Efficient isolation and cultivation of endosymbiotic Chlorella from Paramecium bursaria on agar plates by co-culture with yeast cells.

Paramecium bursaria is a ciliate that harbors Chlorella-like unicellular green algae as endosymbionts. The relationship between the host P. bursaria and the endosymbiotic Chlorella is facultative; therefore, both partners can be cultured independently and re-combined to re-establish symbiosis, making this system suitable for studying algal endosymbiosis. However, despite many previous studies, cultivation of endosymbiotic Chlorella remains difficult, particularly on agar plates. Here we describe a simple agar plate method for efficiently isolating and culturing cells of the endosymbiotic alga Chlorella variabilis from an individual P. bursaria cell, by co-culturing them with yeast Saccharomyces cerevisiae. The co-culture with the yeast significantly improved the colony-forming efficiency of the alga on agar. Growth assays suggest that the main role of the co-cultured yeast cells is not to provide nutrients for the algal cells, but to protect the algal cells from some environmental stresses on the agar surface. Using the algal cells grown on the plates and a set of specially designed primers, direct colony PCR can be performed for screening of multiple endosymbiont clones isolated from a single host ciliate. These methods may provide a useful tool for studying endosymbiotic Chlorella species within P. bursaria and various other protists.

Photosynthetic Endosymbionts Benefit from Host's Phagotrophy, Including Predation on Potential Competitors.

In many endosymbioses, hosts have been shown to benefit from symbiosis, but it remains unclear whether intracellular endosymbionts benefit from their association with hosts [1, 2]. This makes it difficult to determine evolutionary mechanisms underlying cooperative behaviors between hosts and intracellular endosymbionts, such as mutual exchange of vital resources. Here, we investigate the fitness effects of symbiosis on the ciliate host Paramecium bursaria and on the algal endosymbiont Chlorella [3, 4], using experimental microcosms that include the free-living alga Chlamydomonas reinhardtii to mimic ecologically realistic conditions. We demonstrate that both host ciliate and the endosymbiotic algae gain fitness benefits from the symbiosis when another alga C. reinhardtii is present in the system. Specifically, the endosymbiotic Chlorella can grow as the host ciliate feeds and grows on C. reinhardtii, whereas the growth of free-living Chlorella is reduced by its competitor, C. reinhardtii. Thus, we propose that the endosymbiotic algae benefit from the host's phagotrophy, which allows the endosymbiont to access particulate nutrient sources and to indirectly prey on the potential competitors competing with its free-living counterparts. Even though the ecological contexts in which each partner receives its benefits differ, both partners would gain net fitness benefits in an ecological timescale. Thus, the cooperative behaviors can evolve through fitness feedback (partner fidelity feedback) between the host and the endosymbiont, without need for special partner control mechanisms. The proposed ecological and evolutionary mechanisms provide a basis for understanding cooperative resource exchanges in endosymbioses, including many photosynthetic endosymbioses widespread in aquatic ecosystems.

Mutations in the SH1 helix alter the thermal properties of myosin II.

The myosin II SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain and possibly plays a key role in the arrangement of the converter/lever arm. Several point mutations within the SH1 helix in human myosin IIs have been shown to cause diseases. To reveal whether these SH1 helix mutations affect not only motile activities but also thermal properties of myosin II, here we introduced the E683K or R686C point mutation into the SH1 helix in Dictyostelium myosin II. Thermal inactivation as well as thermal aggregation rates of these mutant proteins demonstrated that these mutations decreased the thermal stability of myosin II. Temperature dependence of sliding velocities of actin filaments showed that these mutations also reduced the activation energy of a rate-limiting process involved in actin movement. Given that these mutations are likely to alter coupling between the subdomains, and thus their thermal fluctuations, we propose that the SH1 helix is a key structural element that determines the flexibility and thermal properties of the myosin motor. These characteristics of the SH1 helix may contribute to the pathogenesis of the human diseases caused by mutations within this structural element.

Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms.

Maintenance of algal endosymbionts in Paramecium bursaria: a simple model based on population dynamics.

