 So good afternoon, my name is Anu Kanandare and I'm from the National Institute of Science Education and Research. It's in the eastern coast of India in a place called Bhubaneshwar and it had been set up as an institute of national importance to encourage research in the basic sciences. So I've been working here this summer in Professor Tom Martin's lab in biochemistry and the title for my project was effective mutations in the monk-13 homology domain of calcium-dependent activated protein per secretion, also known as CAHPS. So the specific interest in this protein that I was studying originated from a research in radiative thesis diagnosis. So exocytosis is a process by which cells package their secretory contents into vesicles and release them into the extracellular medium. While most cells have a pathway which is called the constitutive exocytosis, cells which are specialized for secretions such as neural cells or endocrine cells have an additional pathway which is called the regulated exocytosis pathway. It has three steps. First is called docking. First is called docking whereby a vesicle is brought close to the plasma membrane. The second is called priming which makes a docked vesicle ready for release. And this involves tethering the vesicle to the plasma membrane by formation of complexes between vesicular snare proteins and the target membrane snare proteins. Then there is a rise in intracellular calcium concentration. The membranes for the vesicle and the plasma membrane merge with each other and lead to the release of the contents of the vesicle. So the third step is done diffusion and the protein of interest, CAHPS, possibly acts as a step of priming and is known to increase the efficiency of vesicle release and it is also an essential protein required for regulated exocytosis but the exact way in which it functions is not completely understood. So I'll take you through a brief history of CAHPS and the questions of the ideas that led to my project. So beginning with the identification of CAHPS, so investigators of regulated exocytosis wanted to be able to reconstitute the process of calcium dependent exocytosis in an in vitro system and to completely understand what are the factors involved in this process. So what was done initially is that cells were taken, secretory cells were taken. They were cracked open and their cytosol was removed and cytosols from cells lacking a regulated secretory pathway or cytosol from bovine brain extract and other cells which have a regulated secretory pathway were supplemented to these cells. Following an addition of calcium, it was found that for those cells which received cytosols from cells lacking a secretory pathway, calcium did not increase, was not the process of regulated exocytosis could not be reconstituted effectively. So when bovine brain extract was added to these cracked cells and calcium was added to them after that, this effectively, the process of regulated exocytosis could be reconstituted in these cells. So this led to an exhaustive search for all protein factors in bovine brain extract that were responsible for these effects. So I had narrowed down to a 155 kilodalton protein which was later termed CAHPS. And it was this which was, it was seen which was responsible for this effect. So since CAHPS is a protein in verdant secretion, it is pertinent to ask whether the activity of CAHPS depends on the activity of snare proteins and investigators wanted to identify that in an in vitro system. And for this reason a lipid fusion assay was used. So two sets of liposomes were made. First one which contained a quench set of fluorescently labeled phospholipids, NBDPE and RHPE. And they also had inserted into their memory the vesicular snare protein bag too. So when these two lipids, the NBDPE and RHPE are placed very close to each other, the fluorescence of NBDPE can be quenched by a phenomenon called FRET or fluorescence resonance energy transfer which is explicitly sensitive to the distance between these two different kinds of lipids. So a second set of liposomes was also made of the same dimensions but lacking fluorescently labeled lipids and into its membrane were inserted the target membrane snare syntax in SNAP-25. So when there's a fusion between these two liposomes, these two sets of liposomes mediated by these snare proteins, the content of lipids from both these kinds of liposomes makes and there's twofold dilution of the fluorescent lipids that were originally present in just one liposome. So because of this dilution, the average distance between the lipids increased and FRET no longer happens because it exceeds the critical distance for FRET. Because of that, the fluorescence due to NBDPE is recovered and we can follow this up as an assay for that zone fusion. From some experiments it was identified that when CAHPS was added to this system there was an increased amount of lipid fusion which was seen and this indicated that CAHPS was accelerating liposome fusion in a snare dependent manner and also led to other additional questions suggest does CAHPS directly interact with snare proteins even though its function is dependent on snare proteins. So the next set of studies were conducted to address this question and they also used an in vitro technique using liposomes. So liposomes with snare proteins inserted to their membranes were taken and CAHPS was incubated along with these liposomes and this was subjected to density gradient centrifugation. So what we observe is that what we expect to see is that if CAHPS directly interacts with snare proteins which are inserted to liposomes it would be found in the liposome bump fractions whereas if it does not interact it would be found in the unbump fractions unbump protein fraction following density gradient centrifugation. Now what we see is that indeed CAHPS is found along with liposomes which indicates that it might interact directly with liposomes and secondly it again in turn raises another question is which region of CAHPS interacts with snare proteins? To address this truncation of the full leg CAHPS protein was created and all of these were again subjected to the same kind of experiment as I have just shown in the previous slide which is they were made to mount to snare proteins inserted into liposomes and it was seen that while most of these CAHPS fragments were incapable of binding to snare proteins there was a segment which indeed had the same binding property than the full leg CAHPS and it's localized to the LHD domain or the monk protein homology domain in the CAHPS protein but given that the length of this was substantially big it's a 69 residue long segment we still don't know the exact identity of residues which are involved in binding snare proteins so this is where my project found its context so the first part of my project was to mutate two consecutive segments within the 69 residue long fragment to other means and then observe the function of these mutant constructs in a secretion assay coming to look at it we find that a sequence that I'm going to be is there are some residues within these five segment regions which are conserved and whose functions are not known so they demand study and the second part of my project was to mutate another conserved five residue segment in the C terminal region of CAHPS which just preceded a dense core medical localization sequence the reason why this was of interest is because again the segment which has been boxed in red is conserved between several species and again we don't know what precise function it performs the experiment design that we adopted to answer these questions involved first constructing mutants of the CAHPS protein and flowing them into a retrosin protein vector TAC RFP by standard side directed mutated essence protocols followed by transfecting these constructs into HEC 293FT cells and then we observed the protein expression mutated retrosin's microscopy so subject to the condition that these proteins wouldn't aggregate when seen in the microscope we would then express we would then clone them again into a construct that would append a C terminal hexase gene tag to it and transfect them into hexase again following that we would purify proteins using standard nickel NTA column chromatography techniques and then test the activity of these in a permeable cell norepinephrine secretion assay a slight detour from the central focus of my experiments we also looked at the expression patterns of some CAHPS MHT1 domain mutants that had registered in the lab had previously made and the reason we were interested or rather he was interested in this part of CAHPS is because it exhibited some similarity to a snare motif and again these were very consumer residues and we didn't exactly know which functions it performed so upon transfection of these mutant constructs to HEC cells we found out that most of them would concentrate into NTA structures in cells so we were not exactly sure whether they were aggregates but given that the protein wasn't exactly soluble we did not go ahead with the NTA assay because effects due to misphoneded protein it could be a potentially misphoneded protein could not be ruled out when we observed all the other results so these are the other six other three mutants that we also tested and these mutants were also made by mutating these residues to alanines and the same kind of pattern was observed for all of them Coming to the mutants that I had talked about making previously so these were the expression patterns of the CAHPS 952-956 and 957-961 mutants both of these are within the macromology domain and it turns out that the CAHPS 952-956 alanine mutant aggregated again so since it was not soluble we did not use it for the RDE analysis but the 957-61 alanine mutant and the C-terminal mutant the RIDEG they seemed to express soluble proteins so we went ahead and analyzed them