scholarly journals An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii

2016 ◽  
Vol 27 (3) ◽  
pp. 549-571 ◽  
Author(s):  
Jun Liu ◽  
Yudou He ◽  
Imaan Benmerzouga ◽  
William J. Sullivan ◽  
Naomi S. Morrissette ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Mitotic Spindle ◽  
Functional Differentiation ◽  
Cortical Microtubules ◽  
Human Parasite ◽  
Cellular Morphogenesis ◽  
Associated Proteins ◽  
Spindle Microtubules ◽  
The Stability ◽  
Directional Transport

Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy.

scholarly journals Cryo-EM structure of cortical microtubules from human parasite Toxoplasma gondii identifies their microtubule inner proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiangli Wang ◽  
Yong Fu ◽  
Wandy L. Beatty ◽  
Meisheng Ma ◽  
Alan Brown ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Ex Vivo ◽  
Structural Basis ◽  
Cortical Microtubules ◽  
Human Pathogen ◽  
Chemical Treatments ◽  
Human Parasite ◽  
Evolutionarily Conserved ◽  
Bona Fide ◽  
The Stability

AbstractIn living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of β-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside.

scholarly journals Publisher Correction: Cryo-EM structure of cortical microtubules from human parasite Toxoplasma gondii identifies their microtubule inner proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiangli Wang ◽  
Yong Fu ◽  
Wandy L. Beatty ◽  
Meisheng Ma ◽  
Alan Brown ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cortical Microtubules ◽  
Human Parasite

scholarly journals The Putative RNA-Binding Protein Nrp1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered Into Heat-Induced Protein Aggregates in Fission Yeast

2021 ◽  
Author(s):  
Masashi Yukawa ◽  
Mitsuki Ohishi ◽  
Yusuke Yamada ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Protein Aggregates ◽  
Associated Proteins ◽  
Spindle Microtubules ◽  
The Stability ◽  
And Function ◽  
Post Transcriptional Regulation

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here we show that deletion of the nrp1 gene, which encodes a putative RNA-binding protein with unknown function, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the nrp1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Nrp1 are essential for its cytoplasmic localization and function. We have also found that a portion of Nrp1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Nrp1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

scholarly journals Stability and function of a putative microtubule-organizing center in the human parasite Toxoplasma gondii

2017 ◽  
Vol 28 (10) ◽  
pp. 1361-1378 ◽  
Author(s):  
Jacqueline M. Leung ◽  
Yudou He ◽  
Fangliang Zhang ◽  
Yu-Chen Hwang ◽  
Eiji Nagayasu ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Structural Integrity ◽  
Lytic Cycle ◽  
Host Cells ◽  
Structural Defects ◽  
Cortical Microtubules ◽  
Microtubule Organizing Center ◽  
Polar Ring ◽  
Human Parasite ◽  
Organizing Center

The organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and show that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 (ΔkinesinAΔapr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live ΔkinesinAΔapr1 parasites and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and invade host cells, as well as decreased secretion of effectors important for these processes. Together the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.

scholarly journals Mitotic Acetylation of Microtubules Promotes Centrosomal PLK1 Recruitment and Is Required to Maintain Bipolar Spindle Homeostasis

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1859
Author(s):  
Sylvia Fenosoa Rasamizafy ◽  
Claude Delsert ◽  
Gabriel Rabeharivelo ◽  
Julien Cau ◽  
Nathalie Morin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mitotic Spindle ◽  
Mitotic Progression ◽  
Post Translational Modifications ◽  
Microtubule Associated Proteins ◽  
Mitotic Kinase ◽  
Tubulin Acetylation ◽  
Associated Proteins ◽  
Spindle Microtubules ◽  
Bipolar Spindles

