Blocking Protein Phosphatase 1 [PP1] Prevents Loss of Tether Elasticity in Anaphase Crane-Fly Spermatocytes

Abstract

In normal anaphase cells, telomeres of each separating chromosome pair are connected to each other by tethers. Tethers are elastic at the start of anaphase: arm fragments cut from anaphase chromosomes in early anaphase move across the equator to the oppositely-moving chromosome, telomere moving toward telomere. Tethers become inelastic later in anaphase as the tethers become longer: arm fragments no longer move to their partners. When early anaphase cells are treated with Calyculin A (CalA), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), at the end of anaphase chromosomes move backward from the poles, with telomeres moving toward partner telomeres. Experiments described herein show that in cells treated with CalA, backwards movements are stopped in a variety of ways, by cutting the tethers of backwards moving chromosomes, by severing arms of backwards moving chromosomes, by severing arms before the chromosomes reach the poles, and by cutting the telomere toward which a chromosome is moving backwards. Measurements of arm-fragment velocities show that CalA prevents tethers from becoming inelastic as they lengthen. Since treatment with CalA causes tethers to remain elastic throughout anaphase and since inhibitors of PP2A do not cause the backwards movements, PP1 activity during anaphase causes the tethers to become inelastic.

Structural evidence for elastic tethers connecting separating chromosomes in crane-fly spermatocytes

“…Although we cannot prove these structures are the "tethers" defined in experiments that followed movements of severed chromosome arms, they are not microtubules and they satisfy what we know about tethers. We thus suggest that these structures represent the tethers inferred from the laser-cutting experiments in living cells (LaFountain et al, 2002;Forer et al, 2017Forer et al, , 2021.…”

“…After a chromosome arm is severed with a laser microbeam in early anaphase, the resultant arm fragment moves rapidly across the equator until it reaches the partner telomere (LaFountain et al, 2002;Forer et al, 2017;Sheykhani et al, 2017). Both telomeres are necessary for arm fragment movement; ablation of either telomere (that of the arm fragment or that of the partner) causes the arm fragment to stop moving (LaFountain et al, 2002;Forer et al, 2021). Furthermore, when the original arm fragment is cut in half, only the portion with the telomere moves to the partner (LaFountain et al, 2002).…”

“…Nor are the movements due to ultrafine DNA bridges extending between separating chromosomes. Although tethers are expected to form between all of the separating telomere pairs (Sheykhani et al, 2017;Forer et al, 2018Forer et al, , 2021, ultrafine DNA bridges are present in far fewer numbers (Barefield & Karlseder, 2012;Gemble et al, 2015Gemble et al, , 2016Nielsen et al, 2015;Kong et al, 2023) (details in the Discussion section). Furthermore, ultrafine DNA bridges slow down anaphase chromosome movements (Su et al, 2016), but tethers do not (Forer et al, 2017;Sheykhani et al, 2017) (details in the Discussion section).…”

Shining Light in Mechanobiology: Optical Tweezers, Scissors, and Beyond

“…Tethers occur between the tips of sister chromosomes and functional assays of the tethers consist of using laser microsurgery to cut a chromosome tip and monitor it as it moves away from its pole toward its sister partner. − Figure demonstrates a chromosome arm that is cut and moves toward its partner. These studies have determined that the tethers are elastic and that the elasticity is dependent on phosphorylation. , Additionally, Forer et al have found that as the tethers elongate and the cell progresses through anaphase, the tethers loose elasticity. , When optical tweezers were used to trap a fragment tip that was created with laser scissors, the force that the tethers imparted on its partner was found to be ∼1.5 pN . The function of the tethers is not completely understood.…”

“…These studies have determined that the tethers are elastic and that the elasticity is dependent on phosphorylation. 107,108 Additionally, Forer et al have found that as the tethers elongate and the cell progresses through anaphase, the tethers loose elasticity. 104,105 When optical tweezers were used to trap a fragment tip that was created with laser scissors, the force that the tethers imparted on its partner was found to be ∼1.5 pN.…”