Cytoskeletal Transport Research Articles

Dopamine internalization via Uptake2 and stimulation of intracellular D5-receptor-dependent calcium mobilization and CDP-diacylglycerol signaling.

Dopamine stimulates CDP-diacylglycerol biosynthesis through D1-like receptors, particularly the D5 subtype most of which is intracellularly localized. CDP-diacylglycerol regulates phosphatidylinositol-4,5-bisphosphate-dependent signaling cascades by serving as obligatory substrate for phosphatidylinositol biosynthesis. Here, we used acute and organotypic brain tissues and cultured cells to explore the mechanism by which extracellular dopamine acts to modulate intracellular CDP-diacylglycerol. Dopamine stimulated CDP-diacylglycerol in organotypic and neural cells lacking the presynaptic dopamine transporter, and this action was selectively mimicked by D1-like receptor agonists SKF38393 and SKF83959. Dopaminergic CDP-diacylglycerol stimulation was blocked by decynium-22 which blocks Uptake2-like transporters and by anti-microtubule disrupters of cytoskeletal transport, suggesting transmembrane uptake and guided transport of the ligands to intracellular sites of CDP-diacylglycerol regulation. Fluorescent or radiolabeled dopamine was saturably transported into primary neurons or B35 neuroblastoma cells expressing the plasmamembrane monoamine transporter, PMAT. Microinjection of 10-nM final concentration of dopamine into human D5-receptor-transfected U2-OS cells rapidly and transiently increased cytosolic calcium concentrations by 316%, whereas non-D5-receptor-expressing U2-OS cells showed no response. Given that U2-OS cells natively express PMAT, bath application of 10μM dopamine slowly increased cytosolic calcium in D5-expressing cells. These observations indicate that dopamine is actively transported by a PMAT-implicated Uptake2-like mechanism into postsynaptic-type dopaminoceptive cells where the monoamine stimulates its intracellular D5-type receptors to mobilize cytosolic calcium and promote CDP-diacylglycerol biosynthesis. This is probably the first demonstration of functional intracellular dopamine receptor coupling in neural tissue, thus challenging the conventional paradigm that postsynaptic dopamine uptake serves merely as a mechanism for deactivating spent or excessive synaptic transmitter.

In-series vs. in-parallel: The role of cargo-motor connectivity in transport by ensembles of cytoskeletal motor proteins.

The movement of the cytoskeletal molecular motor proteins dynein and kinesin on microtubule networks facilitates the complex intracellular organization of eukaryotic cells. Ensembles of these motors are a prevalent feature of cytoskeletal transport, indicating possible synergistic effects of their use for intracellular organization. While the force output of individual motors is well studied, the mechanisms governing emergent force generated by multiple motors acting as an ensemble remains unclear. Determining the fundamental mechanisms controlling the interaction and integration of the forces produced by these motors at the nanoscale is essential to understanding the broader system of intracellular transport. Using total internal reflection fluorescence (TIRF) microscopy, we are investigating the role of the spatial arrangement of motors in an ensemble with a particular emphasis on the connectivity between motors and their cargo. Using motors purified from yeast and the nanoscale construction techniques of DNA origami, we create programmable chassis structures in which multiple cytoplasmic dyneins or Kip2 kinesins are arranged with independent, “in parallel” connections to a resistive cargo load or, in contrast, are arranged with a single, “in series” shared cargo connection. We expect the use of these chassis will allow us to determine the mechanisms behind the structural integration of the piconewton forces produced by cytoskeletal molecular motors within ensembles, ultimately providing insight on how the individual connectivity of motors to one another and their cargo affects load-sharing, force integration, and the resultant emergent motility of the motor ensemble.

Identification of molecular network of gut-brain axis associated with neuroprotective effects of PPARδ-ligand erucic acid in rotenone-induced Parkinson's disease model in zebrafish.

