An Sfi1p-like centrin-binding protein mediates centrin-based Ca2+ -dependent contractility in Paramecium tetraurelia - PubMed (original) (raw)

An Sfi1p-like centrin-binding protein mediates centrin-based Ca2+ -dependent contractility in Paramecium tetraurelia

Delphine Gogendeau et al. Eukaryot Cell. 2007 Nov.

Abstract

The previous characterization and structural analyses of Sfi1p, a Saccharomyces cerevisiae centrin-binding protein essential for spindle pole body duplication, have suggested molecular models to account for centrin-mediated, Ca2+-dependent contractility processes (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877, 2006). Such processes can be analyzed by using Paramecium tetraurelia, which harbors a large Ca2+ -dependent contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetic studies have shown that the ICL is composed of diverse centrin isoforms and a high-molecular-mass centrin-associated protein, whose reduced size in the démaillé (dem1) mutant correlates with defective organization of the ICL. Using sequences derived from the high-molecular-mass protein to probe the Paramecium genome sequence, we characterized the PtCenBP1 gene, which encodes a 460-kDa protein. PtCenBP1p displays six almost perfect repeats of ca. 427 amino acids (aa) and harbors 89 potential centrin-binding sites with the consensus motif LLX11F/LX2WK/R, similar to the centrin-binding sites of ScSfi1p. The smaller (260-kDa) protein encoded by the dem1 mutant PtCenBP1 allele comprises only two repeats of 427 aa and 46 centrin-binding sites. By using RNA interference and green fluorescent protein fusion experiments, we showed that PtCenBP1p forms the backbone of the ICL and plays an essential role in its assembly and contractility. This study provides the first in vivo demonstration of the role of Sfi1p-like proteins in centrin-mediated Ca2+-dependent contractile processes.

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Figures

FIG. 1.

FIG. 1.

The Ca2+-dependent contractility of the ICL. Cells were immunolabeled with the monoclonal anticentrin antibody 1A9 (7). (Left panel) Ventral side of a wild-type resting cell. (Right panel) Cell labeled during the Ca2+ influx induced by AED treatment (see Materials and Methods), which triggers the contraction of the ICL, resulting in a general contraction of the cell. Scale bar, 10 μm.

FIG. 2.

FIG. 2.

Phenotypical and biochemical features of the dem1 mutant. (Left panel) Wild-type (wt; left) and dem1 mutant (right) cells were immunolabeled with the monoclonal anticentrin antibody 1A9. Scale bar, 10 μm. The ICL of the dem1 mutant is characterized by a reduced number of meshes and an apparent deficit in branching. This phenotype is exhibited only by cells in growing populations, while cells in stationary phase reach a wild-type phenotype. (Right panel) Sodium dodecyl sulfate-PAGE pattern of purified ICL from wild-type and dem1 mutant cells. The gel is a 6 to 15% acrylamide gradient stained with Coomassie blue. A striking difference in the bands corresponding to the high-molecular-mass protein of wild-type and dem1 mutant cells, at 450 and 250 kDa (stars), respectively, is observed, whereas the same pattern for the low-molecular-mass bands (bracket), which correspond to centrins, is seen for both cell types. The presence of a doublet at the PtCenBP1-dem1p position may be due to proteolysis or to an unknown posttranslational modification.

FIG. 3.

FIG. 3.

Analysis of the PtCenBP1p protein in the wild-type and dem1 mutant cells. The wild-type (Wt) PtCenBP1p sequence (orange rectangles) is composed of six repeats (P1 to P6) plus a divergent repeat (P0) and an incomplete repeat (P7). The mutant PtCenBP1-dem1p (CenBP1p-dem1) sequence derives from the wild-type sequence by the deletion of repeats P3 through P5. For both sequences, the positions of the sequenced peptides are indicated by colored boxes, red, black, blue, and green for the peptides KFERTLDILFRV/S(K)LKVSFDPLKE(I)YMXALNIK(T)M(G)LKKLF, DXAQALKKRAIXLMIKLQ, ETQLNKFTLXI, and DSNLRYFFMK, respectively. Vertical bars represent the centrin-binding motifs as defined by Kilmartin (19) and indicate whether the diagnostic hydrophobic amino acid is a tryptophan, a leucine, a tyrosine, a phenylalanine, or a methionine or valine.

FIG. 4.

FIG. 4.

Sequence logos of the Sfi1 repeats in S. cerevisiae and homolog in P. tetraurelia. Among the potential centrin-binding sites identified in PtCenBP1p, 44 present a diagnostic W at position 22 in the 23-aa sequences defined as centrin-binding sites by Kilmartin (19). A less stringent definition of the consensus sequence, allowing L, Y, F, M, or V in addition to W at the diagnostic position 22, led to the identification of a total of 89 potential centrin-binding sites. The two sets of centrin-binding sites (44 and 89 consensus sites, respectively) were submitted to WebLogo. For comparision, logos corresponding to two sets of consensus sequences from S. cerevisiae are shown: a first set of 17, each with a W or F at position 22 (19), and a second set including all 21 centrin-binding sites recognized in Sfi1p (22) and showing more variability at position 22.

