Centrosomes: Sfi1p and Centrin Dispatch Unravel a Structural Riddle

Jeffrey L. Salisbury centrin functions in these diverse processes has remained enigmatic. Centrin’s action within the centrosome has come The discovery of Sfi1p as a novel binding partner for into focus with Kilmartin’s discovery [5] of a novel the Ca2+-binding protein centrin has provided new centrin-binding protein in budding yeast and human insight into the dynamic behavior of centrosomes. cells. Kilmartin [5] conducted ‘pull-down’ experiments Sfi1 binds to multiple centrin molecules along a series in which a recombinant-tagged version of the yeast of internal repeats, and the complex forms Ca2+-sen- centrin Cdc31p was used to assay yeast cell extracts sitive contractile fibers that function to reorient cen- for centrin-binding partners. Earlier studies [12–14] on trioles and alter centrosome structure. the yeast equivalent of the centrosome, the spindle pole body (SPB), revealed two other proteins that interact with Cdc31p, Kar1p and Mps3, but these The centrosome has fascinated cell biologists ever were not picked up in Kilmartin’s [5] pull-down assay, since it was first realised that it plays a fundamental which was designed to select interacting proteins role in the organization of the cytoplasm and in the that bind to Cdc31p under low free Ca2+ conditions. process of cell division [1]. The two defining features Several other proteins, however, were enriched by common to centrosomes of all eukaryotic cells are the this procedure, and one of these, Sfi1p, immediately ability to nucleate microtubules, and duplication once piqued Kilmartin’s [5] interest. in each cell cycle to yield two centrosomes that act as Earlier genetic studies in yeast [15] had shown that spindle poles during mitosis. Beyond these properties, SFI1 is required for proper mitotic spindle assembly, centrosomes are also remarkable for the striking range and deletion of this gene resulted in G2/M cell-cycle of alternative forms they take in different cell types and arrest. Kilmartin [5] extended these observations and the ability to undergo rapid changes in various struc- established that various conditional sfi1 alleles result tural and functional attributes [2]. Studies over the past in a failure of SPB duplication, and that this phenotype twenty years have indicated that centrin, a ubiquitous can be suppressed by overexpression of CDC31. Ca2+-binding protein of centrosomes, plays a role in Mutant sfi1 phenotypes also mimicked those seen for these dynamic processes [3], though precisely how conditional cdc31 mutations [9], and together with the has until now been unclear [4]. In a recent study com- pull-down studies, these observations suggested that bining yeast genetics, genome database mining and Cdc31p and Sfi1p interact functionally and play an clever cell biology, John Kilmartin [5] has discovered a important role in SPB duplication. novel centrosome protein, Sfi1p, with a striking series Analysis of the Sfi1p sequence revealed a remark- of internal centrin-binding repeats. Analysis of the able series of seventeen internal repeats with the con- properties of the Sfi1p–centrin complex, and the cen- sensus motif AX7LLX3F/LX2WK/R, with clusters of trosome fibers that they form, has shed fresh light on repeats separated by gaps of between 23 and 35 the remarkable structural and behavioral diversity of amino acids (Figure 1). Genome database mining iden- centrosomes of eukaryotic cells. tified candidate homologs with similar repeats in other Centrin was first discovered as a major protein 2+ component of Ca -sensitive contractile fibers asso- AX7LLX3F/LX2WK/R ciated with the centrioles of algal cells [6,7]. Centrin is a member of the EF-hand family of small Ca2+- WWWWW W WWWW W WWWW W W S.c. Sfi1p binding proteins, and is known to undergo conforma- 2+ tional changes upon binding Ca ions [8]. Centrins WWWWWWWWWWWWWWW WWW W W are characterized by their ubiquitous presence at the S.p. Sfi1p centrosome, where they are components of both the WWWWWWWWW L WWW W WWWWWWWWW centrioles themselves and of an assortment of fibers H.s. Sfi1p that link the two centrioles to each other, to the sur- 200 400 600 800 1200 rounding pericentriolar material and, in some organ- isms, to the cell and nuclear membranes. Studies in Current Biology ‘lower’ eukaryotes, and more recently in animal cells, have established that centrin-containing fibers play a Figure 1. The general structure of Sfi1p. role in the dynamic behavior of centrosomes, through The picture outlines the domain organization of Sfi1p orthologs control of the position and orientation of centrosomal of the yeasts Saccharomyces cerevisiae (S.c. Sfi1p) and structures, and also in the control of centriole Schizosaccharomyces pombe (S.p. Sfi1p), and of humans duplication [9–11]. But the mechanism by which (H.s. Sfi1p). W indicates the position of a consensus repeat for centrin binding (with the sequence at the top). The yeast Sfi1p sequences have seventeen and twenty consensus repeats, and Tumor Biology Program, Mayo Clinic School of Medicine, the human sequence has twenty-three. A model representing a Rochester, Minnesota 55905, USA. portion of the Sfi1 protein with three bound centrin molecules E-mail: [email protected] (red) is shown below. Dispatch R28

