Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini

The norepinephrine transporter (NET) mediates reuptake of synaptically released norepinephrine in central and peripheral noradrenergic neurons. The molecular processes governing availability of NET in the plasma membrane are poorly understood. Here we use the fluorescent cocaine analogue JHC 1-64, as well as several other approaches, to investigate the trafficking itinerary of NET in live noradrenergic neurons. Confocal imaging revealed extensive constitutive internalization of JHC 1-64-labeled NET in the neuronal somata, proximal extensions and presynaptic boutons. Phorbol 12-myristate 13-acetate increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in uptake capacity. Internalized NET strongly colocalized with the “long loop” recycling marker Rab11, whereas less overlap was seen with the “short loop” recycling marker Rab4 and the late endosomal marker Rab7. Moreover, mitigating Rab11 function by overexpression of dominant negative Rab11 impaired NET function. Sorting of NET to the Rab11 recycling compartment was further supported by confocal imaging and reversible biotinylation experiments in transfected differentiated CATH.a cells. In contrast to NET, the dopamine transporter displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the differentiated CATH.a cells. Exchange of domains between the two homologous transporters revealed that this difference was determined by non-conserved structural elements in the intracellular N terminus. We conclude that NET displays a distinct trafficking itinerary characterized by continuous shuffling between the plasma membrane and the Rab11 recycling compartment and that the functional integrity of the Rab11 compartment is critical for maintaining proper presynaptic NET function

Retrieval of the conductivity spectrum of tissues in vitro with novel multimodal tomography

Objective: Imaging of tissue engineered three-dimensional (3D) specimens is challenging due to their thickness. We propose a novel multimodal imaging technique to obtain multi-physical 3D images and the electrical conductivity spectrum of tissue engineered specimens in vitro. Approach: We combine simultaneous recording of rotational multifrequency electrical impedance tomography (R-mfEIT) with optical projection tomography (OPT). Structural details of the specimen provided by OPT are used here as geometrical priors for R-mfEIT. Main results: This data fusion enables accurate retrieval of the conductivity spectrum of the specimen. We demonstrate experimentally the feasibility of the proposed technique using a potato phantom, adipose and liver tissues, and stem cells in biomaterial spheroids. The results indicate that the proposed technique can distinguish between viable and dead tissues and detect the presence of stem cells. Significance: This technique is expected to become a valuable tool for monitoring tissue engineered specimens' growth and viability in vitro.publishedVersionPeer reviewe