Tubulin post-translational modifications regulate microtubule properties and functions. Mitotic spindle microtubules are highly modified. While tubulin detyrosination promotes proper mitotic progression by recruiting specific microtubule-associated proteins motors, tubulin acetylation that occurs on specific microtubule subsets during mitosis is less well understood. Here, we show that siRNA-mediated depletion of the tubulin acetyltransferase ATAT1 in epithelial cells leads to a prolonged prometaphase arrest and the formation of monopolar spindles. This results from collapse of bipolar spindles, as previously described in cells deficient for the mitotic kinase PLK1. ATAT1-depleted mitotic cells have defective recruitment of PLK1 to centrosomes, defects in centrosome maturation and thus microtubule nucleation, as well as labile microtubule-kinetochore attachments. Spindle bipolarity could be restored, in the absence of ATAT1, by stabilizing microtubule plus-ends or by increasing PLK1 activity at centrosomes, demonstrating that the phenotype is not just a consequence of lack of K-fiber stability. We propose that microtubule acetylation of K-fibers is required for a recently evidenced cross talk between centrosomes and kinetochores.

scholarly journals Stability and function of a putative microtubule organizing center in the human parasite Toxoplasma gondii

2017 ◽  
Author(s):  
Jacqueline M. Leung ◽  
Yudou He ◽  
Fangliang Zhang ◽  
Yu-Chen Hwang ◽  
Eiji Nagayasu ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Structural Integrity ◽  
Lytic Cycle ◽  
Host Cells ◽  
Structural Defects ◽  
Cortical Microtubules ◽  
Microtubule Organizing Center ◽  
Polar Ring ◽  
Human Parasite ◽  
Organizing Center

ABSTRACTThe organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here, we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and discovered that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 (ΔkinesinAΔapr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live ΔkinesinAΔapr1 parasites, and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and to invade host cells, as well as decreased secretion of effectors important for these processes. Together, the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.

scholarly journals The Putative RNA-Binding Protein Dri1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered into Heat-Induced Protein Aggregates in Fission Yeast

2021 ◽  
Vol 22 (9) ◽  
pp. 4795
Author(s):  
Masashi Yukawa ◽  
Mitsuki Ohishi ◽  
Yusuke Yamada ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Protein Aggregates ◽  
Associated Proteins ◽  
Spindle Microtubules ◽  
The Stability ◽  
And Function ◽  
Post Transcriptional Regulation

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here, we show that deletion of the dri1 gene, which encodes a putative RNA-binding protein, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the dri1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Dri1 are essential for its cytoplasmic localization and function. We have also found that a portion of Dri1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Dri1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

scholarly journals A TOGL domain specifically targets yeast CLASP to kinetochores to stabilize kinetochore microtubules

2014 ◽  
Vol 205 (4) ◽  
pp. 555-571 ◽  
Author(s):  
Caroline Funk ◽  
Verena Schmeiser ◽  
Jennifer Ortiz ◽  
Johannes Lechner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Associated Proteins ◽  
Spindle Midzone ◽  
Spindle Microtubules ◽  
The Stability ◽  
Overexpressed Gene

Cytoplasmic linker–associated proteins (CLASPs) are proposed to function in cell division based on their ability to bind tubulin via arrayed tumor overexpressed gene (TOG)–like (TOGL) domains. Structure predictions suggest that CLASPs have at least two TOGL domains. We show that only TOGL2 of Saccharomyces cerevisiae CLASP Stu1 binds to tubulin and is required for polymerization of spindle microtubules (MTs) in vivo. In contrast, TOGL1 recruits Stu1 to kinetochores (KTs), where it is essential for the stability and tension-dependent regulation of KT MTs. Stu1 is also recruited to spindle MTs by different mechanisms depending on the mitotic phase: in metaphase, Stu1 binds directly to the MT lattice, whereas in anaphase, it is localized indirectly to the spindle midzone. In both phases, the activity of TOGL2 is essential for interpolar MT stability, whereas TOGL1 is not involved. Thus, the two TOGL domains of yeast CLASP have different activities and execute distinct mitotic functions.

scholarly journals Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein.

1985 ◽  
Vol 101 (5) ◽  
pp. 1858-1870 ◽  
Author(s):  
N Hirokawa ◽  
R Takemura ◽  
S Hisanaga
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Mitotic Spindle ◽  
Cytoplasmic Dynein ◽  
High Salt ◽  
Mitotic Spindles ◽  
Mitotic Apparatus ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Spindle Microtubules

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three-dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button-like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule-associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75-kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)

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