Structural Study of the Ventral Disc of Giardia lamblia

Joanna Ruth Brown, University of Colorado Boulder

Abstract

Giardia lamblia is single celled eukaryotic parasite. It infects humans, and is one of the most common parasitic infections contracted in the USA. Attachment to the host's intestinal epithelium is essential to the feeding form of Giardia, the trophozoite. Central to attachment is the microtubule (MT) cytoskeleton, the principle structure of which is the ventral disc. The ventral disc is a large array of highly decorated microtubules that forms a domed right-handed spiral. The use of large area montages of multiple tomograms of negatively stained, extracted, ventral discs has provided information on the exact path of each microtubule in the array. This study has provided new information on the total number, length and organization of MTs within this large structure. It was also discovered that the ventral disc is composed of six structurally distinct regions, which vary in terms of microtubule spacing, microtubule curvature, microribbon height, and crossbridge length and morphology. The microtubules of the ventral disc are continuous and pass through multiple regions along their length. The protein decoration along a single microtubule can undergo multiple structural transitions and rearrangements. Cryo-electron tomography and subvolume averaging were used to obtain 3D density maps from four of the newly identified ventral disc regions.

These experiments revealed the presence of smaller protein densities present in all four regions of the ventral disc. These densities are located on both the outside and inside surface of the MT and were found to localize to specific protofilaments. The arrangement and size of these densities was found to vary both within and between the ventral disc regions. One density located on the inner surface of the microtubule, known as Giardia microtubule inner protein 5 (GMIP5), did not vary within or between the regions. It was found that this density occurred at every 8nm repeat along the microtubules and was approximately the size of a tubulin monomer. This structural information obtained was used to narrow the list of possible protein components of GMIP5 from seventeen proteins to only six. These six proteins include: alpha-2 annexin, alpha-3 annexin, alpha-5 annexin, alpha-17 annexin, DAP5374 and DAP13766.