In vitro biocompatibility evaluation of functional electrically stimulating microelectrodes on primary glia.

Neural interfacing devices interact with the central nervous system to alleviate functional deficits arising from disease or injury. This often entails the use of invasive microelectrode implants that elicit inflammatory responses from glial cells and leads to loss of device function. Previous work focused on improving implant biocompatibility by modifying electrode composition; here, we investigated the direct effects of electrical stimulation on glial cells at the electrode interface. A high-throughput in vitro system that assesses primary glial cell response to biphasic stimulation waveforms at 0 mA, 0.15 mA, and 1.5 mA was developed and optimized. Primary mixed glial cell cultures were generated from heterozygous CX3CR-1+/EGFP mice, electrically stimulated for 4 h/day over 3 days using 75 µm platinum-iridium microelectrodes, and biomarker immunofluorescence was measured. Electrodes were then imaged on a scanning electron microscope to assess sustained electrode damage. Fluorescence and electron microscopy analyses suggest varying degrees of localized responses for each biomarker assayed (Hoescht, EGFP, GFAP, and IL-1ß), a result that expands on comparable in vivo models. This system allows for the comparison of a breadth of electrical stimulation parameters, and opens another avenue through which neural interfacing device developers can improve biocompatibility and longevity of electrodes in tissue.

Short-chain fatty and carboxylic acid changes associated with fecal microbiota transplant communally influence microglial inflammation.

The intestinal microbiota has been proposed to influence human mental health and cognition through the gut-brain axis. Individuals experiencing recurrent Clostridioides difficile infection (rCDI) frequently report depressive symptoms, which are improved after fecal microbiota transplantation (FMT); however, mechanisms underlying this association are poorly understood. Short-chain fatty acids and carboxylic acids (SCCA) produced by the intestinal microbiota cross the blood brain barrier and have been proposed to contribute to gut-brain communication. We hypothesized that changes in serum SCCA measured before and after successful FMT for rCDI influences the inflammatory response of microglia, the resident immune cells of the central nervous system. Serum SCCA were quantified using gas chromatography-mass spectroscopy from 38 patients who participated in a randomized trial comparing oral capsule-vs colonoscopy-delivered FMT for rCDI, and quality of life was assessed by SF-36 at baseline, 4, and 12 weeks after FMT treatment. Successful FMT was associated with improvements in mental and physical health, as well as significant changes in a number of circulating SCCA, including increased butyrate, 2-methylbutyrate, valerate, and isovalerate, and decreased 2-hydroxybutyrate. Primary cultured microglia were treated with SCCA and the response to a pro-inflammatory stimulus was measured. Treatment with a combination of SCCA based on the post-FMT serum profile, but not single SCCA species, resulted in significantly reduced inflammatory response including reduced cytokine release, reduced nitric oxide release, and accumulation of intracellular lipid droplets. This suggests that both levels and diversity of SCCA may be an important contributor to gut-brain communication.

Pleiotrophin Signals Through ALK Receptor to Enhance the Growth of Neurons in the Presence of Inhibitory Chondroitin Sulfate Proteoglycans.

Chondroitin sulfate proteoglycans (CSPGs), one of the major extracellular matrix components of the glial scar that surrounds central nervous system (CNS) injuries, are known to inhibit the regeneration of neurons. This study investigated whether pleiotrophin (PTN), a growth factor upregulated during early CNS development, can overcome the inhibition mediated by CSPGs and promote the neurite outgrowth of neurons in vitro. The data showed that a CSPG matrix inhibited the outgrowth of neurites in primary cortical neuron cultures compared to a control matrix. PTN elicited a dose-dependent increase in the neurite outgrowth even in the presence of the growth inhibitory CSPG matrix, with optimal growth at 15 ng mL-1 of PTN (114.8% of neuronal outgrowth relative to laminin control). The growth-promoting effect of PTN was blocked by inhibition of the receptor anaplastic lymphoma kinase (ALK) by alectinib in a dose-dependent manner. Neurite outgrowth in the presence of this CSPG matrix was induced by activation of the protein kinase B (AKT) pathway, a key downstream mediator of ALK activation. This study identified PTN as a dose-dependent regulator of neurite outgrowth in primary cortical neurons cultured in the presence of a CSPG matrix and identified ALK activation as a key driver of PTN-induced growth.

