Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

Paredez, Alexander R.; Nayeri, Arash; Xu, Jennifer; Krtková, Jana; Cande, W. Zacheus

doi:10.1128/ec.00041-14

Cited by 35 publications

(57 citation statements)

References 58 publications

(74 reference statements)

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“…In agreement with our previous studies, actin was enriched around nuclei and forming spindles at the onset of mitosis, and remained enriched around the spindles and the developing axonemes throughout mitosis (Fig. 3A) (31,62). The enrichment of actin around microtubule structures is consistent with actin's role in positioning them (31).…”

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

confidence: 91%

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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.

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138

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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.

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

confidence: 91%

“…Dataset S2 provides sequences and workflow. Note that N-terminal fusions result in morpholino-resistant constructs (62), and therefore we included the first 27 bp of the coding sequence to restore morpholino sensitivity (79) to the 3HA-Rab11 MS construct.…”

Section: Methodsmentioning

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.

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138

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“…While the actin machinery mediates amoeboid movement, it apparently functions without the normally associated motor protein myosin, for which no homologs are encoded by T. vaginalis [10]. The closely related excavate Giardia lamblia does not undergo morphogenesis either and not only lacks myosin, but all known accessory proteins of the actin cytoskeleton [70,71]. A protein phosphatase, TvPP1␥, is also associated with host cell adhesion and proliferation, both of which were blocked by calyculin A, a specific inhibitor of this phosphatase [72].…”

Section: Morphogenesismentioning

confidence: 99%

The biology of Trichomonas vaginalis in the light of urogenital tract infection

Kusdian

Gould

2014

Molecular and Biochemical Parasitology

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“…The coverslips were mounted with ProLong Gold anti-fade plus DAPI, (Thermo Fisher Scientific, Rockford, IL). Fluorescence deconvolution microscopy images were collected as described [34]. A minimum of 150 cells were examined and 30 imaged per experiment.…”

Section: Methodsmentioning

confidence: 99%

Identification and Validation of Small-Gatekeeper Kinases as Drug Targets in Giardia lamblia

Hennessey

Smith

et al. 2016

PLoS Negl Trop Dis

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Giardiasis is widely acknowledged to be a neglected disease in need of new therapeutics to address toxicity and resistance issues associated with the limited available treatment options. We examined seven protein kinases in the Giardia lamblia genome that are predicted to share an unusual structural feature in their active site. This feature, an expanded active site pocket resulting from an atypically small gatekeeper residue, confers sensitivity to “bumped” kinase inhibitors (BKIs), a class of compounds that has previously shown good pharmacological properties and minimal toxicity. An initial phenotypic screen for biological activity using a subset of an in-house BKI library found that 5 of the 36 compounds tested reduced trophozoite growth by at least 50% at a concentration of 5 μM. The cellular localization and the relative expression levels of the seven protein kinases of interest were determined after endogenously tagging the kinases. Essentiality of these kinases for parasite growth and infectivity were evaluated genetically using morpholino knockdown of protein expression to establish those that could be attractive targets for drug design. Two of the kinases were critical for trophozoite growth and attachment. Therefore, recombinant enzymes were expressed, purified and screened against a BKI library of >400 compounds in thermal stability assays in order to identify high affinity compounds. Compounds with substantial thermal stabilization effects on recombinant protein were shown to have good inhibition of cell growth in wild-type G. lamblia and metronidazole-resistant strains of G. lamblia. Our data suggest that BKIs are a promising starting point for the development of new anti-giardiasis therapeutics that do not overlap in mechanism with current drugs.

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Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

Cited by 35 publications

References 58 publications

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

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

The biology of Trichomonas vaginalis in the light of urogenital tract infection

Identification and Validation of Small-Gatekeeper Kinases as Drug Targets in Giardia lamblia

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