Amoebae of P. polycephalum (ATCC #24466 and #24467) were grown on corn-meal agar at 24˚C. The two strains of amoebae were plated together on an SM-2 agar plate so that they fused to form plasmodia [12]. For the subcultivation of amoebae, they were grown on lawns of live Escherichia coli on SM-1 agar plates at 24˚C [13]. Microplasmodia were grown at 24˚C in liquid semi-defined medium [14]. Plasmodia were cultured on corn-meal agar plates. Alternatively, for differentiation into sclerotia, plasmodia were grown on filter papers at 24˚C. Subsequently, the filter papers crawling plasmodia were dried in desiccators [15], inducing the formation of sclerotia. Fruiting bodies were formed from starved plasmodia under light irradiation [16].

The tectonin I sequence from the initiation to the termination codon was amplified by PCR. The PCR product was ligated into the pQE30 vector (Qiagen, CA, USA). E. coli were then transformed with the ligation product and ampicillin-resistant colonies were selected. The transformed cells were grown at 37˚C in LB medium supplemented with ampicillin until the OD₆₀₀ reached nearly 0.6. The expression of recombinant proteins was derived by 1 mM isopropyl-β-D-thiogalactoside. Recombinant proteins were purified on a Ni-NTA agarose column (Invitrogen, CA, USA). The purified protein was emulsified with an equal volume of Freund’s complete adjuvant and injected into a rabbit. Blood was then collected, and the IgG fraction was prepared by ammonium sulfate fractionation and Protein G-Sepharose 4 Fast Flow (GE Healthcare UK Ltd., Buckinghamshire, UK) column chromatography according to the general method of Harlow and Lane [17].

SDS-PAGE was performed in a 12.5% polyacrylamide gel. Proteins were separated on the gel and electrophoretically transferred onto a polyvinylidene fluoride membrane (Millipore, MA, USA). The proteins on the membrane were reacted with tectonin I-specific antibody, diluted 1:50,000, and then with a horseradish peroxidase-conjugated antirabbit IgG antibody (Cell Signaling Technology, Inc., MA, USA). Detection of the desired proteins was achieved with an ECL Plus kit (GE Healthcare UK Ltd.) and visualized with an LAS-1000 Plus camera system (Fujifilm, Tokyo, Japan).

For RNA preparation, each stage-specific cell preparation was lyophilized. A total of 1 mg (dry weight) of each type of cell, except for spores, was suspended in 0.5 ml of RNAwiz (Applied Biosystems, CA, USA) and total RNA samples were then purified according to the RNAwiz manuscript. Spores (approximately 50 mg) were suspended in extraction solution [0.3 ml of phenol and 0.7 ml of buffer (0.1 M sodium acetate, pH 6.0, 10 mM EDTA, 1% w/v SDS, 0.1 M LiCl)] preheated to 80˚C. After the spores were destroyed using a ZircoPrep Mini (Nippon Genetics Co. Ltd., Tokyo, Japan), total RNA was purified according to the method of Melera and Rusch [18]. These RNA samples were subjected to 1% formaldehyde-MOPS agarose gel electrophoresis. RNAs were then transferred from the gel to a Hybond N⁺ membrane (GE Healthcare UK Ltd.). For producing the probes, the cDNA fragments for tectonin I (27 - 681 bp) and II (13 - 434 bp) were amplified by PCR and labeled using an AlkPhos Direct Kit (GE Healthcare UK Ltd.). The labeled cDNAs were hybridized to the membrane containing the RNA for 15 h according to the AlkPhos Direct kit manuscript. RNAs were detected with CDP-Star (GE Healthcare UK Ltd.) detection reagent and the signals were visualized on X-ray film.

Microplasmodia (approximately 2 g wet weight) were suspended in 20 ml of 20 mM Tris-HCl buffer (pH 7.5) and disrupted by sonication. The crude extract was centrifuged at 1000 × g for 15 min. The supernatant was again centrifuged at 10,000 × g for 15 min and was separated into precipitate and supernatant. This supernatant was then ultracentrifuged at 200,000 × g for 1 h. The precipitate obtained at each centrifugation was suspended in 20 ml of the same buffer. Finally, each supernatant and precipitate was analyzed by Western blotting.

Microplasmodia (approximately 3 g wet weight) were disrupted by sonication as described in the preceding section. The suspension was then subjected to ultracentrifugation, and the precipitate was suspended in 40 ml of 20 mM Tris-HCl buffer (pH 7.5). The suspension was separated into eight equal quantities and 5 ml of a buffer containing NaCl (final concentration, 1 M), urea (5 M), Na₂CO₃ (5 M), Triton X-100 (1% v/v), proteinase K (1 mg/ml), or FeCl₃ (10 mM) was added to each suspension. These suspensions were then incubated on ice for 1 h and were again subjected to ultracentrifugation. Each precipitate was suspended in 10 ml of the same buffer. All samples including the precipitates and supernatants were analyzed by Western blotting.