The effects of electrical stimulation on glial cell behaviour.

Neural interface devices interact with the central nervous system (CNS) to substitute for some sort of functional deficit and improve quality of life for persons with disabilities. Design of safe, biocompatible neural interface devices is a fast-emerging field of neuroscience research. Development of invasive implant materials designed to directly interface with brain or spinal cord tissue has focussed on mitigation of glial scar reactivity toward the implant itself, but little exists in the literature that directly documents the effects of electrical stimulation on glial cells. In this review, a survey of studies documenting such effects has been compiled and categorized based on the various types of stimulation paradigms used and their observed effects on glia. A hybrid neuroscience cell biology-engineering perspective is offered to highlight considerations that must be made in both disciplines in the development of a safe implant. To advance knowledge on how electrical stimulation affects glia, we also suggest experiments elucidating electrochemical reactions that may occur as a result of electrical stimulation and how such reactions may affect glia. Designing a biocompatible stimulation paradigm should be a forefront consideration in the development of a device with improved safety and longevity.

Inflammatory Cytokine Profile and Plasticity of Brain and Spinal Microglia in Response to ATP and Glutamate.

Microglia are the primary cells in the central nervous system that identify and respond to injury or damage. Such a perturbation in the nervous system induces the release of molecules including ATP and glutamate that act as damage-associated molecular patterns (DAMPs). DAMPs are detected by microglia, which then regulate the inflammatory response in a manner sensitive to their surrounding environment. The available data indicates that ATP and glutamate can induce the release of pro inflammatory factors TNF (tumor necrosis factor), IL-1ß (interleukin 1 beta), and NO (nitric oxide) from microglia. However, non-physiological concentrations of ATP and glutamate were often used to derive these insights. Here, we have compared the response of spinal cord microglia (SM) relative to brain microglia (BM) using physiologically relevant concentrations of glutamate and ATP that mimic injured conditions in the central nervous system. The data show that ATP and glutamate are not significant modulators of the release of cytokines from either BM or SM. Consistent with previous studies, spinal microglia exhibited a general trend toward reduced release of inflammatory cytokines relative to brain-derived microglia. Moreover, we demonstrate that the responses of microglia to these DAMPs can be altered by modifying the biochemical milieu in their surrounding environment. Preconditioning brain derived microglia with media from spinal cord derived mixed glial cultures shifted their release of IL-1ß and IL-6 to a less inflammatory phenotype consistent with spinal microglia.

Applying a novel 3D hydrogel cell culture to investigate activation of microglia due to rotational kinematics associated with mild traumatic brain injury.

Many investigations on mild traumatic brain injury (mTBI) aim to further understand how cells in the brain react to the mechanical forces associated with the injury. While it is known that rapid head rotation is a mechanism contributing to mTBI, establishing definitive thresholds for head rotation has proved challenging. One way to advance determining mechanisms and thresholds for injury is through in vitro models. Here, an apparatus has been designed that is capable of delivering rotational forces to three-dimensional (3D) hydrogel cell cultures. Using an in vitro model, we test the hypothesis that rotational kinematics can activate microglia suspended in a 3-dimensional mixed glia environment (absent neurons). The impact apparatus was able to deliver peak angular velocities of approximately 45 rad/s, a magnitude for angular velocity that in select literature is associated with diffuse brain injury. However, no measurable glial cell reactivity was observed in response to the rotational kinematics through any of the chosen metrics (nitric oxide, pro-inflammatory cytokine release and proportion of amoeboid activated microglia). The results generated from this study suggest that rotation of the glia alone did not cause activation - in future work we will investigate the effect of neuronal contributions in activating glia.

In Vitro Priming and Hyper-Activation of Brain Microglia: an Assessment of Phenotypes.

