GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway.

Chaperone-assisted proteasome degradation of oncogenic protein acts as an upstream signal controlling tumorigenesis and progression. The understanding of the co-regulation of chaperone and oncoprotein of endocytosis pathways is extremely limited. In this study, we showed for the first time that proto-Dbl (dbl proto-oncogene product) is co-enriched with mitochondrial chaperone GRP75 in endocytosis vesicles from ovarian cancer cells. onco-Dbl, produced by oncogenic mutation/degradation of proto-Dbl, markedly enhanced cellular macropinocytosis but suppressed clathrin-mediated endocytosis and clathrin-independent endocytosis pathways, presenting a derailed endocytosis phenotype. GRP75 was associated with proto-Dbl inside cells and modulated Dbl-driven endocytosis derailed by a co-regulatory mode. In spite of not being a component of the Hsc70/Hsp90/proto-Dbl complex, the degradation of proto-Dbl was promoted by GRP75 through the CHIP-mediated ubiquitin-proteasome pathway, of which GRP75 acts as a cooperator with CHIP but also acts as a competitor to Hsc70 and Hsp90 in the multiple chaperones-assisted pro-folding/pro-degradation machinery. Knockdown or inhibition of GRP75 attenuated proto-Dbl degradation and reduced the onco-Dbl level, which differentially impaired Rho GTPases activation and therefore shifted the endocytosis-derailed phenotype. Our data uncovered a novel GRP75-Dbl endocytosis regulatory axis and provided an alternative using chaperone inhibitor to shut down the oncoprotein-driven endocytosis derailment mechanism.

Quantitative Co-Localization and Pattern Analysis of Endo-Lysosomal Cargo in Subcellular Image Cytometry and Validation on Synthetic Image Sets.

Late endosomes and lysosomes (LE/LYSs) play a central role in trafficking of endocytic cargo, secretion of exosomes, and hydrolysis of ingested proteins and lipids. Failure in such processes can lead to lysosomal storage disorders in which a particular metabolite accumulates within LE/LYSs. Analysis of endocytic trafficking relies heavily on quantitative fluorescence microscopy, but evaluation of the huge image data sets is challenging and demands computer-assisted statistical tools. Here, we describe how to use SpatTrack ( www.sdu.dk/bmb/spattrack ), an imaging toolbox, which we developed for quantification of the distribution and dynamics of endo-lysosomal cargo from fluorescence images of living cells. First, we explain how to analyze experimental images of endocytic processes in Niemann Pick C2 disease fibroblasts using SpatTrack. We demonstrate how to quantify the location of the sterol-binding protein NPC2 in LE/LYSs relative to cholesterol -rich lysosomal storage organelles (LSOs) stained with filipin. Second, we show how to simulate realistic vesicle patterns in the cell geometry using Markov Chain Monte Carlo and suitable inter-vesicle and cell-vesicle interaction potentials. Finally, we use such synthetic vesicle patterns as "ground truth" for validation of two-channel analysis tools in SpatTrack, revealing their high reliability. An improved version of SpatTrack for microscopy-based quantification of cargo transport through the endo-lysosomal system accompanies this protocol.

Physiologically-based pharmacokinetic modeling to predict the clinical pharmacokinetics of monoclonal antibodies.

