MyTH4, independent of its companion FERM domain, affects the organization of an intramacronuclear microtubule array and is involved in elongation of the macronucleus in Tetrahymena thermophila.

Myo1 is a class XIV Tetrahymena myosin involved in amitotic elongation and constriction of the macronucleus into two subnuclei at cell division. Elongation of the macronucleus is accompanied by elongation of an intramacronuclear microtubule array, which is oriented parallel to the axis of nuclear elongation. Elongation of the macronucleus often fails to occur or is only partially completed in a MYO1 knockout, and division of the macronucleus is frequently uncoupled from cytokinesis. Myo1 contains a myosin tail homology 4 (MyTH4) and a band 4.1, ezrin, radixin, moesin homology (FERM) domain. Recently, we used green fluorescent protein (GFP) fusions to demonstrate that the entire FERM domain, independent of MyTH4, is essential for localization of FERM to the cytoskeleton and does not appear to directly affect nuclear division. Antiactin coprecipitates GFP-FERM, tubulin, actin, and Myo1. The immunoprecipitated GFP-FERM cosediments with either exogenous F-actin or exogenous microtubules. Here, we show that overexpressed GFP-MyTH4 colocalized with antitubulin to intramacronuclear microtubules. Ninety percent of overexpressing cells assembled intramacronuclear microtubules that did not become organized into a parallel array. Amitosis did not advance in the absence of the parallel array of intramacronuclear microtubules. Five percent of overexpressing cells organized the parallel array, but the microtubules and the macronucleus did not achieve full elongation. Partially elongated macronuclei constricted without cytokinesis. Antiactin coprecipitated GFP-MyTH4, tubulin, and actin. AntiGFP pulled down GFP-MyTH4, tubulin, and actin. GFP-MyTH4 cosedimented with either exogenous microtubules or exogenous F-actin. A novel finding from this study is that MyTH4 and FERM have overlapping and distinct roles in the function of a myosin.

A FERM domain in a class XIV myosin interacts with actin and tubulin and localizes to the cytoskeleton, phagosomes, and nucleus in Tetrahymena thermophila.

Previous studies have shown that Myo1(myosin class XIV) localizes to the cytoskeleton and is involved in amitosis of the macronucleus and trafficking of phagosomes. Myo1 contains a FERM domain that could be a site for interaction between Myo1 and the cytoskeleton. Here, we explore the function of FERM by investigating its cytoskeleton binding partners and involvement in localization of Myo1. Alignment of Myo1 FERM with a talin actin-binding sequence, a MAP-2 tubulin-binding sequence, the radixin FERM dimerization motif, and the SV40 nuclear localization sequence (NLS) revealed putative actin- and tubulin-binding sequences, a putative FERM dimerization motif, and NLS-like sequences in both the N-terminal and C-terminal regions of Myo1 FERM. Alignment of Myo1 with an ERM C-terminal motif revealed a similar sequence in the Myo1 motor domain. GFP-FERM and two truncated FERM domains were separately expressed in Tetrahymena. GFP-FERM contained the entire Myo1 FERM. Truncated Myo1 FERM domains contained either the N-terminal or the C-terminal region of FERM and one putative sequence for actin-binding, one for tubulin-binding, a putative dimerization motif, and a NLS-like sequence. Actin antibody coprecipitated GFP-fusion polypeptides and tubulin from lysate of cells expressing GFP-fusions. Cosedimentation assays performed with either whole cell extracts or anti-actin immunoprecipitation pellets revealed that F-actin (independent of ATP) and microtubules cosedimented with GFP-fusion polypeptides. GFP-FERM localized to the cytoskeleton, phagosomes, and nucleus. Truncated GFP-FERM domains localized to phagosomes but not to the cytoskeleton or nucleus.

Lack of agreement between gas exchange variables measured by two metabolic systems.