Disruption of the gut-brain axis in Parkinson's disease (PD) may lead to motor symptoms and PD pathogenesis. Recently, the neuroprotective potential of different PPARδ-agonists has been shown. We aimed to reveal the effects of erucic acid, peroxisome proliferator-activated receptors (PPARs)-ligand in rotenone-induced PD model in zebrafish, focusing on the gut-brain axis. Adult zebrafish were exposed to rotenone and erucic acid for 30 days. Liquid chromatography-mass spectrometry and tandem mass spectrometry (LC-MS/MS) analysis was performed. Raw files were analysed by Proteome Discoverer 2.4 software; peptide lists were searched against Danio rerio proteins. STRING database was used for protein annotations or interactions. Lipid peroxidation (LPO), nitric oxide (No), alkaline phosphatase, superoxide dismutase, glutathione S-transferase (GST), acetylcholinesterase and the expressions of PD-related genes were determined. Immunohistochemical tyrosine hydroxylase (TH) staining was performed. LC-MS/MS analyses allowed identification of over 2000 proteins in each sample. The 2502 and 2707 proteins overlapped for intestine and brain. The 196 and 243 significantly dysregulated proteins in the brain and intestines were found in rotenone groups. Erucic acid treatment corrected the changes in the expression of proteins associated with cytoskeletal organisation, transport and localisation and improved locomotor activity, expressions of TH, PD-related genes (lrrk2, park2, park7, pink1) and oxidant-damage in brain and intestines in the rotenone group as evidenced by decreased LPO, No and increased GST. Our results showed beneficial effects of erucic acid as a PPARδ-ligand in neurotoxin-induced PD model in zebrafish. We believe that our study will shed light on the mechanism of the effects of PPARδ agonists and ω9-fatty acids in the gut-brain axis of PD.

Mode of Action of the Natural Product Allicin in a Plant Model: Influence on the Cytoskeleton and Subsequent Shift in Auxin Localization

Allicin is a defense substance produced by garlic cells when they are injured. It is a redox-active thiosulfinate showing redox-activity with a broad range of dose-dependent antimicrobial and biocidal activity. It is known that allicin efficiently oxidizes thiol-groups, and it has been described as a redox toxin because it alters the redox homeostasis in cells and triggers oxidative stress responses. Allicin can therefore be used as a model substance to investigate the action of thiol-specific prooxidants. In order to learn more about the effect of allicin on plants, we used pure synthetized allicin, and studied the influence of allicin on organelle movement in Tradescantia fluminensis as a cytoskeleton-dependent process. Furthermore, we investigated cytoplasmic streaming in sterile filaments of Tradescantia fluminensis, organelle movement using transgenic Arabidopsis with organelle-specifics GFP-tags, and effects on actin and tubulin in the cytoskeleton using GFP-tagged lines. Tubulin and actin were visualized by GFP-tagging in transgenic lines of Arabidopsis thaliana to visualize the influence of allicin on the cytoskeleton. Since auxin transport depends on recycling and turnover of the PIN protein involving cytoskeletal transport to and from the membrane localization sites, auxin distribution in roots was investigated using of transgenic PIN1–GFP, PIN3–GFP, DR5–GFP and DII–VENUS Arabidopsis reporter lines. Allicin inhibited cytoplasmic streaming in T. fluminensis, organelle movement of peroxi-somesperoxisomes, and the Golgi apparatus in a concentration-dependent manner. It also destroyed the correct root tip distribution of auxin, which probably contributed to the observed inhibition of root growth. These observations of the disruption of cytoskeleton-dependent transport processes in plant cells add a new facet to the mechanism of action of allicin as a redox toxin in cells.

The Cytoskeletal Transport Protein, Secretagogin, Is Essential for Diurnal Glucagon-like Peptide-1 Secretion in Mice.

The intestinal L-cell incretin, glucagon-like peptide-1 (GLP-1), exhibits a circadian pattern of secretion, thereby entraining diurnal insulin release. Secretagogin (Scgn), an actin-binding regulatory protein, is essential for the temporal peak of GLP-1 secretion in vitro. To interrogate the role of Scgn in diurnal GLP-1 secretion in vivo, peak and trough GLP-1 release were evaluated in knockout mice (Scgn-/-, Gcg-CreERT2/+; Scgnfl/fl and Vil-CreERT2/+; Scgnfl/fl), and RNA sequencing (RNA-Seq) was conducted in Scgn knockdown L-cells. All 3 knockout models demonstrated loss of the diurnal rhythm of GLP-1 secretion in response to oral glucose. Gcg-CreERT2/+; Scgnfl/fl mice also lost the normal pattern in glucagon secretion, while Scgn-/- and Vil-CreERT2/+; Scgnfl/fl animals demonstrated impaired diurnal secretion of the related incretin, glucose-dependent insulinotrophic polypeptide. RNA-Seq of mGLUTag L-cells showed decreased pathways regulating vesicle transport, transport and binding, and protein-protein interaction at synapse, as well as pathways related to proteasome-mediated degradation including chaperone-mediated protein complex assembly following Scgn knockdown. Scgn is therefore essential for diurnal L-cell GLP-1 secretion in vivo, likely mediated through effects on secretory granule dynamics.

