Coomassie staining provides routine (sub)femtomole in‐gel detection of intact proteoforms: Expanding opportunities for genuine Top‐down Proteomics

Modified colloidal Coomassie Brilliant Blue (cCBB) staining utilising a novel destain protocol and near‐infrared fluorescence detection (nIRFD) rivals the in‐gel protein detection sensitivity (DS) of SYPRO Ruby. However, established DS estimates are likely inaccurate in terms of 2DE‐resolved proteoform ‘spots’ since DS is routinely measured from comparatively diffuse protein ‘bands’ following wide‐well 1DE. Here, cCBB DS for 2DE‐based proteomics was more accurately determined using narrow‐well 1DE. As precise estimates of protein standard monomer concentrations are essential for accurate quantitation, coupling UV absorbance with gel‐based purity assessments is described. Further, as cCBB is compatible with both nIRFD and densitometry, the impacts of imaging method (and image resolution) on DS were assessed. Narrow‐well 1DE enabled more accurate quantitation of cCBB DS for 2DE, achieving (sub)femtomole DS with either nIRFD or densitometry. While densitometry offers comparative simplicity and affordability, nIRFD has the unique potential for enhanced DS with Deep Imaging. Higher‐resolution nIRFD also improved analysis of a 2DE‐resolved proteome, surpassing the DS of standard nIRFD and densitometry, with nIRFD Deep Imaging further maximising proteome coverage. cCBB DS for intact proteins rivals that of mass spectrometry (MS) for peptides in complex mixtures, reaffirming that 2DE‐MS currently provides the most routine, broadly applicable, robust, and information‐rich Top‐down approach to Discovery Proteomics.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141479/1/elps6323.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141479/2/elps6323_am.pd

Examining phospho-modulation of regulated exocytosis and the release-ready state of native secretory vesicles

Regulated exocytosis enables numerous critical cellular functions such as wound healing, fertilisation, and neurotransmission. In most eukaryotic cells, proteins and other compounds that need to be secreted into the extracellular space are sorted into secretory vesicles, which then translocate to the plasma membrane where they dock and undergo priming reactions. Upon elevation of intracellular calcium levels (caused by a physiological stimulus), release-ready secretory vesicles fuse with the plasma membrane and release their contents. The late, calcium regulated steps of exocytosis (including docking, priming, and fusion) have been studied using a variety of model systems; in particular, studies utilising sea urchin eggs, cell surface complexes (large sheets of plasma membrane with endogenous, docked vesicles) and isolated cortical vesicles have provided numerous original insights into fundamental, conserved molecular mechanisms (Reviewed in Chapter One, and methods pertaining to the use of the model are detailed in Chapter Two). In Chapter Three, using intact eggs and cell surface complexes, I test the hypothesis that the fully primed, release-ready state is one of stable hemifusion (i.e. with the contacting monolayers of the vesicle and plasma membrane having already merged). Studies utilising fluorescent dyes with well-defined membrane properties and membrane permeable cationic amphiphiles capable of disrupting hemifusion intermediates indicate that, contrary to current opinion, the release-ready state is dynamic, and capable of transiently transitioning between hemifusion and close bilayer apposition. The phosphorylation of proteins and lipids has been implicated in modulating all stages of regulated exocytosis, and in Chapter Four I use cortical vesicles and minimised docking and membrane fusion assays to screen a range of well characterised small molecule modulators of phosphorylation. Data from the screen implicate phosphatases and casein kinase 2 in modulating the calcium sensitivity of fusion, and sphingosine kinase in modulating the ability to fuse. As detailed in Chapter Five, combined molecular and functional analysis of cell surface complexes and cortical vesicles with altered membrane sphingolipid levels confirmed that a critical balance of sphingolipids and sphingolipid metabolites is needed to maintain efficient docking, calcium sensitivity, and capacity to fuse

Application of High-Throughput Assays to Examine Phospho-Modulation of the Late Steps of Regulated Exocytosis

Abstract: Regulated exocytosis enables a range of physiological functions including neurotransmission, and the late steps (i.e., docking, priming and Ca2+-triggered membrane fusion) are modulated by a highly conserved set of proteins and lipids. Many of the molecular components and biochemical interactions required have been identified; the precise mechanistic steps they modulate and the biochemical interactions that need to occur across steps are still the subject of intense investigation. Particularly, although the involvement of phosphorylation in modulating exocytosis has been intensively investigated over the past three decades, it is unclear which phosphorylation events are a conserved part of the fundamental fusion mechanism and/or serve as part of the physiological fusion machine (e.g., to modulate Ca2+ sensitivity). Here, the homotypic fusion of cortical vesicles was monitored by utilizing new high-throughput, cost-effective assays to assess the influence of 17 small molecule phospho-modulators on docking/priming, Ca2+ sensitivity and membrane fusion. Specific phosphatases and casein kinase 2 are implicated in modulating the Ca2+ sensitivity of fusion, whereas sphingosine kinase is implicated in modulating the ability of vesicles to fuse. These results indicate the presence of multiple kinases and phosphatases on the vesicles and critical phosphorylation sites on vesicle membrane proteins and lipids that directly influence late steps of regulated exocytosis