The purpose of this study was to assess the agreement and consistency between gas exchange variables measured by two online metabolic systems during an incremental exercise test. After obtaining local ethics approval and informed consent, 15 healthy subjects performed an incremental exercise test to volitional fatigue using the Bruce protocol. The Innocor (Innovision, Denmark) and CardiO2 (Medical Graphics, USA) systems were placed in series, with the Innocor mouthpiece attached to the pneumotach of the CardiO2. Metabolic data were analysed during the last 30 seconds of each stage and at peak exercise. There were non- significant differences (p > 0.05) between the two systems in estimation of oxygen consumption (VO2) and in minute ventilation (VE). Mean Cronbach's alpha for VO2 and VE were 0.88 and 0.92. The Bland-Altman analysis revealed that limits of agreement were -0.52 to 0.55 l.min(-1) for VO2, and -8.74 to 10.66 l.min(-1) for VE. Carbon dioxide production (VCO2) and consequently respiratory exchange ratio (RER) measured by the Innocor were significantly lower (p < 0.05) through all stages. The CardiO2 measured fraction of expired carbon dioxide (FeCO2) significantly higher (p < 0.05). The limits of agreement for VO2 and VE are wide and unacceptable in cardio-pulmonary exercise testing. The Innocor reported VCO2 systematically lower. Therefore the Innocor and CardiO2 metabolic systems cannot be used interchangeably without affecting the diagnosis of an individual patient. Results from the present study support previous suggestion that considerable care is needed when comparing metabolic data obtained from different automated metabolic systems. Key pointsThere is general concern regarding the limited knowledge available about the accuracy of a number of commercially available systems.Demonstrated limits of agreement between key gas exchange variables (oxygen consumption and minute ventilation) as measured by the two metabolic systems were wide and unacceptable in cardio-pulmonary exercise testing.Considerable care is needed when comparing metabolic data obtained from different automated metabolic systems.

Myo1 localizes to phagosomes, some of which traffic to the nucleus in a Myo1-dependent manner in Tetrahymena thermophila.

Myo1 is one of 13 myosins in Tetrahymena thermophila. Initially, twelve of the myosins in Tetrahymena were assigned to Class XX in the myosin superfamily but recently re-assigned to a subclass within Class XIV. In a previous study, we reported that genomic knockout of MYO1 affected phagocytosis and macronuclear amitosis. These two phenotypes have appeared disparate because a possible mechanism linking phagocytosis and amitosis was unknown. In the present study, Myo1 localization was investigated in order to further link machinery for phagocytosis and amitosis. Antibodies directed against the Myo1 motor domain detected an immunospecific polypeptide at 175-180 kDa on immunoblots of wild-type proteins. The 175-180 kDa polypeptide was not detected on immunoblots of proteins from the knockout strain. For immunofluorescence microscopy, cells were allowed to internalize fluorescent beads as markers for phagosomes. In wild-type cells, anti-Myo1 and anti-actin antibodies co-localized to the periphery of phagosomes and the macronucleus. In the MYO1-knockout strain only background fluorescence was observed with anti-Myo1 antibody. Confocal x-z series through macronuclei revealed fluorescent beads within the nucleoplasm. Statistical analysis showed a significant difference between the mean distributions of fluorescent beads in the nucleoplasm of wild-type and MYO1-knockout cells. A fluorescent dye was used to label plasma membrane in living cells. Dye-labeled vacuoles trafficked to the macronucleus. Trafficking of phagosomes to the macronucleus in a myosin-dependent manner is a novel finding and a possible mechanism for targeting myosin and actin to the nucleus.

Myosin genes in Tetrahymena.

This report presents an initial comparison of motor, neck, and tail domains of myosin genes in Tetrahymena thermophila. An unrooted phylogenetic tree drawn from alignment of predicted amino acid translations determined the relationship among 13 myosins in Tetrahymena and their relationship to the myosin superfamily. The myosins in Tetrahymena did not align with any of the previously named myosin classes. Twelve of the Tetrahymena myosins form a new class designated as XX. The other Tetrahymena myosin is divergent from the twelve. Surprisingly, none of the myosins in Tetrahymena aligned with either class I, class II, or class V myosins. Apparent absence of a class II myosin is an indication that cytokinesis in Tetrahymena either utilizes an unconventional myosin or does not require a myosin motor.

Directed motility of phagosomes in Tetrahymena thermophila requires actin and Myo1p, a novel unconventional myosin.