Accurate prediction of the clinical pharmacokinetics of new therapeutic entities facilitates decision making during drug discovery, and increases the probability of success for early clinical trials. Standard strategies employed for predicting the pharmacokinetics of small-molecule drugs (e.g., allometric scaling) are often not useful for predicting the disposition monoclonal antibodies (mAbs), as mAbs frequently demonstrate species-specific non-linear pharmacokinetics that is related to mAb-target binding (i.e., target-mediated drug disposition, TMDD). The saturable kinetics of TMDD are known to be influenced by a variety of factors, including the sites of target expression (which determines the accessibility of target to mAb), the extent of target expression, the rate of target turnover, and the fate of mAb-target complexes. In most cases, quantitative information on the determinants of TMDD is not available during early phases of drug discovery, and this has complicated attempts to employ mechanistic mathematical models to predict the clinical pharmacokinetics of mAbs. In this report, we introduce a simple strategy, employing physiologically-based modeling, to predict mAb disposition in humans. The approach employs estimates of inter-antibody variability in rate processes of extravasation in tissues and fluid-phase endocytosis, estimates for target concentrations in tissues derived through use of categorical immunohistochemical scores, and in vitro measures of the turnover of target and target-mAb complexes. Monte Carlo simulations were performed for four mAbs (cetuximab, figitumumab, dalotuzumab, trastuzumab) directed against three targets (epidermal growth factor receptor, insulin-like growth factor receptor 1, human epidermal growth factor receptor 2). The proposed modeling strategy was able to predict well the pharmacokinetics of cetuximab, dalotuzumab, and trastuzumab at a range of doses, but trended towards underprediction of figitumumab concentrations, particularly at high doses. The general agreement between model predictions and experimental observations suggests that PBPK modeling may be useful for the a priori prediction of the clinical pharmacokinetics of mAb therapeutics.

Flow cytometric assessment of reactive oxygen species generations that are directly related to cellular ZnO nanoparticle uptake.

In this study, a simple flow cytometry protocol to evaluate nanoparticle associated biological response was proposed. Particularly, we have evaluated the effect of surface charge on the cellular nanoparticle associations and nanoparticle-induced apoptosis. Significant enhancement in side scattering intensity was observed for the HeLa cells treated with positively charged (PLL)ZnO nanoparticles, suggesting that the (PLL)ZnO nanoparticles may induce cell death via adsorption and endocytosis of the nanoparticles. On the other hand, the negatively charged (PAA)ZnO nanoparticle seems to cause cell death process indirectly via the released Zn ions, with less contribution from cellular association of nanoparticles. Time- and dose-dependent studies on cellular association of ZnO nanoparticles, and ZnO associated reactive oxygen species generation were also performed for the HeLa cells exposed to the (PLL)ZnO nanoparticle. For those cells associated with (PLL)ZnO nanoparticle, a significant enhancement in reactive oxygen species generation was observed even at a lower concentration (10 ppm), which was not observable for the results with the whole cell population. By using this approach, we are able to distinguish biological responses (e.g., reactive oxygen species (ROS) generation) directly related to the cellular associations of NPs from those indirectly related to the cellular associations of NPs, such as the cytotoxicity caused by the NP released metal ions.

Chronic methadone treatment shows a better cost/benefit ratio than chronic morphine in mice.

Chronic treatment of pain with opiate drugs can lead to analgesic tolerance and drug dependence. Although all opiate drugs can promote tolerance and dependence in practice, the severity of those unwanted side effects differs depending on the drug used. Although each opiate drug has its own unique set of pharmacological profiles, methadone is the only clinically used opioid drug that produces substantial receptor endocytosis at analgesic doses. Here, we examined whether moderate doses of methadone carry any benefits over chronic use of equianalgesic morphine, the prototypical opioid. Our data show that chronic administration of methadone produces significantly less analgesic tolerance than morphine. Furthermore, we found significantly reduced precipitated withdrawal symptoms after chronic methadone treatment than after chronic morphine treatment. Finally, using a novel animal model with a degrading µ-opioid receptor we showed that, although endocytosis seems to protect against tolerance development, endocytosis followed by receptor degradation produces a rapid onset of analgesic tolerance to methadone. Together, these data indicated that opioid drugs that promote receptor endocytosis and recycling, such as methadone, may be a better choice for chronic pain treatment than morphine and its derivatives that do not.

Assessment of the recycling of the membrane-bound chemokine, CX3CL1.

