Discovery of the fastest myosin, its amino acid sequence, and structural features

Cytoplasmic streaming with extremely high velocity (~70 μm s−1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by organelle-associated myosin XI sliding along actin filaments, it has been suggested that a myosin XI, which has a velocity of 70 μm s−1, the fastest myosin measured so far, exists in Chara cells. However, the previously cloned Chara corallina myosin XI (CcXI) moved actin filaments at a velocity of around 20 μm s−1, suggesting that an unknown myosin XI with a velocity of 70 μm −1 may be present in Chara. Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. In the work reported in this paper, we cloned these four myosin XIs (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 were similar to that of CcXI, the velocities of CbXI-1 and CbXI-2 were estimated to be 73 and 66 μm s−1, respectively, suggesting that CbXI-1 and CbXI-2 are the main contributors to cytoplasmic streaming in Chara cells and showing that CbXI-1 is the fastest myosin yet found. We also report the first atomic structure (2.8 Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-EM structure of acto-myosin XI at low resolution (4.3 Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loop 2 support this hypothesis.

Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and the charophyte Chara corallina

Background: Pumilio RNA-binding proteins are evolutionarily conserved throughout eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Although a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. The species of the genus Chara have been known to be most closely related to land plants, as they share several characteristics with modern Embryophyta. Results: In this study, we identified two putative nucleolar Pumilio protein genes, namely, ChPUM2 and ChPUM3, from the transcriptome of Chara corallina. Of the two ChPUM proteins, ChPUM2 was most similar in amino acid sequence (27% identity and 45% homology) and predicted protein structure to Arabidopsis APUM23, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of 35S:APUM23-GFP and 35S:APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23, but 35S:ChPUM3/apum24+/- plants did not rescue that of apum24. Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GGAAUUGACGG) for APUM23 was conserved in Arabidopsis and C. corallina, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in C. corallina than in A. thaliana. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain, which had a longer random coil in ChPUM3 than in APUM24. Conclusions: ChPUM2 of C. corallina was functional in Arabidopsis, similar to APUM23, but ChPUM3 did not substitute for APUM24 in Arabidopsis. Protein homology modeling showed high coverage between APUM23 and ChPUM2, but displayed structural differences between APUM24 and ChPUM3. Together with the protein structure of ChPUM3 itself, a short ITS2 of Arabidopsis pre-rRNA may interrupt the binding of ChPUM3 to 3’-extended 5.8S pre-rRNA.

Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and the charophyte Chara corallina

Background: Pumilio RNA-binding proteins are evolutionarily conserved throughout eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Although a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. The species of the genus Chara have been known to be most closely related to land plants, as they share several characteristics with modern Embryophyta.Results: In this study, we identified two putative nucleolar Pumilio protein genes, namely, ChPUM2 and ChPUM3, from the transcriptome of Chara corallina. Of the two ChPUM proteins, ChPUM2 was most similar in amino acid sequence (27% identity and 45% homology) and predicted protein structure to Arabidopsis APUM23, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of 35S:APUM23-GFP and 35S:APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23, but 35S:ChPUM3/apum24+/- plants did not rescue that of apum24. Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GGAAUUGACGG) for APUM23 was conserved in Arabidopsis and C. corallina, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in C. corallina than in A. thaliana. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain, which had a longer random coil in ChPUM3 than in APUM24.Conclusions: ChPUM2 of C. corallina was functional in Arabidopsis, similar to APUM23, but ChPUM3 did not substitute for APUM24 in Arabidopsis. Protein homology modeling showed high coverage between APUM23 and ChPUM2, but displayed structural differences between APUM24 and ChPUM3. Together with the protein structure of ChPUM3 itself, a short ITS2 of Arabidopsis pre-rRNA may interrupt the binding of ChPUM3 to 3’-extended 5.8S pre-rRNA.

Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and Charophyte Chara corallina

Background: Pumilio RNA-binding proteins are evolutionarily conserved throughout all eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Even though a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. Chara is known to be most closely related to land plants as it shares several characteristics with modern embryophytes. Results: In this study, we identified two putative nucleolar Pumilio protein genes, ChPUM2 and ChPUM3 , from the transcriptome of Chara corallina . Of the two ChPUM proteins, ChPUM2 was the most similar to Arabidopsis APUM23 in the amino acid sequence (27% identity and 45% homology) and the predicted protein structure, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of APUM23-GFP and APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23 , but 35S:ChPUM3/apum24 +/- plants did not rescue that of apum24 . Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GAAUUGACGG) for APUM23 was conserved in Arabidopsis and Chara, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in Chara than in Arabidopsis. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain as ChPUM3 has a long random coil in this domain. Conclusions: Our results indicate that ChPUM2 has evolutionarily maintained functions in Arabidopsis, while ChPUM3 is not functional in Arabidopsis, most likely due to the long target ITS2 sequence of Arabidopsis and the distinct tertiary structure from Arabidopsis homologue APUM24.

Prolonged oxygen depletion in microwounded cells of Chara corallina detected with novel oxygen nanosensors

The use of a novel nanoelectrode shows that mechanical microinjury of the cell wall of Chara corallina with a pointed instrument induces localized anoxia and alkalinization near the cell surface.

A Statistical Model of Cell Wall Dynamics during Expansive Growth

ABSTRACTExpansive growth is a process by which walled cells found in plants, algae and fungi, use turgor pressure to mediate irreversible wall deformation and regulate their shape and volume. The molecular structure of the primary cell wall must therefore be able to perform multiple function simultaneously such as providing structural support by a combining elastic and irreversible deformation and facilitate the deposition of new material during growth. This is accomplished by a network of microfibrils and tethers composed of complex polysaccharides and proteins that are able to dynamically mediate the network topology via constant detachment and reattachment events. Global biophysical models such as those of Lockhart and Ortega have provided crucial macroscopic understanding of the expansive growth process, but they lack the connection to molecular processes that trigger network rearrangements in the wall. In this context, we propose a statistical approach that describes the population behavior of tethers that have elastic properties and the ability to break and re-form in time. Tether properties such as bond lifetimes and stiffness, are then shown to govern global cell wall mechanics such as creep and stress relaxation. The model predictions are compared with experiments of stress relaxation and turgor pressure step-up from existing literature, for the growing cells of incised pea (Pisum sativus L.), algaeChara corallinaand the sporangiophores of the fungus,Phycomyces blakesleeanus. The molecular parameters are estimated from fits to experimental measurements combined with the information on the dimensionless number Πpethat is unique to each species. To our knowledge, this research is the first attempt to use a statistical approach to model the cell wall during expansive growth and we believe it will provide a better understanding of the cell wall dynamics at a molecular level.

Cyclosis-mediated long distance communications of chloroplasts in giant cells of Characeae

Long-distance communications in giant characean internodal cells involve cytoplasmic streaming as an effective means for transportation of regulatory substances. The local illumination of Chara corallina Klein ex C.L.Willdenow internodal cells with an intense 30 s pulse of white light caused a transient increase of modulated chlorophyll fluorescence in cell regions positioned downstream the cytoplasmic flow after a delay whose duration increased with the axial distance from the light source. No changes in fluorescence were observed in cell regions residing upstream of the light spot. The transient increase in actual fluorescence Fʹ in cell areas exposed to constant dim illumination at large distances from the brightly lit area indicates the transmission of photosynthetically active metabolite between chloroplasts separated by 1–5 mm distances. The shapes of fluorescence transients were sensitive to retardation of cytoplasmic streaming by cytochalasin D and to variations in cyclosis velocity during gradual recovery of streaming after an instant arrest of cyclosis by elicitation of the action potential. Furthermore, the analysed fluorescence transients were skewed on the ascending or descending fronts depending on the position of light-modulated cytoplasmic package at the moment of streaming cessation with respect to the point of measurements. The observations are simulated in qualitative terms with a simplified streaming–diffusion model.