genetic and biochemical studies, suggest an intimate functional interaction between the two proteins. Revisiting the striated flagellar roots of the green algae, where centrin was first discovered [7], provides >[Ca2+] insight into the mechanism of centrin function in centrosomes of diverse cell types. The striated flagellar root is a centriole-associated fiber system which Current Biology shows Ca2+-sensitive contractile behavior and is composed largely, but not exclusively, of centrin [6,7]. Elec- Figure 2. A two-component molecular thread model for tron microscopy revealed that striated flagellar roots Sfi1p–centrin contraction. consist of parallel linear arrays of fine fibers, approxi- In this model, the Sfi1p (blue rod) serves as a flexible elastic mately 5–7 nm in diameter and 0.2 µm long. Following backbone along which multiple centrin molecules (red) are Ca2+-stimulation, these fibers bind Ca2+ ions and 2+ tightly bound. When free [Ca ] increases, the centrin mol- shorten by acquiring bends, twists and kinks, much ecules bind Ca2+ ions and undergo a change in conformation while remaining bound to the Sfi1p backbone. The resulting like the rubber band engine of a model airplane that torsion forces along the length of the Sfi1p–centrin complex has been wound tightly before flight [18]. cause the assembly to bend or twist, shortening the overall Shortening of the striated flagellar root draws the cen- length of the molecular thread. trioles deeper into the cell towards the nucleus. When the intracellular Ca2+ concentration returns to the basal yeast species, as well as in humans and mice. Two level, the flagellar roots re-extend, coincident with versions of human Sfi1p (hSfi1p) were identified with phosphorylation of centrin. Similar structural features almost identical sequences containing twenty three are also seen in the contractile spasmoneme of the vor- internal consensus repeats, but with stop codons after ticellid ciliates [19]. Striated flagellar roots represent an either 968 or 1242 amino acids. Other notable features extraordinary elaboration of centrosome structure. of the Sfi1p sequences include low proline content Nonetheless, centrosomes of diverse cell types show a within the repeat regions and a lack of homology in the wide variety of centrin-containing fibers that link centri- amino- and carboxy-terminal domains of Sfi1p from oles to one another and to other elements of the peri- different species. centriolar material. Centrosomes exhibit a range of The unusual series of internal repeats in Sfi1p looked morphologies in different eukaryote species, but these good as candidates for being the sites of centrin are essentially variations on a common structural theme, binding. To investigate this possibility, Kilmartin [5] and so a molecular model based on Sfi1p–centrin used a modification of the pull-down assay in which protein complexes can be proposed to explain the GST-fusions containing only a portion of yeast or dynamic behavior of centrosomes and their associated human Sfi1p were systematically assessed for their fibrous structures. ability to bind to their species’ centrin. GST-fusions with Picture the Sfi1p–centrin complex as a two- the amino- or carboxy-terminal regions of Sfi1p, lacking component molecular thread as depicted in Figure 2, the consensus repeats, failed to bind centrin. But Sfi1p with half of the thread composed of repeating units of GST-fusions that contained even only a single repeat a Ca2+-sensitive regulator — centrin — and half sequence bound to centrin with a molar ratio close to 1. composed of a flexible or elastic backbone — Sfi1p. Larger fusions with multiple repeats showed saturable Kilmartin’s [5] observations suggest a model in which, binding with stoichiometries closely corresponding to under conditions of low free Ca2+, the individual regu- the number of consensus repeats present. These find- lator molecules tightly associate with the thread back- ings