- Email: [email protected]
Chromosome Segregation: A Kinetochore Missing Link Is Found
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here? It is interesting to speculate whether the presence of these bizarre additional illusory contours might make sense in terms of Bayesian model averaging. Indeed, this is not as different from the authors’ own explanation as it may sound. Essentially, when multiple models that impose different constraints are brought into conflict, sometimes the globally most probable solution (the solution that gets the right answer most of the time) will produce the ‘wrong’ answer (a relatively improbable answer) in a specific given case. Thus, as Anderson et al. [2] argue, the presence of the spurious contours results from higher-level constraints than those captured by a single Bayesian model concerned only with contour completion. Developing theories that explain how the brain resolves conflicts between different kinds of explanation will not just shed
light on improbable contours. It will also help us to understand the brain’s supreme flexibility more generally. References 1. Wade, N.J. (2000). A Natural History of Vision (Cambridge, MA: MIT Press). 2. Anderson, B.L., O’Vari, J., and Barth, H. (2011). Improbable percepts. Curr. Biol. 21, 492–496. 3. Schumann, F. (1900). Beitra¨ge zur Analyse der Gesichtswahrnehmungen. Erste Abhandlung. Einige Beobachtungen u¨ber die Zusammenfassung von Gesichtseindru¨cken zu Einheiten. Zeitschrift Psychol. Physiol. Sinnesorg. 23, 1–32. 4. Ehrenstein, W. (1941). U¨ber Abwandlungen der L. Hermannschen Helligkeitserscheinung. Zeitschrift Psychol. 150, 83–91. 5. Kanizsa, G. (1955). Margini quasi-percettivi in campi con stimolazione omogenea. Rivista Psicol. 49, 7–30. 6. von Helmholtz, H. (1867/1962). Helmholtz’s Treatise on Physiological Optics. J.P.C. Southall, ed. (New York: Dover). 7. Knill, D.C., and Richards, W. (1996). Perception as Bayesian Inference (Cambridge: Cambridge University Press). 8. Elder, J.H., and Goldberg, R.M. (2002). Ecological statistics of Gestalt laws for the perceptual organization of contours. J. Vis. 2 4, 324–353.
Chromosome Segregation: A Kinetochore Missing Link Is Found During mitosis the kinetochore assembles on centromeric chromatin. The component that connects the chromatin-associated inner domain to the microtubule-binding outer domain has eluded researchers. Two new studies identify a conserved molecular linkage between the inner and outer kinetochore. Thomas J. Maresca The ultimate measure of a successful cell division event is equal distribution of the genetic material into two daughter cells. At its most fundamental level and in all eukaryotes, this requires that replicated DNA physically interacts with spindle microtubules in a configuration that best ensures each daughter receives a copy. This is no simple task. For one, the interaction between the DNA and microtubules cannot be static but rather capable of channeling the energy of microtubule dynamics and microtubule-associated motor proteins into the alignment and segregation of chromosomes. The attachment site must also be able to ‘communicate’ with the cell if problems in chromosome alignment arise so that division can be halted and errors repaired. These demanding requirements are achieved by an
extraordinary macro-molecular complex called the kinetochore. The kinetochore is a large multi-protein assemblage that localizes to specialized chromatin regions called centromeres (reviewed by [1]). Molecularly speaking, a subset of kinetochore components, which are referred to as the constitutive centromere-associated network (CCAN), are present at centromeres throughout the cell cycle while other kinetochore proteins assemble at the centromere only during mitosis. From a structural perspective, the kinetochore has spatially distinct domains that were initially identified as separate electron-dense regions or plates by electron microscopy. The inner kinetochore is the DNA-proximal interface of the kinetochore while the outer kinetochore is the microtubule-binding surface. Fittingly, numerous CCAN components localize
9. Ernst, M.O., and Banks, M.S. (2002). Humans integrate visual and haptic information in a statistically optimal fashion. Nature 415, 429–433. 10. Hillis, J.M., Ernst, M.O., Banks, M.S., and Landy, M.S. (2002). Combining sensory information: mandatory fusion within, but not between senses. Science 298, 1627–1630. 11. Geisler, W.S., and Diehl, R.L. (2003). A Bayesian approach to the evolution of perceptual and cognitive systems. Cogn. Sci. 27, 379–402. 12. Knill, D.C., and Pouget, A. (2004). The Bayesian brain: The role of uncertainty in neural coding and computation. Trends Neurosci. 27, 712–719. 13. Kersten, D., Mamassian, P., and Yuille, A. (2004). Object perception as Bayesian inference. Annu. Rev. Psychol. 55, 271–304. 14. Welchman, A.E., Lam, J.M., and Bu¨lthoff, H.H. (2008). Bayesian motion estimation accounts for a surprising bias in 3D vision. Proc. Natl. Acad. Sci. USA 105, 12087–12092.
