William H. Humphries

Endo-lysosomal vesicles positive for Rab7 and LAMP1 are terminal vesicles for the transport of dextran.

The endo-lysosomal pathway is essential for intracellular transport and the degradation of extracellular cargo. The relationship between three populations of endo-lysosomal vesicles—Rab7-positive, LAMP1-positive, and both Rab7- and LAMP1-postive—was probed with fluorescence microscopy and single particle tracking. Of specific interest was determining if these vesicles were intermediate or terminal vesicles in the transport of extracellular cargo. We find that the major organelle in the endo-lysosomal pathway, both in terms of population and cargo transport, is positive for Rab7 and LAMP1. Dextran, a fluid phase cargo, shifts from localization within all three populations of vesicles at 30 minutes and 1 hour to primarily LAMP1- and Rab7/LAMP1-vesicles at longer times. This demonstrates that LAMP1- and Rab7/LAMP1-vesicles are terminal vesicles in the endo-lysosomal pathway. We tested two possible mechanisms for this distribution of cargo, delivery to mannose 6-phosphate receptor (M6PR)-negative vesicles and the fusion dynamics of individual vesicles. We find no correlation with M6PR but do find that Rab7-vesicles undergo significantly fewer fusion events than LAMP1- or Rab7/LAMP1-vesicles suggesting that the distribution of fluid phase cargo is driven by vesicle dynamics.

Fluorescent coumarin thiols measure biological redox couples.

In this report we present a new chemical probe, 3-HTC, that can reversibly and ratiometrically measure the thiol-disulfide equilibrium of biological systems. 3-HTC is composed of a coumarin that has a thiolate directly conjugated to its extended aromatic π system while formation of a disulfide attenuates this conjugation. The fluorescence and absorption properties of 3-HTC are therefore very sensitive to the redox state of its thiol. 3-HTC reacts reversibly with thiols and disulfides enabling its use to measure dynamic GSH/GSSH ratios in vitro as well as to monitor the reversible redox status of whole cell lysates.

Pyrenebutyrate-Mediated Delivery of Quantum Dots across the Plasma Membrane of Living Cells

Quantum dots have been delivered directly across the plasma membrane to the cytosol of living cells using a combination of a cationic peptide, polyarginine, and a hydrophobic counterion, pyrenebutyrate. Quantum dot delivery did not disrupt the plasma membrane and bypassed the barrier of endocytic vesicles. Cellular uptake was independent of temperature but highly dependent on the surface charge of the quantum dot and the membrane potential of the cell, suggesting a direct translocation across the membrane. This method of delivery can find immediate application for quantum dots and may be broadly applicable to other nanoparticles.

The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine

Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.

Imaging lysosomal enzyme activity in live cells using self-quenched substrates

Endocytosis, the internalization and transport of extracellular cargo, is an essential cellular process. The ultimate step in endocytosis is the intracellular degradation of extracellular cargo for use by the cell. While live cell imaging and single particle tracking have been well-utilized to study the internalization and transport of cargo, the final degradation step has required separate biochemical assays. We describe the use of self-quenched endocytic cargo to image the intracellular transport and degradation of endocytic cargo directly in live cells. We first outline the fluorescent labeling and quantification of two common endocytic cargos: a protein, bovine serum albumin, and a lipid nanoparticle, low-density lipoprotein. In vitro measurements confirm that self-quenching is a function of the number of fluorophores bound to the protein or particle and that recovery of the fluorescent signal occurs in response to enzymatic degradation. We then use confocal fluorescence microscopy and flow cytometry to demonstrate the use of self-quenched bovine serum albumin with standard fluorescence techniques. Using live cell imaging and single particle tracking, we find that the degradation of bovine serum albumin occurs in an endo-lysosomal vesicle that is positive for LAMP1.

P‐Glycoprotein‐Dependent Trafficking of Nanoparticle‐Drug Conjugates

Resistance to chemotherapy contributes to treatment failure in over 90% of patients with metastatic cancer.[1] Although, many tumors initially respond well to chemotherapies, selective pressure can lead to the proliferation and dissemination of cell subpopulations exhibiting either de novo or adaptive drug resistance, often accompanied by cross-resistance to a range of structurally diverse small molecule drugs. MDR1 P-glycoprotein (P-gp) is considered to be the most prevalent and single most important cause of multidrug-resistance (MDR) in humans,[2] where the protein facilitates recognition, intracellular trafficking, sequestration, [3] and/or cellular efflux of up to 50% of all cytotoxic chemotherapeutics (e.g. doxorubicin, paclitaxel, vinblastine, etoposide), as well as antibiotics (e.g. erythromycin, azithromycin, ketolides), and other therapeutic small molecules.

Late Endosomal Degradation of Low-Density Lipoprotein Probed with Multi-Color Single Particle Tracking Fluorescence Microscopy

The vesicle-mediated degradation of low-density lipoprotein (LDL) is an essential cellular function due to its role in cellular membrane biosynthesis. Using multi-color single particle tracking fluorescence microscopy, we have probed the intracellular degradation of low-density lipoprotein in living cells. The unique aspect of our experiments is the direct observation of LDL degradation using an LDL-based probe that increases fluorescence intensity upon degradation. Specifically, individual LDL particles are labeled with multiple fluorophores resulting in a quenched fluorescent signal. Control experiments demonstrate that enzymatic degradation of the LDL particle results in an increase in fluorescence. The ability to directly observe LDL degradation allows us to determine which vesicle is responsible for degradation and quantify the vesicle dynamics involved in LDL degradation. Visualization of early endosomes, late endosomes and lysosomes is accomplished by fluorescently labeling vesicles with variants of GFP. Transient colocalization of LDL with specific vesicles and the intensity of the LDL particle are measured simultaneously. The measured colocalization durations are then correlated with changes in fluorescence intensity due to LDL degradation. We observe that degradation of LDL occurs in the late endosome. While there are a broad distribution of colocalization durations of LDL with Rab7, a late endosomal protein, only relatively long (>420 s) colocalization leads to the degradation of LDL. These studies, which are the first to directly observe the degradation of LDL within a cell, support a model in which late endosomes are the site of degradation with lysosomes serving as enzyme storage vesicles.