Structure of the lens MP20 mediated adhesive junction

Abstract

Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C- termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency.

Keywords: Lens membrane protein; MicroED; cryoEM; ion-beam milling; junction; lipidic cubic phase.

Figure 1:. Expression, purification and crystallization of human lens MP20.

a. FPLC trace of MP20 with accompanying SDS-PAGE gel (right lane) and western blot (left lane). Arrows indicate the MP20 eluted as a uniform peak consistent in size with an MP20 octamer (8 x MP20 plus two detergent micelles). Inset SDS-PAGE of the peak showing purified MP20 and faint bands for its higher molecular weight oligomers (Lane 1) and the corresponding western blot (Lane 2). b. Cross-polarized light image of a typical drop of LCP with small MP20 crystals appearing as tiny puncta (red arrowheads). c. Region of a whole grid fluorescent atlas showing the LCP with JaneliaFluor 550 conjugated crystals (yellow signal). d. Same crystal boxed out in red in panel c. but imaged in the SEM with the iFLM (red arrowhead). FIBucial markers created with the FIB beam are also visible (green dots and arrows). e-g. SEM top-down image (e) and FIB grazing incidence image (f) of the same field of view (FOV) as in (d) with reprojected coordinates of the crystal (red markers and arrowhead) and the FIBucials (green arrowheads). f. shows an inset of the boxed-out region after milling was performed to create a lamella on the targeted crystal. g. Overlay of the SEM view of the final lamella with the fluorescence signal emanating from the crystal on the milled lamella, confirming presence of the crystal. i. An example of a diffraction pattern from the MicroED dataset collected on the crystal lamella shown in panel g. Blue dashed ring represents diffraction limit for this dataset (3.25Å).

Figure 2 :. MicroED structure of the lens MP20

a. Structure of lens MP20 in rainbow with the N terminus in blue and C-terminus in red. Both termini are cytoplasmic. The loops are indicated as ECL1a, ECL1b, ECL2 and ICL. b and c. Space filling model (half is clipped in c.) of the MP20 monomer showing that no channel or pathway can be seen in the protein. The black lines indicate the position of the lipid bilayer with the extracellular and intracellular sides as indicated.

Figure 3 :. MicroED structure of the lens MP20 junctions

a. MP20 mediated membrane junction. Two MP20 tetramers interact in a head to head fashion (yellow and blue). The adhesive interactions are mediated by the extracellular loops. b. MicroED density map around one yellow and one blue MP20 monomer as they form the adhesive interaction. c. two monomers shown in rainbow with the N terminus in blue and C terminus in red. The black lines indicate the position of the lipid bilayer with the extracellular and intracellular sides as indicated. d and e. Vesicles without and with MP20, respectively. Vesicles without MP20 are evenly distributed on the EM grid while the vesicles with MP20 aggregated. Scale bar = 200nm

Figure 4 :. Insertion of MP20 into fiber cell membranes correlates with the constriction of the extracellular space in vivo

Human lenses labelled with a. WGA b. MP20 c. extracellular dye. d. overlay of b and c. Image montage of the outer cortical region of cryosection taken through the equator of a human donor lenses organ cultured in the extracellular space marker Texas Red-dextran for 6 hours. a. WGA-TRITC labelling to highlight the change in membrane morphology as fiber cells differentiate and become internalized into the adult nucleus. b. MP20 labelling showing the shift form the cytoplasm to the membrane. c. Texas Red-dextran labelling of the extracellular space. White line showing where the dye stops and the extracellular spaces become restricted. d. Double labelling of MP20 (green) and Texas Red-dextran (red) showing the formation of the extracellular diffusion barrier corelates with the membrane insertion of MP20 into the membranes of fiber cells in the adult nucleus. e. Schematic diagram (not drawn to scale) summarizing changes in the morphology of differentiating fiber cells in the outer cortex of the human lens. DF = differentiating fiber cells; RZ = remodeling zone; TZ = transition zon