Microglia are the resident immune cells of the central nervous system that mediate the life and death of nervous tissue. During normal function, they exhibit a surveying phenotype and maintain vital functions in nervous tissue. In the event of injury or disease, chronic inflammation can result, wherein microglia develop a hyper-activated phenotype, shed their regenerative function, actively kill contiguous cells, and can partition injured tissue by initiating scar formation. With recoverable injury, microglia can develop a primed phenotype, where they appear to recover from an inflammatory event, but are limited in their support functions and show inappropriate responses to future injury often associated with neurodegenerative disorders. These microglial phenotypes were acutely recreated in vitro with potent pro- and anti-inflammatory treatments. Primary cultured microglia or mixed glia (microglia, astrocytes, and oligodendrocytes) were treated for 6 h with lipopolysaccharide (LPS). Recovery from an inflammatory state was modeled with 18-h treatment of the anti-inflammatory steroid dexamethasone. The cells were then treated for 24 h with interferon gamma (IFNγ) to detect inflammatory memory after recovery. Surveying was best represented in the untreated vehicle (Veh) cases and was characterized by negligible secretion of pro-inflammatory factors, limited expression of immune proteins such as induced nitric oxide synthase (iNOS), major histocompatibility complex class II (MHCII), relatively high expression of brain-derived and glial-derived neurotrophic factors (BDNF and GDNF), and thinly branched smaller microglia. Activation was noted in the LPS- and IFNγ-treated microglia with increased cytokines, NO, NGF, iNOS, proliferation, phagocytosis, reduced BDNF, and flattened round amoeboid-shaped microglia. Priming was observed to be an incomplete surveying restoration using dexamethasone from an activation comparison of LPS, IFNγ, and LPS/IFNγ. Dexamethasone treatments resulted in the most profound dysregulation of expression of NO, TNF, IL-1ß, NGF, CD68, and MHCII as well as ramified morphology and uptake of myelin. These findings suggest microglial priming and hyper-activation may be effectively modeled in vitro to allow mechanistic investigations into these key cellular phenotypes.

Fluoxetine and its metabolite norfluoxetine induce microglial apoptosis.

Inflammatory insult to the central nervous system (CNS) can lead to development of depression, and subsequently depression is the most frequent psychiatric comorbidity following ischemic stroke, often limiting recovery and rehabilitation in patients. The initiators of inflammatory pathways in the CNS are microglia activated in response to acute ischemic stress, and anti-depressants have been shown to have anti-inflammatory effects in the CNS, promoting neuronal survival following ischemic insult. We have previously shown that the selective serotonin reuptake inhibitors (SSRIs) fluoxetine and citalopram promote neuronal survival after oxygen-glucose deprivation, an in vitro model of ischemia, by attenuating the release of glutamate and D-serine from activated microglia. Interestingly, we found that fluoxetine-treated microglial cultures contained fewer numbers of cells compared to other groups and hypothesized that fluoxetine and citalopram attenuated the release of glutamate and D-serine by promoting the apoptosis of microglia. The present study aimed to test and compare antidepressants from three distinct classes (tricyclics, monoamine oxidase inhibitors, and SSRIs) on microglial apoptosis. Primary microglia were treated with 1 µg/mL lipopolysaccharide and/or 10 µM antidepressants, and various apoptotic markers were assayed. Fluoxetine and its metabolite norfluoxetine decreased protein levels in cell lysates, decreased cell viability of microglia, and increased the expression of the apoptotic marker cleaved-caspase 3 in microglia. Live/dead nuclear staining also showed that fluoxetine- or norfluoxetine-treated cultures contained greater numbers of dying microglial cells compared to vehicle-treated cultures. Cultures treated with citalopram, phenelzine, or imipramine showed no evidence of inducing microglial apoptosis. Our results demonstrate that fluoxetine and norfluoxetine induce the apoptotic death of microglia, which may serve as a mechanism to attenuate the release of glutamate and D-serine from activated microglia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Microglial Function during Glucose Deprivation: Inflammatory and Neuropsychiatric Implications.