A model of minimal entropy generation for cytoskeletal transport systems with multiple interacting motors

We study the steady-state rate of entropy generation for multiple interacting particles. The description used is based on the partially asymmetric exclusion process in a lattice with periodic boundary conditions. Our methodology shows that in the steady-state, the rate of entropy generation is directly proportional to the bulk drift and the applied driving force. Since in many cases the driving force is unknown or hard to determine. We circumvent this by deriving a lower bound for the entropy, resulting in an extended thermodynamic uncertainty relation for the asymmetric simple exclusion process. We systematically compared this bound with the actual entropy generation. Thus, we identify the force regimes, and particles’ density conditions where the entropy bound derived from this extended thermodynamic uncertainty relation is meaningful.

An Model of Minimal Entropy Generation for Cytoskeletal Transport Systems with Multiple Interacting Motors

An Model of Minimal Entropy Generation for Cytoskeletal Transport Systems with Multiple Interacting Motors

Dipteran endoparasitoid infestation actively suppressed host defense components in hemocytes of silkworm Bombyx mori for successful parasitism

Infestation of commercially important silkworm Bombyx mori by dipteran endoparasitoid Exorista bombycis causes ~20% revenue loss. Though invasion has induced hemocyte encapsulation and melanisation of invaded larva, strategies to compromise with host-defense system by dipteran endoparasitoids is unknown. In the present study variation in genome wide gene expression was analyzed by utilizing microarray data acquired from hemocytes collected at 6 h after invasion that showed significant (P < 0.05) up-regulation of 423 genes and down-regulation of 587 genes. Among them 11 detoxification genes and 34 immune genes down-regulated showing repressed defense reactions substantiated by reduced activity of detoxification enzymes esterase and FMO in early stage of infestation. The down-regulated genes were enriched after comparing with control and by filtering unreliable probes. GO annotation of significantly (P < 0.05) down-regulated genes was used for clustering and categorization of functionally related defense processes. Networking of interacting partners of down-regulated defense genes showed relatedness among the down- regulated biological processes viz., Wnt signalling, DNA repair, transcription, catabolism, glutamine metabolism, components of cytoskeletal structure and transport. These clusters perform immune responses, signalling, stress responses, cell redox homeostasis, cytoskeletal organization, glutamine, glycogen and chitin metabolism, protein, lipid and carbohydrate metabolism and transmembrane transport. Autophagy, apoptosis, cell adhesion, cell growth and cellular lipid metabolism also showed down-regulation independently. Down-regulation of defense genes, interacting components and low detoxification enzyme activity in early infestation revealed active suppression of host defense at genic, enzymic and interactome level which is an unknown anti-defense mechanism of dipteran endoparasitoids.

The cross-talk between RAGE and DIAPH1 in neurological complications of diabetes: A review.

Neuropathy, or dysfunction of peripheral nerve, is one of the most common neurological manifestation in patients with diabetes mellitus (DM). DM is typically associated with a hyperglycaemic milieu, which promotes non-enzymatic glycation of proteins. Proteins with advanced glycation are known to engage a cell-surface receptor called the receptor for advanced glycation end products (RAGE). Thus, it is reasonable to assume that RAGE and its associated molecule-mediated cellular signalling may contribute to DM-induced symmetrical axonal (length-dependent) neuropathy. Of particular interest is diaphanous related formin 1 (DIAPH1), a cytoskeletal organizing molecule, which interacts with the cytosolic domain of RAGE and whose dysfunction may precipitate axonopathy/neuropathy. Indeed, it has been demonstrated that both RAGE and DIAPH1 are expressed in the motor and sensory fibres of nerve harvested from DM animal models. Although the detailed molecular role of RAGE and DIAPH1 in diabetic neurological complications remains unclear, here we will discuss available evidence of their involvement in peripheral diabetic neuropathy. Specifically, we will discuss how a hyperglycaemic environment is not only likely to elevate advanced glycation end products (ligands of RAGE) and induce a pro-inflammatory environment but also alter signalling via RAGE and DIAPH1. Further, hyperglycaemia may regulate epigenetic mechanisms that interacts with RAGE signalling. We suggest the cumulative effect of hyperglycaemia on RAGE-DIAPH1-mediated signalling may be disruptive to axonal cytoskeletal organization and transport and is therefore likely to play a key role in pathogenesis of diabetic symmetrical axonal neuropathy.