Algal endosymbiosis is widely distributed in eukaryotes including many protists and metazoans, and plays important roles in aquatic ecosystems, combining phagotrophy and phototrophy. To maintain a stable symbiotic relationship, endosymbiont population size in the host must be properly regulated and maintained at a constant level; however, the mechanisms underlying the maintenance of algal endosymbionts are still largely unknown. Here we investigate the population dynamics of the unicellular ciliate Paramecium bursaria and its Chlorella-like algal endosymbiont under various experimental conditions in a simple culture system. Our results suggest that endosymbiont population size in P. bursaria was not regulated by active processes such as cell division coupling between the two organisms, or partitioning of the endosymbionts at host cell division. Regardless, endosymbiont population size was eventually adjusted to a nearly constant level once cells were grown with light and nutrients. To explain this apparent regulation of population size, we propose a simple mechanism based on the different growth properties (specifically the nutrient requirements) of the two organisms, and based from this develop a mathematical model to describe the population dynamics of host and endosymbiont. The proposed mechanism and model may provide a basis for understanding the maintenance of algal endosymbionts.

Myosin-actin interaction in Dictyostelium cells revealed by GFP-based strain sensor and validated linear spectral unmixing.

Myosin is an actin-based motor protein that is involved in a wide range of cellular motile processes. Although in vitro properties of the myosin-actin interaction have been extensively studied, the interaction in vivo remains poorly understood. Recently, we developed a GFP-based strain sensor termed PriSSM (PRIM-based strain sensor module), by using the proximity imaging (PRIM) technique, which detects spectral changes of two GFP molecules that are in direct contact. Using PriSSM-myosin II fusion proteins, the interaction between myosin II and F-actin can be detected in Dictyostelium cells. In the spectroscopic measurements of PriSSM, to decompose the measured spectra of the cells expressing the sensor proteins into the contributions from the sensor and the background autofluorescence, we applied the linear spectral unmixing approach, which was based on the assumption that the errors at each wavelength were independent. Cellular autofluorescence, however, often includes systematic errors, so that the unmixing procedures might lead to biased estimates. Here, to validate our spectral unmixing procedures, we estimate the possible maximum errors in the fluorescence ratio values that are obtained by unmixing spectra including such systematic errors. This estimation provided a general criterion to validate the results obtained by linear unmixing of spectra including serially correlated error terms. Using the proposed criterion and PriSSM-myosin II fusion proteins, we examined the interaction between myosin II and F-actin in Dictyostelium cells under several different conditions. The spectroscopic results, together with the microscopic observations of the cells expressing the proteins, suggest that the formation of myosin filaments through the tail region has only a slight effect on binding to F-actin but has significant effects on the cortical localization of myosin II.

Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments.

Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations.

Photoregulated assembly/disassembly of DNA-templated protein arrays using modified oligonucleotide carrying azobenzene side chains.

DNA-templated self-assembly of nanomaterials provides great potential for applications including biosensors, nanoelectronics, and programmable and autonomous molecular machines. To switch or regulate the activities of those nanobiotechnological devices, non-invasive methods to assemble and disassemble specific nanoscale components are needed. Here, we describe photocontrol of assembly of DNA-templated protein arrays in solution, by using photo-controlled duplex formation of oligonucleotides carrying azobenzene. As a proof of concept prototype, we designed a one-dimensional protein array system that consists of a scaffold of DNA and two kinds of anchor DNA that were conjugated with fluorescent proteins (CFP and YFP, respectively). The scaffold DNA was modified to carry multiple azobenzene side chains so that the hybridization involving the scaffold DNA is regulated by photoirradiation through conformational changes of the azobenzene moieties. Melting temperatures of duplex made of the modified DNA scaffold and an anchor oligonucleotide were shifted significantly and reversibly by UV and visible photoirradiation (difference of T(m) was 34.8 degrees C in 150 mM potassium acetate). Measurements of Förster resonance energy transfer between CFP and YFP showed that the assembly of the protein array system was also changed by photoirradiation. Such non-invasive and reversible method to control assembly/disassembly of multiple, specific proteins in a DNA-templated protein array system would provide many functions for nanobiotechnological devices such as on/off switches and the ability to change the configuration reversibly.

Visualizing myosin-actin interaction with a genetically-encoded fluorescent strain sensor.