clearly demonstrated that Sfi1p binds multiple bone, and may also interact with adjacent regulator molecules of centrin through its conserved repeat molecules. Binding of Ca2+ ions to the regulator mol- sequences. Further analysis of the gap distance ecules induces small conformational changes that between centrin-binding repeats along Sfi1p, and con- recur along the length of the thread. In this model, the sideration of structural features of the centrin molecule, two-component molecular thread is subject to torsion indicated that adjacent centrins might also interact with force along its length caused by a greater conforma- one another. tional change in the regulator molecule than in the Kilmartin [5] also showed that the Sfi1p–centrin backbone to which it remains bound. Given the geo- complex is concentrated in the centrosome. Light metric demands of such a system, the complex would microscopy of yeast cells expressing recombinant Sfi1p bend or twist much like a bimetallic strip in a thermo- fused to the green fluorescent protein (GFP) and paral- stat after a change in temperature. When the free Ca2+ lel immuno-labeling studies at electron micro-scope concentration returns to the low basal level, the regu- resolution confirmed that Sfi1p occurs at the SPB half- lator protein, now no longer Ca2+-bound, would be bridge where centrin is also found [5,16]. The half- free to return to its original conformation. The thread bridge is a structure adjacent to the central plaque, would then ‘relax’ through the elastic properties of the where SPB duplication is initiated [17]. Likewise, recom- backbone molecule itself, possibly assisted by phos- binant GFP–hSfi1p showed centrosome localization in phorylation events or by antagonistic forces placed on HeLa cells, and co-localization with either centrin or glu- the system from the outside. tamylated tubulin, a marker for centrioles. These obser- This model was borrowed, more or less intact, from vations show that Sfi1p–centrin complexes occur at the an early proposal by Albert Szent-György [20] for the centrosome of diverse cell types and, together with the mechanism of muscle contraction (it was obviously Current Biology R29

wrong for muscle!). If correct, this model would explain 15. Ma, P., Winderickx, J., Nauwelaers, D., Dumortier, F., De Doncker, how fibers composed of Sfi1p and centrin act as con- A., Thevelein, J.M., and Van Dijck, P. (1999). Deletion of SFI1, a novel suppressor of partial Ras-cAMP pathway deficiency in the tractile or elastic connections between the various ele- yeast Saccharomyces cerevisiae, causes G(2) arrest. Yeast 15, ments of the centrosome to mediate dynamic changes 1097–1109. in its overall structure. Some of these changes may be 16. Spang, A., Courtney, I., Fackler, U., Matzner, M., and Schiebel, E. (1993). The calcium-binding protein cell division cycle 31 of Sac- as tangible as the re-orientation of centrioles during the charomyces cerevisiae is a component of the half bridge of the cell cycle, while others may occur as subtle tugs on the spindle pole body. J. Cell Biol. 123, 405–416. pericentriolar material which alter its shape and 17. Kilmartin, J.V. (1994). Genetic and biochemical approaches to spindle function and chromosome segregation in eukaryotic perhaps translate pulling forces to the microtubules. microorganisms. Curr. Opin. Cell Biol. 6, 50–54. The precise role of Sfi1p–centrin fibers in centriole 18. Salisbury, J.L. (1998). Roots. J. Eukaryot. Microbiol. 45, 28–32. duplication remains an unsolved mystery. But one 19. Amos, W.B. (1975). Contraction and calcium binding in the vorticel- possibility is that Sfi1p–centrin fibers mediate a key lid ciliates. Soc. Gen. Physiol. Ser. 30, 411–436. licensing step necessary for centriole duplication 20. Szent-Györgyi, A. (1947). Chemistry of Muscle Contraction (New York: Academic Press). involving disorientation of the centriole pair which reveals previously cryptic sites along the centriole wall that template assembly of nascent centrioles. This may occur, at a key moment in the cell cycle, either directly through mechanical pulling forces as described here, or perhaps through the disassembly of Sfi1p–centrin complexes that previously linked the centrioles to one another. Kilmartin’s [5] observations, together with the model presented here, offer a simple yet elegant solution to a riddle that has perplexed cell biologists ever since centrosomes were first described. It is also satisfying, because it indicates that at the very center of every eukaryotic cell, there beats a heart of Sfi1p–centrin.

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