A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc

Abstract

Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is a widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications.

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

“…We found that adjacent microtubules of Giardia’s adhesive disc exhibited a spacing of 54 ± 1 nm by ExSIM, which agrees excellently with our SMLM measurements on unexpanded specimens (53 ± 6 nm) and cryoelectron tomography measurements (53 ± 2 nm) 21 from similar regions of the disc. Based on Gaussian-fitting of adhesive disc microtubules, which had a FWHM of 33 ± 3 nm, we estimate a spatial resolution of ~30 nm, a value consistent with the ~110 nm resolution of SIM divided by the 3.5× expansion factor.…”

“…At the core of the Giardia cell body (~10 µm × 15 µm) is the adhesive disc, a “suction-cup” composed of a tightly packed, right-handed semiconical-spiral array of microtubules and associated microribbons with a concentric spacing of ~40–70 nm (Figure 1). 21 Many of the disc microtubules originate from a nucleation zone near to the bare region at the center of the disc where eight basal bodies are also located. Eight flagella flank the disc, and each flagellum contains an axoneme consisting of a canonical ~200–250 nm diameter array of nine microtubule doublets separated from each other by ~70 nm and surrounding a central pair of microtubules (“9 + 2” geometry, Figure 1f and Figure 4j).…”

“…Some microtubules were clearly observed to originate near the inner margin of the disc, rather than at the nucleation zone, and to terminate near the outer margin (Supporting Information, Figure S1), both of which have been previously observed by cryo-electron tomography. 21 …”

“…21 Additionally, since giardiasis presently afflicts hundreds of millions of people worldwide, particularly in developing nations, the study of recently identified novel Giardia proteins, termed disc-associated proteins (DAPs), may help identify badly needed targets for new therapies. 22,23 …”

Giardiasis Epidemiology

“…The adhesive disc is essential for atachment and appears to play a major role in the virulence of Giardia [15]. Several disc-associated proteins have been identiied using proteomics [16], and it is clear that the disc is an advanced cytoskeletal structure [17]. At the jejunum, the trophozoites start to encyst forming the wall that enables the parasite to survive outside the host for several weeks in cold water.…”