Transient expression of botulinum neurotoxin C1 light chain differentially inhibits calcium and glucose induced insulin secretion in clonal β‐cells

Lang, Jochen; Zhang, Hui; Vaidyanathan, Vadakkanchery-Viswanathan; Sadoul, Karin; Niemann, Heiner; Wollheim, Claes B.

doi:10.1016/s0014-5793(97)01411-7

Cited by 48 publications

(50 citation statements)

References 39 publications

(69 reference statements)

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“…That the effect of glucose is not related to exocytosis is supported by the observa- tion that it persists in ␤-cells transfected with botulinus toxin, which prevents secretion by Ͼ90%, as previously shown (31) and confirmed in the current study. These experiments also indicate that spreading is not the result of presentation of adhesion molecules at the cell surface during exocytosis.…”

Section: Discussionsupporting

confidence: 91%

Increased Intracellular Calcium Is Required for Spreading of Rat Islet β-Cells on Extracellular Matrix

Bosco¹,

Gonelle‐Gispert²,

Wollheim

et al. 2001

Diabetes

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Rat islet ␤-cells spread in response to glucose when attached on the matrix produced by a rat bladder carcinoma cell line (804G). Furthermore, in a mixed population of cells, it has been observed previously that spread cells secrete more insulin acutely in response to glucose, compared with cells that remain rounded. These results suggest bi-directional signaling between the islet ␤-cell and the extracellular matrix. In the present study, the role of increased intracellular free Ca E xtracellular matrix (ECM) surrounds pancreatic islets and forms the basal lamina, which separates endocrine cells from capillaries. Although the molecular components of these basal laminae remain to be fully investigated in islets, there is evidence indicating that some of the molecules generally found in epithelial basal lamina play a role in the growth and differentiation of ␤-cells. Fetal pancreatic epithelia cultured in Matrigel or collagen I gel differentiated into endocrine cells and form islet structures (1); neonatal rat islet cells embedded in collagen I formed well-organized islet-like organoids (2); and adult human ␤-cells or associated islet cells proliferated when attached on 804G matrix produced by a rat bladder carcinoma cell line (3,4). More recently, it has been shown that laminin I specifically promotes differentiation of mouse ␤-cells from precursor cells in vitro. This finding is of physiological relevance because laminin I is expressed in basement membranes of the mouse pancreas from ϳ15 days of intrauterine life (5).The importance of cell-matrix interactions for an optimal regulated insulin secretion is supported by a number of studies on ␤-cells cultured on various crude matrixes or their purified extracts (6 -14). In a recent study, we observed that ␤-cells attached to a matrix produced by a rat bladder carcinoma cell line 804G (hereafter referred to as 804G matrix) secreted more insulin than control cells. We also noticed that on 804G matrix, cells that had spread expressed higher amounts of ␣6␤1 integrins and secreted more insulin than cells that had remained round (15). The importance of cell-matrix interactions for regulated insulin secretion was further demonstrated by the fact that secretagogues, including glucose, increased the number of spread cells (15).The mechanism leading stimulated ␤-cell to spread on 804G matrix is totally unknown. In the present study, we raise the hypothesis that some of the intracellular pathways governing stimulation-secretion coupling of ␤-cells might be shared for coupling stimulation and spreading. Intracellular free Ca 2ϩ concentration [Ca 2ϩ ] i plays a central role in the consensus model for stimulus-secretion coupling, which may be summarized as follows: in response to glucose, ␤-cell metabolism is stimulated, resulting in increased levels of ATP produced by the mitochondria. ATP-sensitive K ϩ (K ATP ) channels then close, thereby triggering membrane depolarization. The subsequent opening of voltage-dependent Ca 2ϩ channels leads to an increase in [Ca 2ϩ ] i , ...

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Section: Discussionsupporting

confidence: 91%

Increased Intracellular Calcium Is Required for Spreading of Rat Islet β-Cells on Extracellular Matrix

Bosco¹,

Gonelle‐Gispert²,

Wollheim

et al. 2001

Diabetes

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“…8). In a previous report, expression of a pEGFP͞BoNT͞C1-LC construct in HIT-T15 cells appeared to yield GFP fluorescence dispersed throughout the cell, including the nucleus (38), similar to that what we have previously reported for BoNT͞B-LC. † Although shorter than serotype A (18), B and C1 do have a long duration of action in neuronal cells, supporting an alternative mechanism for these serotypes other than plasma membrane localization for the persistence of the endopeptidase activity.…”

