -D2 and -D3 in human pancreatic samples recovered either from heart-beating donors or post mortem. Immunofluorescence methods were used to reveal the cellular localisation of cyclin-D1 and -D3. Results: the distribution of cyclin-D2 was invariably cytosolic in islet cells, whereas the localisation of cyclin-D1 and -D3 varied according to the status of the donor. In pancreatic sections from heart-beating donors these molecules were primarily nuclear. By contrast, in samples collected post mortem, they were mainly cytosolic. Cyclin-D1 was detected only in beta cells whereas cyclin-D3 was detected in both alpha and beta cells. The proportion of donors who were immunopositive for cyclin-D1 declined from 71% in controls to 30% in those with type 1 diabetes. Cyclin-D3 was present in the islets of the majority of donors in both groups. Conclusions/interpretation: the subcellular localisation of cyclin-D molecules varies according to the status of the donor. Both cyclin-D1 and -D3 can be found in the nuclei of human islet cells in situ.

Aims/hypothesis: Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes. Methods: Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. Results: Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1. Conclusions/interpretation: When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression. © 2013 Springer-Verlag Berlin Heidelberg.

Considerable efforts have been invested to understand the mechanisms by which pro-inflammatory cytokines mediate the demise of β-cells in type 1 diabetes but much less attention has been paid to the role of anti-inflammatory cytokines as potential cytoprotective agents in these cells. Despite this, there is increasing evidence that anti-inflammatory molecules such as interleukin (IL)-4, IL-10 and IL-13 can exert a direct influence of β-cell function and viability and that the circulating levels of these cytokines may be reduced in type 1 diabetes. Thus, it seems possible that targeting of anti-inflammatory pathways might offer therapeutic potential in this disease. In the present review, we consider the evidence implicating IL-4, IL-10 and IL-13 as cytoprotective agents in the β-cell and discuss the receptor components and downstream signaling pathways that mediate these effects.

Pro-inflammatory cytokines are important mediators of β-cell demise in type 1 diabetes, and similar mechanisms are increasingly implicated in type 2 diabetes, where a state of chronic inflammation may persist. It is likely that the actions of anti-inflammatory cytokines are also altered in diabetes. Cytokines are released from immune cells, which may be recruited to the islets in diabetes, but they can also be produced by islet endocrine cells in response to environmental factors, including enteroviral infection. Since enteroviral infection of islet cells may influence the development of diabetes in humans, we examined the actions of two cytokines, IL-13 and IL-6, whose expression are reported to be altered in β-cells during enteroviral infection. Human and rodent islet cells were shown to express receptors for both IL-13 and IL-6, and treatment with either cytokine resulted in the rapid phosphorylation of STAT3 and STAT6. However, while β-cells were protected against a range of cytotoxic insults during exposure to IL-13, treatment with IL-6 enhanced cytotoxicity and western blotting revealed that IL-13 induced one specific isoform of phospho-STAT6 preferentially. Upon incubation with both cytokines together, the isoform of STAT6 that was upregulated by IL-13 alone was again induced, and the effects of IL-6 on β-cell viability were attenuated. Overall, the results suggest that induction of specific isoforms of STAT family transcription factors may underlie the cytoprotective actions of IL-13, and they imply that selective targeting of specific STAT-mediated signaling components could provide a means to ameliorate the loss of β-cell viability in diabetes.

Common polymorphisms within the FTO (fat mass and obesity-associated) gene correlate with increased BMI (body mass index) and a rising risk of Type 2 diabetes. FTO is highly expressed in the brain but has also been detected in peripheral tissues, including the endocrine pancreas, although its function there is unclear. The aim of the present study was to investigate the role of FTO protein in pancreatic β-cells using a conditional expression system developed in INS-1 cells. INS-1 cells were stably transfected with FTO-HA (haemagluttinin) incorporated under the control of a tetracycline-inducible promoter. Induction of FTO protein resulted in localization of the tagged protein to the nucleus. The level of FTO-HA protein achieved in transfected cells was tightly regulated, and experiments with selective inhibitors revealed that FTO-HA is rapidly degraded via the ubiquitin/proteasome pathway. The nuclear localization was not altered by proteasome inhibitors, although following treatment with PYR-41, an inhibitor of ubiquitination, some of the protein adopted a perinuclear localization. Unexpectedly, modestly increased expression of FTO-HA selectively enhanced the first phase of insulin secretion when INS-1 monolayers or pseudoislets were stimulated with 20 mM glucose, whereas the second phase remained unchanged. The mechanism responsible for the potentiation of glucose-induced insulin secretion is unclear; however, further experiments revealed that it did not involve an increase in insulin biosynthesis or any changes in STAT3 (signal transducer and activator of transcription 3) expression. Taken together, these results suggest that the FTO protein may play a hitherto unrecognized role in the control of first-phase insulin secretion in pancreatic β-cells.

The effects on pancreatic beta-cell viability and function of three microbial secondary metabolites pyrrolnitrin, phenazine and patulin were investigated, using the rat clonal pancreatic beta-cell line, INS-1. Cells were exposed to 10-fold serial dilutions (range 0-10 mug mL(-1)) of the purified compounds for 2, 24 and 72 h. After 2 h exposure, only patulin (10 mug mL(-1)) was cytotoxic. All compounds showed significant cytotoxicity after 24 h. None of the compounds altered insulin secretion with 2 and 20 mM glucose after 2 h. However, after 24 h treatment, phenazine and pyrrolnitrin (10 and 100 ng mL(-1)) potentiated insulin production and glucose-stimulated insulin secretion, whereas patulin had no effect. Exposure (24 h) to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 ng mL(-1)) caused similar increases in the Ca(2+) content of INS-1 cells. The outward membrane current was inhibited after 24 h exposure to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 or 100 ng mL(-1)). This study presents novel data suggesting that high concentrations of pyrrolnitrin and phenazine are cytotoxic to pancreatic beta-cells and thus possibly diabetogenic, whereas at lower concentrations these agents are nontoxic and may be insulinotropic. The possible role of such agents in the development of cystic fibrosis-related diabetes is discussed.