The phagosome cycle was investigated in Tetrahymena thermophila, which had internalized fluorescent latex beads. Confocal microscopy of cells from a GFP-actin strain revealed actin filaments that extended 3-5 mum from the periphery of fluorescent phagosomes. In GFP-actin cells and in wild-type cells, motility of fluorescent phagosomes was directed from the oral cavity to the posterior end of the cell. Although 60% of fluorescent phagosomes in the MYO1-knockout strain were motile, movement of phagosomes was not directed toward the posterior end of the cell and was random. Forty percent of fluorescent phagosomes in knockout cells were non-motile in contrast to only 20% non-motile phagosomes in wild-type cells. The increased incidence of non-motile phagosomes in the knockout strain could reflect absence of Myo1p as a motor. Another myosin or other molecular motors could power random movement of phagosomes in the MYO1-knockout strain. In latrunculin-treated GFP-actin cells, movement of fluorescent phagosomes was random. Average velocity of random movement of fluorescent phagosomes in the knockout strain and in latrunculin-treated cells was statistically the same as the average velocity (2.0 +/- 1.9 microm/min) of phagosomes in GFP-actin cells. These findings are an indication that dynamic actin and Myo1p are required for directed motility of phagosomes.

Myosins in protists.

This review focuses on selected papers that illustrate an historical perspective and the current knowledge of myosin structure and function in protists. The review contains a general description of myosin structure, a phylogenetic tree of the myosin classes, and descriptions of myosin isoforms identified in protists. Each myosin is discussed within the context of the taxonomic group of the organism in which the myosin has been identified. Domain structure, cellular location, function, and regulation are described for each myosin.

Putative myosin heavy and light chains in Tetrahymena: co-localization to the basal body-cage complex and association of the heavy chain with skeletal muscle actin filaments in vitro.

The basal body cage is a fibrillar chamber which surrounds each basal body in the ciliate cytoskeleton. The function of this chamber is unknown. In Tetrahymena, the cage contains actin filaments which connect the cage to triplet microtubules. In this study, we have examined the cage for the presence of myosin. Skeletal muscle myosin-II heavy and light chains were used to affinity-purify anti-MHC and anti-MLC antibodies, respectively, from an antiserum raised against Tetrahymena oral apparatus proteins. On western immunoblots of ATP-solubilized Tetrahymena proteins, the anti-MHC antibody detected a putative myosin heavy (180 kDa) chain, and the anti-MLC antibody detected a putative myosin light (18 kDa) chain. The anti-MHC antibody specifically labeled the AI zone of sarcomeres. In cosedimentation assays with an ATP-solubilized protein fraction, the 180 kDa polypeptide associated with skeletal muscle actin filaments in an ATP-dependent manner. The sedimented actin filaments appeared to be organized into bundles. Immunodepletion of the 180 kDa rendered the ATP-solubilized protein fraction ineffective in bundling actin filaments in a cosedimentation assay. ATP-solubilized Tetrahymena proteins, which included the 180 kDa polypeptide, exhibited F-actin-stimulated, Mg2+ ATPase activity and K+, EDTA ATPase activity which are characteristic of myosin ATPases. Immunodepletion of the 180 kDa polypeptide reduced the F-actin, Mg2+ ATPase activity of the ATP-solubilized protein fraction by more than 80%. Based on these various observations, we conclude that the 180 kDa polypeptide is a putative myosin heavy chain, probably a myosin-II and that the 18 kDa polypeptide is probably a myosin-II light chain. We have used the affinity-purified, anti-myosin antibodies with immunofluorescence microscopy and immunogold electron microscopy to map the location of the putative myosin heavy and light chains in Tetrahymena. Immunofluorescence microscopy showed that the anti-myosin antibodies localized to Tetrahymena somatic and oral region basal bodies. At the ultrastructural level, the anti-myosin antibodies localized to filaments in the basal body-cage complex. The labeling patterns with both anti-myosin antibodies were identical to the labeling pattern observed with an anti-actin antibody reported in a previous study. The co-localization of myosin and actin argue for a motility system within the basal body-cage complex.

Characteristics of basal body cartwheel reassembly.

Cartwheel complexes reassembled in a fraction derived by treating isolated oral apparatuses from Tetrahymena with 1.0 M KCl for 12 h. Approximately 40% of the KCl-soluble protein reassembled into cartwheel complexes. The reassembly reaction was protein-concentration dependent, and reassembled cartwheels were stable at 3 degrees C. Sucrose gradient centrifugation resolved 3 high molecular mass protein complexes from the KCl-soluble fraction. Each of the 3 complexes has a different mass, but each contains the same 5 polypeptides, 2 of which are probably tubulins. When these complexes were removed from the KCl-soluble fraction by high speed centrifugation, cartwheel reassembly did not occur. The 5 polypeptides in the high molecular mass complexes were among several other polypeptides resolved from reassembled cartwheels by 2-dimensional gel electrophoresis. The high molecular mass complexes are probably essential for cartwheel formation. The electrophoretic data also show that several polypeptides in the KCL-soluble fraction do not appear to be incorporated into cartwheels. These polypeptides are probably non-essential for cartwheel formation.

