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 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.The diffusional mobility of DNA fragments in cytoplasm is thought to be an important determinant of the efficacy of DNA delivery in gene therapy and antisense oligonucleotide therapy (1-3). Liposome-mediated gene transfer involves endocytic uptake, release from endosomes, dissociation of DNA from lipid, diffusion through cytoplasm, transport across nuclear pores, and diffusion to nuclear target sites (4 -7). Although considerable attention has been given to the mechanisms of cellular DNA internalization, nuclear uptake, and subsequent molecular events, little is known about the diffusive properties of introduced DNA fragments in cytoplasm and nucleus. It is not known whether the diffusion of DNA fragments is hindered by binding and steric interactions or how the size and physical structure of DNA affect its diffusional properties.Recent studies have provided information about the diffusional mobilities of small and macromolecule-sized solutes in cytoplasm and nucleus. Spot photobleaching measurements indicated that small solutes diffuse freely and rapidly in cytoplasm and nucleus, with diffusion coefficients only 3-4 times lower than that in water (8, 9). Analysis of the individual factors slowing solute diffusion, including fluid-phase viscosity, binding, and collisional interactions, indicated that the principal barrier for diffusion of small solutes was collisional interactions due to macromolecular crowding (8). The "fluid-phase" viscosity of cytoplasm and nucleus, defined as the viscosity sensed by a small probe that does not interact with cellular components, was determined by time-resolved anisotropy (10) and ratio imaging of a viscosity-sensitive fluorescent probe (11) to be only 1.2-1.4 times greater than the viscosity of water. The translational diffusion of larger, macromolecule-sized solutes (FITC 1 -labeled dextrans and Ficolls) in cytoplasm and nucleus was only 3-4-fold slower than in water for solutes Ͻ500 -750 kDa (12) but was markedly slowed for larger solutes (11, 12). The diffusional mobilities of targeted green fluorescent protein chimeras have been measured recently i...

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 s 100 mM for Cl 3 . Indicators with improved sensitivities are needed for cell transport studies, particularly in drug discovery by high-throughput screening, and for measurement of Cl 3 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 3 (I152L), 40 mM for Cl 3 (V163S), and 10 mM for NO 3 3 (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. ß

We report the application of a targetable green fluorescent protein-based cellular halide indicator. Fluorescence titrations of the purified recombinant yellow fluorescent protein YFP-H148Q indicated a pK a of 7.14 in the absence of Cl

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 (M r 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 Ͼ10-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.

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause cystic fibrosis. To investigate interactions of CFTR in living cells, we measured the diffusion of quantum dot-labeled CFTR molecules by single particle tracking. In multiple cell lines, including airway epithelia, CFTR diffused little in the plasma membrane, generally not moving beyond 100-200 nm. However, CFTR became mobile over micrometer distances after 1) truncations of the carboxy terminus, which contains a C-terminal PDZ (PSD95/Dlg/ZO-1) binding motif; 2) blocking PDZ binding by C-terminal green fluorescent protein fusion; 3) disrupting CFTR association with actin by expression of a mutant EBP50/NHERF1 lacking its ezrin binding domain; or 4) skeletal disruption by latrunculin. CFTR also became mobile when the cytoskeletal adaptor protein binding capacity was saturated by overexpressing CFTR or its C terminus. Our data demonstrate remarkable and previously unrecognized immobilization of CFTR in the plasma membrane and provide direct evidence that C-terminal coupling to the actin skeleton via EBP50/ezrin is responsible for its immobility.