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Cytoskeleton: Anatomy of an Organizing Center Laura G View metadata, citation and similar papers at core.ac.uk brought to you by CORE R754 Dispatch provided by Elsevier - Publisher Connector Cytoskeleton: Anatomy of an organizing center Laura G. Marschall and Tim Stearns One component of the yeast spindle pole body, Spc42p, SPB are duplicated each cell cycle, such that a new has been found to form a crystalline array within one of organelle is created next to the existing one. Unlike DNA the central layers of the structure; the Spc42p crystal replication, where the structure of the molecule immedi- might provide a scaffold around which the spindle pole ately suggests a templated mechanism of replication, body is assembled, and could be involved in regulating there is no obvious template in either the centrosome or the size of the spindle pole body. SPB. Two recent papers suggest that the SPB might be assembled from the inside out, starting with a central Address: Department of Biological Sciences, Stanford University, Stanford, California 94305, USA. crystalline array of a single protein [1], to which other components are added [2]. Current Biology 1997, 7:R754–R756 http://biomednet.com/elecref/09609822007R0754 Early studies showed that, when viewed by thin-section © Current Biology Ltd ISSN 0960-9822 electron microscopy, the SBP appears to consist of three layers (reviewed in [3]). The central plaque is embedded Most cells in your body have an extensive microtubule in the nuclear envelope, the inner plaque is on the nuclear network that is busily transporting organelles and vesicles side of the central plaque, and the outer plaque is on the from one place to another, and separating chromosomes in cytoplasmic side of the central plaque. Microtubules mitosis and meiosis. For these microtubule ‘tracks’ to be nucleated from the outer plaque of the SPB extend into effective, they must be present in the right number and the cytoplasm and are involved in orienting the nucleus orientation at the right time. The temporal and spatial during cell division and mating (reviewed in [4]). Micro- organization of microtubules is determined, in part, by the tubules nucleated from the inner plaque of the SPB form microtubule organizing center, a specialized organelle that the mitotic and meiotic spindles [4]. exists in one copy per cell. In fungi, the microtubule organizing center is the spindle pole body (SPB), a laminar Recent cryoelectron microscopy studies by Bullitt and structure embedded in the nuclear envelope. In animal coworkers [1] have now been able to resolve at least six cells, the main microtubule organizing center is the cen- layers within the SPB. Starting from the cytoplasmic face trosome, a nucleus-associated organelle that consists of of the SPB and moving toward the nuclear face, these centrioles and pericentriolar material. Although the SPB layers are as follows. The first layer is the outer plaque, and centrosome are structurally different, they are func- which interfaces with the cytoplasmic microtubules. Then tionally equivalent: both nucleate the polymerization of come two intermediate layers, IL1 and IL2. The fourth microtubules, anchor those microtubules, and duplicate layer is the central plaque, which spans the nuclear once per cell cycle. envelope. Fifth is a beaded layer of the inner plaque (IP1). Sixth and last is the capped microtubule layer (IP2), Two aspects of the assembly of these organizing centers which anchors the nuclear microtubules (Figure 1). An are remarkable. First, both the centrosome and the SPB unexpected finding was that there is considerable have the capacity to change size and nucleation capacity variation in the lateral dimension — in the plane of the during the course of the cell cycle, and during develop- nuclear envelope — of the SPB, whereas the vertical ment. For example, the centrosome nucleates a radial dimension — through the layers — remained constant. array of microtubules in interphase, becomes larger and nucleates more microtubules at the onset of mitosis, then To get more information about SPB structure from their becomes smaller and nucleates few microtubules at samples, Bullitt et al. [1] used tomographic reconstruction telophase, finally returning to its interphase state. The to examine heparin-extracted SPBs. In this technique, a Saccharomyces cerevisiae SPB does not appear to go through series of electron microscope images is taken, each with such radical changes in size and nucleation capacity over the sample tilted a different amount with respect to the the course of a cell cycle, but it can change size in electron beam. These images are then reconstructed by response to cell ploidy. The SPB in diploid cells is computer, giving a three-dimensional representation of approximately twice as large as that in haploid cells, pre- the object. The reconstructed object can be ‘sectioned’ by sumably because the SPB must organize more