Xenopus marginal band disassembly by calcium-activated cytoplasmic factors.

The marginal band microtubules of isolated Xenopus erythrocyte cytoskeletons possess the stability properties of non-steady-state microtubules. They are unperturbed by low temperatures, a variety of microtubule inhibitors, hypotonic treatment and the direct action of calcium. These microtubules can be rapidly depolymerized by erythrocyte lysis in the presence of calcium or by exposure of cytoskeletons obtained and washed in calcium-free media to calcium-containing supernatants of other cell lysates. Thus, marginal band microtubules are calcium-sensitive only in the presence of cytoplasm. The calcium-activated disassembly of the marginal band does not appear to be the result of general or tubulin-specific proteolysis and is prevented by the calmodulin inhibitor, trifluoperazine. On sodium dodecyl sulphate/polyacrylamide gels, samples of calcium-induced, marginal band disassembled cytoskeletons are always tubulin-depleted and also possess a new high molecular weight polypeptide doublet that is believed to constitute stable partial degradation products of spectrin. In the presence of calcium, addition of calmodulin and ATP to cytoskeletons washed free of cytoplasm does not initiate marginal band disassembly. Therefore, if calmodulin mediates marginal band disassembly, it requires cytoplasmic binding proteins or cytoplasmic cofactors.

Studies on the cytoskeletal and nuclear architecture of Xenopus erythrocytes.

A proteinaceous cytoskeletal network is present in nucleated erythrocytes, which is obscured ultrastructurally in whole cells due to the presence of haemoglobin. Lysis of Xenopus erythrocytes in solutions containing Triton X-100 reveals a cytoskeleton that contains a centrally positioned nucleus, which is linked to the cell surface-associated cytoskeleton by intermediate filaments. The marginal band microtubules are also preserved in these structures. In addition, a single or a pair of perinuclear centrioles is frequently observed in thin sections. These structures are surrounded by a mass of intermediate filaments and fibrogranular material. In contrast to the centrioles in invertebrate erythrocytes those in Xenopus erythrocytes are not associated with the marginal band. Cytonuclear skeletons were obtained by DNase I digestion and subsequent high-salt extraction of cytoskeletons. The resulting structures were chromatin-depleted and consisted of a nuclear lamina that was maintained in the same overall shape and position as that of intact nuclei. With the exception of the marginal band, the remaining cytoskeletal elements persisted after these treatments. Although marginal bands were not detectable by electron microscopy, the cytonuclear skeletons contained roughly the same amount of tubulin as cytoskeletons, as indicated by immunoblotting with affinity-purified anti-tubulin antibodies. When intact erythrocytes were exposed to the ionophore A23187 in the presence of calcium, the cell shape and centric nuclear position were altered. Nuclear dislodgement may be attributable to the disruption of intermediate filament associations with the subsurface cytoskeletal shell. Indirect immunofluorescent staining of cytoskeletons lysed in buffers containing either EGTA or calcium indicates that in the absence of calcium, the intermediate filament network extends to the cell periphery. In structures lysed in calcium, however, the filaments are restricted to the vicinity of the nucleus.

The oral apparatus of Tetrahymena. V. Oral apparatus polypeptides and their distribution.

Two-dimensional electrophoresis was used to resolve approximately 162 polypeptides from the isolated oral apparatus of Tetrahymena thermophila. The molecular weight range was between 110 000 and 15 000 Daltons. The polypeptides had apparent isoelectric points between pH 3.3 and pH 7.2. Electrophoretic analysis of isolated ciliary axonemes and fractionated oral apparatuses made possible the assignment of polypeptides to structures within the oral apparatus. Approximately 24 polypeptides, including alpha and beta tubulins, are probable components of the basal body-basal plate complex. At least 5 of the oral apparatus polypeptides, including alpha and beta tubulin, are components of the oral apparatus ciliary axonemes. Approximately 138 polypeptides are components of the oral apparatus framework.