Dopamine tone regulates D1 receptor trafficking and delivery in striatal neurons in dopamine transporter-deficient mice

In vivo, G protein-coupled receptors (GPCR) for neurotransmitters undergo complex intracellular trafficking that contribute to regulate their abundance at the cell surface. Here, we report a previously undescribed alteration in the subcellular localization of D1 dopamine receptor (D1R) that occurs in vivo in striatal dopaminoceptive neurons in response to chronic and constitutive hyperdopaminergia. Indeed, in mice lacking the dopamine transporter, D1R is in abnormally low abundance at the plasma membrane of cell bodies and dendrites and is largely accumulated in rough endoplasmic reticulum and Golgi apparatus. Decrease of striatal extracellular dopamine concentration with 6-hydroxydopamine (6- OHDA) in heterozygous mice restores delivery of the receptor from the cytoplasm to the plasma membrane in cell bodies. These results demonstrate that, in vivo, in the central nervous system, the storage in cytoplasmic compartments involved in synthesis and the membrane delivery contribute to regulate GPCR availability and abundance at the surface of the neurons under control of the neurotransmitter tone. Such regulation may contribute to modulate receptivity of neurons to their endogenous ligands and related exogenous drugs.

Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion

A method for cell–cell and cell–liposome fusion at the single-cell level is described. Individual cells or liposomes were first selected and manipulated either by optical trapping or by adhesion to a micromanipulator-controlled ultramicroelectrode. Spatially selective fusion of the cell–cell or cell–liposome pair was achieved by the application of a highly focused electric field through a pair of 5-μm o.d. carbon-fiber ultramicroelectrodes. The ability to fuse together single cells opens new possibilities in the manipulation of the genetic and cellular makeup of individual cells in a controlled manner. In the study of cellular networks, for example, the alteration of the biochemical identity of a selected cell can have a profound effect on the behavior of the entire network. Fusion of a single liposome with a target cell allows the introduction of the liposomal content into the cell interior as well as the addition of lipids and membrane proteins onto the cell surface. This cell–liposome fusion represents an approach to the manipulation of the cytoplasmic contents and surface properties of single cells. As an example, we have introduced a membrane protein (γ-glutamyltransferase) reconstituted in liposomes into the cell plasma membrane.

Heterothermal acclimation: An experimental paradigm for studying the control of thermal acclimation in crabs

A method for the study of the control of the attainment of thermal acclimation has been applied to the crabs, Cancer pagurus and Carcinus maenas. Crabs were heterothermally acclimated by using an anterior–posterior partition between two compartments, one at 8°C and the other at 22°C. One compartment held a three-quarter section of the crab including the central nervous system (CNS), eye stalks, and ipsilateral legs; the other held a quarter section including the contralateral legs. Criteria used to assess the acclimation responses were comparisons of muscle plasma membrane fatty acid composition and “fluidity.” In both species, the major fatty acids of phosphatidylcholine were 16:0, 18:1, 20:5, and 22:6, whereas phosphatidylethanolamine contained significantly less 16:0 but more 18:0; these fatty acids comprised 80% of the total. Differences in fatty acid composition were demonstrated between fractions obtained from the ipsilateral and contralateral legs from the same heterothermally acclimated individual. In all acclimation states (except 22CNS, phosphatidylcholine fraction), membrane lipid saturation was significantly increased with acclimation at 22° as compared with 8°C. Membrane fluidity was determined by using 1,3-diphenyl-1,3,5 hexatriene (DPH) fluorescence polarization. In both species, membranes from legs held at 8° were more fluid than from legs held at 22°C irrespective of the acclimation temperature of the CNS. Heterothermal acclimation demonstrated that leg muscle membrane composition and fluidity respond primarily to local temperature and were not predominately under central direction. The responses between 8°C- and 22°C-acclimated legs were more pronounced when the CNS was cold-acclimated, so a central influence cannot be excluded.

A deoxyribonucleotidase in mitochondria: Involvement in regulation of dNTP pools and possible link to genetic disease

Three cytosolic and one plasma membrane-bound 5′-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5′-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5′- and 2′(3′)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith–Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.