Fractalkine (CX(3)CL1) is a membrane-anchored chemokine whose N-terminus contains a unique CX(3)C motif that is cleaved and released. The membrane-bound form functions as an adhesion molecule and the secreted form as a chemotactic factor. Like other chemokines, CX(3)CL1 is regulated at the levels of transcription and translation. Recent evidence points to additional functional regulation by cellular trafficking owing to the unique transmembrane structure. CX(3)CL1 is the only chemokine known to undergo constitutive internalization. To understand mechanisms governing the regulation and processing of such membrane-bound proteins, it is vital to study their subcellular distribution and transport. The methods outlined in this chapter describe (1) transfection of mammalian cells with plasmids encoding the expression of green fluorescent protein-tagged CX(3)CL1; (2) immunofluorescence antibody labeling as well as fluorescence recovery after photobleaching to study internalization of CX(3)CL1 by endocytosis; and (3) acid-stripping assays to study the recycling of internalized CX(3)CL1 back to the plasma membrane. Together, these methods allow for the examination of subcellular distribution and traffic of recycling membrane proteins.

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts.

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.

Synaptic vesicle retrieval time is a cell-wide rather than individual-synapse property.

Although individual nerve terminals from the same neuron often differ in neurotransmitter release characteristics, the extent to which endocytic retrieval of synaptic vesicle components differs is unknown. We used high-fidelity optical recordings to undertake a large-scale analysis of endocytosis kinetics of individual boutons in hippocampal rat neurons. Our data indicate that endocytosis kinetics do not differ substantially across boutons from the same cell but instead appear to be controlled at a cell-wide level.

Synaptic vesicle endocytosis: get two for the price of one?

Tight coupling between synaptic vesicle exocytosis and endocytosis is critical for the maintenance of neurotransmission. In this issue of Neuron, Zhu et al. reveal a surprising facet of this coupling by showing that, at low frequencies, fusion of a single vesicle leads to retrieval of two vesicles with dissimilar attributes.

A novel high-throughput assay for the quantitative assessment of receptor trafficking.

1. Receptor transport between intracellular compartments has important consequences for receptor function and is an exciting area of current study. Existing methods for studying receptor trafficking often require labour-intensive techniques or are difficult to quantify reliably. We report a novel high-throughput method that uses automated imaging and analysis tools to accurately quantify cannabinoid CB1 receptor trafficking. 2. Haemagglutinin (HA)-tagged CB1 was stably expressed in HEK-293 cells and cell surface or total receptors were detected immunocytochemically. Images of receptor and nuclear staining were acquired with an automated fluorescent microscope (Discovery-1; Molecular Devices, Sunnyvale, CA, USA) and quantified at high throughput with MetaMorph (Molecular Devices) software. The 'Granularity' assay measured internalization by counting receptor clusters that appear during receptor endocytosis, a well-established approach. Our assay, referred to as 'Total Grey Value per Cell' (TGVC), measures the total fluorescence above background, normalized to cell count. 3. Incubation with the cannabinoid agonist HU-210 (100 nmol/L) resulted in rapid CB1 internalization, reaching a maximum within 20 min. Whether quantified by Granularity or TGVC, the time-course of endocytosis could be modelled with exponentially derived curves and with similar half-lives. We demonstrate the sensitivity of our TGVC method by measuring the concentration dependence of CB1 internalization and its versatility by measuring downregulation following chronic agonist exposure, whereby total CB1 was reduced to approximately 55% of basal after 3 h. 4. The TGVC quantification method described is efficient, accurate and versatile and is likely to provide a valuable tool in receptor trafficking studies.

Analysis of spatial dependencies of endocytic proteins based on temporal random sets.

Statistical image analysis has emerged as a basic methodology in the study of many real phenomena, which can be represented by sequences of binary images. Recent techniques, such as total internal reflection fluorescent microscopy, allow us to image simultaneously two fluorescent-tagged proteins which are relevant in important cellular processes, such as endocytosis. Here, we model these biological pairs of image sequences as realizations of a 3D non-isotropic bivariate random set, in which one dimension corresponds to time. We analyze their second-order properties by means of the cross-covariance and the pair correlation function in order to study colocalization between proteins. Results show the proposed methodology allows us to study spatial dependencies in a formal and robust way.