Department of Psychology, University of Giessen, Otto-Behaghel-Str. 10/F, 35394, Giessen, Germany. E-mail: roland.w.fleming@psychol. uni-giessen.de
DOI: 10.1016/j.cub.2011.02.012
to the inner kinetochore and directly interact with DNA and/or centromeric nucleosomes while the core kinetochore microtubule attachment complex (called the KMN complex) assembles during mitosis and is found in the outer plate. A glut of studies over the past decade has yielded a lengthy kinetochore ‘parts list’ as well as a comprehensive network of molecular interdependencies that are required for the localization of many of these parts. With this strong foundation in place, the field is quickly advancing towards an extensive characterization of kinetochore structure and function. Two studies that appeared in a recent issue of Current Biology [2,3] have filled a significant gap in our understanding of kinetochore biology by identifying the CCAN component CENP-C as the missing molecular link between the centromeric chromatin in the inner kinetochore and the core microtubule-attachment complex in the outer kinetochore. Both Przewloka et al. [2] and Screpanti et al. [3] discovered that the amino terminus of CENP-C mediates interaction with the KMN network via association with the Mis12 complex (M in KMN) in Drosophila and human, respectively. CENP-C was initially identified as a component of the inner
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Figure 1. The path from the chromatin to the microtubule. (A) The central players discussed in two papers appearing in a recent issue of Current Biology drawn to scale and noting flexibility based on electron microscopic analysis [3,8,17,18]. The length and flexibility of CENP-C is unknown but, based on light microscopy, it is likely longer than 14 nm [19]. (B) The components drawn at the same scale as (A) but organized geometrically to correspond with distance measurements made by high resolution imaging of human (left side) and Drosophila (right side) kinetochores [19,20]. The path of contact points from the microtubule to the centromeric DNA are: (1) Ndc80/Nuf2 binds the microtubule, (2) and (3) the Mis12 complex contacts Spc24/25 (KNL-1 not shown) through Nsl1 and the amino terminus of CENP-C via Nnf1, and (4) CENP-C associates with both CENP-A and H3 nucleosomes. CENP-C is shown to loop back because microscopic analyses placed the carboxyl terminus 14 nm from its amino terminus and w25 nm from CENP-A. Non CENP-C CCAN components may provide another conserved linkage between the inner and outer kinetochore, although homologues have not be found in Drosophila or C. elegans.
