Abstract
The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 "long" (M1) and "short" (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Single-molecule step photobleaching and intensity analysis of GFP-labeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37 degrees C indicated that OAPs diffuse slowly (D approximately 10(-12) cm(2)/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.
Highlights
Neuromyelitis optica [6], in which AQP4 autoantibodies are involved in the pathogenesis of central nervous system neuroinflammation [7]
Single-molecule measurements of the diffusion of various AQP4 mutants and chimeras in live cells showed that OAP formation by M23 involves hydrophobic intermolecular interactions of N-terminal AQP4 residues just downstream of Met-23 and that lack of OAP formation by M1 results from nonspecific blocking of N-terminal interactions by residues just upstream of Met-23 [15]
Characterization of green fluorescent protein (GFP)-labeled AQP4—Cells expressing GFP-labeled AQP4 isoforms were characterized to ensure that the GFP insertions did not interfere with AQP4 plasma membrane targeting or OAP formation
Summary
AQP4 Constructs—DNA sequences encoding AQP4 fragments and GFP were generated by PCR using pCMV6.ratAQP4 and pEGFP-N1 (Clontech) as templates, respectively. AQP4exGFP was labeled with rabbit anti-GFP antibody (Santa Cruz Biotechnology) and quantum dot-conjugated goat anti-rabbit IgG (Invitrogen). AQP4 containing C-terminal GFP was labeled with a recombinant neuromyelitis optica monoclonal autoantibody [7, 17] and quantum dot-conjugated goat anti-human IgG (Invitrogen). Myc-tagged AQP4 was labeled with mouse anti-Myc antibody (Covance) and quantum dot-conjugated goat anti-mouse IgG (Invitrogen). Time-lapse images of AQP4 OAPs in live cells were acquired at three frames/min for up to 3 h, with the excitation laser shuttered between image acquisitions. Immunoblots were blocked with Tris-buffered saline/Tween 20 containing 5% nonfat milk and incubated with rabbit anti-AQP4 antibody (Santa Cruz Biotechnology) or rabbit anti-GFP polyclonal antibody (Abcam), followed by peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories), and signals were detected using an ECL Plus chemiluminescence kit (Amersham Biosciences)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