The provision of parenteral nutrition (PN) to 'stressed' patients often results in hyperglycaemia, which may be detrimental. In animal models limited amounts of enteral nutrition (EN) improve intestinal integrity and stimulate intestinal incretin production, which may lead to improved glucose control. We set out to assess if combining EN with PN results in improved glucose homeostasis rather than PN given alone. We conducted a randomised trial in a university teaching hospital of patients undergoing a 'curative' oesophagectomy for adenocarcinoma. Differences between the two intervention groups were assessed for continuous glucose measurement, insulin sensitivity using insulin tolerance tests (ITT) and homeostasis model analysis (HOMA), the incretin glucose-dependent insulinotropic polypeptide (GIP) and intestinal permeability. The combination of PN with EN resulted in lower interstitial glucose concentrations (P = 0.002), reduced insulin resistance, improved insulin sensitivity (HOMA-insulin resistance (IR) P = 0.045; HOMA beta P = 0.037; ITT P = 0.006), improved intestinal permeability (P < 0.001) and increased GIP (P = 0.01) when compared with PN alone. The combination of EN with PN, when compared with PN alone, results in reduced glucose concentrations, reduced insulin resistance, increased incretins and improvements in intestinal permeability.

This study has assessed the expression and functional significance of cGMP-dependent signalling components in BRIN-BD11 cells. RT-PCR analysis revealed the expression of two subunits of soluble guanylate cyclase (sGC) suggesting the presence of an α2/β1 heterodimer. The expression of three particulate guanylate cyclases (pGC) was also detected (GC-A, GC-B and GC-C), as well as two cGMP-selective PDE isoforms (PDE5A and PDE9). Stimulation of BRIN-BD11 cells with agonists selective for sGC (NO, YC-1 and BAY 41-2272), GC-A (atrial natriuretic peptide (ANP) or GC-C (guanylin) caused an elevation in cGMP, and in the case of sGC, this was blocked by the selective inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). The stimulatory effects of each activator on cGMP levels were further potentiated by the PDE5A inhibitor, zaprinast. Treatment of cells with sGC activators induced a loss of viability and increased insulin secretion. However these effects were not attenuated by ODQ suggesting that they were independent of a rise in cGMP. A modest increase in β-cell death and insulin secretion were also observed in guanylin and ANP treated cells, although the latter only reduced cell viability in the presence of a PDE5A inhibitor. Taken together, the data reveal that BRIN-BD11 cells express several functionally active enzymes capable of modulating cGMP levels, and they imply that signalling through these proteins may impact upon β-cell viability. The results further suggest that pGC isozymes can also regulate insulin secretion but that the pool of cGMP controlling insulin release is small relative to the global cGMP concentration in the cell.

This chapter describes a method to measure the viability of isolated intact islets of Langerhans from rat pancreas and considers the use of isolated islets and of pancreatic beta-cell lines to study cell viability following culture. The islet isolation method is based on the use of preparations of collagenase to selectively digest the bulk of the exocrine tissue while leaving the endocrine islets intact and separated from their surrounding acini. The islets can be obtained in relatively pure form and are suitable for use in hormone secretion assays as well as for measurement of cell viability. They can be cultured if required and viability maintained for periods of days to weeks. Beta-cell lines are useful for study of the control of cell viability although their secretory capacity is usually altered compared to primary islet cells. Islet cell death can be estimated in a number of ways using either direct or more indirect means. Some methods will distinguish between apoptotic and necrotic death while others offer a more generic index of changes in viability.

The formation and secretion of heterococcoliths by the non-motile life phase of the coccolithophore Coccolithus pelagicus was investigated using electron microscopy and time-lapse bright field imaging. Coccolithogenesis in C. pelagicus exhibited sequential mineralization of single coccoliths in Golgi-derived and nuclear-associated vesicles, a pattern similar to the formation of heterococcoliths in Emiliania huxleyi. Our TEM data show that only on maturation does the single coccolith vesicle migrate away from the nucleus before secretion. A reticular body, distinct from the Golgi body, was also clearly visible at the distal surface of the developing coccolith vesicle, suggesting this is a common structural feature in placolith cells that mineralize and secrete coccoliths one at a time. Time-lapse imaging revealed that the coccolith secretion process is rapid, taking 60-190 seconds, and involves considerable contractile activity to eject and position the coccolith on the surface of the cell. An intact flagellar root apparatus was discovered at the anterior pole of this non-motile cell from which polarized secretion of coccoliths occurs, which may indicate a novel role for such cytoskeletal structures. Freeze-fracture preparations revealed columnar deposits and adhesions linking the scales and coccolith baseplates to the cell, across the periplasmic space providing points of attachment for cellular movement. Rotatory movements of the cell relative to external coccoliths were exhibited by all actively calcifying cells. These movements enable the cell, while exhibiting morphologically polarized secretion, to locate and secrete a mature coccolith in a spatially well-defined manner. Finally, the time-lapse imaging approach described here provides an opportunity to quantify the regulation of coccolith production in single cells with high temporal resolution allowing responses of calcification to rapidly fluctuating environmental conditions such as light-dark transitions to be examined in detail, which has not been possible with bulk calcification studies.