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First published online 12 February 2003
doi: 10.1242/jcs.00333

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Cytoskeletal proteins with N-terminal signal peptides: plateins in the ciliate Euplotes define a new family of articulins

¹ Department of Biological Sciences, University of Maryland Baltimore County (UMBC), Baltimore, MD 21250, USA
² Laboratoire de Biologie Cellulaire 4, Université Paris-Sud, 91405 Orsay, France
³ Biologia MCA, Università di Camerino, 62032 Camerino, Italy
⁴ Kimball Research Institute, New York Blood Center, 310 E. 67th St, New York, NY 10021, USA

View larger version (99K): [in a new window]   Fig. 1. General features of Euplotes. Projection of optical sections passing through the ventral (A) or dorsal (B) surfaces of a cell decorated (in green) with anti-E antiserum directed against proteins of the alveolar plates (Williams et al., 1989), and decorated (in red) with the 20H5 antibody directed against centrins at the bases of the cilia (Sanders and Salisbury, 1994). On the ventral surface, clusters of cilia form the oral ciliature (OC) and the locomotory cirri (C); alveolar plates on this surface are irregular in shape. On the dorsal surface, the cilia are aligned into antero-posterior rows (in red), between which alveolar plates are regularly patterned.

View larger version (52K): [in a new window]   Fig. 3. Amino acid sequences of three plateins, derived from their respective nucleotide sequences. The proteins are arrayed to display their respective repetitive domain structures. N-terminal signal sequences (predicted by SignalP V1.1 for 1 and 2; directly determined for ß/) are highlighted in yellow. The primary repetitive sequences (VP-rich 12-mers, with some degeneracy) are indicated in red; the secondary proline-rich pentameric repeats are shown in blue. Note the inversion of primary and secondary repeat domains between the - and ß/-plateins. Sequences found within one of the -isoforms, but missing from the other, are highlighted in light gray; the corresponding gaps in the other isoform (representing deletions from the aligned sequences) are indicated with lines. Sequences corresponding to the tryptic peptides from -platein (Table 1) are underlined. For ß/-platein, the ß-tryptic peptides are in double underline, and the -peptides (and actual N-terminal sequence) in single underline; sequences common to both are in wavy underline. Residues that show very high prediction values as phosphorylation substrates (0.9 output values using NetPhos 2.0) are highlighted in darker gray.

View larger version (59K): [in a new window]   Fig. 2. Analysis of the expression of the platein genes. (A) Products of the -platein RT-PCR experiments (see text), stained with ethidium bromide. Lane 1: RT-PCR products obtained using oligonucleotides AP11 and AP12 at low annealing stringency so that they bind to both genes; the two bands correspond in size to the expected fragments of the two genes, considering the deletion within the 2-platein gene. Lane 2: RT-PCR product obtained using oligonucleotide AP11 (common to both genes) and AP12 at a stringency condition specific for the 1-platein gene. Lane 3: RT-PCR product obtained as in lane 2 except for the use of oligonucleotide AP13, instead of AP12, at a stringency condition specific to the 2-platein gene. Lane M: molecular size markers (reported in base pairs on the left of the gel). (B) RT-PCR amplification product from total RNA obtained with primers designed from the ß/-platein gene sequence. M-MLV(+): RNA sample incubated with the reverse transcriptase enzyme. M-MLV(-): a control RNA sample treated in the same way but without reverse transcriptase. DNA size markers are shown to the left.

View larger version (110K): [in a new window]   Fig. 4. Immunofluorescence localization of - and ß/-plateins in whole permeabilized cells. The dorsal surface of cells decorated with anti--platein (A and C) or anti-ß/-platein (B and D) are shown. Before staining, cells were permeabilized/extracted with either mild (0.25%; A and B) or stronger (1%; C and D) concentrations of Triton X-100. After mild permeabilization, plates are clearly visible after decoration with the -platein antibody (A), while appearing irregularly decorated with the anti-ß/-platein (B). However, after strong extraction, the plates are no longer detected with the anti--platein (C); many small vesicles appear just beneath the cell cortex, as seen in the center of this cell. With the same treatment, the plates are fully and strongly decorated with the anti-ß/-platein (D). Bars, 10 µm.

View larger version (83K): [in a new window]   Fig. 5. Immunofluorescence localization of - and ß/-plateins in dividing cells. Shown are the dorsal surface of cells decorated with anti-- (A) and anti-ß/-plateins (B), with enlargements (large white arrows). The general appearance after decoration with the anti--platein (A) is as described in Fig. 4A, except in the area of ciliary duplication at the equator of the cell (black arrow), where the staining appears more punctuate (black circle). At higher magnification (top), the small new plates that appear concomitantly with the new cilia are virtually unlabeled (small white arrows); plates surrounding this area appear irregularly decorated (black, white circles). After treatment with anti-ß/-platein (B), only a partial staining of mature plates is seen, as in Fig. 4B. However, the new plates that appear along with the new cilia (black arrow in B, and small white arrows in the enlargement, bottom), as well as those surrounding this area (black circle in B, white circle in the enlargement), are fully decorated. Bars, 10 µm (A, B); bars, 1 µm (for the enlargements).