Kinetochore fibre dynamics outside the context of the spindle during anaphase

Abstract

Chromosomes move polewards as kinetochore fibres shorten during anaphase. Fibre dynamics and force production have been studied extensively, but little is known about these processes in the absence of the spindle matrix. Here we show that laser-microbeam-severed kinetochore fibres in the cytoplasm of grasshopper spermatocytes maintain a constant length while turning over in a polarized manner. Tubulin incorporates at or near the kinetochore and translocates towards severed ends without shortening the fibre. Consequently, the chromosome cannot move polewards unless the severed fibre reattaches to the pole through microtubules. A potential seclusion artefact has been ruled out, as fibres severed inside spindles behave identically despite being surrounded by the spindle matrix. Our data suggest that kinetochore microtubules constantly treadmill during anaphase in insect cells. Treadmilling is an intrinsic property of microtubules in the kinetochore fibre, independent of the context and attachment of the spindle. The machinery that depolymerizes minus ends of kinetochore microtubules is functional in a non-spindle context. Attachment to the pole, however, is required to cause net kinetochore fibre shortening to generate polewards forces during anaphase.

Spindle microtubules in flux

“…Grasshopper and crane fly spermatocytes offer exceptional systems for the study of flux: during anaphase, their kMT plus-ends do not disassemble and flux appears to be the sole means to move chromosomes poleward (Wilson et al, 1994;Chen and Zhang, 2004). In fact, their kMT plus-ends polymerize during anaphase A at a rate identical to the chromosome-to-pole velocity (Chen and Zhang, 2004).…”

“…Although not mutually exclusive, two popular models describe how this movement is accomplished utilizing either a MT depolymerization or sliding mechanism Arrows within the red and blue lines indicate the direction of continuous ATP-dependent polymer movement. kMT plus-end assembly stops at the transition to anaphase (although there are exceptions to this rule) (Chen and Zhang, 2004;LaFountain et al, 2004) (see text). Astral MTs (green lines), whose minus-ends are embedded in the centrosomes, do not flux.…”

“…Grasshopper and crane fly spermatocytes offer exceptional systems for the study of flux: during anaphase, their kMT plus-ends do not disassemble and flux appears to be the sole means to move chromosomes poleward (Wilson et al, 1994;Chen and Zhang, 2004). In fact, their kMT plus-ends polymerize during anaphase A at a rate identical to the chromosome-to-pole velocity (Chen and Zhang, 2004). Although this should hinder anaphase chromosome-to-pole movement, crane fly spermatocytes accomplish chromosome segregation by fluxing kMTs at rates almost twice that of chromosome velocity (LaFountain et al, 2004).…”

Anaphase A: Melting Microtubules Move Chromosomes toward Spindle Poles

“…Furthermore, flux continues to occur even when the spindles are monopolar, and therefore lacking antiparallel microtubules [179], indicating that neither kinesin-5 nor antiparallel microtubules are required for flux in these cells. Likewise, detached and isolated kinetochore-associated microtubule fibers in grasshopper spermatocytes flux in the apparent absence of antiparallel neighboring microtubules [182]. Thus, it seems that the flux of kinetochore-attached microtubules is driven by another mechanism, independent of the kinesin-5-dependent sliding of neighboring, antiparallel (inter-polar) microtubules.…”

The perpetual movements of anaphase

“…At the entry of anaphase, CDK1 activity drops abruptly, which is known to promote microtubule stabilization in vitro and in vivo [154,155], whereas prevention of CDK1 inactivation by expression of non-degradable cyclin B, caused a 20% reduction in the velocity of anaphase A in Xenopus egg extracts [156]. Moreover, in some systems, like insect spermatocytes, kinetochore microtubules polymerize rather then depolymerize at their plus ends during anaphase [56,157]. Curiously, a similar phenomenon has been reported in Ptk1 cells injected with tubulin but only during early anaphase, without affecting normal chromosome-to-pole velocity [158].…”