scholarly journals The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins

2021 ◽  
Vol 17 (5) ◽  
pp. e1009588
Author(s):  
Amy N. Sinclair ◽  
Christine T. Huynh ◽  
Thomas E. Sladewski ◽  
Jenna L. Zuromski ◽  
Amanda E. Ruiz ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Single Layer ◽  
Microtubule Array ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Asymmetric Shape ◽  
Local Properties ◽  
Parasite Viability ◽  
Spindle Microtubules ◽  
Flagellar Axoneme

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.

scholarly journals The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins

2020 ◽  
Author(s):  
Amy N. Sinclair ◽  
Christine T. Huynh ◽  
Thomas E. Sladewski ◽  
Jenna L. Zuromski ◽  
Amanda E. Ruiz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Brucei ◽  
Causative Agent ◽  
Microtubule Array ◽  
Wide Range ◽  
Associated Proteins ◽  
Asymmetric Shape ◽  
Local Properties ◽  
And Function ◽  
Viscous Solutions

AbstractMicrotubules are inherently dynamic cytoskeletal polymers whose length and activity can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle, comprising a single microtubule layer that is crosslinked to each other and the plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins. We characterize the localization and function of the array-associated protein PAVE1, which is a component of the inter-microtubule crosslinking fibrils present within the posterior subdomain. PAVE1 functions to stabilize these microtubules to produce the tapered cell posterior. PAVE1 and the newly identified PAVE2 form a complex that binds directly to the microtubule lattice. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for retaining PAVE1 within the posterior subdomain, and also maintains array-associated proteins in the middle and anterior subdomains of the array. The arrangement of proteins within the array is likely to tune the local properties of the array microtubules and create the asymmetric shape of the cell, which is essential for parasite viability.Author summaryMany parasitic protists use arrays of microtubules that contact the inner leaflet of the plasma membrane, typically known as subpellicular microtubules, to shape their cells into forms that allow them to efficiently infect their hosts. While subpellicular arrays are found in a wide range of parasites, very little is known about how they are assembled and maintained. Trypanosoma brucei, which is the causative agent of human African trypanosomiasis, has an elaborate subpellicular array that produces the helical shape of the parasite, which is essential for its ability to move within crowded and viscous solutions. We have identified a series of proteins that have a range of localization patterns within the array, which suggests that the array is regulated by subdomains of array-associated proteins that may tune the local properties of the microtubules to suit the stresses found at different parts of the cell body. Among these proteins are the first known components of the inter-microtubule crosslinks that are thought to stabilize array microtubules, as well as a potential regulator of the array subdomains. These results establish a foundation to understand how subpellicular arrays are built, shaped, and maintained, which has not previously been appreciated.

scholarly journals Two Related Subpellicular Cytoskeleton-associated Proteins inTrypanosoma brucei Stabilize Microtubules

2002 ◽  
Vol 13 (3) ◽  
pp. 1058-1070 ◽  
Author(s):  
Cécile Vedrenne ◽  
Christiane Giroud ◽  
Derrick R. Robinson ◽  
Sébastien Besteiro ◽  
Christophe Bosc ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Trypanosoma Brucei ◽  
Cold Resistance ◽  
Chinese Hamster ◽  
Low Molecular Weight ◽  
Microtubule Bundle ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Asymmetric Cytokinesis

The subpellicular microtubules of the trypanosome cytoskeleton are cross-linked to each other and the plasma membrane, creating a cage-like structure. We have isolated, from Trypanosoma brucei, two related low-molecular-weight cytoskeleton-associated proteins (15- and 17-kDa), called CAP15 and CAP17, which are differentially expressed during the life cycle. Immunolabeling shows a corset-like colocalization of both CAPs and tubulin. Western blot and electron microscope analyses show CAP15 and CAP17 labeling on detergent-extracted cytoskeletons. However, the localization of both proteins is restricted to the anterior, microtubule minus, and less dynamic half of the corset. CAP15 and CAP17 share properties of microtubule-associated proteins when expressed in heterologous cells (Chinese hamster ovary and HeLa), colocalization with their microtubules, induction of microtubule bundle formation, cold resistance, and insensitivity to nocodazole. When overexpressed inT. brucei, both CAP15 and CAP17 cover the whole subpellicular corset and induce morphological disorders, cell cycle-based abnormalities, and subsequent asymmetric cytokinesis.

