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

Arthur Forer,Aisha Adil,Michael W Berns

doi:10.3389/fmolb.2021.636746

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.

Highlights

Tethers are elastic connections between telomeres of separating anaphase chromosomes
The major conclusions from our experiments are (1) that inhibition of phosphatases 1 (PP1) by Calyculin A (CalA) causes tethers to remain elastic throughout anaphase instead of becoming inelastic in later anaphase; (2) that elastic tethers pull chromosomes backwards toward each other at the end of anaphase after pole-directed forces weaken; and (3) that inhibition of PP1 preserves tether elasticity but does not increase tether elasticity
Cutting tethers stops backwards movements (Figure 2), cutting arms that lead the backwards moving chromosomes stops their backwards movement (Figure 3), cutting arms from chromosomes before the chromosomes reach the poles stops subsequent backwards movements (Table 2), ablating the telomere of the arm to which backwards chromosomes are moving stops the backwards movements (Figure 4), and tethers in CalA cells are elastic throughout 80–100% of their length, for all tether lengths up to 17 μm, maintaining elasticity at lengths much longer than tethers in control cells remain elastic (Figure 9)

Summary

Introduction

Tethers are elastic connections between telomeres of separating anaphase chromosomes. They produce tension between the separating chromosome arms, stretching them by ∼10% (Forer et al, 2017). Tethers were originally described (operationally) in crane-fly spermatocytes (and named as such) by LaFountain et al (2002). Their presence has been determined in other animal cells (Forer et al, 2017). In fixed and stained cells, nonDNA connections between the telomeres of separating anaphase chromosomes have been described/illustrated in large numbers of animal cell types (Paliulis and Forer, 2018), presumably representing tethers. A range of evidence (discussed, for example, in Forer et al, 2017; Forer and Berns, 2020) shows that the movements of arm fragments are not microtubule-based and require direct physical connections between separating telomeres

Methods

Results

Conclusion