Peter M. Haggie

Size-dependent DNA Mobility in Cytoplasm and Nucleus

The diffusion of DNA in cytoplasm is thought to be an important determinant of the efficacy of gene delivery and antisense therapy. We have measured the translational diffusion of fluorescein-labeled double-stranded DNA fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into cytoplasm and nucleus of HeLa cells. Diffusion was measured by spot photobleaching using a focused argon laser spot (488 nm). In aqueous solutions, diffusion coefficients of the DNA fragments in water (D w ) decreased from 53 × 10−8 to 0.81 × 10−8 cm2/s for sizes of 21–6000 bp; D w was related empirically to DNA size:D w = 4.9 × 10−6cm2/s·[bp size]−0.72. DNA diffusion coefficients in cytoplasm (D cyto) were lower than D w and depended strongly on DNA size.D cyto/D w decreased from 0.19 for a 100-bp DNA fragment to 0.06 for a 250-bp DNA fragment and was <0.01 for >2000 bp. Diffusion of microinjected fluorescein isothiocyanate (FITC) dextrans was faster than that of comparably sized DNA fragments of 250 bp and greater. In nucleus, all DNA fragments were nearly immobile, whereas FITC dextrans of molecular size up to 580 kDa were fully mobile. These results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding. The diffusion of DNA in cytoplasm may thus be an important rate-limiting barrier in gene delivery utilizing non-viral vectors.

Green fluorescent protein-based halide indicators with improved chloride and iodide affinities.

The green fluorescent protein YFP‐H148Q is sensitive to halides by a mechanism involving halide binding and a shift in pK a. However, a limitation of YFP‐H148Q is its low halide sensitivity, with K d>100 mM for Cl−. Indicators with improved sensitivities are needed for cell transport studies, particularly in drug discovery by high‐throughput screening, and for measurement of Cl− concentration in subcellular organelles. YFP‐H148Q libraries were generated in which pairs of residues in the vicinity of the halide binding site were randomly mutated. An automated procedure was developed to screen bacterial colonies for improved halide sensitivity. Analysis of 1536 clones revealed improved anion sensitivities with K d down to 2 mM for I− (I152L), 40 mM for Cl− (V163S), and 10 mM for NO3 − (I152L). The anion‐sensitive mechanism of these indicators was established and their utility in cells was demonstrated using transfected cells expressing the cystic fibrosis transmembrane conductance regulator chloride channel.

In Vivo Measurement of Brain Extracellular Space Diffusion by Cortical Surface Photobleaching

Molecular diffusion in the brain extracellular space (ECS) is an important determinant of neural function. We developed a brain surface photobleaching method to measure the diffusion of fluorescently labeled macromolecules in the ECS of the cerebral cortex. The ECS in mouse brain was labeled by exposure of the intact dura to fluorescein-dextrans (Mr 4, 70, and 500 kDa). Fluorescein-dextran diffusion, detected by fluorescence recovery after laser-induced cortical photobleaching using confocal optics, was slowed approximately threefold in the brain ECS relative to solution. Cytotoxic brain edema (produced by water intoxication) or seizure activity (produced by convulsants) slowed diffusion by >10-fold and created dead-space microdomains in which free diffusion was prevented. The hindrance to diffusion was greater for the larger fluorescein-dextrans. Interestingly, slowed ECS diffusion preceded electroencephalographic seizure activity. In contrast to the slowed diffusion produced by brain edema and seizure activity, diffusion in the ECS was faster in mice lacking aquaporin-4 (AQP4), an astroglial water channel that facilitates fluid movement between cells and the ECS. Our results establish a minimally invasive method to quantify diffusion in the brain ECS in vivo, revealing stimulus-induced changes in molecular diffusion in the ECS with unprecedented spatial and temporal resolution. The in vivo mouse data provide evidence for: (1) dead-space ECS microdomains after brain swelling; (2) slowed molecular diffusion in the ECS as an early predictor of impending seizure activity; and (3) a novel role for AQP4 as a regulator of brain ECS.