Inflammation is increasingly recognized as a contributor to the pathophysiology of neuropsychiatric disorders, including depression, anxiety disorders and autism, though the factors leading to contextually inappropriate or sustained inflammation in pathological conditions are yet to be elucidated. Microglia, as the key mediators of inflammation in the CNS, serve as likely candidates in initiating pathological inflammation and as an ideal point of therapeutic intervention. Glucose deprivation, as a component of the pathophysiology of ischemia or occurring transiently in diabetes, may serve to modify microglial function contributing to inflammatory injury. To this end, primary microglia were cultured from postnatal rat brain and subject to glucose deprivation in vitro. Microglia were characterized for their proliferation, phagocytic function and secretion of inflammatory factors, and tested for their capacity to respond to a potent inflammatory stimulus. In the absence of glucose, microglia remained capable of proliferation, phagocytosis and inflammatory activation and showed increased release of inflammatory factors after presentation of an inflammatory stimulus. Glucose-deprived microglia demonstrated increased phagocytic activity and decreased accumulation of lipids in lipid droplets over a 48-h timecourse, suggesting they may use scavenged lipids as a key alternate energy source during metabolic stress. In the present manuscript, we present novel findings that glucose deprivation may sensitize microglial release of inflammatory mediators and prime microglial functions for both survival and inflammatory roles, which may contribute to psychiatric comorbidities of ischemia, diabetes and/or metabolic disorder.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Peptide modification of polyimide-insulated microwires: Towards improved biocompatibility through reduced glial scarring.

The goal of this study is to improve the integration of implanted microdevices with tissue in the central nervous system (CNS). The long-term utility of neuroprosthetic devices implanted in the CNS is affected by the formation of a scar by resident glial cells (astrocytes and microglia), limiting the viability and functional stability of the devices. Reduction in the proliferation of glial cells is expected to enhance the biocompatibility of devices. We demonstrate the modification of polyimide-insulated microelectrodes with a bioactive peptide KHIFSDDSSE. Microelectrode wires were functionalized with (3-aminopropyl) triethoxy silane (APTES); the peptide was then covalently bonded to the APTES. The soluble peptide was tested in 2D mixed cultures of astrocytes and microglia, and reduced the proliferation of both cell types. The interactions of glial cells with the peptide-modified wires was then examined in 3D cell-laden hydrogels by immunofluorescence microscopy. As expected for uncoated wires, the microglia were first attracted to the wire (7days) followed by astrocyte recruitment and hypertrophy (14days). For the peptide-treated wires, astrocytes coated the wires directly (24h), and formed a thin, stable coating without evidence of hypertrophy, and the attraction of microglia to the wire was significantly reduced. The results suggest a mechanism to improve tissue integration by promoting uniform coating of astrocytes on a foreign body while lessening the reactive response of microglia. We conclude that the bioactive peptide KHIFSDDSSE may be effective in improving the biocompatibility of neural interfaces by both reducing acute glial reactivity and generating stable integration with tissue. STATEMENT OF SIGNIFICANCE: The peptide KHIFSDDSSE has previously been shown in vitro to both reduce the proliferation of astrocytes, and to increase the adhesion of astrocyte to glass substrates. Here, we demonstrate a method to apply uniform coatings of peptides to microwires, which could readily be generalized to other peptides and surfaces. We then show that when peptide-modified wires are inserted into 3D cell-laden hydrogels, the normal cellular reaction (microglial activation followed by astrocyte recruitment and hypertrophy) does not occur, rather astrocytes are attracted directly to the surface of the wire, forming a relatively thin and uniform coating. This suggests a method to improve tissue integration of implanted devices to reduce glial scarring and ultimately reduce failure of neural interfaces.

Statin treatment affects cytokine release and phagocytic activity in primary cultured microglia through two separable mechanisms.

