Spatial organisation of the SNARE proteins: the role of munc18-1

Regulated exocytosis is a highly conserved process in all eukaryotes that is mediated by conserved protein families. Central to this process are the SNARE (soluble Nethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) proteins that are targeted either to the target plasma membrane (t-SNAREs) or to the vesicle membrane (v-SNAREs). The fusion of the two phospholipids bilayers is dependent on the formation of the ternary SNARE complex, in which the plasma membraneassociated SNAP-25 and syntaxin la interact with the vesicle-associated membrane protein (VAMP). The formation of this SNARE complex is essential to drive membrane fusion and thus the sites of SNARE complex formation are strictly regulated both temporally and spatially. Many accessory factors have been identified that are essential to control the availability of these proteins to form the ternary SNARE complex. Syntaxin 1 a, a component of the SNARE complex in neurons and neuroendocrine cells, is directly regulated by muncl8-l, its cognate Secl/Muncl8 (SM) protein, although the cellular functions have remained controversial.In this study I show using in vitro and in vivo approaches that in neuroendocrine cells muncl8-l has two modes of binding to syntaxin la. I demonstrate that muncl8-l plays a vital role in binding to a closed-form of syntaxin la, keeping syntaxin la inactive and permitting its trafficking to the plasma membrane. Muncl8-1 is also able to bind to an active open form of syntaxin la, allowing it to bind to other SNAREs. Using Forster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) I demonstrate that in the absence of muncl8-l, syntaxin la and SNAP-25 can readily interact in the Golgi complex, forming reactive SNARE complexes that fail to traffic to the plasma membrane. The exocytotic SNARE proteins are highly promiscuous in their interactions with other SNAREs, and thus it is essential to traffic the exocytotic SNARE proteins through intracellular compartments while avoiding ectopic interactions between non-cognate SNARE proteins. Muncl8-1 has a vital regulatory role in preventing the formation of the binary complex between syntaxin la and SNAP-25 before the proteins reach their target destination of the plasma membrane. Upon delivery to the plasma membrane 1 the t-SNAREs have been shown to form cholesterol dependent clusters in several cell types of between 60-750 nm, depending on the imaging approach employed. Recently, it was shown that syntaxin la forms dynamic clusters by virtue of self association. However, the factors that regulate SNAP-25 cluster stability are unknown. Here 1 present evidence to show that once at the plasma membrane syntaxin la and SNAP-25 are concentrated in clusters of high local concentration that co-localise in neuroendocrine cells. Interestingly, despite reducing the affinity of SNAP-25 for syntaxin la using mutagenesis, these proteins still co-cluster and interact on the plasma membrane. The newly delivered t-SNAREs, less than 48 hours old, can form interaction-heterogeneous clusters with interactions modulated by 9+ elevated Ca levels. Furthermore, quantitative changes in the lipid microenvironment by cholesterol depletion play a role in cluster integrity. In summary, the pathway for SNARE cluster formation involves muncl8-l regulation of syntaxin la, cholesterol association and SNARE protein interactions. Muncl8-1 plays a vital role in trafficking syntaxin 1 a through the secretory pathway, preventing ectopic interactions between SNARE proteins. This is essential to present syntaxin la and SNAP-25 in dynamic clusters at the plasma membrane and represents a mechanism of membrane patterning that is based on favourable local membrane composition and protein-protein interactions. Muncl8-1 has spatially and functionally distinct roles that are defined by its binding mode to syntaxin

Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays

Robust generation of hepatocyte-like cells from human embryonic stem cell populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies

Sodium channel current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient

Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G>A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1-45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression system

DC: Three-dimensional culture of human embryonic stem cell derived hepatic endoderm and its role in bioartificial liver construction

The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays

Stem cell differentiation and human liver disease

Human stem cells are scalable cell populations capable of cellular differentiation. This makes them a very attractive in vitro cellular resource and in theory provides unlimited amounts of primary cells. Such an approach has the potential to improve our understanding of human biology and treating disease. In the future it may be possible to deploy novel stem cell-based approaches to treat human liver diseases. In recent years, efficient hepatic differentiation from human stem cells has been achieved by several research groups including our own. In this review we provide an overview of the field and discuss the future potential and limitations of stem cell technology