Cenp-F is a nuclear matrix component that localizes to kinetochores during mitosis and is then rapidly degraded after mitosis [1]. Unusually, both the localization and degradation of Cenp-F require it to be farnesylated [2]. Five studies recently demonstrated that Cenp-F is required for kinetochore-microtubule interactions and spindle checkpoint function [3-7]; however, the underlying molecular mechanisms have yet to be defined. Here, we show that Cenp-F interacts with Ndel1 and Nde1, two human NudE-related proteins implicated in regulating Lis1/Dynein motor complexes (reviewed in [8]). We show that Ndel1, Nde1, and Lis1 localize to kinetochores in a Cenp-F-dependent manner. In addition, Nde1, but not Ndel1, is required for kinetochore localization of Dynein. Accordingly, suppression of Nde1 inhibits metaphase chromosome alignment and activates the spindle checkpoint. By contrast, inhibition of Ndel1 results in malorientations that are not detected by the spindle checkpoint; Ndel1-deficient cells consequently enter anaphase in a timely manner but lagging chromosomes then manifest. A major function of Cenp-F, therefore, is to link the Ndel1/Nde1/Lis1/Dynein pathway to kinetochores. Furthermore, our data demonstrate that Ndel1 and Nde1 play distinct roles to ensure chromosome alignment and segregation.

Cenp-F is an unusual kinetochore protein in that it localizes to the nuclear matrix in interphase and the nuclear envelope at the G2/M transition; it is farnesylated and rapidly degraded after mitosis. We have recently shown that farnesylation of Cenp-F is required for G2/M progression, its localization to kinetochores, and its degradation. However, the role Cenp-F plays in mitosis has remained enigmatic. Here we show that, following repression of Cenp-F by RNA interference (RNAi), the processes of metaphase chromosome alignment, anaphase chromosome segregation and cytokinesis all fail. Although kinetochores attach to microtubules in Cenp-F-deficient cells, the oscillatory movements that normally occur following K-fibre formation are severely dampened. Consistently, inter-kinetochore distances are reduced. In addition, merotelic associations are observed, suggesting that whereas kinetochores can attach microtubules in the absence of Cenp-F, resolving inappropriate interactions is inhibited. Repression of Cenp-F does not appear to compromise the spindle checkpoint. Rather, the chromosome alignment defect induced by Cenp-F RNA interference is accompanied by a prolonged mitosis, indicating checkpoint activation. Indeed, the prolonged mitosis induced by Cenp-F RNAi is dependent on the spindle checkpoint kinase BubR1. Surprisingly, chromosomes in Cenp-F-deficient cells frequently show a premature loss of chromatid cohesion. Thus, in addition to regulating kinetochore-microtubule interactions, Cenp-F might be required to protect centromeric cohesion prior to anaphase commitment. Intriguingly, whereas most of the sister-less kinetochores cluster near the spindle poles, some align at the spindle equator, possibly through merotelic or lateral orientations.

Cytoplasmic dynein 1 is a minus-end-directed microtubule motor that is required for a wide range of activities involving movement or anchoring of cellular structures (Vallee et al., 2004). These activities include separation of chromosomes during mitosis, cell migration, and the movement and localisation of substrates such as mRNAs, signalling complexes and membrane organelles. To understand how dynein function over such a diverse range of activities is regulated, much attention has focussed on the composition of the dynein motor complex and the roles of accessory proteins.Dynein can be purified as a 1.6 MDa complex. This contains two copies of a motor subunit (dynein heavy chain; DHC), each of which comprises an AAA ATPase region involved in force generation and a stem region that connects the ATPase to the remaining, regulatory and cargo-binding subunits of the dynein complex. These subunits include dynein intermediate chain (DIC), dynein light intermediate chain (DLIC), and dynein light chains (DLCs) (Pfister et al., 2006). Dynein engages several accessory proteins or protein complexes, which might regulate its motor and/or cargo-binding activities. The best characterised of these is dynactin, a multiprotein complex that is almost universally associated with dynein-dependent functions (Schroer, 2004). More recently, several additional dynein-interacting proteins have been identified using a screen for Aspergillus nidulans mutants that are defective for nuclear distribution. These include NudF (Xiang et al., 1995) and NudE (Efimov and Morris, 2000), the higher eukaryote counterparts of which are Lissencephaly1 (LIS1) and NDE1, respectively. An NDE1-related protein, NDEL1 (also referred to as NudEL) has also been identified Sasaki et al., 2000). LIS1 interacts directly with DHC via sites on the first AAA domain and the stem region (Sasaki et al., 2000;Tai et al., 2002), with DIC (Tai et al., 2002), and with the p50 subunit of dynactin (Tai et al., 2002). NDEL1 and NDE1 also bind to dynein. However, the mode of binding might not be conserved here, because NDEL1 has been reported to bind HC (Sasaki et al., 2000), whereas NDE1 binds to DIC and the LC8 isoform of DLC (Stehman et al., 2007). In addition, NDE1 (Efimov and Morris, 2000;Feng et al., 2000) and NDEL1 (Derewenda et al., 2007;Niethammer et al., 2000;Sasaki et al., 2000) bind to LIS1 directly, and to each other (Bradshaw et al., 2009). There are currently two models to explain how these effectors modulate dynein function. They might regulate dynein motor activity directly (Mesngon et al., 2006;Yamada et al., 2008), or they might have a role in targeting dynein to cargoes (Liang et al., 2007;Vergnolle and Taylor, 2007).LIS1, NDE1 and NDEL1 have been implicated in many dyneinmediated activities, including cell migration, (Ding et al., 2009;Dujardin et al., 2003;Feng and Walsh, 2004;Kholmanskikh et al., 2003;Sasaki et al., 2005;Shu et al., 2004;Tanaka et al., 2004;Tsai et al., 2007;Tsai et al., 2005) , 2008). Mutations in or haplo-insufficiency of mammalian...

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Progression through mitosis and cytokinesis requires the sequential proteolysis of several cell-cycle regulators. This proteolysis is mediated by the ubiquitin-proteasome system, with the E3 ligase being the anaphase-promoting complex, also known as the cyclosome (APC/C). The APC/C is regulated by two activators, namely Cdc20 and Cdh1. The current view is that prior to anaphase, the APC/C is activated by Cdc20, but that following anaphase, APC/C switches to Cdh1-dependent activation. However, here we present an analysis of the kinetochore protein Cenp-F that is inconsistent with this notion. Although it has long been appreciated that Cenp-F is degraded sometime during or after mitosis, exactly when and how has not been clear. Here we show that degradation of Cenp-F initiates about six minutes after anaphase, and that this is dependent on a C-terminal KEN-box. Although these two observations are consistent with Cenp-F being a substrate of Cdh1-activated APC/C, Cenp-F is degraded normally in Cdh1-null cells. By contrast, RNAi-mediated repression of APC/C subunits or Cdc20 does inhibit Cenp-F degradation. These findings therefore suggest that the APC/C does not simply ‘switch’ upon anaphase onset; rather, our observations indicate that Cdc20 also contributes to post-anaphase activation of the APC/C. We also show that the post-anaphase, KEN-box-dependent degradation of Cenp-F requires it to be farnesylated, a post-translational modification usually linked to membrane association. Because so many of the behaviours of Cenp-F are farnesylation-dependent, we suggest that this modification plays a more global role in Cenp-F function.