Gene Knockouts Reveal Separate Functions for Two Cytoplasmic Dyneins inTetrahymena thermophila

Abstract

In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena. INTRODUCTIONDynein is a molecular motor that transduces chemical energy into mechanical motion along microtubules. There are two functional classes of dynein: axonemal dynein that produces the propagated bending of cilia and of eukaryotic flagella and nonaxonemal or "cytoplasmic" dynein that performs functions other than ciliary beating. Cytoplasmic dynein is implicated in a variety of intracellular movements, including fast retrograde transport (Schroer et al., 1989;Muresan et al., 1996), slow axonal transport (Cleveland and Hoffman, 1991;Dillman et al., 1996), mediation of the trafficking of membrane-bounded organelles (Corthesy-Theulaz et al., 1992;Aniento et al., 1993;Fath et al., 1994;Oda et al., 1995;Holleran et al., 1996), the organization and separation of the bipolar mitotic spindle (Verde et al., 1991;Vaisberg et al., 1993;Saunders et al., 1995;Echeverri et al., 1996), and postmitotic nuclear migrations in yeast and filamentous fungi (Eshel et al., 1993;Li et al., 1993;Plamann et al., 1994;Xiang et al., 1994;Inoue et al., 1998).The realization that dynein performs so many different tasks and the discovery that many organisms express multiple dynein heavy chain genes lead to the hypothesis that different dynein isoforms perform different cellular tasks (reviewed in Asai, 1996). Cilia and eukaryotic flagella provide clear examples of dynein functional specialization (Piperno, 1990). The close coordination of ϳ12 distinct axonemal dynein heavy chains produces axonemal bending (e.g., Piperno and Ramanis, 1991;Mastronarde et al., 1992; reviewed in Brokaw, 1994).The functional specialization of the cytoplasmic dyneins is not well understood. All eukaryotes examined express homologues of the ubiquitous Dyh1 (also called MAP1C, cDHC1, DHC1a), and some organisms, including Saccharomyces, Aspergillus, Neurospora, and Dictyostelium, apparently have only this dynein. In the analysis of the family of dynein gen...

Targeted gene disruption of dynein heavy chain 7 ofTetrahymena thermophilaresults in altered ciliary waveform and reduced swim speed

“…The templates used were prepared from mutant cell lines that had been cultured in the absence of selective agents for more than 2 months. Previous studies, as well as our own observations, have shown that Tetrahymena dynein mutant genotypes that have not been fully assorted to fixation tend to rapidly revert back to wild type in the absence of selective agents (Angus et al, 2001;Lee et al, 1999). No genotypic reversion is observed in this DYH7neo3 mutant cell line.…”

“…Previous studies in which gene disruptions were made in cytoplasmic dyneins of Tetrahymena resulted in a mutant in which the disrupted allele could not be brought to fixation even after prolonged selection in paromomycin (Lee et al, 1999). This incomplete dynein knockout displayed reversion to the wild-type genotype within a span of days if the selective agent was removed from cell culture.…”

Calmodulin and Ca2+/Calmodulin-Binding Proteins are Involved in Tetrahymena thermophila Phagocytosis.

“…The phagocytosis of Kupffer cells (Watanabe et al, 1988) and macrophage line J774.16 (Horwitz et al, 1981) cells was prevented by Ca 2+ /CaM inhibitors TFP and W7, suggesting that Ca 2+ /CaM signal regulates the phagocytosis of these cells. In Dictyostelium, the Ca 2+ -binding and actin-crosslinking protein, a-actinin, is localized in the phagocytic cup and is thought to be involved in the phagocytosis (Furukawa and Fechheimer, 1994 (Lee et al, 1999). Dynein is a molecular motor that transduces the chemical energy of ATP hydrolysis into mechanical motion along microtubules.…”

“…Therefore, Dyh1 may function in the formation of food vacuoles in T. thermophila cells, as well as in Ca 2+ /CaM signaling. Lee et al did not discuss the molecular mechanisms of phagocytosis in detail (Lee et al, 1999), and the relation between Dyh1 and CaM in the vacuole formation is unknown. It is important to elucidate the relation among Dhy1, CaM, and Ca 2+ /CaM-binding proteins in the formation of food vacuoles in T. thermophila.…”

The Dilute Locus and Griscelli Syndrome: Gateways Towards a Better Understanding of Melanosome Transport

“…a different affinity towards certain organelles. With regard to this phenomenon, kinesin (64,65) and dynein (66,67) alternative splice variants were looked at by several groups and found to be involved in a range of different organellar functions. The elucidation of the function of the different MYO5A splice variants in human melanocytes to see which splice variant is involved in melanosome transport is currently being investigated by our group.…”