kinetochore plate [4]. It has subsequently been shown that the middle portion of CENP-C contains a DNA-binding and CENP-A (centromere-specific histone H3 variant) nucleosome-associating domain and that this domain in combination with a carboxy-terminal motif are responsible for targeting CENP-C to the centromere [5–7]. Thus, CENP-C must bridge the inner and outer kinetochore, with its amino terminus contacting the Mis12 complex and its middle and perhaps carboxyl terminus interacting with the centromeric chromatin. The Mis12 complex is part of the KMN network that binds to microtubules and contains the Mis12 complex, KNL-1/Blinkin (K in KMN) and the four subunit Ndc80 complex (N in KMN) (reviewed by [1]). Importantly, Screpanti et al. [3] showed that the association of CENP-C with the Mis12 complex did not disrupt its other essential associations with KNL-1 or the Ndc80 complex. These data, in combination with previous findings by the Musacchio group [8], as well as in vitro pull-down experiments by
Przewloka et al. [2] using Drosophila Mis12 complex components, suggest that one end of the Mis12 complex interacts with CENP-C via the Nnf1 subunit while the other end of the complex associates with both KNL-1 and the Ndc80 complex through the Nsl1 subunit (Figure 1). Screpanti et al. [3] also investigated the structure of their purified KMN–CENP-C complexes by electron microscopy. The CENP-C–Mis12– Ndc80 complex assembled into a long and flexible 80 nm structure that appeared as a ‘whip’ with a thicker ‘handle’ at one end. Intriguingly, the Mis12 complex alone was quite bendy while the addition of CENP-C caused a striking rigidification of the Mis12 complex into a straight w20–25 nm rod with a distinct globular head at one end. It is noteworthy that the addition of CENP-C did not yield additional density to the electron micrographs, suggesting that the amino-terminal 400 amino acids of CENP-C (representing w40% of the full-length protein) is flexible relative to the Mis12 complex. With a firmer grasp on the structure of these important kinetochore
complexes in hand we now move on to questions of function. Experiments in Drosophila and human tissue culture cells by both groups clearly demonstrated that the amino terminus of CENP-C plays an essential role in localizing outer kinetochore components [2,3]. Przewloka et al. [2] implemented a clever cell-based assay in which artificially targeting the amino terminus of CENP-C to centrosomes was sufficient to recruit the KMN network along with it. In fact, the centrosomal localization of KMN components was so robust in this assay that centromeres often failed to recruit the KMN network and consequently chromosomes became severely misaligned. Both groups also found that overexpression of the amino terminus had a dominant-negative effect on outer kinetochore assembly resulting in major chromosome alignment defects [2,3]. Another essential kinetochore function is communicating to the cell when problems in chromosome attachment and alignment occur (reviewed by [9]). To achieve this, the kinetochore serves as the hub for a signaling pathway called the spindle assembly checkpoint (SAC) that delays anaphase onset in the presence of unattached and misaligned chromosomes. This ‘wait-anaphase’ signal is dependent upon the recruitment of checkpoint proteins to the kinetochore through mechanisms and interactions that are not clearly understood. Normally, the kinetochores of unattached and misaligned chromosomes exhibit elevated levels of checkpoint proteins; however, Screpanti et al. [3] found that kinetochores in cells over-expressing CENP-C1-71 (amino terminus) failed to properly recruit the checkpoint proteins Bub1, BubR1 and Mad1. In support of these findings, Przewloka et al. [2] found that targeting the amino terminus of CENP-C to centrosomes was also sufficient to localize the checkpoint proteins Mad2 (a Mad1-binding partner) and BubR1, albeit at lower levels, along with KMN components to the centrosomes. Thus, both groups provide data to support previous studies showing that the outer kinetochore, specifically the KMN complex, is required for efficient localization of checkpoint components to the kinetochore [10,11].