The Repetitive Microtubule-Associated Proteins MARP-1 and MARP-2 of Trypanosoma brucei

1994 ◽  
Vol 112 (3) ◽  
pp. 241-251 ◽  
Author(s):  
Marianne Affolter ◽  
Andrew Hemphill ◽  
Isabel Roditi ◽  
Norbert Müller ◽  
Thomas Seebeck
Keyword(s):  
Trypanosoma Brucei ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

scholarly journals Microtubule polyglutamylation is important for regulating cytoskeletal architecture and motility in Trypanosoma brucei

2020 ◽  
Vol 133 (18) ◽  
pp. jcs248047 ◽  
Author(s):  
Jana Jentzsch ◽  
Adal Sabri ◽  
Konstantin Speckner ◽  
Gertrud Lallinger-Kube ◽  
Matthias Weiss ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Dynamic Behaviour ◽  
Cell Movement ◽  
Post Translational Modifications ◽  
Life Cycle Stages ◽  
Associated Proteins ◽  
Flagellar Axoneme

ABSTRACTThe shape of kinetoplastids, such as Trypanosoma brucei, is precisely defined during the stages of the life cycle and governed by a stable subpellicular microtubule cytoskeleton. During the cell cycle and transitions between life cycle stages, this stability has to transiently give way to a dynamic behaviour to enable cell division and morphological rearrangements. How these opposing requirements of the cytoskeleton are regulated is poorly understood. Two possible levels of regulation are activities of cytoskeleton-associated proteins and microtubule post-translational modifications (PTMs). Here, we investigate the functions of two putative tubulin polyglutamylases in T. brucei, TTLL6A and TTLL12B. Depletion of both proteins leads to a reduction in tubulin polyglutamylation in situ and is associated with disintegration of the posterior cell pole, loss of the microtubule plus-end-binding protein EB1 and alterations of microtubule dynamics. We also observe a reduced polyglutamylation of the flagellar axoneme. Quantitative motility analysis reveals that the PTM imbalance correlates with a transition from directional to diffusive cell movement. These data show that microtubule polyglutamylation has an important role in regulating cytoskeletal architecture and motility in the parasite T. brucei.This article has an associated First Person interview with the first author of the paper.

scholarly journals A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei

1992 ◽  
Vol 117 (1) ◽  
pp. 95-103 ◽  
Author(s):  
A Hemphill ◽  
M Affolter ◽  
T Seebeck
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Binding Motif ◽  
Amino Acid Repeat ◽  
Microtubule Binding ◽  
Microtubule Associated Proteins ◽  
Repeat Units ◽  
Associated Proteins

The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule-associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins.

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.

A high molecular mass phosphoprotein defined by a novel monoclonal antibody is closely associated with the intermicrotubule cross bridges in the Trypanosoma brucei cytoskeleton

1992 ◽  
Vol 103 (3) ◽  
pp. 665-675 ◽  
Author(s):  
A. Woods ◽  
A.J. Baines ◽  
K. Gull
Keyword(s):  
Molecular Mass ◽  
Trypanosoma Brucei ◽  
Minor Component ◽  
Relative Molecular Mass ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Cytoplasmic Face ◽  
Associated Proteins ◽  
Cross Bridges ◽  
Main Component

The main component of the cell body cytoskeleton of Trypanosoma brucei is the highly organised array of stable, subpellicular microtubules on the cytoplasmic face of the plasma membrane. Although several microtubule associated proteins (MAPs) have been shown to be associated with this array, the mechanisms by which individual microtubules interact with one another and with the membrane are still largely undetermined. In this study we have used the T. brucei cytoskeleton as a complex immunogen for the production of monoclonal antibodies to define novel cytoskeletal antigens. Screening by immunofluorescence enabled the selection of an antibody, WCB-1, which detects an antigen associated specifically with the subpellicular microtubules and not with the flagellum microtubules. The antigen (WCB210) was shown to have a relative molecular mass of 210,000 by western blotting. Immunogold studies showed the epitope to be located on the membrane-facing side of the subpellicular cage; it appears to be closely associated with the cross-bridges lying between the microtubules. Unlike many MAPs this protein was shown not to be heat stable and is predicted to be a roughly globular monomer. Even though WCB210 is a very minor component of the cytoskeleton it is heavily phosphorylated. It is possible that this protein is involved in regulation of the subpellicular microtubule crossbridges by interaction with other proteins.

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