Centrin Scaffold in Chlamydomonas reinhardtii Revealed by Immunoelectron Microscopy

Abstract

In the flagellate green alga Chlamydomonas reinhardtii the Ca 2؉ -binding EF-hand protein centrin is encoded by a single-copy gene. Previous studies have localized the protein to four distinct structures in the flagellar apparatus: the nucleus-basal body connector, the distal connecting fiber, the flagellar transitional region, and the axoneme. To explain the disjunctive distribution of centrin, the interaction of centrin with as yet unknown specific centrin-binding proteins has been implied. Here, we demonstrate using serial section postembedding immunoelectron microscopy of isolated cytoskeletons that centrin is located in additional structures (transitional fibers and basal body lumen) and that the centrin-containing structures of the basal apparatus are likely part of a continuous filamentous scaffold that extends from the nucleus to the flagellar bases. In addition, we show that centrin is located in the distal lumen of the basal body in a rotationally asymmetric structure, the V-shaped filament system. This novel centrin-containing structure has also been detected near the distal end of the probasal bodies. Taken together, these results suggest a role for a rotationally asymmetric centrin "seed" in the growth and development of the centrin scaffold following replication of the basal apparatus.Centrin (also known as caltractin) is a member of the highly conserved superfamily of calcium-binding EF-hand proteins and occurs in all eukaryotic cells, being mostly associated with centrosomes (58, 64). It was first discovered in flagellate green algae, where it is located in the basal apparatus (the part of the cytoskeleton anchoring the flagella) and involved in the contraction of calcium-sensitive filaments (42, 60) which, in general, play an important role in the dynamic behavior of centrosomes by controlling the position and orientation of centrosomal structures (58, 64). Analysis of centrin mutants and RNA interference strains deficient in centrin in the green algal model system Chlamydomonas reinhardtii suggested that centrin is essential for basal body duplication and segregation during cell division (28,39,73), functions also deduced for centrin homologues in vertebrates (46, 62) and yeast spp. (3,50,69). More recently, novel centrin functions have been discovered such as involvement in recombinational DNA repair through modulation of the nucleotide excision repair pathway in humans and plants (2, 47), a role in the nuclear mRNA export machinery in yeast (11), regulation of calcium-dependent ciliary activities in ciliates (14, 15), and signal transduction in vertebrate photoreceptors (53). How centrin exerts these different functions at the molecular level is, however, still unknown although considerable progress has recently been made through in vitro studies of centrin and its interaction with target peptides (8,19,20,40,75).In some organisms, the functional diversity of centrin may be related to the presence of different centrin isotypes, e.g., four centrin proteins (centrin1p to centrin4p), which are d...

Functional diversification of centrins and cell morphological complexity

“…This diversity of situations suggests that the ancestral centrin-based contractile system was localised at the central MTOC and controlled its duplication (Ruiz et al, 2005) and cell division. Interesting support for such a view comes from the characterisation in Chlamydomonas of a centrin scaffold linking the various 'centrin-containing organelles' (Geimer and Melkonian, 2005). The existence of such centrinbased links between cell organelles and cytoskeletal networks remains to be demonstrated.…”

The Neomuran Revolution and Phagotrophic Origin of Eukaryotes and Cilia in the Light of Intracellular Coevolution and a Revised Tree of Life

“…3); Apusozoa, Cercozoa, and the heterokont Caecitellus reevolved posterior ciliary gliding, presumably using kinesin-2. The V-fiber, with associated acorn-base attached distally to centriole 1 -2 triplets (at least in neokaryotes: Geimer and Melkonian 2005), demarcates the centriole from the transition zone and perhaps evolved in the cenancestral eukaryote; its rotational asymmetry and that of centriolar root attachments to specific triplets probably reflect an asymmetric doublet "numbering machinery" that probably evolved in the earliest gliding protocilium (B).…”

Reconstructing the evolutionary history of the centriole from protein components

“…SAS-4 has been proposed to be involved in the attachment of microtubules to the central centriolar cylinder (Pelletier et al, 2006) and in the elongation of centriolar microtubules (Kohlmaier et al, 2009;Schmidt et al, 2009;Tang et al, 2009); SAS-6 has been implicated in cartwheel formation (Kilburn et al, 2007;Nakazawa et al, 2007;Rodrigues-Martins et al, 2007). Centrin 2 is a well-characterised centriolar component that is essential for centriole duplication (Geimer and Melkonian, 2005;Koblenz et al, 2003;Salisbury et al, 2002). Although the wider centrin family is involved in multiple cellular processes and is present in non-ciliated species, there is a clade termed CrCentype centrin 2, which has a distribution restricted to ciliated species (Bornens and Azimzadeh, 2007).…”