On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force.

Abstract

Abstract. As anaphase began, mitotic PtK~ and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATE ATE GTP, or ATP-~-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (,x,16~ or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent.Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.N 'o consensus currently exists regarding the role of ATP during anaphase A (chromosome-to-pole movement) and it is difficult to evaluate the diverse data on this subject. We (Pickett-Heaps and Spurck, 1982; Spurck et al., 1986a, b) have found that the concentration of metabolic inhibitors needed to cause rapid (within 1 min) and completely reversible cessation of cellular activity (presumably by depleting cellular ATP levels) in live cells is critical; 2,4 dinitrophenol (DNP) 1 is effective in both diatoms and mammalian cells only at the seemingly high concentration of 1 mM, and lowering the concentration even to 0.5 mM results in a significantly delayed and often incomplete response. In contrast, far lower concentrations of metabolic inhibitors were used in most previous work (reviewed by Spurck et al., 1986a, b; Hepler and Palevitz, 1986). Furthermore, many who work with animal cell systems use inhibitors of oxidative phosphorylation alone. We (Spurck et al., 1986a) found that glycolysis must also be blocked by using 2-deoxyglucose (DOG) in conjunction with DNP or other metabolic inhibitors. Without DOG, cellular activity may be maintained in DNP, and glycolysis, we believe, keeps cells alive for the long periods of DNP treatment alone (1--4 h or more) repo...

Microtubule treadmilling: what goes around comes around

“…(38) It is evident that the poleward flux could be due either to intrinsic dynamics or to associated motor activity or to a combination of both. In this respect, it is interesting to note that recent evidence from in vitro model systems has established that chromosome migration toward microtubule (-) ends can occur without ATP hydrolysis, (46,47) thus demonstrating that motor activity is not an absolute requirement for this motility.…”

The nuclear-mitotic apparatus protein is important in the establishment and maintenance of the bipolar mitotic spindle apparatus.

“…These include the generation of force by the action of various microtubule motors that are thought to be located at the kinetochores and along spindle fibers (Leslie et al, 1987;Pfarr et al, 1990;Steuer et al, 1990;Hoyt et al, 1992;Roof et al, 1992), and also possibly by forces exerted from the assembly and disassembly of microtubules themselves (Rieder et al, 1986;Spurck and Pickett-Heaps, 1987;Koshland et al, 1988). Although this chromosome movement pauses during metaphase, the observation of microtubule flux during this stage suggests that the apparent stability of the metaphase spindle apparatus may in fact represent a complex but balanced interplay of the several mechanisms acting along microtubules (Gorbsky and Borisy, 1989;Mitchison, 1989).…”

The perpetual movements of anaphase

“…In 1988, Koshland and colleagues showed that microtubule shortening was able to move chromosomes in vitro by tubulin dissociation from the kinetochore [118]. Importantly, the rate of microtubule depolymerization following dilution of tubulin was the same in the absence of ATP, suggesting that the poleward force at the kinetochore may be generated by some form of energy stored in the microtubule lattice [119], such as that provided by a conformational change associated with GTP-hydrolysis and/or curling of protofilaments [118]. This became known as the "conformational-wave" model.…”

“…On this regard, the work of Spurck and Pickett-Heaps might have shed light into the problem [119]. By using permeabilized Ptk1 and newt lung cells, these authors noted that anaphase A could be resumed either by the addition of ATP or by treatments that promote microtubule disassembly, such as cold or calcium.…”