BACKGROUND: As the primary immune cells of the central nervous system, microglia contribute to development, homeostasis, and plasticity of the central nervous system, in addition to their well characterized roles in the foreign body and inflammatory responses. Increasingly, inappropriate activation of microglia is being reported as a component of inflammation in neurodegenerative and neuropsychiatric disorders. The statin class of cholesterol-lowering drugs have been observed to have anti-inflammatory and protective effects in both neurodegenerative diseases and ischemic stroke, and are suggested to act by attenuating microglial activity. RESULTS: We sought to investigate the effects of simvastatin treatment on the secretory profile and phagocytic activity of primary cultured rat microglia, and to dissect the mechanism of action of simvastatin on microglial activity. Simvastatin treatment altered the release of cytokines and trophic factors from microglia, including interleukin-1-ß, tumour necrosis factor-α, and brain derived neurotrophic factor in a cholesterol-dependent manner. Conversely, simvastatin inhibited phagocytosis in microglia in a cholesterol-independent manner. CONCLUSIONS: The disparity in cholesterol dependence of cytokine release and phagocytosis suggests the two effects occur through distinct molecular mechanisms. These two pathways may provide an opportunity for further refinement of pharmacotherapies for neuroinflammatory, neurodegenerative, and neuropsychiatric disorders.

Hyaluronic acid-based 3D culture model for in vitro testing of electrode biocompatibility.

This work describes the development of a robust and repeatable in vitro 3D culture model of glial scarring, which may be used to evaluate the foreign body response to electrodes and other implants in the central nervous system. The model is based on methacrylated hyaluronic acid, a hydrogel that may be photopolymerized to form an insoluble network. Hydrogel scaffolds were formed at four different macromer concentrations (0.50, 0.75, 1.00, and 1.50% (w/v)). As expected, the elastic modulus of the scaffolds increased with increasing macromer weight fraction. Adult rat brain tested under identical conditions had an elastic modulus range that spanned the elastic modulus of both the 0.50 and 0.75% (w/v) hydrogel samples. Gels formed with higher macromer weight fraction had decreased equilibrium swelling ratio and visibly thicker pore walls relative to gels formed with lower macromer weight fractions. Mixed glial cells (microglia and astrocytes) were then encapsulated in the HA scaffolds. Viability of the mixed cultures was most stable at a cell density of 1 × 10(7) cells/mL. Cell viability at the highest macromer weight fraction tested (1.50% (w/v)) was significantly lower than other tested gels (0.50, 0.75 and 1.00% (w/v)). The inflammatory response of microglia and astrocytes to a microelectrode inserted into the scaffold was assessed over a period of 2 weeks and closely represented that reported in vivo. Microglia responded first to the electrode (increased cell density at the electrode, and activated morphology) followed by astrocytes (appeared to line the electrode in a manner similar to glial scarring). All together, these results demonstrate the potential of the 3D in vitro model system to assess glial scarring in a robust and repeatable manner.

Anionic lipids in Ca(2+)-triggered fusion.

Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca(2+)-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca(2+)-sensing and even bind Ca(2+) directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca(2+) sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined.

Cholesterol, regulated exocytosis and the physiological fusion machine.

Exocytosis is a highly conserved and essential process. Although numerous proteins are involved throughout the exocytotic process, the defining membrane fusion step appears to occur through a lipid-dominated mechanism. Here we review and integrate the current literature on protein and lipid roles in exocytosis, with emphasis on the multiple roles of cholesterol in exocytosis and membrane fusion, in an effort to promote a more molecular systems-level view of the as yet poorly understood process of Ca2+-triggered membrane mergers.

Identifying critical components of native Ca2+-triggered membrane fusion. Integrating studies of proteins and lipids.

Ca(2+)-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca(2+)-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayer merger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca(2+) sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca(2+) sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca(2+) sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca(2+)-triggered membrane fusion.

Specific lipids supply critical negative spontaneous curvature--an essential component of native Ca2+-triggered membrane fusion.