Sphingolipids modulate docking, Ca2+ sensitivity and membrane fusion of native cortical vesicles

Docking, priming, and membrane fusion of secretory vesicles (i.e. regulated exocytosis) requires lipids and proteins. Sphingolipids, in particular, sphingosineand sphingosine-1-phosphate, have been implicated in the modulation of exocytosis. However, the specific exocytotic steps that sphingolipids modulate and the enzymes that regulate sphingolipid concentrations on native secretory vesicle membranes remain unknown. Here we use tightly coupled functional and molecular analyses of fusion-ready cell surface complexes and cortical vesicles isolated from oocytes to assess the role of sphingolipids in the late, Ca2+-triggered steps of exocytosis. The molecular changes resulting from treatments with sphingolipid modifying compounds coupled with immunoblotting analysis revealed the presence of sphingosine kinase on native vesicles; the presence of a sphingosine-1-phosphate phosphatase is also indicated. Changes in sphingolipid concentrations on vesicles altered their docking/priming, Ca2+-sensitivity, and ability to fuse, indicating that sphingolipid concentrations are tightly regulated and maintained at optimal levels and ratios to ensure efficient exocytosis

Critical role of cortical vesicles in dissecting regulated exocytosis : overview of insights into fundamental molecular mechanisms

Regulated exocytosis is one of the defining features of eukaryotic cells, underlying many conserved and essential functions. Definitively assigning specific roles to proteins and lipids in this fundamental mechanism is most effectively accomplished using a model system in which distinct stages of exocytosis can be effectively separated. Here we discuss the establishment of sea urchin cortical vesicle fusion as a model to study regulated exocytosis-a system in which the docked, release-ready, and late Ca2+-triggered steps of exocytosis are isolated and can be quan-titatively assessed using the rigorous coupling of functional and molecular assays. We provide an overview of the in-sights this has provided into conserved molecular mecha-nisms and how these have led to and integrate with findings from other regulated exocytotic cells

The sea urchin egg and cortical vesicles as model systems to dissect the fast, Ca2+-triggered steps of regulated exocytosis

Exocytosis is a fundamental process utilized by all eukaryotic organisms; this elegantly efficient process mediates such diverse functions as fertilization, synaptic transmission, and wound healing. Membrane fusion, the defining step of this process, has been well conserved through evolution. However, the mechanisms defining the priming, docking, and merger of two apposed native bilayer membranes have not been fully elucidated. Sea urchin cortical vesicles are locked at a stage just prior to Ca2+-triggered membrane fusion and are thus an ideal system for fully defining the mechanisms underlying this process. Here we describe detailed methods to isolate these native secretory vesicles, monitor the fusion process, assess the minimal essential biochemical components, and identify their ultrastructural interactions that define the triggered exocytotic pathway

Drosophila development, physiology, behavior, and lifespan are influenced by altered dietary composition

© 2017 Taylor & Francis. Diet profoundly influences the behavior of animals across many phyla. Despite this, most laboratories using model organisms, such as Drosophila, use multiple, different, commercial or custom-made media for rearing their animals. In addition to measuring growth, fecundity and longevity, we used several behavioral and physiological assays to determine if and how altering food media influence wild-type (Canton S) Drosophila melanogaster, at larval, pupal, and adult stages. Comparing 2 commonly used commercial food media we observed several key developmental and morphological differences. Third-instar larvae and pupae developmental timing, body weight and size, and even lifespan significantly differed between the 2 diets, and some of these differences persisted into adulthood. Diet was also found to produce significantly different thermal preference, locomotory capacity for geotaxis, feeding rates, and lower muscle response to hormonal stimulation. There were no differences, however, in adult thermal preferences, in the number or viability of eggs laid, or in olfactory learning and memory between the diets. We characterized the composition of the 2 diets and found particularly significant differences in cholesterol and (phospho)lipids between them. Notably, diacylglycerol (DAG) concentrations vary substantially between the 2 diets, and may contribute to key phenotypic differences, including lifespan. Overall, the data confirm that 2 different diets can profoundly influence the behavior, physiology, morphology and development of wild-type Drosophila, with greater behavioral and physiologic differences occurring during the larval stages

Vesicle cholesterol controls exocytotic fusion pore

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.</p