Longitudinal stability of urinary extracellular vesicle protein patterns within and between individuals

The protein content of urinary extracellular vesicles (EVs) is considered to be an attractive non-invasive biomarker source. However, little is known about the consistency and variability of urinary EV proteins within and between individuals over a longer time-period. Here, we evaluated the stability of the urinary EV proteomes of 8 healthy individuals at 9 timepoints over 6 months using data-independent-acquisition mass spectrometry. The 1802 identified proteins had a high correlation amongst all samples, with 40% of the proteome detected in every sample and 90% detected in more than 1 individual at all timepoints. Unsupervised analysis of top 10% most variable proteins yielded person-specific profiles. The core EV-protein-interaction network of 516 proteins detected in all measured samples revealed sub-clusters involved in the biological processes of G-protein signaling, cytoskeletal transport, cellular energy metabolism and immunity. Furthermore, gender-specific expression patterns were detected in the urinary EV proteome. Our findings indicate that the urinary EV proteome is stable in longitudinal samples of healthy subjects over a prolonged time-period, further underscoring its potential for reliable non-invasive diagnostic/prognostic biomarkers.

Cell scientists to watch – Zdeněk Lánský and Marcus Braun

ABSTRACT Zdeněk Lánský received his PhD in physics from the Charles University in Prague in 2006. He then moved to The Netherlands for his postdoc, where he studied the biophysics of molecular motors – first at the Vrije Universiteit in Amsterdam with Erwin Peterman, and then at Wageningen University with Marcel Janson. In 2011 he joined the lab of Stefan Diez at the B CUBE Center for Molecular Bioengineering in Dresden, where his research focused on crosslinker-mediated force generation in the cytoskeleton. Zdeněk set up his lab at the Institute of Biotechnology, Czech Academy of Sciences, BIOCEV in 2015, which he has run together with Marcus Braun since 2017. Marcus Braun received his PhD in biochemistry from King's College London in 2009 for his work on microtubule-based molecular motors in the labs of Andrew McAinsh and Rob Cross. He then moved to Dresden for his postdoc to study cytoskeletal transport, with a particular focus on microtubule crosslinkers, in Stefan Diez's lab – first at the Max Planck Institute of Molecular Cell Biology and Genetics, then later at B CUBE. In 2017, he joined Zdeněk Lánský at the Institute of Biotechnology, Czech Academy of Sciences, BIOCEV, as a principal investigator. The Lánský and Braun lab investigates ensemble dynamics of cytoskeletal proteins.

Influence of lipid rafts on pattern formation during T-cell adhesion

Adhesion of T cells to antigen presenting cells is mediated by the TCR–MHCp and LFA1–ICAM1 protein complexes. These intercellular protein complexes segregate and form characteristic special patterns in the cell contact zone. Previous studies have attempted to explain the mechanisms of formation of these patterns. While emphasis has been put on membrane elasticity and active cytoskeletal transport, it remains unclear whether and how the pattern formation process is related to lipid rafts, which are nanoscale molecular clusters enriched in cholesterol and saturated phospholipids in cell membranes. Using Monte Carlo simulations of a statistical mechanical model for T-cell adhesion, we find that lipid rafts can lead to the formation of intermediate pattern with a ring of LFA1–ICAM1 complexes around a central domain of TCR–MHCp complexes even in the absence of active transport of T-cell receptor (TCR) molecules toward the center of the contact zone. In the presence of active TCR transport, lipid rafts can accelerate the formation of this monocentric pattern. We also find that lipid rafts have a strong stabilizing effect on the monocentric pattern after removal of the active TCR transport. Our results not only help to explain recent experimental observations, but also demonstrate that lipid rafts can cooperate with active cytoskeletal transport during the immunological synapse formation.

Nuclear crowding and nonlinear diffusion during interkinetic nuclear migration in the zebrafish retina.