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It is hypothesized that structural changes within the kinetochore complex impact microtubuleattachment stability and SAC function (reviewed by [12]). Specifically, an increase in the distance between the inner and outer kinetochore, deemed intrakinetochore stretch, occurs upon binding to dynamic microtubules [13,14]. Introduction of intrakinetochore stretch correlates with inactivation of the wait-anaphase signal and is postulated to promote a higher affinity interaction between the outer kinetochore and microtubules by regulating phosphorylation of the KMN complex. At the time this Dispatch was being prepared, no known compliant or ‘stretchable’ component(s) between the inner and outer kinetochore had been characterized. With help from Przewloka et al. [2] and Screpanti et al. [3] we now know that CENP-C is in the right place; the next question is whether it (or perhaps CENP-Cassociated chromatin) is being stretched. The ability of the amino terminus of CENP-C to contact the Mis12 complex is clearly conserved between Drosophila and humans. However, what is happening outside the amino terminus of the molecule is murky. For example, in chicken cells, immunoprecipitated CENP-C exclusively interacted with histone-H3containing chromatin [15]; however, human CENP-C was found to directly interact with CENP-A-containing nucleosomes but not H3 nucleosomes in vitro [5]. Thus, it has been proposed, and is reiterated by Screpanti et al. [3], that CENP-C could interact with both CENP-A and H3 nucleosomes, thereby crosslinking distinct blocks of centromeric chromatin [16]. Obviously this issue remains to be resolved. Even the role of CENP-C as a bridge between the inner and outer kinetochore is not entirely conserved. Recent work in budding yeast found that the Mis12 complex did not interact with CENP-C but rather with another CCAN complex that is generally conserved from yeast to man but has not been identified in either Drosophila or Caenorhabditis elegans [17]. It is exciting to imagine that there are additional, and potentially novel, molecular connections between the inner and outer kinetochore that remain to be characterized. After all, what’s great about missing links is that there
are always more out there just waiting to be found.
12.
References 1. Cheeseman, I.M., and Desai, A. (2008). Molecular architecture of the kinetochore-microtubule interface. Nat. Rev. Mol. Cell Biol. 9, 33–46. 2. Przewloka, M.R., Zsolt, V., BolanosGarcia, V.M., Debski, J., Dadlez, M., and Glover, D.M. (2011). CENP-C is a structural platform for kinetochore assembly. Curr. Biol. 21, 399–405. 3. Screpanti, E., De Antoni, A., Alushin, G.M., Petrovic, A., Melis, T., Nogales, E., and Musacchio, A. (2011). Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore. Curr. Biol. 21, 391–398. 4. Saitoh, H., Tomkiel, J., Cooke, C.A., Ratrie, H., 3rd, Maurer, M., Rothfield, N.F., and Earnshaw, W.C. (1992). CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate. Cell 70, 115–125. 5. Carroll, C.W., Milks, K.J., and Straight, A.F. (2010). Dual recognition of CENP-A nucleosomes is required for centromere assembly. J. Cell Biol. 189, 1143–1155. 6. Milks, K.J., Moree, B., and Straight, A.F. (2009). Dissection of CENP-C-directed centromere and kinetochore assembly. Mol. Biol. Cell 20, 4246–4255. 7. Yang, C.H., Tomkiel, J., Saitoh, H., Johnson, D.H., and Earnshaw, W.C. (1996). Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C. Mol. Cell Biol. 16, 3576–3586. 8. Petrovic, A., Pasqualato, S., Dube, P., Krenn, V., Santaguida, S., Cittaro, D., Monzani, S., Massimiliano, L., Keller, J., Tarricone, A., et al. (2010). The MIS12 complex is a protein interaction hub for outer kinetochore assembly. J. Cell Biol. 190, 835–852. 9. Musacchio, A., and Salmon, E.D. (2007). The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8, 379–393. 10. DeLuca, J.G., Howell, B.J., Canman, J.C., Hickey, J.M., Fang, G., and Salmon, E.D. (2003). Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores. Curr. Biol. 13, 2103–2109. 11. Kiyomitsu, T., Obuse, C., and Yanagida, M. (2007). Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic
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checkpoint through direct interaction with Bub1 and BubR1. Dev. Cell 13, 663–676. Maresca, T.J., and Salmon, E.D. (2010). Welcome to a new kind of tension: translating kinetochore mechanics into a wait-anaphase signal. J. Cell Sci. 123, 825–835. Maresca, T.J., and Salmon, E.D. (2009). Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity. J. Cell Biol. 184, 373–381. Uchida, K.S., Takagaki, K., Kumada, K., Hirayama, Y., Noda, T., and Hirota, T. (2009). Kinetochore stretching inactivates the spindle assembly checkpoint. J. Cell Biol. 184, 383–390. Hori, T., Amano, M., Suzuki, A., Backer, C.B., Welburn, J.P., Dong, Y., McEwen, B.F., Shang, W.H., Suzuki, E., Okawa, K., et al. (2008). CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore. Cell 135, 1039–1052. Ribeiro, S.A., Vagnarelli, P., Dong, Y., Hori, T., McEwen, B.F., Fukagawa, T., Flors, C., and Earnshaw, W.C. (2010). A super-resolution map of the vertebrate kinetochore. Proc. Natl. Acad. Sci. USA 107, 10484–10489. Hornung, P., Maier, M., Alushin, G.M., Lander, G.C., Nogales, E., and Westermann, S. (2011). Molecular architecture and connectivity of the budding yeast Mtw1 kinetochore complex. J. Mol. Biol. 405, 548–559. Maskell, D.P., Hu, X.W., and Singleton, M.R. (2010). Molecular architecture and assembly of the yeast kinetochore MIND complex. J. Cell Biol. 190, 823–834. Schittenhelm, R.B., Heeger, S., Althoff, F., Walter, A., Heidmann, S., Mechtler, K., and Lehner, C.F. (2007). Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes. Chromosoma 116, 385–402. Wan, X., O’Quinn, R.P., Pierce, H.L., Joglekar, A.P., Gall, W.E., DeLuca, J.G., Carroll, C.W., Liu, S.T., Yen, T.J., McEwen, B.F., et al. (2009). Protein architecture of the human kinetochore microtubule attachment site. Cell 137, 672–684.
Biology Department, University of Massachusetts Amherst, Amherst, MA 01003, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2011.02.026
Auditory Neuroscience: How to Stop Tinnitus by Buzzing the Vagus Recent observations linking the vagus nerve to plasticity in the central nervous system could pave the way to new treatments for one of the most common and intractable disorders of the auditory system. Jan Schnupp Many millions of people (an estimated 14% of the population) suffer from persistent tinnitus, a constant ‘ringing in their ears’, and about 2% find their tinnitus very disruptive, as it interferes with their ability to follow conversations, to concentrate, or to enjoy beautiful music or a quiet
night’s sleep. Tinnitus is therefore a major public health issue, but treatment options remain limited. While the causes and symptoms of tinnitus may be quite diverse, tinnitus often arises when the central auditory pathway struggles to adapt to focal damage to the sensory structures of the cochlea, typically by excessive noise exposure.
here? It is interesting to speculate whether the presence of these bizarre additional illusory contours might make sense in terms of Bayesian model averaging. Indeed, this is not as different from the authors’ own explanation as it may sound. Essentially, when multiple models that impose different constraints are brought into conflict, sometimes the globally most probable solution (the solution that gets the right answer most of the time) will produce the ‘wrong’ answer (a relatively improbable answer) in a specific given case. Thus, as Anderson et al. [2] argue, the presence of the spurious contours results from higher-level constraints than those captured by a single Bayesian model concerned only with contour completion. Developing theories that explain how the brain resolves conflicts between different kinds of explanation will not just shed
light on improbable contours. It will also help us to understand the brain’s supreme flexibility more generally. References 1. Wade, N.J. (2000). A Natural History of Vision (Cambridge, MA: MIT Press). 2. Anderson, B.L., O’Vari, J., and Barth, H. (2011). Improbable percepts. Curr. Biol. 21, 492–496. 3. Schumann, F. (1900). Beitra¨ge zur Analyse der Gesichtswahrnehmungen. Erste Abhandlung. Einige Beobachtungen u¨ber die Zusammenfassung von Gesichtseindru¨cken zu Einheiten. Zeitschrift Psychol. Physiol. Sinnesorg. 23, 1–32. 4. Ehrenstein, W. (1941). U¨ber Abwandlungen der L. Hermannschen Helligkeitserscheinung. Zeitschrift Psychol. 150, 83–91. 5. Kanizsa, G. (1955). Margini quasi-percettivi in campi con stimolazione omogenea. Rivista Psicol. 49, 7–30. 6. von Helmholtz, H. (1867/1962). Helmholtz’s Treatise on Physiological Optics. J.P.C. Southall, ed. (New York: Dover). 7. Knill, D.C., and Richards, W. (1996). Perception as Bayesian Inference (Cambridge: Cambridge University Press). 8. Elder, J.H., and Goldberg, R.M. (2002). Ecological statistics of Gestalt laws for the perceptual organization of contours. J. Vis. 2 4, 324–353.