Many proteins have been shown to undergo conformational changes in response to externally applied force in vitro, but whether the force-induced protein conformational changes occur in vivo remains unclear. To reveal the force-induced conformational changes, or strains, within proteins in living cells, we have developed a genetically encoded fluorescent "strain sensor," by combining the proximity imaging (PRIM) technique, which uses spectral changes of 2 GFP molecules that are in direct contact, and myosin-actin as a model system. The developed PRIM-based strain sensor module (PriSSM) consists of the tandem fusion of a normal and circularly permuted GFP. To apply strain to PriSSM, it was inserted between 2 motor domains of Dictyostelium myosin II. In the absence of strain, the 2 GFP moieties in PriSSM are in contact, whereas when the motor domains are bound to F-actin, PriSSM has a strained conformation, leading to the loss of contact and a concomitant spectral change. Using the sensor system, we found that the position of the lever arm in the rigor state was affected by mutations within the motor domain. Moreover, the sensor was used to visualize the interaction between myosin II and F-actin in Dictyostelium cells. In normal cells, myosin was largely detached from F-actin, whereas ATP depletion or hyperosmotic stress increased the fraction of myosin bound to F-actin. The PRIM-based strain sensor may provide a general approach for studying force-induced protein conformational changes in cells.

Mutation in the SH1 helix reduces the activation energy of the ATP-induced conformational transition of myosin.

The SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain. Recently, we showed that a mutation within the SH1 helix in Dictyostelium myosin II (R689H) reduced the elasticity and thermal stability of the protein. To reveal the involvement of the SH1 helix in ATP-dependent conformational changes of the motor domain, we have investigated the effects of the R689H mutation on the conformational changes of the converter, using a GFP-based fluorescence resonance energy transfer method. Although the mutation does not seem to strongly affect conformations, we found that it significantly reduced the activation energy required for the ATP-induced conformational transition corresponding to the recovery stroke. Given the effects of the mutation on the mechanical properties of myosin, we propose that the SH1 helix plays an important role in the mechanochemical energy conversion underlying the conformational change of the myosin motor domain.

A point mutation in the SH1 helix alters elasticity and thermal stability of myosin II.

Movement generated by the myosin motor is generally thought to be driven by distortion of an elastic element within the myosin molecule and subsequent release of the resulting strain. However, the location of this elastic element in myosin remains unclear. The myosin motor domain consists of four major subdomains connected by flexible joints. The SH1 helix is the joint that connects the converter subdomain to the other domains, and is thought to play an important role in arrangements of the converter relative to the motor. To investigate the involvement of the SH1 helix in elastic distortion in myosin, we have introduced a point mutation into the SH1 helix of Dictyostelium myosin II (R689H), which in human nonmuscle myosin IIA causes nonsyndromic hereditary deafness, DFNA17. The mutation resulted in a significant impairment in motile activities, whereas actin-activated ATPase activity was only slightly affected. Single molecule mechanical measurements using optical trap showed that the step size was not shortened by the mutation, suggesting that the slower motility is caused by altered kinetics. The single molecule measurements demonstrated that the mutation significantly reduced cross-bridge stiffness. Motile activities produced by mixtures of wild-type and mutant myosins also suggested that the mutation affected the elasticity of myosin. These results suggest that the SH1 helix is involved in modulation of myosin elasticity, presumably by modulating the converter flexibility. Consistent with this, the mutation was also shown to reduce thermal stability and induce thermal aggregation of the protein, which might be implicated in the disease process.

A novel actin-bundling kinesin-related protein from Dictyostelium discoideum.

Actin filaments and microtubules are two major cytoskeletal systems involved in wide cellular processes, and the organizations of their filamentous networks are regulated by a large number of associated proteins. Recently, evidence has accumulated for the functional cooperation between the two filament systems via associated proteins. However, little is known about the interactions of the kinesin superfamily proteins, a class of microtubule-based motor proteins, with actin filaments. Here, we describe the identification and characterization of a novel kinesin-related protein named DdKin5 from Dictyostelium. DdKin5 consists of an N-terminal conserved motor domain, a central stalk region, and a C-terminal tail domain. The motor domain showed binding to microtubules in an ATP-dependent manner that is characteristic of kinesin-related proteins. We found that the C-terminal tail domain directly interacts with actin filaments and bundles them in vitro. Immunofluorescence studies showed that DdKin5 is specifically enriched at the actin-rich surface protrusions in cells. Overexpression of the DdKin5 protein affected the organization of actin filaments in cells. We propose that a kinesin-related protein, DdKin5, is a novel actin-bundling protein and a potential cross-linker of actin filaments and microtubules associated with specific actin-based structures in Dictyostelium.