Chromosome movement in lysed mitotic cells is inhibited by vanadate.

Abstract

Mitotic PtK1 cells, lysed at anaphase into a carbowax 20 M Brij 58 solution, continue to move chromosomes toward the spindle poles and to move the spindle poles apart at 50% in vivo rates for 10 min. Chromosome movements can be blocked by adding metabolic inhibitors to the lysis medium and inhibition of movement can be reversed by adding ATP to the medium. Vanadate at micromolar levels reversibly inhibits dynein ATPase activity and movement of demembranated flagella and cilia. It does not affect glycerinated myofibril contraction or myosin ATPase activity at less than millimolar concentrations. Vanadate at 10-100/xM reversibly inhibits anaphase movement of chromosomes and spindle elongation. After lysis in vanadate, spindles lose their fusiform appearance and become more barrel shaped. In vitro microtubule polymerization is insensitive to vanadate.

A permeabilized cell model for studying cytokinesis using mammalian tissue culture cells

“…A and F were taken before lysis; B and G, 1 min after lysis ; C and H, 2 min after lysis ; D and 1, 4 min after lysis ; and E and 1, 6 min after lysis . Bar,5 ,um . and cytochalasin B, but in 10 mM ATP and in NEM-S,, furrow diameter increased slowly and relaxation often did not occur until several minutes after lysis. These observations suggest that cytochalasin B, alkaline pH, and high calcium act directly on the contractile ring to disorganize and relax the furrow, and that NEWS, and high ATP have an indirect effect on furrow integrity.…”

“…PtK, cells were used in all experiments and were maintained and handled for light microscopy as described previously (5). Coverslips were mounted on slides with coverslip fragments as spacers.…”

“…The Brij 58-polyethylene glycol lysis procedure is similar to the one previously described (5), which maintains anaphase chromosome movement in permeabilized PtK1 cells . After lysis, the plasma membrane, mitochondria, and other membranebounded organelles are partially disrupted but the ground substance and the cytoskeletons of these cells appear to be similar to those of unlysed cells (Cande, Meeusen, and McDonald, manuscript submitted for publication).…”

The perpetual movements of anaphase

“…The addition of vanadate into lysed mitotic cells in the presence of EGTA to buffer the concentration of free Ca 2+ , caused only an impact on anaphase B [87,88], which was subsequently confirmed after inhibition of dynein ATPase activity with erythro-9-[3-(2-hydroxynonyl)] adenine [89]. Curiously, milimolar concentrations of vanadate or anti-dynein antibody injections did not affect anaphase movements in unlysed Ptk1 cells [90,91].…”

“…These data indicated that in some systems, pulling forces acting on astral microtubules are the main drivers of spindle elongation, whereas spindle midzone microtubules resist or govern the rate of pole separation. Cytoplasmic dynein located at the cell cortex has been implicated in this astral MT pulling mechanism [89,90,96,[233][234][235]. On the other hand, proteins required for microtubule polymerization and growth/stabilization of interpolar microtubules [236], such as CLASPs [219], coordinated with the activity of kinesin-5 at the spindle midzone, might work as governors [237].…”