Chromosome Segregation: A Kinetochore Missing Link Is Found During mitosis the kinetochore assembles on centromeric chromatin. The component that connects the chromatin-associated inner domain to the microtubule-binding outer domain has eluded researchers. Two new studies identify a conserved molecular linkage between the inner and outer kinetochore. Thomas J. Maresca The ultimate measure of a successful cell division event is equal distribution of the genetic material into two daughter cells. At its most fundamental level and in all eukaryotes, this requires that replicated DNA physically interacts with spindle microtubules in a configuration that best ensures each daughter receives a copy. This is no simple task. For one, the interaction between the DNA and microtubules cannot be static but rather capable of channeling the energy of microtubule dynamics and microtubule-associated motor proteins into the alignment and segregation of chromosomes. The attachment site must also be able to ‘communicate’ with the cell if problems in chromosome alignment arise so that division can be halted and errors repaired. These demanding requirements are achieved by an
extraordinary macro-molecular complex called the kinetochore. The kinetochore is a large multi-protein assemblage that localizes to specialized chromatin regions called centromeres (reviewed by [1]). Molecularly speaking, a subset of kinetochore components, which are referred to as the constitutive centromere-associated network (CCAN), are present at centromeres throughout the cell cycle while other kinetochore proteins assemble at the centromere only during mitosis. From a structural perspective, the kinetochore has spatially distinct domains that were initially identified as separate electron-dense regions or plates by electron microscopy. The inner kinetochore is the DNA-proximal interface of the kinetochore while the outer kinetochore is the microtubule-binding surface. Fittingly, numerous CCAN components localize
9. Ernst, M.O., and Banks, M.S. (2002). Humans integrate visual and haptic information in a statistically optimal fashion. Nature 415, 429–433. 10. Hillis, J.M., Ernst, M.O., Banks, M.S., and Landy, M.S. (2002). Combining sensory information: mandatory fusion within, but not between senses. Science 298, 1627–1630. 11. Geisler, W.S., and Diehl, R.L. (2003). A Bayesian approach to the evolution of perceptual and cognitive systems. Cogn. Sci. 27, 379–402. 12. Knill, D.C., and Pouget, A. (2004). The Bayesian brain: The role of uncertainty in neural coding and computation. Trends Neurosci. 27, 712–719. 13. Kersten, D., Mamassian, P., and Yuille, A. (2004). Object perception as Bayesian inference. Annu. Rev. Psychol. 55, 271–304. 14. Welchman, A.E., Lam, J.M., and Bu¨lthoff, H.H. (2008). Bayesian motion estimation accounts for a surprising bias in 3D vision. Proc. Natl. Acad. Sci. USA 105, 12087–12092.