An important question in early neural development is the origin of stochastic nuclear movement between apical and basal surfaces of neuroepithelia during interkinetic nuclear migration. Tracking of nuclear subpopulations has shown evidence of diffusion - mean squared displacements growing linearly in time - and suggested crowding from cell division at the apical surface drives basalward motion. Yet, this hypothesis has not yet been tested, and the forces involved not quantified. We employ long-term, rapid light-sheet and two-photon imaging of early zebrafish retinogenesis to track entire populations of nuclei within the tissue. The time-varying concentration profiles show clear evidence of crowding as nuclei reach close-packing and are quantitatively described by a nonlinear diffusion model. Considerations of nuclear motion constrained inside the enveloping cell membrane show that concentration-dependent stochastic forces inside cells, compatible in magnitude to those found in cytoskeletal transport, can explain the observed magnitude of the diffusion constant.

Modelling cytoskeletal transport by clusters of non-processive molecular motors with limited binding sites.

Molecular motors are responsible for intracellular transport of a variety of biological cargo. We consider the collective behaviour of a finite number of motors attached on a cargo. We extend previous analytical work on processive motors to the case of non-processive motors, which stochastically bind on and off cytoskeletal filaments with a limited number of binding sites available. Physically, motors attached to a cargo cannot bind anywhere along the filaments, so the number of accessible binding sites on the filament should be limited. Thus, we analytically study the distribution and the velocity of a cluster of non-processive motors with limited number of binding sites. To validate our analytical results and to go beyond the level of detail possible analytically, we perform Monte Carlo latticed based stochastic simulations. In particular, in our simulations, we include sequence preservation of motors performing stepping and binding obeying a simple exclusion process. We find that limiting the number of binding sites reduces the probability of non-processive motors binding but has a relatively small effect on force–velocity relations. Our analytical and stochastic simulation results compare well to published data from in vitro and in vivo experiments.

Temperature-dependent activity of kinesins is regulable

Cytoskeletal transport in cells is driven by enzymes whose activity shows sensitive, typically Arrhenius, dependence on temperature. Often, the duration and outcome of cargo transport is determined by the relative success of kinesin vs. dynein motors, which can simultaneously bind to individual cargos and move in opposite direction on microtubules. The question of how kinesin and dynein activity remain coupled over the large temperature ranges experienced by some cells is one of clear biological relevance. We report a break in the Arrhenius behavior of both kinesin-1 and kinesin-3 enzymatic activity at 4.7 °C and 10.5 °C, respectively. Further, we report that this transition temperature significantly changes as a function of chemical background: addition of 200 mM TMAO increases transition temperatures by ∼6 °C in all cases. Our results show that Arrhenius trend breaks are common to all cytoskeletal motors and open a broad question of how such activity transitions are regulated in vivo. Statement of significanceMany cytoskeletal motors studied to date follow Arrhenius kinetics, at least from room temperature up to mammalian body temperature. However the thermal dynamic range is typically finite, and breaks in Arrhenius trends are commonly observed at biologically relevant temperatures. Here we report that the thermal dynamic range of kinesins is also limited and moreover that the location of the Arrhenius break for kinesins can shift significantly based on chemical backgrounds. This implies that the balance of multiple motor cargo transport along the cytoskeleton is far more tunable as a function of temperature than previously appreciated.

Regulation of cytoskeletal dynamics and transport.

Regulation of cytoskeletal dynamics and transport.

Ontogenesis of the tear drainage system requires Prickle1-driven polarized basement membrane deposition.

In terrestrial animals, the lacrimal drainage apparatus evolved to serve as conduits for tear flow; however, little is known about the ontogenesis of this system. Here, we define the anatomy of the fully formed tear duct in mice, characterize crucial morphogenetic events for the development of tear duct components and identify the site for primordial tear duct (PTD) initiation. We report that the PTD originates from the orbital lacrimal lamina, a junction formed by the epithelia of the maxillary and lateral nasal processes. We demonstrate that Prickle1, a key component of planar cell polarity signaling, is expressed in progenitors of the PTD and throughout tear duct morphogenesis. Disruption of Prickle1 stalls tear duct elongation; in particular, the loss of basement membrane deposition and aberrant cytoplasmic accumulation of laminin are salient. Altered cell adhesion, cytoskeletal transport systems, vesicular transport systems and cell axis orientation in Prickle1 mutants support the role of Prickle1 in planar cell polarity. Taken together, our results highlight a crucial role of Prickle1-mediated polarized basement membrane secretion and deposition in PTD elongation.

Temporal Control of Axonal Transport: The Extreme Case of Organismal Ageing.

