Anaphase Chromosomes in Crane-Fly Spermatocytes Treated With Taxol (Paclitaxel) Accelerate When Their Kinetochore Microtubules Are Cut: Evidence for Spindle Matrix Involvement With Spindle Forces

Abstract

Various experiments have indicated that anaphase chromosomes continue to move after their kinetochore microtubules are severed. The chromosomes move poleward at an accelerated rate after the microtubules are cut but they slow down 1–3 min later and move poleward at near the original speed. There are two published interpretations of chromosome movements with severed kinetochore microtubules. One interpretation is that dynein relocates to the severed microtubule ends and propels them poleward by pushing against non-kinetochore microtubules. The other interpretation is that components of a putative “spindle matrix” normally push kinetochore microtubules poleward and continue to do so after the microtubules are severed from the pole. In this study we distinguish between these interpretations by treating cells with taxol. Taxol eliminates microtubule dynamics, alters spindle microtubule arrangements, and inhibits dynein motor activity in vivo. If the dynein interpretation is correct, taxol should interfere with chromosome movements after kinetochore microtubules are severed because it alters the arrangements of spindle microtubules and because it blocks dynein activity. If the “spindle matrix” interpretation is correct, on the other hand, taxol should not interfere with the accelerated movements. Our results support the spindle matrix interpretation: anaphase chromosomes in taxol-treated crane-fly spermatocytes accelerated after their kinetochore microtubules were severed.

Elastic Tethers Between Separating Anaphase Chromosomes Regulate the Poleward Speeds of the Attached Chromosomes in Crane-Fly Spermatocytes

“…This model is silent on why dynein relocates to the tips of severed kinetochore microtubules, but it predicts that the movement slows down when the stub encounters an obstacle ( Spurck et al, 1997 ). The different interpretations remain unresolved, but since chromosomes in Mesostoma spermatocytes move poleward very rapidly (up to 200 μm/min) in the absence of kinetochore microtubules ( Fegaras and Forer, 2018 ) and since chromosomes in crane-fly spermatocytes with severed kinetochore microtubules (and severed tethers) accelerate even in the presence of taxol ( Forer et al, 2018 ), it seems likely that forces external to spindle microtubules produce forces for movement. Regardless of which models give correct representations of the poleward forces on chromosomes, models of mitotic spindle forces as a whole must include forces from tethers, forces that act on chromosome arms in the opposite direction to those that propel anaphase chromosomes poleward.…”

“…Further, when the chromosome fragment is cut in half only the part with the telomere moves ( LaFountain et al, 2002 ). Yet further, arm fragments produced in taxol-treated cells move at the same speeds as in normal cells ( Forer et al, 2018 ) even though taxol stabilises spindle microtubules and blocks spindle transport of akinetic material ( LaFountain et al, 2001 ). Thus, arm-fragment movements are not due to microtubules and seem to require connection between telomeres.…”

“…Further, ultra-fine DNA threads are not elastic. They slow anaphase chromosomes ( Su et al, 2016 ) whereas tethers do not: anaphase velocities are unchanged when tethers are cut ( Sheykhani et al, 2017 ; Forer et al, 2018 ). Except for data indicating that tethers are not microtubules or ultra-fine DNA bridges, the composition of tethers is not known.…”

“…We can deduce that we have severed tethers by using a visible-light laser to first sever an arm and then to irradiate between the arm fragment and the partner telomere to stop the motion of the arm fragment (e.g., Figure 4 of Sheykhani et al, 2017 ). However, we cannot use a visible light laser both to cut tethers and to stop chromosome movement: visible-light laser microbeam irradiations sever kinetochore fibre microtubules in crane-fly spermatocytes ( Forer et al, 2013 , 2018 ; Sheykhani et al, 2017 ) in grasshopper spermatocytes ( Chen and Zhang, 2004 ) and in PtK cells ( Elting et al, 2014 ) but they do not stop anaphase chromosomes from moving. Thus we cannot use visible-light laser irradiation to do this experiment.…”

Laser Scissors and Tweezers to Study Chromosomes: A Review

“…In collaboration with Professor Arthur Forer, York University, Toronto, we conducted a multiyear series of studies on the forces involved in chromosome and spindle pole movement, in meiotic cells from several different invertebrates as well as in mitotic vertebrate PTK (Potorous tridactylus) cells. These studies suggested that in addition to the normal poleward forces exerted by kinetochore-attached microtubules, there are external non-spindle forces involved in chromosome movements (Forer et al, 2018). In particular, one source of force regulation on the chromosomes in meiosis of crane-fly spermatocytes appeared to be caused by tethers attached to the tips of separating chromosomes (Sheykhani et al, 2017).…”