* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Association of voltage-dependent calcium channels with docked
Survey
Document related concepts
Node of Ranvier wikipedia , lookup
Theories of general anaesthetic action wikipedia , lookup
Magnesium transporter wikipedia , lookup
Action potential wikipedia , lookup
Cyclic nucleotide–gated ion channel wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Membrane potential wikipedia , lookup
Cytokinesis wikipedia , lookup
SNARE (protein) wikipedia , lookup
Cell membrane wikipedia , lookup
Mechanosensitive channels wikipedia , lookup
Endomembrane system wikipedia , lookup
Transcript
Association of voltage-dependent calcium channels with docked insulin granules and Role of Nterminal domains for binding of syntaxin-1a clusters to secretory granules. All secreting cells transmit information out of cells via molecules in order to begin or complete a response. Any such cell is able to secrete low amounts of product continuously without external stimulus. However, in the case of certain neuronal and endocrine secretions, voltage gated calcium channels need to be activated for regulated secretion to occur. Using rat insulin secreting cell lines as a model for such regulated emissions or exocytosis, we study the machinery which aids the phenomenon by visualisation methods such as Total Internal Reflection Fluorescence microscopy- a high resolution imaging technique. Though currents across the voltage gated calcium channels have been recorded and studied by electrophysiological methods, these channels have never been visualized. It has been possible not only image the channel with an attached enhanced green fluorescence protein but also classify their tendency to cluster. Further aiding to the study the channel was stabalized by over expressing the pore forming α1 subunit along with the peripheral β3 subunit. This lead to an increased number of insulin granules colocalizing at the site of calcium channels. With some evidence it was also possible to confirm the function of synaptic protein interaction site (synprint) by over expressing it simultaneously with the channel. The docking site of the granules at the membrane could have been further away from the calcium channel determined by lowered colocalisation with the docking site. This could confirm biochemical evidence that the synprint plays a role in the location of the docking at the plasma membrane. The exocytotic machinery consists of proteins that are located on the plasma membrane, granule vesicular membrane and within the cytoplasm. The syntaxin- 1a protein located at the plasma membrane has an N’ terminus that consists of four major α helices. These helices interact with a cytosolic protein- Munc 18 and modify it structurally so as to allow the assembly of SNARE (Soluble NSF attachment protein receptor) proteins, which are important downstream in fusion with the plasma membrane. We hypothesize that one of the first three helices of the Habc domain can be sufficient to render the Munc 18 open. By creating deletions in the wild type syntaxin-1a we visualize and try to ascertain which of the delete mutants can restore clustering of syntaxin along with colocalization at the docking site. Though all mutants clustered, it has been difficult to adjudge with preliminary experiments which of the helices is the prime candidate. Swati Arora Degree project in applied biotechnology, Master of Science (2 years), 2012 Examensarbete i tillämpad bioteknik 45 hp till masterexamen, 2012 Biology Education Centre and Department of Medical Cell Biology Uppsala University Supervisor: Dr. Sebastian Barg