More than Microtubules: The Structure and Function of the Subpellicular Array in Trypanosomatids

Abstract

The subpellicular microtubule array defines the wide range of cellular morphologies found in parasitic kinetoplastids (trypanosomatids). Morphological studies have characterized array organization, but little progress has been made towards identifying the molecular mechanisms that are responsible for array differentiation during the trypanosomatid life cycle, or the apparent stability and longevity of array microtubules. In this review, we outline what is known about the structure and biogenesis of the array, with emphasis on Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, which cause life-threatening diseases in humans and livestock. We highlight unanswered questions about this remarkable cellular structure that merit new consideration in light of our recently improved understanding of how the 'tubulin code' influences microtubule dynamics to generate complex cellular structures.

Keywords: Trypanosoma; corset; cytokinesis; microtubules; morphology; subpellicular array.

Figure 1, Key Figure.. The microtubules of the subpellicular array are extensively crosslinked.

(a) Schematics of the arrangement of the subpellicular microtubules in a Trypanosoma brucei procyclic-form trypomastigote (top), a Trypanosoma cruzi epimastigote (middle), and a Leishmania mexicana promastigote (bottom). The growing plus-ends of the microtubules are oriented towards the cell posterior. In T. brucei and T. cruzi trypomastigotes and epimastigotes, the array microtubules bend around the flagellar pocket as the flagellum exits onto the cell surface. In T. cruzi, the array microtubules also curve around the cytostome, which is an endocytic organelle that forms an opening at the cell surface. In Leishmania, the array ends at the flagellar pocket located at the cell anterior. Based on [2,3,11,27,38]. (b) Schematic of the fibrils that crosslink the microtubules of the array together from the boxed region in (a). (c) Tomogram (z = 50 nm) of an extracted cytoskeleton of T. brucei in which the crosslinks between the array microtubules are visible (arrow). The microtubules of the array are separated from each other by a space of 18–22 nm. Image kindly contributed by Dr. Sue Vaughan and Mr. Timm Mohr. (d) Original transmission electron micrograph of a positively-stained procyclic-form T. brucei trypomastigote cytoskeleton. The cytoskeleton was critical-point dried and cleaved to remove half of the array. (Inset) Higher magnification image of the cell posterior, in which the inter-microtubule crosslinks are clearly shown (arrows). Where a microtubule ends in the array (asterisk), the microtubules on either side of it continue and become crosslinked to each other.

Figure 2.. The microtubules of the array are tightly associated with the plasma membrane.

(a) Schematic of a trypomastigote cross-section, looking from posterior-to-anterior. The array microtubules are located just 10 nm from the plasma membrane. The microtubule quartet (MtQ) has the opposite polarity to the other array microtubules. The flagellum attachment zone (FAZ) filament interrupts the array and attaches the flagellum to the cell surface. PFR; para-flagellar rod. (b) Thin-section transmission electron micrograph of a T. brucei cell in the same orientation as the schematic in (a). Kindly contributed by Dr. Sue Vaughan and Mr. Timm Mohr.

Figure 3.. The primary morphologies of human-infectious trypanosomatid parasites.

(Left) Schematic of cell structure and topological order of single-copy organelles in each morphology. The trypomastigote form is characterized by the posterior localization of the mitochondrial DNA aggregate, called the kinetoplast (K), to that of the nucleus (N). An attached flagellum exits from the flagellar pocket (FP) near the cell posterior. In the epimastigote form, the kinetoplast is anterior to the nucleus. While the flagellum is still attached to the cell surface, it also has a long, cell-free overhang. The promastigote form has a similar arrangement of DNA-containing organelles as the epimastigote, but the flagellum is unattached after exiting the flagellar pocket at the cell anterior. The kinetoplast is also positioned anterior to the nucleus in the smaller, more-spherical amastigote form. The flagellum is short and non-motile, and barely protrudes from the flagellar pocket. (Right) A list of the life-cycle stages in which the human-infectious trypanosomatids adopt these morphologies.

Figure 4.. Cell division in trypanosomatid parasites.

(a) In a Trypanosoma brucei trypomastigote, the kinetoplast replicates prior to the nucleus. The array enlarges to accommodate the increase in cell volume, after which cytokinesis initiates at the cell anterior and progresses towards the cell posterior. Microtubule segregation and bundling separates the subpellicular array, leading to abscission. The division is asymmetric, producing daughter cells with either a rounded or pointed posterior. The old and new flagellum are indicated in dark and light grey, respectively. Based on [40]. (b) The cell body of a Leishmania mexicana promastigote lengthens before undergoing a dramatic reduction in cell length while increasing in cell width during replication. The nucleus replicates prior to the kinetoplast. Cytokinesis also proceeds towards the cell posterior. Based on [38]. (c) In a Trypanosoma cruzi epimastigote, the kinetoplast replicates prior to the nucleus, after which cytokinesis initiates at the cell anterior. The daughter cells are attached end-to-end in the final stage of cytokinesis, but it is currently unknown how T. cruzi epimastigotes resolve the array to complete abscission. Based on [44].

Figure 5.. Microtubule modifications found in the trypanosomatid subpellicular array.

(a) (Top) A microtubule is a hollow tube made up of tubulin protofilaments, which are comprised of α- and β-tubulin heterodimers (green and purple, respectively. (Bottom) Both α- and β-tubulin subunits have long, intrinsically disordered and highly-charged C-terminal tails that face the cytoplasm, and are the most common site of post-translational modifications. Trypanosomatid microtubules are highly glutamylated on both α- and β-tubulin subunits. The α- and β-tubulin subunits also undergo detyrosination in trypanosomatids, in which the terminal tyrosine residue is cleaved. Unlike most tubulin post-translational modifications, acetylation occurs at an inward-facing site on the α-tubulin subunit. The amino acid sequence of the tails in this schematic is from Trypanosoma brucei. (b) The amino acid sequences of the cytoplasmic C-terminal tails of α- and β-tubulin from Trypanosoma brucei, Trypanosoma cruzi (Y-strain), and Leishmania mexicana. The human and yeast sequences are listed beneath for comparison, and acidic and aromatic charges are indicated. Sequences obtained from [93,119].