Total internal reflection fluorescence microscopy: technical innovations and novel applications.

Recent years have seen the introduction of novel techniques and applications of total internal reflection fluorescence microscopy (TIRFM). Key technical achievements include miniaturization, enhanced depth resolution, reduction of detection volumes and the combination of TIRFM with other microscopic techniques. Novel applications have concentrated on single-molecule detection (e.g. of cellular receptors), imaging of exocytosis or endocytosis, measurements of adhesion foci of microtubules, and studies of the localization, activity and structural arrangement of specific ion channels. In addition to conventional fluorescent dyes, genetically engineered fluorescent proteins are increasingly being used to measure molecular conformations or intermolecular distances by fluorescence resonance energy transfer.

Targeted gene therapy: frontiers in the development of 'smart drugs'.

Chronic diseases, particularly malignancies and immune-mediated inflammatory diseases (IMIDs), are a challenging frontier for clinical diagnosis and treatment, as well as for biomedical research. Current treatment regimens are frequently insufficient and thus new treatment strategies are needed. Novel therapies for disabling such diseases should provide improvements with respect to safety, efficacy and cost. To fulfill these three key criteria, recent research efforts have focused on the development of 'smart drugs'. This review highlights some examples of the rapidly expanding possibilities that current biotechnology has to offer in the development of novel therapeutic strategies for complex diseases such as IMIDs. Special attention is given to advances in, and limitations of, controlled and targeted gene product application in inflammatory diseases.

Multimodal quantal release at individual hippocampal synapses: evidence for no lateral inhibition.

Most CNS synapses investigated thus far contain a large number of vesicles docked at the active zone, possibly forming individual release sites. At the present time, it is unclear whether these vesicles can be discharged independently of one another. To investigate this problem, we recorded miniature excitatory currents by whole-cell and single-synapse recordings from CA3-CA1 hippocampal neurons and analyzed their stochastic properties. In addition, spontaneous release was investigated by ultrastructural analysis of quickly frozen synapses, revealing vesicle intermediates in docking and spontaneous fusion states. In these experiments, no signs of inhibitory interactions between quanta could be detected up to 1 msec from the previous discharge. This suggests that exocytosis at one site does not per se inhibit vesicular fusion at neighboring sites. At longer intervals, the output of quanta diverged from a random memoryless Poisson process because of the presence of a bursting component. The latter, which could not be accounted for by random coincidences, was independent of Ca2+ elevations in the cytosol, whether from Ca2+ flux through the plasma membrane or release from internal stores. Results of these experiments, together with the observation of spontaneous pairs of omega profiles at the active zone, suggest that multimodal release is produced by an enduring activation of an integrated cluster of release sites.

Yolk sac failure in embryopathy due to hyperglycemia: horseradish peroxidase uptake in the assessment of yolk sac function.

We described previously the morphologic alterations of the visceral endodermal yolk sac cells of rat conceptuses cultured under hyperglycemic conditions which occurred concomitantly with major embryonic malformations. To determine whether the transport function of the yolk sac was impaired simultaneously as a result of these hyperglycemic conditions, horseradish peroxidase was used as a tracer protein to assess the transport function of the visceral endodermal yolk sac cells of conceptuses cultured in both control and hyperglycemic media. Cellular uptake of peroxidase, which was added to the culture medium for 3 or 24 hours, was observed in controls. This differed from the marked diminution in peroxidase uptake seen in conceptuses cultured in hyperglycemic medium. These results demonstrate that during hyperglycemia-induced embryopathy, there is concomitant yolk sac failure evidenced by morphologic alterations and impaired endocytosis. These findings therefore strengthen our hypothesis that diabetes-related malformations, as demonstrated experimentally in rat conceptuses, are associated with impairment in the structure and functions of the visceral yolk sac cells during a critical period of organogenesis.