Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle.

Abstract

Abstract. Trypanosoma brucei has a precisely ordered microtubule cytoskeleton whose morphogenesis is central to cell cycle events such as organelle positioning, segregation, mitosis, and cytokinesis. We have defined microtubule polarity and show the + ends of the cortical microtubules to be at the posterior end of the cell. Measurements of organelle positions through the cell cycle reveal a high degree of coordinate movement and a relationship with overall cell extension. Quantitative analysis of the segregation of the replicated mitochondrial genome (the kinetoplast) by the flagellar basal bodies identifies a new G2 cell cycle event marker. The subsequent mitosis then positions one "daughter" nucleus into the gap between the segregated basal bodies/kinetoplasts. The anterior daughter nucleus maintains its position relative to the anterior of the cell, suggesting an effective yet cryptic nuclear positioning mechanism. Inhibition of microtubule dynamics by rhizoxin results in a phenomenon whereby cells, which have segregated their kinetoplasts yet are compromised in mitosis, cleave into a nucleated portion and a flagellated, anucleate, cytoplast. We term these cytoplasts "zoids" and show that they contain the posterior (new) flagellum and associated basal-body/ kinetoplast complex. Examination of zoids suggests a role for the flagellum attachment zone (FAZ) in defining the position for the axis of cleavage in trypanosomes. Progression through cytokinesis, (zoid formation) while mitosis is compromised, suggests that the dependency relationships leading to the classical cell cycle check points may be altered in trypanosomes, to take account of the need to segregate two unit genomes (nuclear and mitochondrial) in this cell.T HE cytoskeleton of eukaryotic cells has been shown to operate in important functions such as cell movement, intracellular transport, division, and the control of cell shape. The basic molecular architecture of the individual components of the cytoskeleton confers particular properties on the final complex high order structure. These properties, such as polarity and polymerization dynamics, provide basic attributes of organelles such as microtubules that are reflected in their modulation during cell cycle shape changes or in the establishment of overall cellular polarity.The African trypanosome, Trypanosoma brucei has a very precisely ordered microtubule cytoskeleton that provides an excellent opportunity to study the relationships between molecular events and acquisition of cellular form. The detergent-extracted cytoskeleton of T. brucei retains the overall form of the cell from which it was derived (Sherwin and Gull, 1989a). The cell shape is maintained by a subpellicular corset of microtubules that are cross-linked to each other and to the plasma membrane (HemphiU et al.,

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

“…bb duplication occurs almost simultaneously with the initiation of kDNA S phase (51). Importantly, multiple studies have demonstrated that bb duplication and positioning are tightly linked with kinetoplast replication and segregation (14,41,42). bb duplication events and inter-bb distance are easily monitored by light microscopy, providing a marker for early and late events of the T. brucei kDNA S phase.…”

The Involvement of Two cdc2-related Kinases (CRKs) in Trypanosoma brucei Cell Cycle Regulation and the Distinctive Stage-specific Phenotypes Caused by CRK3 Depletion

“…Kinetoplast segregation is presumably well coordinated with mitosis initiating and leading the cell through cytokinesis and eventual division (8). Recent experimental evidence suggested, however, that these two events may not be inter-dependent of each other in procyclic form trypanosome, because okadaic acid treatment prevented the kinetoplast from replicating but resulted in multinucleated cells (9). Treatment with the nuclear DNA synthesis inhibitor aphidicolin or the antimicrotubule agent rhizoxin resulted in blocked mitosis without inhibiting kinetoplast segregation, which led to cytokinesis and cell division, generating anucleated daughter cells designated as the zoids (7,10).…”

A High-OrderTrans-Membrane Structural Linkage Is Responsible for Mitochondrial Genome Positioning and Segregation by Flagellar Basal Bodies in Trypanosomes

“…Thus, although we have a rapidly growing appreciation of kinetoplast replication we have very little insight to the features responsible for the segregation of the daughter kinetoplasts. We do know that segregation is mediated by the separation of the flagellar basal bodies (Robinson and Gull, 1991;Robinson et al, 1995). In essence, we know how the process functions but we have no information on the structures involved, even though unequivocal evidence for the existence of some form of high-order structural link between the kinetoplast and flagellum has been demonstrated (Robinson and Gull, 1991).…”