Plasmodia were placed on a glass slide and fixed with 10% (v/v) TCA as described previously [19]. After washing, the cells were incubated at room temperature for 1 h with TBS containing 1% (w/v) BSA and 1000-fold diluted anti-tectonin I antibody. They were then incubated with 2000-fold diluted Alexa Fluor 488 goat anti-rabbit IgG (H + L) (Molecular Probes, OR, USA). The immunostained cells were observed with an LSM410 confocal laser microscope (Carl Zeiss Co. Ltd., Jena, Germany). Fluorescence labeling was observed at a wavelength of 488 nm. Images were expressed as the projections of a series of 1-nm optical sections.

Purified recombinant tectonin I that was expressed as a His-tag fusion protein in E. coli was injected into rabbits as antigen. As shown in Figure 1(b), the antibody specific for tectonin reacted with both tectonins I and II in the crude extract from plasmodia. This specific antibody was used for the subsequent experiments.

The expression of tectonins I and II at each stage of the life cycle was detected by Western and Northern blotting (Figure 2). Tectonins I and II were detected in the plasmodia, microplasmodia cultured in liquid medium, and dehydrated, dormant-stage sclerotia. However, these proteins were not detected in spores. Interestingly, only tectonin I was expressed in haploid amoebae, indicating that tectonin II is not essential to amoebal cells and may have a different function from tectonin I.

During differentiation from haploid amoebae to plasmodia, tectonin II was detected in cells by both Western and Northern blotting (Figure 3). When small zygotes were observed after 48 h, the RNA expression of tec tonin II had already begun (Figure 3(c)). After 96 h from the start of mating, when cytoplasmic streaming in the small yellowish plasmodia was visible under microscopy, the expression of tectonin II protein could be detected in the cells. From that point onward, the tectonin proteins in the cells increased with the RNA levels. When complete plasmodia were observed after 120 h, the tectonin II content in the cells was maximal. Thus, tectonin II appears to mediate some functions in plasmodia but not in amoebae.

Neither tectonin I nor II was found in spores (Figure 2). To examine the change of tectonins I and II during differentiation from plasmodia to spores, the expression level was evaluated by Western and Northern blotting. RNA synthesis of tectonins I and II in the cells was not detected even when many protuberances in the lamellipodia of cells were seen, as shown in photograph 2 of Figure 4. However, cells at this stage were able to return to typical plasmodia by the interruption of the condition for differentiation (e.g. light, starvation, and static culture) and to recover the expression of tectonins I and II. Thus,

although the RNA level in the cells decreased during the early stages, tectonin expression persisted in the cells until the formation of spores was complete.

Diverse proteins that contain tectonin domains in their structures have been identified, and their functions have been reported [6-9]. However, no function has yet been assigned to the tectonin first found in P. polycephalum, and it is speculated that tectonin resides on the surface of the plasma membrane and is involved in phagocytosis [1]. We examined the association of tectonins I and II with the cellular membrane. The crude extract obtained after sonication of microplasmodia was centrifuged at 1000, 10,000, and 200,000 × g, and both tectonins I and II were detected in the precipitates obtained by centrifugation at 200,000 × g (Figure 5). This finding suggested that tectonins I and II were associated with relatively light membranes such as intracellular vesicles and were easily released from the membranes. Because hydropathy analysis revealed that both tectonins are hydrophilic (results not shown), they may be associated with the membranes through other membrane proteins.

Tectonins I and II could be released from the membrane fraction by chemical treatment. Tectonin I was released

by urea, Na₂CO₃, or Triton X-100 treatment, but tectonin II was released only by urea (Figure 6). These results showed that tectonins I and II associate with membranes with different conformations. Further, when the membrane fraction was digested with proteinase K, tectonin II disappeared completely, whereas tectonin I remained in the membrane. This result agrees with that of Huh et al. [1], who examined the sensitivity of tectonin to trypsin digestion in microplasmodia. Tectonin II, unlike tectonin I, may thus be exposed on the membrane surface. Because it is thought that intracellular membranes form vesicles upon sonication, the orientations of tectonins I and II may be different in the vesicles.

Figure 7 shows an immunofluorescence micrograph of plasmodia reacted with anti-tectonin I antibody. The micrograph shows that tectonins I and II are located on the lamellipodium of the plasmodia. Treatment with a tectonin II-specific antibody showed that the localization of tectonin II coincided completely with that of tectonin I (data not shown). As discussed by Huh et al. [1], tectonin proteins may be involved in phagocytosis in the lamellipodia of plasmodia.