A fundamental question in cell biology is how cellular components are delivered to their destination with spatial and temporal precision within the crowded cytoplasmic environment. The long processes of neurons represent a significant spatial challenge and make these cells particularly dependent on mechanisms for long-range cytoskeletal transport of proteins, RNA and organelles. Although many studies have substantiated a role for defective transport of axonal cargoes in the pathogenesis of neurodevelopmental and neurodegenerative diseases, remarkably little is known about how transport is regulated throughout ageing. The scale of the challenge posed by ageing is considerable because, in this case, the temporal regulation of transport is ultimately dictated by the length of organismal lifespan, which can extend to days, years or decades. Recent methodological advances to study live axonal transport during ageing in situ have provided new tools to scratch beneath the surface of this complex problem and revealed that age-dependent decline in the transport of mitochondria is a common feature across different neuronal populations of several model organisms. In certain instances, the molecular pathways that affect transport in ageing animals have begun to emerge. However, the functional implications of these observations are still not fully understood. Whether transport decline is a significant determinant of neuronal ageing or a mere consequence of decreased cellular fitness remains an open question. In this review, we discuss the latest developments in axonal trafficking in the ageing nervous system, along with the early studies that inaugurated this new area of research. We explore the possibility that the interplay between mitochondrial function and motility represents a crucial driver of ageing in neurons and put forward the hypothesis that declining axonal transport may be legitimately considered a hallmark of neuronal ageing.

Optical Control of Fast and Processive Engineered Myosins In Vitro and in Living Cells

Spatiotemporal control of cytoskeletal transport can provide new possibilities for dissecting cellular processes or for constructing complex artificial devices. Optogenetic approaches have been used for both controlled recruitment of motors to cellular cargos [1] and direct modulation of motor speed and direction [2]. Here we have worked to create optimized and diversified engineered myosin motors with velocities that can be optically controlled using dynamic changes in lever arm geometry. Previous designs for light-activated gearshifting [2] were non-processive, and suffered from either low velocities (< 10 nm/s) or modest degrees of velocity modulation (∼15%) in response to light. These limitations preclude many applications in cell biology, devices, and reconstituted systems. We have now engineered (i) non-processive myosin motors that combine large optical modulation depths with high velocities and (ii) processive myosin motors with optically controllable directionality. We have characterized a series of optimized constructs using in vitro motility assays of propelled actin filaments, single-molecule tracking of processive complexes, and live-cell imaging of individual motors tagged with fluorescent protein arrays [3]. For non-processive plus-end directed myosins, the most responsive constructs are approximately stopped in the dark and reach several microns/second in the presence of blue light. We also report on speed modulation in minus-end directed motors. For processive myosins, we demonstrate controllable bidirectional transport, and observe subcellular localization at filopodial tips under optical stimulation. An extended set of optogenetic motors will contribute to an expanded toolkit for programmable control of nanoscale transport and force generation. [1] P. van Bergeijk, et al. (2015) Nature 518, 7537. [2] M. Nakamura et al. (2014) Nat. Nanotechnol 9, 693. [3] R.P. Ghosh et al. (2017) BioRxiv https://doi.org/10.1101/159004.

Redox-Regulated, Targeted Affinity Isolation of NADH-Dependent Protein Interactions with the Branched Chain Aminotransferase Proteins.

Isolation and identification of protein targets for redox-active proteins is challenging. The human branched chain aminotransferase (hBCAT) proteins are redox active transaminases that can be regulated through oxidation, S-nitrosation and S-glutathionylation. This metabolic protein was shown to associate with the E1 decarboxylase component of the branched-chain α-keto acid dehydrogenase complex in a NADH-dependent manner, where mutation of the CXXC center was shown to prevent complex formation. To determine if the redox state of the CXXC motif can influence other NADH-dependent protein-protein interactions, proteins were extracted from neuronal cells treated under reduced and oxidized conditions and then isolated using targeted affinity chromatography, resolved using 2D electrophoresis. Select proteins spots were excised and identified using a quadrupole time of flight mass spectrometer (Thermo) with a precursor tolerance of 10ppm and subsequently analyzed using Proteome Discoverer 2.1 with Swiss-Prot human DB. Mass tolerances for precursor/product were set to 10ppm/0.6Da and data were filtered by peptide confidence with PD2.1. It was determined that the protein profile considerably altered in both number and abundance dependent on the redox state of the cell and also on the availability of the redox active thiol groups. The biological relevance of the newly identified partners was determined using DAVID, the bioinformatics database, which indicated that proteins important to cytoskeletal function, protein transport, protein synthesis, chaperone activity, and cell signaling.