A small molecule CFTR inhibitor produces cystic fibrosis-like submucosal gland fluid secretions in normal airways

Airway submucosal glands have been proposed as a primary site for initiating and sustaining airway disease in cystic fibrosis (CF). However, it has been difficult to define the role of CFTR in gland fluid secretion because of concerns in interpreting experiments on diseased CF human airways subjected to chronic infection and inflammation. Here, we test the role of CFTR in gland fluid secretion by using a selective CFTR inhibitor (CFTRinh‐172) in pig and human airways. Measurements of single‐gland fluid secretion rates showed inhibition of both cholinergic and cAMP‐stimulated fluid secretion by CFTRinh‐172. Secreted fluid [Na+] and [Cl–] measured by fluorescence ratio imaging were 101 and 116 mM, respectively, and not significantly altered by secretory agonists or CFTR inhibition. Gland fluid pH was 7.1 and reduced by 0.4 units after CFTR inhibition. Gland fluid viscosity, determined by photobleaching of FITC‐dextran, was threefold increased in pilocarpine‐stimulated gland fluid after CFTR inhibition, and protein concentration was increased from 12 to 20 mg/ml. Our data provide strong evidence that gland fluid secretion is CFTR‐dependent. The relatively hyper‐viscous and acidic fluid secretions produced by acute CFTR inhibition support a role for defective gland function in CF lung disease and provide a rational basis for pharmacological creation of a large animal model of CF.

Cystic Fibrosis Transmembrane Conductance Regulator-independent Phagosomal Acidification in Macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933–944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green® 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH ∼5.1 at 45 min), murine alveolar macrophages (pH ∼5.3), and human alveolar macrophages (pH ∼5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTRinh-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for ΔF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within ∼15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTRinh-172 and was not different in alveolar macrophages from wild-type versus ΔF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.

Increased Diffusional Mobility of CFTR at the Plasma Membrane after Deletion of Its C-terminal PDZ Binding Motif

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel expressed at the apical plasma membrane. It has been proposed that the C-terminal PDZ binding motif of CFTR is required for its apical membrane targeting and that PDZ-domain interactions may tether CFTR to the actin cytoskeleton via soluble proteins including EBP50/NHERF1 and ezrin. We measured the diffusional mobility of human CFTR in the plasma membrane of Madin-Darby canine kidney cells by photobleaching of green fluorescent protein (GFP)-CFTR chimeras. After bleaching by a focused laser beam, GFP-CFTR fluorescence in the bleached membrane region recovered to ∼90% of its initial level, indicating that nearly all of the CFTR was mobile. The GFP-CFTR diffusion coefficient (D) was 0.99 ± 0.09 × 10-10 cm2/s at 37 °C, similar to that of other membrane proteins. GFP-CFTR diffusion was not altered by protein kinase A or C activators but was blocked by paraformaldehyde and filipin. CFTR mutants lacking functional PDZ-binding domains (GFPCFTR-ΔTRL and GFP-CFTR-ΔTRA) were also mobile with D significantly increased by ∼60% compared with GFP-CFTR. However, GFP-CFTR, GFP-CFTR-Δ TRL, and GFP-CFTR-ΔTRA had similar mobilities (D ∼12 × 10-10 cm2/s) at the endoplasmic reticulum in brefeldin A-treated cells. Agents that modulate the actin cytoskeleton (cytochalasin D and jasplakinolide) altered the plasma membrane mobility of CFTR but not CFTR- ΔTRL. EBP50 (NHERF1), a PDZ domain-containing protein that interacts with the C terminus of CFTR, diffused freely in the cytoplasm with a diffusion coefficient of 0.9 ± 0.1 × 10-7 cm2/s. EBP50 diffusion increased by ∼2-fold after deletion of its ezrin-binding domain. These results indicate that wild-type CFTR is not tethered statically at the plasma membrane but that its diffusion is dependent on PDZ-domain interactions and an intact actin skeleton. PDZ-domain interactions of CFTR are thus dynamic and occur on a time scale of seconds or faster.

Submucosal gland dysfunction as a primary defect in cystic fibrosis

It has been proposed that defective submucosal gland function in CF airways is a major determinant of CF airway disease. We tested the hypothesis that submucosal gland function is defective early in CF subjects with minimal clinical disease. Functional assays of gland fluid secretion rate and viscosity were performed on freshly obtained nasal biopsies from 6 CF subjects and 5 non‐CF controls (age range 2–22 years). Secretions from individual submucosal glands were visualized by light/fluorescence microscopy after orienting and immobilizing biopsy specimens in a custom chamber. The viscosity of freshly secreted gland fluid after pilocarpine, measured by fluorescence recovery after photobleaching of microinjected FITC‐dextran, was 4.9 ± 0.2‐ vs. 2.2 ± 0.2‐fold greater than water viscosity in CF vs. non‐CF specimens, respectively (se, P<10−4). Gland fluid secretion rate in CF specimens, measured by video imaging (4.5±0.5 nL/min/gland, n=6), was 2.7–fold reduced compared to non‐CF specimens (n=3, P<0.05). Quantitative histology revealed similar size and morphology of submucosal glands in CF and non‐CF specimens. Our results suggest that defective airway submucosal gland function is an early, primary defect in CF. Therapies directed at normalizing gland fluid secretion early in CF may thus reduce lung disease.