Department of Psychology, University of Giessen, Otto-Behaghel-Str. 10/F, 35394, Giessen, Germany. E-mail: roland.w.fleming@psychol. uni-giessen.de
DOI: 10.1016/j.cub.2011.02.012
to the inner kinetochore and directly interact with DNA and/or centromeric nucleosomes while the core kinetochore microtubule attachment complex (called the KMN complex) assembles during mitosis and is found in the outer plate. A glut of studies over the past decade has yielded a lengthy kinetochore ‘parts list’ as well as a comprehensive network of molecular interdependencies that are required for the localization of many of these parts. With this strong foundation in place, the field is quickly advancing towards an extensive characterization of kinetochore structure and function. Two studies that appeared in a recent issue of Current Biology [2,3] have filled a significant gap in our understanding of kinetochore biology by identifying the CCAN component CENP-C as the missing molecular link between the centromeric chromatin in the inner kinetochore and the core microtubule-attachment complex in the outer kinetochore. Both Przewloka et al. [2] and Screpanti et al. [3] discovered that the amino terminus of CENP-C mediates interaction with the KMN network via association with the Mis12 complex (M in KMN) in Drosophila and human, respectively. CENP-C was initially identified as a component of the inner
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Figure 1. The path from the chromatin to the microtubule. (A) The central players discussed in two papers appearing in a recent issue of Current Biology drawn to scale and noting flexibility based on electron microscopic analysis [3,8,17,18]. The length and flexibility of CENP-C is unknown but, based on light microscopy, it is likely longer than 14 nm [19]. (B) The components drawn at the same scale as (A) but organized geometrically to correspond with distance measurements made by high resolution imaging of human (left side) and Drosophila (right side) kinetochores [19,20]. The path of contact points from the microtubule to the centromeric DNA are: (1) Ndc80/Nuf2 binds the microtubule, (2) and (3) the Mis12 complex contacts Spc24/25 (KNL-1 not shown) through Nsl1 and the amino terminus of CENP-C via Nnf1, and (4) CENP-C associates with both CENP-A and H3 nucleosomes. CENP-C is shown to loop back because microscopic analyses placed the carboxyl terminus 14 nm from its amino terminus and w25 nm from CENP-A. Non CENP-C CCAN components may provide another conserved linkage between the inner and outer kinetochore, although homologues have not be found in Drosophila or C. elegans.
kinetochore plate [4]. It has subsequently been shown that the middle portion of CENP-C contains a DNA-binding and CENP-A (centromere-specific histone H3 variant) nucleosome-associating domain and that this domain in combination with a carboxy-terminal motif are responsible for targeting CENP-C to the centromere [5–7]. Thus, CENP-C must bridge the inner and outer kinetochore, with its amino terminus contacting the Mis12 complex and its middle and perhaps carboxyl terminus interacting with the centromeric chromatin. The Mis12 complex is part of the KMN network that binds to microtubules and contains the Mis12 complex, KNL-1/Blinkin (K in KMN) and the four subunit Ndc80 complex (N in KMN) (reviewed by [1]). Importantly, Screpanti et al. [3] showed that the association of CENP-C with the Mis12 complex did not disrupt its other essential associations with KNL-1 or the Ndc80 complex. These data, in combination with previous findings by the Musacchio group [8], as well as in vitro pull-down experiments by
Przewloka et al. [2] using Drosophila Mis12 complex components, suggest that one end of the Mis12 complex interacts with CENP-C via the Nnf1 subunit while the other end of the complex associates with both KNL-1 and the Ndc80 complex through the Nsl1 subunit (Figure 1). Screpanti et al. [3] also investigated the structure of their purified KMN–CENP-C complexes by electron microscopy. The CENP-C–Mis12– Ndc80 complex assembled into a long and flexible 80 nm structure that appeared as a ‘whip’ with a thicker ‘handle’ at one end. Intriguingly, the Mis12 complex alone was quite bendy while the addition of CENP-C caused a striking rigidification of the Mis12 complex into a straight w20–25 nm rod with a distinct globular head at one end. It is noteworthy that the addition of CENP-C did not yield additional density to the electron micrographs, suggesting that the amino-terminal 400 amino acids of CENP-C (representing w40% of the full-length protein) is flexible relative to the Mis12 complex. With a firmer grasp on the structure of these important kinetochore
complexes in hand we now move on to questions of function. Experiments in Drosophila and human tissue culture cells by both groups clearly demonstrated that the amino terminus of CENP-C plays an essential role in localizing outer kinetochore components [2,3]. Przewloka et al. [2] implemented a clever cell-based assay in which artificially targeting the amino terminus of CENP-C to centrosomes was sufficient to recruit the KMN network along with it. In fact, the centrosomal localization of KMN components was so robust in this assay that centromeres often failed to recruit the KMN network and consequently chromosomes became severely misaligned. Both groups also found that overexpression of the amino terminus had a dominant-negative effect on outer kinetochore assembly resulting in major chromosome alignment defects [2,3]. Another essential kinetochore function is communicating to the cell when problems in chromosome attachment and alignment occur (reviewed by [9]). To achieve this, the kinetochore serves as the hub for a signaling pathway called the spindle assembly checkpoint (SAC) that delays anaphase onset in the presence of unattached and misaligned chromosomes. This ‘wait-anaphase’ signal is dependent upon the recruitment of checkpoint proteins to the kinetochore through mechanisms and interactions that are not clearly understood. Normally, the kinetochores of unattached and misaligned chromosomes exhibit elevated levels of checkpoint proteins; however, Screpanti et al. [3] found that kinetochores in cells over-expressing CENP-C1-71 (amino terminus) failed to properly recruit the checkpoint proteins Bub1, BubR1 and Mad1. In support of these findings, Przewloka et al. [2] found that targeting the amino terminus of CENP-C to centrosomes was also sufficient to localize the checkpoint proteins Mad2 (a Mad1-binding partner) and BubR1, albeit at lower levels, along with KMN components to the centrosomes. Thus, both groups provide data to support previous studies showing that the outer kinetochore, specifically the KMN complex, is required for efficient localization of checkpoint components to the kinetochore [10,11].
