Using a Hand-Held Gene Gun for Genetic Transformation of Tetrahymena thermophila.

Biolistic bombardment is widely used as a means of delivering vector-coated microparticles into microorganisms, cultured cells, and tissues. The first particle delivery system contained a helium propulsion unit (the gun) mounted in a vacuum-controlled chamber. In contrast, the hand-held gene gun does not operate within a chamber. It is completely hand-held, easy, and efficient to use, and it requires minimal space on the laboratory bench top. This chapter describes protocols for using a hand-held gene gun to deliver transformation vectors for overexpression of genes or gene replacement into the macronucleus of Tetrahymena thermophila. The protocols provide helpful information for preparing Tetrahymena for biolistic bombardment, preparation of vector-coated microcarriers, and basic gene gun operating procedures.

Heparin-fibronectin interactions in the development of extracellular matrix insolubility.

During extracellular matrix (ECM) assembly, fibronectin (FN) fibrils are irreversibly converted into a detergent-insoluble form which, through FN's multi-domain structure, can interact with collagens, matricellular proteins, and growth factors to build a definitive matrix. FN also has heparin/heparan sulfate (HS) binding sites. Using HS-deficient CHO cells, we show that the addition of soluble heparin significantly increased the amount of FN matrix that these cells assemble. Sulfated HS glycosaminoglycan (GAG) mimetics similarly increased FN assembly and demonstrated a dependence on GAG sulfation. The length of the heparin chains also plays a role in assembly. Chains of sufficient length to bind to two FN molecules gave maximal stimulation of assembly whereas shorter heparin had less of an effect. Using a decellularized fibroblast matrix for proteolysis, detergent fractionation, and mass spectrometry, we found that the predominant domain within insoluble fibril fragments is FN's major heparin-binding domain HepII (modules III12-14). Multiple HepII domains bind simultaneously to a single heparin chain in size exclusion chromatography analyses. We propose a model in which heparin/HS binding to the HepII domain connects multiple FNs together to facilitate the formation of protein interactions for insoluble fibril assembly.

Using a Handheld Gene Gun for Genetic Transformation of Tetrahymena thermophila.

This chapter describes protocols for using a handheld gene gun to deliver transformation vectors for overexpression of genes or gene replacement in the macronucleus of Tetrahymena thermophila. The protocols provide helpful information for preparing Tetrahymena for biolistic bombardment, preparation of vector-coated microcarriers, and basic gene gun operating procedures.

A shared mechanism of adhesion modulation for tenascin-C and fibulin-1.

Adhesion modulatory proteins are important effectors of cell-matrix interactions during tissue remodeling and regeneration. They comprise a diverse group of matricellular proteins that confer antiadhesive properties to the extracellular matrix (ECM). We compared the inhibitory effects of two adhesion modulatory proteins, fibulin-1 and tenascin-C, both of which bind to the C-terminal heparin-binding (HepII) domain of fibronectin (FN) but are structurally distinct. Here, we report that, like tenascin-C, fibulin-1 inhibits fibroblast spreading and cell-mediated contraction of a fibrin-FN matrix. These proteins act by modulation of focal adhesion kinase and extracellular signal-regulated kinase signaling. The inhibitory effects were bypassed by lysophosphatidic acid, an activator of RhoA GTPase. Fibroblast response to fibulin-1, similar to tenascin-C, was dependent on expression of the heparan sulfate proteoglycan syndecan-4, which also binds to the HepII domain. Therefore, blockade of HepII-mediated signaling by competitive binding of fibulin-1 or tenascin-C represents a shared mechanism of adhesion modulation among disparate modulatory proteins.

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.

Expression of GFP-actin leads to failure of nuclear elongation and cytokinesis in Tetrahymena thermophila.

Green fluorescent protein (GFP)-tagged actin was used to investigate the distribution and function of actin in Tetrahymena. A strain that expresses both GFP-actin and endogenous actin was developed by transformation of Tetrahymena thermophila with a ribosomal DNA-based replicative vector. Confocal microscopy of living cells and immunogold electron microscopy confirmed localization of GFP-actin to basal bodies and the contractile ring. Incorporation of the fusion protein into these and other actin-related structures correlated with severe impairment of macronuclear elongation and cytokinesis. At 30 degrees C macronuclear elongation failed to occur in 25% of the transformants despite completion of micronuclear division. At 20 degrees C macronuclear elongation failed to occur in 2% of the population. Arrest of cytokinesis coincided with failure of macronuclear elongation. Arrested cells developed into homopolar doublets with two sets of oral structures. This study indicates a requirement for actin in nuclear elongation and cytokinesis. Although GFP-actin can interfere with the functioning of actin-containing structures, the GFP-actin transformant strain can be used to monitor actin distribution and dynamics and is therefore an important new tool for further studies of Tetrahymena actin.