Ciliary signaling systems for motility and growth control in Tetrahymena

Abstract

Using Tetrahymena as a model system, we examined the sensory aspect of the cilium in terms of fast behavioral responses and its effect on growth and cell division control. Prior research in Paramecium established that an outer arm dynein regulatory light chain, p29, is activated by cAMP to induce an increase in dynein activity, and therefore increase ciliary beat frequency and fast forward swimming. We have identified the Tetrahymena ortholog of p29, p34, using biochemical and genomic analyses. These two proteins are interchangeable in in-vitro assays. Mass spectrometry of 2D gel isolated p34 identified 2 unique peptide sequences that allowed us to identify the gene and its complete translation sequence from the Tetrahymena genome. In a second fast response, ciliary beat form is altered via Ca2+ influx. We identified centrin as an EF-hand containing Ca2+ binding protein that co-purifies with the inner dynein arms and is localized along the entire axoneme. Using in-vitro assays, we show that centrin activates inner dynein arm upon binding Ca2+.;Growth and division responses are typically initiated via receptor tyrosine kinases and tyrosine phosphorylation cascades. A potential receptor tyrosine kinase subunit (TtPTK1), a 66 kDa protein, has been identified in Tetrahymena ciliary membranes. TtPTK1 might participate in signal transduction cascades involving ciliary growth responses. Using several techniques downstream effectors including PIK3, Mek1/2, an SH2 containing protein and protein tyrosine phosphatase have been identified within the cilium. Additional downstream partners were also identified not within the cilium.;We have developed a unifying hypothesis for both rapid behavioral and growth and cell division responses where by transduction begins in the cilia. Evolutionarily it would be of selective advantage to tie ciliary signaling systems into cell cycle control. Our results show that second messengers effect rapid behavioral changes by acting on axonemal dynein to change doublet sliding rates. For growth and division control, responses must not only be turned on and transduced into the nucleus, but also reversible and turned off. For this control, Tetrahymena has evolved a tyrosine phosphorylation cascade, originating in the cilium with parallels to mammalian insulin signaling.