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It is hypothesized that structural changes within the kinetochore complex impact microtubuleattachment stability and SAC function (reviewed by [12]). Specifically, an increase in the distance between the inner and outer kinetochore, deemed intrakinetochore stretch, occurs upon binding to dynamic microtubules [13,14]. Introduction of intrakinetochore stretch correlates with inactivation of the wait-anaphase signal and is postulated to promote a higher affinity interaction between the outer kinetochore and microtubules by regulating phosphorylation of the KMN complex. At the time this Dispatch was being prepared, no known compliant or ‘stretchable’ component(s) between the inner and outer kinetochore had been characterized. With help from Przewloka et al. [2] and Screpanti et al. [3] we now know that CENP-C is in the right place; the next question is whether it (or perhaps CENP-Cassociated chromatin) is being stretched. The ability of the amino terminus of CENP-C to contact the Mis12 complex is clearly conserved between Drosophila and humans. However, what is happening outside the amino terminus of the molecule is murky. For example, in chicken cells, immunoprecipitated CENP-C exclusively interacted with histone-H3containing chromatin [15]; however, human CENP-C was found to directly interact with CENP-A-containing nucleosomes but not H3 nucleosomes in vitro [5]. Thus, it has been proposed, and is reiterated by Screpanti et al. [3], that CENP-C could interact with both CENP-A and H3 nucleosomes, thereby crosslinking distinct blocks of centromeric chromatin [16]. Obviously this issue remains to be resolved. Even the role of CENP-C as a bridge between the inner and outer kinetochore is not entirely conserved. Recent work in budding yeast found that the Mis12 complex did not interact with CENP-C but rather with another CCAN complex that is generally conserved from yeast to man but has not been identified in either Drosophila or Caenorhabditis elegans [17]. It is exciting to imagine that there are additional, and potentially novel, molecular connections between the inner and outer kinetochore that remain to be characterized. After all, what’s great about missing links is that there
are always more out there just waiting to be found.
12.
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Biology Department, University of Massachusetts Amherst, Amherst, MA 01003, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2011.02.026
Auditory Neuroscience: How to Stop Tinnitus by Buzzing the Vagus Recent observations linking the vagus nerve to plasticity in the central nervous system could pave the way to new treatments for one of the most common and intractable disorders of the auditory system. Jan Schnupp Many millions of people (an estimated 14% of the population) suffer from persistent tinnitus, a constant ‘ringing in their ears’, and about 2% find their tinnitus very disruptive, as it interferes with their ability to follow conversations, to concentrate, or to enjoy beautiful music or a quiet
night’s sleep. Tinnitus is therefore a major public health issue, but treatment options remain limited. While the causes and symptoms of tinnitus may be quite diverse, tinnitus often arises when the central auditory pathway struggles to adapt to focal damage to the sensory structures of the cochlea, typically by excessive noise exposure.