Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement

Abstract

Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise.

Figure 1

Schematic diagram showing the relationships between myosin and its activators and inhibitors. In order for myosin to be active, its regulatory light chain (MRLC) has to be phosphorylated either by myosin light chain kinase (MLCK) or by Rho-associated kinase (Rho-K). Dephosphorylation of MRLC is done by myosin light chain phosphatase (MLCPase). Various inhibitors interfere with myosin activity: Rho-K is inhibited by Y-27632, MLCPase is inhibited by Calyculin A and myosin is inhibited by BDM.

Figure 2

Plots of chromosome separation versus time during anaphase (A) and cell diameter at the site of cleavage furrow during cytokinesis (B) in a control crane-fly spermatocyte. One half-bivalent pair is shown in (A), illustrating constant separation velocity during anaphase.

Figure 3

Calyculin A effects on anaphase chromosome movement and cytokinesis in crane-fly spermatocytes. (A) CalA added during anaphase (arrow) causes chromosomes to accelerate and to move backwards after they reached the poles (one half-bivalent pair is shown). Dashed line represents linear regression through the crosses before addition of CalA and the solid line after addition of CalA. (B) CalA added during metaphase (arrow), a few minutes before anaphase onset, causes chromosomes to move fast during subsequent anaphase and to move backwards after reaching the poles (one half-bivalent pair is shown). (C) CalA added during cytokinesis (downward arrow) stops furrow ingression and reverses cytokinesis. Cells do not resume cytokinesis after washing out CalA (upward arrow).

Figure 4

(A) Chromosome movement in a cell treated with CalA (left arrow) and with Y-27632 in CalA (right arrow) during anaphase, illustrating one pair of half-bivalents that accelerated after CalA, did not slow after Y-27632, and moved backwards after reaching the poles. The dashed line represents linear regression through the circles with dots before addition of CalA and the solid line through the circles with dots after addition of Y27632 in CalA. (B) Chromosome movement in a cell treated with Y-27632 (left arrow) and with CalA in Y27632 (right arrow) illustrating one pair of half-bivalents that stopped after Y-27632, accelerated after CalA and moved backwards after reaching the poles. The dashed line represents linear regression through the circles with dots before addition of Y27632 and the solid line through the circles with dots after addition of CalA in Y27632. (C) Chromosome movement in a cell treated with CalA (left arrow), followed by BDM in CalA (mid arrow) and then by CalA again (right arrow) illustrating one pair of half-bivalents that accelerated after Calyculin, slowed after BDM, accelerated again after washing out BDM and moved backwards after reaching the poles. The dashed line represents linear regression through the circles with dots before addition of CalA and the solid lines regression through the circles with dots after addition of CalA (left line) and after washing out BDM (right line).

Figure 5

Distribution of phosphorylated myosin RLC (p-squash) in crane-fly spermatocytes. (A) Confocal fluorescence micrograph of p-squash in a control crane-fly spermatocyte at metaphase. P-squash localizes to the spindle, at the poles and, with lower intensities, in the chromosomes. (B) Confocal fluorescence micrograph of p-squash in a crane-fly spermatocyte treated with 50 nM CalA. P-squash concentrates around the chromosomes and at the poles. From the timing and appearance of the cell, this cell is in the stage after the half-bivalents moved backwards and reformed a pseudo-nucleus. (C) Fluorescence micrograph illustrating localisation of p-squash at the kinetochores of the two sex chromosomes, during autosomal anaphase, after CalA treatment. P-squash also localizes at the kinetochores of autosomes. (D) DIC image of the cell illustrated in (C) showing the two sex chromosomes and their kinetochores (asterisks). (E) Fluorescence micrograph illustrating localisation of p-squash in the midbody and in the daughter nuclei during cytokinesis. (F) Fluorescence micrograph illustrating increased staining with antibody against p-squash in the cleavage furrow and in the chromosomes after CalA treatment. Scale bar = 5 μm.

Figure 6

Distribution of various spindle proteins in crane-fly spermatocytes. Control spermatocytes are illustrated in (A,C,D,F,G,I,K,M). Calyculin-treated spermatocytes are illustrated in (B,B',B",C',E,F',H,J,L,N). (A): Fluorescence image of tubulin distribution in a control cell in early anaphase. (B, B', B"): Fluorescence images of tubulin distribution in CalA-treated cells in early anaphase (B) and metaphase (B',B"). Individual microtubules "peel off" from the kinetochore bundles (arrowheads), split along their length and surround the chromosomes (open arrowheads) after CalA treatment. (C): DIC image of a control spermatocyte showing kinetochore spindle fibres terminating at the kinetochores. (C'): Fluorescence image merged with the DIC image (orange-green) showing that CalA added during prometaphase causes chromosomes to loose attachment to the spindle fibres. (D): Fluorescence image of filamentous actin distribution in a control cell in metaphase.(E): Fluorescence images of filamentous actin distribution in a CalA-treated cell in early anaphase. Actin filaments become more prominent in the kinetochore fibres. Two half-bivalents from the same autosomal pair (open arrowheads), one univalent sex chromosome (arrow) and sex chromosome spindle fibres (closed arrowheads) are indicated (F): Fluorescence image of a control cell in cytokinesis. (F'): Fluorescence image of a cell in cytokinesis after CalA treatment. CalA causes formation of actin aggregates in the mid body. (G, I, M): Fluorescence images of skeletor (G), titin (I) and myosin (M) in control cells in prometaphase. (H, J, N): Fluorescence images of skeletor (H), titin (J) and myosin (N) in CalA-treated cells. There is less of these proteins in the spindle region. (K,L): Titin is present in the interzone (arrowheads) between the arms of separating half-bivalents (arrows), both in control cells (K) and in CalA-treated cells (L). Scale bar in A (for panels A-J, M-N) = 5 μm. Scale bar in (B',B",K,L) = 1 μm.

Figure 7

Chromosome movement in a cell treated with LatB (left arrow) and with CalA in LatB (right arrow) illustrating two pairs of half-bivalents that drastically slowed after LatB and accelerated after CalA in LatB. One moved backwards after reaching the poles (+), while the other stayed at the poles (o). The dashed lines represents linear regression through the dotted circles and crosses before addition of LatB and the solid lines regression through the dotted circles and crosses after addition of CalA in LatB.

Figure 8

Backwards chromosome movements in crane-fly spermatocytes treated with CalA are not actin or myosin dependent. (A) Chromosome movements in a cell treated with CalA in anaphase (left arrow), and with BDM in CalA added after the chromosomes started to move backwards (right arrow). Backwards movements are not affected by the myosin inhibitor BDM. The dashed line represents linear regression through the circles with dots before addition of CalA and the solid line through the circles with dots after addition of CalA. (B) Chromosome movements in a cell treated with CalA in anaphase (left arrow), and with LatB in CalA added after the chromosomes reached the poles, at the time when they started to move backwards (right arrow). Backwards movements are not affected by actin inhibitor LatB. The dashed line represents linear regression through the circles with dots before addition of CalA and the solid line through the circles with dots after addition of CalA.