19F NMR measurements of the rotational mobility of proteins in vivo.

Three glycolytic enzymes, hexokinase, phosphoglycerate kinase, and pyruvate kinase, were fluorine labeled in the yeast Saccharomyces cerevisiae by biosynthetic incorporation of 5-fluorotryptophan. 19F NMR longitudinal relaxation time measurements on the labeled enzymes were used to assess their rotational mobility in the intact cell. Comparison with the results obtained from relaxation time measurements of the purified enzymes in vitro and from theoretical calculations showed that two of the labeled enzymes, phosphoglycerate kinase and hexokinase, were tumbling in a cytoplasm that had a viscosity approximately twice that of water. There were no detectable signals from pyruvate kinase in vivo, although it could be detected in diluted cell extracts, indicating that there was some degree of motional restriction of the enzyme in the intact cell.

Diffusion of tricarboxylic acid cycle enzymes in the mitochondrial matrix in vivo. Evidence for restricted mobility of a multienzyme complex.

It has been proposed that enzymes in many metabolic pathways, including the tricarboxylic acid cycle in the mitochondrial matrix, are physically associated to facilitate substrate channeling and overcome diffusive barriers. We have used fluorescence recovery after photobleaching to measure the diffusional mobilities of chimeras consisting of green fluorescent protein (GFP) fused to the C terminus of four tricarboxylic acid cycle enzymes: malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinyl-CoA synthetase. The GFP-enzyme chimeras were localized selectively in the mitochondrial matrix in transfected Chinese hamster ovary (CHO) and COS7 cells. Laser photobleaching using a 0.7-μm diameter spot demonstrated restricted diffusion of the GFP-enzyme chimeras. Interestingly, all four chimeras had similar diffusional characteristics, ∼45% of each chimera was mobile and had a diffusion coefficient of 4 × 10−8 cm2/s. In contrast, unconjugated GFP in the mitochondrial matrix (targeted using COX8 leader sequence) diffused freely (nearly 100% mobility) with a greater diffusion coefficient of 20 × 10−8cm2/s. The mobility of the GFP-enzyme chimeras was insensitive to substrate source, ATP depletion, or inhibition of the adenine nucleotide translocase. These results indicate similar mobility characteristics of unrelated tricarboxylic acid cycle enzymes having different sizes and physical properties, providing biophysical evidence for a diffusible multienzyme complex in the mitochondrial matrix.

Defective organellar acidification as a cause of cystic fibrosis lung disease: reexamination of a recurring hypothesis

The cellular mechanisms by which loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel produce cystic fibrosis (CF) lung disease remain uncertain. Defective organellar function has been proposed as an important determinant in the pathogenesis of CF lung disease. According to one hypothesis, reduced CFTR chloride conductance in organelles in CF impairs their acidification by preventing chloride entry into the organelle lumen, which is needed to balance the positive charge produced by proton entry. According to a different hypothesis, CFTR mutation hyperacidifies organelles by an indirect mechanism involving unregulated sodium efflux through epithelial sodium channels. There are reports of defective Golgi, endosomal and lysosomal acidification in CF epithelial cells, defective phagolysosomal acidification in CF alveolar macrophages, and organellar hyperacidification in CF respiratory epithelial cells. The common theme relating too high or low organellar pH to cellular dysfunction and CF pathogenesis is impaired functioning of organellar enzymes, such as those involved in ceramide metabolism and protein processing in epithelial cells and antimicrobial activity in alveolar macrophages. We review here the evidence for defective organellar acidification in CF. Significant technical and conceptual concerns are discussed regarding the validity of data showing too high/low organellar pH in CF cells, and rigorous measurements of organellar pH in CF cells are reviewed that fail to support defective organellar acidification in CF. Indeed, there is an expanding body of evidence supporting the involvement of non-CFTR chloride channels in organellar acidification. We conclude that biologically significant involvement of CFTR in organellar acidification is unlikely.