FIG. 5.

FIG. 5.

Disassembly of the ICL upon Pt_CenBP1_ silencing by RNAi as visualized by using immunofluorescence. Disassembly triggered by PtCenBP1p depletion was monitored using the anticentrin antibody 1A9. (A) Ventral side of a wild-type cell before the start of the experiment. (B) Ventral side of a cell after 24 h (ca. three cell divisions) of gene silencing. The cell presents a disrupted ICL in which only the longitudinal part of the meshes is still present and the transversal branches are missing. (C) Ventral side of a cell after 48 h (ca. six to seven fissions) of CenBP1p depletion. The disassembly is complete. Double labeling with a polyclonal anticentrin antibody (26/14-1) and a monoclonal antitubulin antibody (ID5) reveals both basal bodies (red) and the remnants of the ICL (green), which form discrete masses of amorphous material, shown to be reactive to anticentrin antibodies and to be the sites of ICL reassembly, the ICLOCs (7). Enlargements of representative areas selected from panels A, B, and C are presented in the lower panels. Scale bar, 10 μm.

FIG. 6.

FIG. 6.

Disassembly of the ICL upon Pt_CenBP1_ silencing by RNAi as shown by electron microscopy. Tangential thin sections of the cortex show the differences between the organization of the ICL in a control cell (A) and that in a cell after the inactivation of the Pt_CenBP1_ gene (B). (A) The polygonal meshes surrounding basal bodies (bb) with their attached ciliary rootlet (cr) and secretory vesicle tip (t) are composed of branched filamentous bundles. At the branching points, note the presence of one or two dense structures (arrows) called posts (1). (B) In the inactivated cells, the meshes of the network are no longer present. Only small pieces of amorphous material (stars) are visible to the right of each basal body: the enclosed posts (arrows) ensure that they are genuine ICL remnants. Note the mitochondria (m), which are always found at the level of the proximal part of basal bodies in the absence of the ICL, while they are found deeper in the cytoplasm in wild-type cells when the ICL is present (our unpublished observations). Scale bar, 0.30 μm.

FIG. 7.

FIG. 7.

GFP-tagged PtCenBP1p localizes at the ICL. Transformants expressing a GFP-tagged fragment of PtCenBP1p comprising aa 59 to 1344 were examined in vivo and after immunolabeling with anti-GFP antibodies. (A) Ventral and dorsal faces of a living cell showing the homogeneous fluorescence of the ICL. (B to D) Transformants after permeabilization and fixation in the presence of 1 mM Ca2+ and double labeling with a polyclonal anti-GFP antibody and the monoclonal anticentrin antibody 1A9. (B) Anti-GFP staining. (C) Anticentrin staining. (D) Merged images. Scale bar, 10 μm.

FIG. 8.

FIG. 8.

Role of CenBP1p in the Ca2+-dependent contractility of the ICL. (A) Schematic representation, based on Fig. 1, of the ICL. The geometry of the polygonal ICL meshwork at resting (10−7 M) and physiologically high (10−6 M) Ca2+ concentrations is shown. The filament bundles that constitute the edges of the polygons are on average 25% shorter in the presence of high levels of Ca2+. (B) Schematic representation of one ICL polygon. Under resting conditions, the unit filament consisting of one PtCenBP1p molecule with associated centrins is likely to be ∼0.3 to 0.5 μm in length (depending on whether or not the Sfi-like backbone is a completely extended α-helix). This is the distance between posts (red triangles) along the shortest polygon edges. The lateral association of unit filaments builds up the bundles. The N- and/or C-terminal coiled-coil domains may be involved in tethering the filament bundles to the posts. At high Ca2+ concentrations, the filament bundles shorten and the tension is relayed through the posts to yield a global reduction in the size of the ICL, and hence of the whole cell, with very little distortion of global cell shape (Fig. 1). (C) Schematic representation of a possible arrangement of PtCenBP1p and centrins. At resting Ca2+ levels, the ICL unit filament is probably a disordered helical array of ∼4-nm centrins (pink) wrapped around an essentially extended α-helical PtCenBP1p molecule (gray), as originally proposed by Kilmartin for yeast Sfi1p-Cdc31p complexes (19). The binding of Ca2+ to the centrin high-affinity sites at in vivo Ca2+ concentrations favors the lateral interactions necessary for filament bundling. At high Ca2+ levels, the occupation of centrin low-affinity Ca2+-binding sites leads to interactions between adjacent centrins along the length of the filament, as seen in the yeast Sfi1p-Cdc31p crystal structures (22). The consequence is the shortening of the filaments, probably owing to reduced distances between adjacent centrins and the consequent bending, twisting, or supercoiling of the Sfi1-like PtCenBP1p backbone.

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