We examined spindle elongation in anaphase in Saccharomyces cerevisiae cells mutated for the kinesin-5 motor proteins Cin8 and Kip1. Cells were deleted for KIP1 and/or expressed one of two motor-domain Cin8 mutants (Cin8-F467A or Cin8-R196K, which differ in their ability to bind microtubules in vitro, with Cin8-F467A having the weakest ability). We found that, in kinesin-5-mutated cells, predominantly in kip1Δ cin8-F467A cells, anaphase spindle elongation was frequently interrupted after the fast phase, resulting in a mid-anaphase pause. Expression of kinesin-5 mutants also caused an asymmetric midzone location and enlarged midzone size, suggesting that proper organization of the midzone is required for continuous spindle elongation. We also examined the effects of components of the FEAR pathway, which is involved in the early-anaphase activation of Cdc14 regulatory phosphatase, on anaphase spindle elongation in kip1Δ cin8-F467A cells. Deletion of SLK19, but not SPO12, eliminated the mid-anaphase pause, caused premature anaphase onset and defects in DNA division during anaphase, and reduced viability in these cells. Finally, overriding of the pre-anaphase checkpoint by overexpression of Cdc20 also eliminated the mid-anaphase pause and caused DNA deformation during anaphase in kip1Δ cin8-F467A cells. We propose that transient activation of the pre-anaphase checkpoint in kinesin-5-mutated cells induces a Slk19-dependent mid-anaphase pause, which might be important for proper DNA segregation.

The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO syntase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating.

We examined protein kinase C (PKC)-dependent regulation of Na+-K+-ATPase in frog mucociliary cells. Activation of PKC by 12- O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl- sn-glycerol (diC8) either in intact cells or isolated membranes resulted in a specific inhibition of Na+-K+-ATPase activity by ∼25–45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximal inhibition concentration (IC50) = 0.5 ± 0.1 nM and 2.4 ± 0.2 μM, respectively, for TPA and diC8] and were not influenced by Ca2+. Analysis of the ouabain inhibition pattern revealed the presence of two Na+-K+-ATPase isoforms with IC50 values for cardiac glycoside of 2.6 ± 0.8 nM and 409 ± 65 nM, respectively. Most importantly, the isoform possessing a higher affinity for ouabain was almost completely inhibited by TPA, whereas its counterpart was hardly sensitive to the PKC activator. The results suggest that, in frog mucociliary cells, PKC regulates Na+-K+-ATPase and that this action is related to the specific Na+-K+-ATPase isoform.