Shift in the function of netrin-1 from axon outgrowth to axon branching in developing cerebral cortical neurons.

Background: Netrin-1, a multifunctional axon guidance cue, elicits axon outgrowth via one of its receptors deleted in colorectal cancer (DCC) in several types of neurons, including cerebral cortical neurons of embryonic mice. However, we and others have observed de novo formation of axon branches without axon outgrowth induced by netrin-1 in cortical culture of neonatal hamsters. These previous reports suggested the possibility that netrin-1 function might alter during development, which we here investigated using dissociated culture prepared from cerebral cortices of embryonic mice.

Netrin-1 elevates the level and induces cluster formation of its receptor DCC at the surface of cortical axon shafts in an exocytosis-dependent manner.

During development, a diffusible axon guidance cue, netrin-1, plays a variety of important roles in the correct wiring of the nervous system by inducing axon outgrowth, attraction, repulsion and/or branching in various types of neurons. It has been reported that translocation of its receptor DCC (deleted in colorectal cancer) from an intracellular pool to the plasma membrane enhances outgrowth of rat spinal commissural axons in response to netrin-1 (Bouchard et al., 2004).

Identification and characterization of p49/STRAP as a novel GLUT4-binding protein.

To identify novel regulatory components involved in the recycling of the insulin-responsive glucose transporter GLUT4, we have used the yeast two-hybrid system to isolate GLUT4-binding proteins from a rat adipose cell cDNA library. We found a 49-kDa protein (p49/STRAP) that specifically interacts with an acidic amino acid motif (Q7IGSEDG) in the N-terminus of GLUT4. Confocal immunofluorescence microscopy of primary rat adipose cells shows co-localization of myc-p49 with GLUT4 and also with the ER-resident protein calnexin.

Demonstration of differential quantitative requirements for NSF among multiple vesicle fusion pathways of GLUT4 using a dominant-negative ATPase-deficient NSF.

In this study, we investigated the relative participation of N-ethylmaleimide-sensitive factor (NSF) in vivo in a complex multistep vesicle trafficking system, the translocation response of GLUT4 to insulin in rat adipose cells. Transfections of rat adipose cells demonstrate that over-expression of wild-type NSF has no effect on total, or basal and insulin-stimulated cell-surface expression of HA-tagged GLUT4. In contrast, a dominant-negative NSF (NSF-D1EQ) can be expressed at a low enough level that it has little effect on total HA-GLUT4, but does reduce both basal and insulin-stimulated cell-surface HA-GLUT4 by approximately 50% without affecting the GLUT4 fold-translocation response to insulin.

Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture.

Involvement of the V2 receptor in vasopressin-stimulated translocation of placental leucine aminopeptidase/oxytocinase in renal cells.

The placental leucine aminopeptidase (P-LAP)/oxytocinase is a membrane-bound enzyme thought to play an important role during pregnancy. In this study, we identified the presence of P-LAP protein in the renal distal tubules and collecting ducts. In rat NRK52E cells derived from renal tubules, P-LAP was localized mainly in the intracellular compartment.