Imaging and Analysis of the Microtubule Cytoskeleton in Giardia

Dawson, Scott C.; House, Susan A.

doi:10.1016/s0091-679x(10)97017-9

Cited by 23 publications

(25 citation statements)

References 80 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ventral disc has been shown to be necessary for attachment of Giardia to different substrates (21), yet the role of specific disc conformational changes in attachment is less understood. Attachment, ventral disc conformational dynamics, and contacts of trophozoites with a glass substrate can be quantified using three-dimensional live imaging (11,25). We have previously demonstrated that attachment of live trophozoites occurs within seconds as a stepwise process characterized by defined contacts of the trophozoite with an inert surface.…”

Section: Discussionmentioning

confidence: 99%

“…For live imaging experiments, trophozoites were also grown on coverslips placed in 8-well dishes in a sealed chamber (PlasLabs) and gassed with 100% N 2 to maintain a low-oxygen atmosphere. This chamber was incubated at 37°C prior to live cell imaging as previously described (11).…”

Section: Methodsmentioning

confidence: 99%

“…Trophozoites were resuspended in 10 ml of chilled medium, and then 3 ml was transferred to custom sample holders capped with thick, circular glass slides. The cells were incubated at 37°C for 1 h in a micro-oxic chamber to allow attachment to the glass slides (11). Sample holders were centrifuged at 37°C in a hanging-bucket centrifuge (Sorvall RC5C HB4 rotor 07) at 10,000 rpm (2.1 nN normal force).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The Giardia Median Body Protein Is a Ventral Disc Protein That Is Critical for Maintaining a Domed Disc Conformation during Attachment

Woessner

Dawson

2012

Eukaryot Cell

Self Cite

View full text Add to dashboard Cite

Giardia has unique microtubule structures, including the ventral disc, the primary organelle of attachment to the host, and the median body, a structure of undefined function. During attachment, the ventral disc has a domed conformation and enables Giardia to attach to the host intestinal epithelia within seconds. The mechanism of attachment via the ventral disc and the overall structure, function, and assembly of the ventral disc are not well understood. Our recent proteomic analysis of the ventral disc indicated that the median body protein (MBP), previously reported to localize exclusively to the median body, was primarily localized to the ventral disc. Using high-resolution light and electron microscopy, we confirm that the median body protein localizes primarily to the overlap zone of the ventral disc. The MBP also occasionally localized to the median body during prophase. To define the contribution of MBP to the ventral disc structure, we depleted MBP using an anti-MBP morpholino. We found that the ventral disc was no longer able to form properly and that the disc structure often had an aberrant nondomed or flattened horseshoe conformation. The ability of attached anti-MBP morpholino-treated trophozoites to withstand shear forces and normal forces was significantly decreased. Most notably, the plasma membrane contacts with the surface, including those of the bare area, were defective after the anti-MBP knockdown. To our knowledge, this is the first ventral disc protein whose depletion directly alters ventral disc structure, confirming that the domed ventral disc conformation is important for robust attachment.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Giardia Median Body Protein Is a Ventral Disc Protein That Is Critical for Maintaining a Domed Disc Conformation during Attachment

Woessner

Dawson

2012

Eukaryot Cell

Self Cite

View full text Add to dashboard Cite

show abstract

“…2 ml of fresh PHEM buffer with 2% triton-X-100 was added to the cells and transferred to an Eppendorf tube. Cytoskeletons were vortexed on low for 30 minutes; water bath sonicated for 1 min and then checked in the light microscope (see also: Dawson and House, 2010A). The vortex/sonication procedure was repeated until the cytoskeletons were adequately extracted.…”

Section: Methodsmentioning

confidence: 99%

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

Brown

Schwartz

Heumann

et al. 2016

Journal of Structural Biology

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Giardia has a highly organized microtubule cytoskeleton (Fig. 1A), including eight flagella, a ventral adhesive disk (formed from an overlapping sheet of parallel microtubules that facilitates attachment to the host), and a median body (a bundled microtubule structure thought to be a reservoir of tubulin and disk components) (35). Expression of mNG-Tub under its endogenous promoter permitted visualization of microtubules in the ventral disk, median body, flagella, and mitotic spindles ( Fig.…”

Section: Giardia Uses a Tubulin Reservoir To Support Rapid Mitosis Anmentioning

confidence: 99%

Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking

Hardin

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

138

View full text Add to dashboard Cite

Devoid of all known canonical actin-binding proteins, the prevalent parasite Giardia lamblia uses an alternative mechanism for cytokinesis. Unique aspects of this mechanism can potentially be leveraged for therapeutic development. Here, live-cell imaging methods were developed for Giardia to establish division kinetics and the core division machinery. Surprisingly, Giardia cytokinesis occurred with a median time that is ∼60 times faster than mammalian cells. In contrast to cells that use a contractile ring, actin was not concentrated in the furrow and was not directly required for furrow progression. Live-cell imaging and morpholino depletion of axonemal Paralyzed Flagella 16 indicated that flagella-based forces initiated daughter cell separation and provided a source for membrane tension. Inhibition of membrane partitioning blocked furrow progression, indicating a requirement for membrane trafficking to support furrow advancement. Rab11 was found to load onto the intracytoplasmic axonemes late in mitosis and to accumulate near the ends of nascent axonemes. These developing axonemes were positioned to coordinate trafficking into the furrow and mark the center of the cell in lieu of a midbody/phragmoplast. We show that flagella motility, Rab11, and actin coordination are necessary for proper abscission. Organisms representing three of the five eukaryotic supergroups lack myosin II of the actomyosin contractile ring. These results support an emerging view that flagella play a central role in cell division among protists that lack myosin II and additionally implicate the broad use of membrane tension as a mechanism to drive abscission.

show abstract

Imaging and Analysis of the Microtubule Cytoskeleton in Giardia

Cited by 23 publications

References 80 publications

The Giardia Median Body Protein Is a Ventral Disc Protein That Is Critical for Maintaining a Domed Disc Conformation during Attachment

The Giardia Median Body Protein Is a Ventral Disc Protein That Is Critical for Maintaining a Domed Disc Conformation during Attachment

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

Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking

Contact Info

Product

Resources

About