Section: Discussionsupporting

confidence: 87%

Plasma membrane localization signals in the light chain of botulinum neurotoxin

Fernández‐Salas

Steward

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

115

103

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Botulinum neurotoxin (BoNT) is a potent biological substance used to treat neuromuscular and pain disorders. Both BoNT type A and BoNT type E display high-affinity uptake into motor neurons and inhibit exocytosis through cleavage of the synaptosome-associated protein of 25 kDa (SNAP25). The therapeutic effects of BoNT͞A last from 3 to 12 months, whereas the effects of BoNT͞E last less than 4 weeks. Using confocal microscopy and site-specific mutagenesis, we have determined that the protease domain of BoNT͞A light chain (BoNT͞ A-LC) localizes in a punctate manner to the plasma membrane, colocalizing with the cleaved product, SNAP25 197. In contrast, the short-duration BoNT͞E serotype is cytoplasmic. Mutations in the BoNT͞A-LC have revealed sequences at the N terminus necessary for plasma membrane localization, and an active dileucine motif in the C terminus that is likely involved in trafficking and interaction with adaptor proteins. These data support sequence-specific signals as determinants of intracellular localization and as a basis for the different durations of action in these two BoNT serotypes.otulinum neurotoxins (BoNTs) are the most potent of all biological substances (1). Although BoNTs are well publicized as a potential biological weapon and as the causative agents in clinical botulism, the potency and myorelaxant actions of BoNTs have been exploited clinically in more than 100 indications, including muscle hyperactivity in cerebral palsy and cervical dystonia, migraines, myofacial pain, and focal hyperhidrosis (2-5). These toxins are specific endoproteases that, collectively, target several distinct proteins in nerve terminals. Motor nerve terminals at neuromuscular junctions are particularly sensitive to these neurotoxins, resulting in a transient and reversible muscle relaxation through inhibition of acetylcholine release. The Clostridium neurotoxin family includes seven serotypes of BoNT (A-G), and a single form of toxin produced by Clostridium tetani (TeNT). These toxins consist of a heavy chain (HC, 100 kDa) and light chain (LC, 50 kDa) linked by a disulfide bond (6, 7). The three-dimensional crystal structures of BoNT͞A (8) and BoNT͞B (9) have been resolved, providing a basis for understanding the structure͞function mechanism of BoNT action. The BoNT-LCs are zinc-dependent endoproteases that specifically cleave one of three soluble Nethylmaleimide-sensitive factor-attachment protein-receptor (SNARE) proteins (10) involved in synaptic vesicle docking and fusion at the nerve terminal (11). The synaptosome-associated protein of 25 kDa (SNAP25) is cleaved at distinct sites near the C terminus by ) and BoNT͞E (R 180 -I 181 ), generating truncated SNAP25 197 (12) and SNAP25 180 (13), respectively.

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“…However, this complex is not essential per se in the triggered fusion steps of exocytosis because in our work higher [Ca 2+ ]free overcame the block to fusion (seen at 'physiological' [Ca 2+ ]free) caused by proteolysis. Such recovery has also been documented in earlier experiments with Clostridial toxins (Dreyer and Schmitt, 1983;Ahnert-Hilger and Weller, 1993;Bittner and Holz, 1993;Lawrence et al, 1994;Glenn and Burgoyne, 1996;Lawrence et al, 1996;Capogna et al, 1997;Land et al, 1997;Fassio et al, 1999), suggesting that the concept of a regulatory complex may extend beyond the present system. In general, the testing and rescue of exocytosis at only one [Ca 2+ ]free after complex disruption tells us a great deal about the modulatory role of the complex.…”

Section: Snares and The Process Of Exocytosissupporting

confidence: 75%

Regulated secretion: SNARE density, vesicle fusion and calcium dependence

Coorssen

Blank

Albertorio

et al. 2003

Journal of Cell Science

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SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion(Ca2+ sensitivity and the catalysis and execution of fusion)require additional proteins that function downstream of SNAREs.

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Transient expression of botulinum neurotoxin C1 light chain differentially inhibits calcium and glucose induced insulin secretion in clonal β‐cells

Cited by 48 publications

References 39 publications

Increased Intracellular Calcium Is Required for Spreading of Rat Islet β-Cells on Extracellular Matrix

Increased Intracellular Calcium Is Required for Spreading of Rat Islet β-Cells on Extracellular Matrix

Plasma membrane localization signals in the light chain of botulinum neurotoxin

Regulated secretion: SNARE density, vesicle fusion and calcium dependence

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