micro- computer, revealing aspects of structure that may not be tubules for mitosis in the diploid cell. visible in standard thin-section electron microscopy. This technique has been used to great effect on the animal The second remarkable aspect of organizing center centrosome [5,6], resulting in the identification of putative assembly is the way that both the centrosome and the microtubule-nucleating structures in the pericentriolar Dispatch R755 Figure 1 Schematic representation of the spindle pole body (SPB). The representation is Cytosol diagrammatic and is not meant to imply stoichiometric relationships amongst the Cytoplasmic (a) (b) microtubules proteins that comprise the SPB. The inset Tub4p, shows a hexagonal layer of Spc42p within the Spc97p, Spc98p SPB core: (a) a slice through the tomographic Outer plaque reconstruction of an SBP core; (b) computed IL1 Spc42p (c) Fourier transform of (a); (c) two-dimensional IL2 projection map of (a) with imposed symmetry. Central plaque 33 (Inset reproduced with permission from [1]). Nuclear envelope Calmodulin Spc110p Inner plaque IP1 Spc97p, Spc98p, IP2 Tub4p Nuclear/spindle microtubules Spc97p Spc98p Nucleus Tub4p Current Biology material. Heparin-extraction of SPBs yields a core changes the distance between the inner and central structure that electron tomographic sectioning showed plaques [8], again indicating a vertical orientation of the contains a hexagonal lattice in the lateral dimension, corre- protein within the layer. This type of organization would sponding to layer IL2 [1] (Figure 1, inset). neatly explain the invariant vertical architecture of the SPB, as the layers and the connections between layers The presence of this crystalline hexagonal lattice immedi- would be defined by the intrinsic length of the major ately suggested to Bullitt et al. [1] that this layer might be protein(s) making them up. Also, the lateral dimension of made up of Spc42p, a SPB protein that had been shown the SPB could easily be varied in response to ploidy by previously to form a two-dimensional sheet when over- adding or subtracting subunits from the layers. The use of expressed in yeast [7]. This Spc42p sheet formed around proteins as biological ‘rulers’ to determine the length of the SPB, as if it were an extension of an existing layer of structures was first indicated in studies of the genetic the SPB. When Bullitt et al. [1] used electron microscopy control of the length of the tail of phage lambda [9], to examine these Spc42p sheets, they found a crystalline although in that case the ruler protein, the gene H protein, lattice with a hexagonal structure very similar to that in is probably not a main structural component of the tail. the IL2 layer, suggesting that IL2 may comprise, at least in part, hexagonally packed Spc42p. The Spc42p crystal There are some unanswered questions, of course. If a resulting from overexpression of the protein could be crystal of Spc42p is the main determinant of the lateral imaged at a much higher resolution than the SPB core, dimension of the SPB, then how is its size controlled? allowing predictions about the orientation of Spc42p in Similarly, new Spc42p subunits would be likely to add the layer. Spc42p is predicted to have a coiled-coil central preferentially to an existing crystalline array of other domain [7], and edge-on views of the SPB showed vertical Spc42p molecules rather than start a new crystal, so how striations, suggesting that the rod-like coiled-coil domain can a new SPB ever be initiated? Bullitt et al. [1] note that is oriented vertically in the crystal. there appears to be a ring of protein in the central plaque outside of the central core of Spc42p, and suggest that What role does this crystalline layer of Spc42p play in the perhaps this regulates crystal growth. This, of course, only assembly of the complex SPB? Bullitt et al. [1] propose pushes the issue back a step — what is this ring made of, that the Spc42p layer is the base structure of the SPB, and and how is its size determined? The duplication question that other layers of the SPB are added to that. Further, is more perplexing. The duplication cycle of the SPB has they propose that each of the visible layers of the SPB is been described in detail [10], and there are specific struc- made up of coiled-coil proteins interacting laterally. This tures associated with duplication, but there is nothing to is supported by work on another SPB component, indicate how it is that a new layer is initiated. It is possible Spc110p, which is also a coiled-coil protein, and which that one of the SPB structures associated with duplication, links the inner plaque to the central plaque. Remarkably, the half-bridge, nucleates a new crystal of Spc42p adjacent changing the length of the coiled-coil domain of Spc110p to the existing SPB. R756 Current Biology, Vol 7 No 12 If we assume that a central crystal of Spc42p is the core of the nuclear side of the central plaque.
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