Ca 2؉ -dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca 2؉ -triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P2 in acceptor liposomes and was independent of Ca 2؉ , but Ca 2؉ dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.vesicle exocytosis ͉ priming ͉ PIP2 ͉ contents mixing V esicle fusion in the secretory pathway employs SNARE proteins, members of a conserved family of membrane-associated proteins that contain approximately 60-aa heptad repeat motifs (1). A donor membrane SNARE protein associates in trans with acceptor membrane SNARE proteins to generate a 4-helix bundle (2, 3). Vesicle exocytosis in neuronal and endocrine cells utilizes VAMP2 (also known as synaptobrevin 2) on the vesicle (v-SNARE) and the target membrane SNAREs (t-SNAREs) syntaxin-1 and SNAP-25. The structure of a 4-helix bundle of neuronal SNAREs suggested how an assembled trans-SNARE complex could promote fusion through close membrane apposition (3). Evidence that neuronal SNARE complexes might directly catalyze membrane fusion was provided by lipid mixing studies with SNAREs reconstituted into proteoliposomes (4). The function of several key SNARE regulatory proteins, including synaptotagmin and Munc18-1, was successfully reconstituted in the lipid-mixing assay (5, 6).Dense-core vesicle exocytosis in cells proceeds through several stages before Ca 2ϩ -triggered fusion, consisting of docking/tethering and priming steps (7). Vesicles engage the plasma membrane in docking/tethering interactions that involve syntaxin-1 and SNAP-25 (8, 9). However, not all docked/tethered vesicles are competent for fusion, and priming reactions are needed to convert docked vesicles to a fusion-ready state (10). Priming involves the progressive assembly of trans-SNARE complexes, likely through the initial assembly of t-SNARE heterodimers with subsequent incorporation of VAMP2 by zippering N-terminal to C-terminal regions of the SNAREs (11-14). SNARE-binding proteins likely catalyze SNARE complex assembly during priming but little is known about how this is achieved.Molecular studies of factors that act in priming are needed to elucidate the pathway of SNARE co...

CAPS (aka CADPS)Peptide and neurotransmitter release from endocrine cells and neurons occurs by the regulated exocytic fusion of secretory vesicles with the plasma membrane (1). Membrane fusion is mediated by trans complexes of SNARE proteins that bridge the vesicle and plasma membrane to promote close membrane apposition and bilayer mixing (2). The SNARE complexes for endocrine and neuronal vesicle fusion consist of parallel bundles of four ␣-helical SNARE motifs with one helix contributed by the vesicle v-SNARE vesicle-associated membrane protein 2 (VAMP-2) 2 (aka synaptobrevin), one helix contributed by the plasma membrane t-SNARE syntaxin-1, and two helices contributed by the plasma membrane t-SNARE SNAP-25 (3). Based on a central layer residue (arginine or glutamine) and helix position in the SNARE bundle, these proteins are also classified as R-, Qa-, and QbQc-SNAREs, respectively (4). In vitro studies suggested a "zipper" model for the assembly of ternary SNARE complexes involving the pairing of syntaxin-1 (Qa) with SNAP-25 (QbQc) followed by the N-to C-terminal insertion of the R-SNARE VAMP-2 (5, 6).

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Heart failure is the leading cause of death in the western world and as such, there is a great need for new therapies. Heart failure has a variable presentation in patients and a complex etiology; however, it is fundamentally a condition that affects the mechanics of cardiac contraction, preventing the heart from generating sufficient cardiac output under normal operating pressures. One of the major issues hindering the development of new therapies has been difficulties in developing appropriate in vitro model systems of human heart failure that recapitulate the essential changes in cardiac mechanics seen in the disease. Recent advances in stem cell technologies, genetic engineering, and tissue engineering have the potential to revolutionize our ability to model and study heart failure in vitro. Here, we review how these technologies are being applied to develop personalized models of heart failure and discover novel therapeutics.

Progress in the development of assays for measuring cardiac action potential is crucial for the discovery of drugs for treating cardiac disease and assessing cardiotoxicity. Recently, high-throughput methods for assessing action potential using induced pluripotent stem cell (iPSC) derived cardiomyocytes in both two-dimensional monolayer cultures and three-dimensional tissues have been developed. We describe an improved method for assessing cardiac action potential using an ultra-fast cost-effective plate reader with commercially available dyes. Our methods improve dramatically the detection of the fluorescence signal from these dyes and make way for the development of more high-throughput methods for cardiac drug discovery and cardiotoxicity.