The Ca(2+)-triggered merger of two apposed membranes is the defining step of regulated exocytosis. CHOL is required at critical levels in secretory vesicle membranes to enable efficient, native membrane fusion: CHOL-sphingomyelin enriched microdomains organize the site and regulate fusion efficiency, and CHOL directly supports the capacity for membrane merger by virtue of its negative spontaneous curvature. Specific, structurally dissimilar lipids substitute for CHOL in supporting the ability of vesicles to fuse: diacylglycerol, alphaT, and phosphatidylethanolamine support triggered fusion in CHOL-depleted vesicles, and this correlates quantitatively with the amount of curvature each imparts to the membrane. Lipids of lesser negative curvature than cholesterol do not support fusion. The fundamental mechanism of regulated bilayer merger requires not only a defined amount of membrane-negative curvature, but this curvature must be provided by molecules having a specific, critical spontaneous curvature. Such a local lipid composition is energetically favorable, ensuring the necessary "spontaneous" lipid rearrangements that must occur during native membrane fusion-Ca(2+)-triggered fusion pore formation and expansion. Thus, different fusion sites or vesicle types can use specific alternate lipidic components, or combinations thereof, to facilitate and modulate the fusion pore.

Copper (II) sulfate charring for high sensitivity on-plate fluorescent detection of lipids and sterols: quantitative analyses of the composition of functional secretory vesicles.

A wide range of methods exist for the on-plate detection of lipids resolved by thin layer chromatography. Fluorescence generally offers improvements in sensitivity over methods that use colorimetric or simple densitometric detection. In this paper, we report that a classic cupric sulfate charring protocol produces a fluorescent signal that sensitively and quantitatively detects a wide range of phospholipids, neutral lipids, and sterols after automated, multi-development high performance thin layer chromatography. The measured lower limits of detection and quantification, respectively, were, on average, 80 and 210 pmol for phospholipids and 43 fmol and 8.7 pmol for sterols. The simple, inexpensive, and highly sensitive approach described here was used to quantitatively analyze the lipid and sterol composition of sea urchin cortical vesicles, a stage-specific model system used to study the mechanism of regulated membrane fusion.

Assessing detection methods for gel-based proteomic analyses.

Proteomic analyses using two-dimensional gel electrophoresis (2DE) depend heavily upon the quality of protein stains for sensitive detection. Indeed, detection rather than protein resolution is likely a current limiting factor in 2DE. The recent development of fluorescent protein stains has dramatically improved the sensitivity of in-gel protein detection and has enabled more accurate protein quantification. Here, we have evaluated the overall quality and relative cost of five commercially available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo, and the most commonly used stain, Sypro Ruby (SR). All stains were found to be statistically comparable with regard to number of protein spots detected, but SR was superior with regard to fluorophore stability (e.g., capacity for repeated use of the stain solution). Notably, colloidal Coomassie Blue was also found to be comparable to SR when detected using an infrared fluorescence imaging system rather than standard densitometry. Thus, depending on available equipment and operating budgets, there are at least two high-sensitivity alternatives to achieve the best currently available in-gel protein detection: Sypro Ruby or Coomassie Blue.

Cholesterol facilitates the native mechanism of Ca2+-triggered membrane fusion.

The process of regulated exocytosis is defined by the Ca2+-triggered fusion of two apposed membranes, enabling the release of vesicular contents. This fusion step involves a number of energetically complex steps and requires both protein and lipid membrane components. The role of cholesterol has been investigated using isolated release-ready native cortical secretory vesicles to analyze the Ca2+-triggered fusion step of exocytosis. Cholesterol is a major component of vesicle membranes and we show here that selective removal from membranes, selective sequestering within membranes, or enzymatic modification causes a significant inhibition of the extent, Ca2+ sensitivity and kinetics of fusion. Depending upon the amount incorporated, addition of exogenous cholesterol to cholesterol-depleted membranes consistently recovers the extent, but not the Ca2+ sensitivity or kinetics of fusion. Membrane components of comparable negative curvature selectively recover the ability to fuse, but are unable to recover the kinetics and Ca2+ sensitivity of vesicle fusion. This indicates at least two specific positive roles for cholesterol in the process of membrane fusion: as a local membrane organizer contributing to the efficiency of fusion, and, by virtue of its intrinsic negative curvature, as a specific molecule working in concert with protein factors to facilitate the minimal molecular machinery for fast Ca2+-triggered fusion.