Trimerisation is important for the function of clathrin at the mitotic spindle

Stephen J. Royle ) 0 1 Leon Lagnado 1 0 Present address: The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool , Liverpool, L69 3BX , UK 1 MRC Laboratory of Molecular Biology , Hills Road, Cambridge, CB2 2QH , UK - Clathrin is a triskelion consisting of three heavy chains each with an associated light chain. During mitosis, clathrin contributes to kinetochore fibre stability. As the N-terminal domain at the foot of each leg can bind to the mitotic spindle, we proposed previously a bridge hypothesis wherein clathrin acts as a brace between two or three microtubules within a kinetochore fibre to increase fibre stability. Here, we have tested this hypothesis by replacing endogenous clathrin heavy chain in human cells e with a panel of clathrin constructs. Mutants designed to cn abolish trimerisation were unable to rescue the mitotic ie defects caused by depletion of endogenous clathrin. By c S l l e C Introduction fo Clathrin is a three-legged molecule, or triskelion, which la consists of three ~190 kDa (1,675 residue) heavy chains each rn with an associated ~25 kDa light chain (Kirchhausen, 2000; u Kirchhausen and Harrison, 1981; Ungewickell and Branton, o J 1981). In mammalian cells, clathrin has two functions. First, during interphase, clathrin plays a key role in membrane trafficking (Kirchhausen, 2000). Second, when the cell enters mitosis, membrane traffic ceases (Warren, 1993) and a portion of clathrin is targeted to the mitotic spindle where it apparently stabilises kinetochore fibres (Mack and Compton, 2001; Maro et al., 1985; Okamoto et al., 2000; Royle et al., 2005; Sutherland et al., 2001). When clathrin heavy chain (CHC) is depleted from cells using RNAi, a number of mitotic defects arise, such as problems in congression (the movement of chromosomes to the metaphase plate), destabilisation of kinetochore fibres and lengthened mitosis as a result of prolonged signalling of the spindle checkpoint (Royle et al., 2005). The organisation of a clathrin triskelion, as defined by a recent molecular model (Fotin et al., 2004) is shown in Fig. 1A. A single CHC molecule consists of an N-terminal sevenbladed -propeller, a linker region, eight clathrin heavy chain repeat (CHCR0-7) segments, a proximal hairpin, a tripod region that is thought to be responsible for trimerisation, and a variable C-terminal segment (residues 1631-1675). Thus, one CHC molecule resembles a human leg: the foot comprises the N-terminal domain, linker and part of CHCR0; the ankle corresponds to the remainder of CHCR0, CHCR1 and CHCR2; and the knee is at CHCR5 (Fotin et al., 2004). As the N-terminal domain at the end of each leg can bind to contrast, stunted triskelia with contracted legs could partially rescue normal mitosis. These results indicate that the key structural features of clathrin that are necessary for its function in mitosis are a trimeric molecule with a spindle interaction domain at each end, supporting the bridge hypothesis for clathrin function in mitosis. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/19/4071/DC1 the mitotic spindle, we proposed a bridge hypothesis wherein clathrin triskelia act as a brace between two or three microtubules within a kinetochore fibre to increase fibre stability (Royle et al., 2005). An alternative view is that clathrin does not act as a bridge, but as a lattice or matrix that can support spindle fibres. In our earlier paper (Royle et al., 2005), we showed that normal mitosis could be rescued by full-length clathrin triskelia and not by the N-terminal domain alone, but this did not allow us to distinguish between these two models. In the present study, we aimed to test these two hypotheses by replacing endogenous clathrin heavy chain (CHC) in human cells with a variety of CHC constructs. These constructs allowed us to ask: is trimerisation essential for the function of clathrin in mitosis? And what are the minimal structural requirements for normal mitosis? Our findings exclude the lattice model and support the bridge hypothesis for clathrin function in mitosis. Results To test whether or not the triskelion structure of clathrin was essential for its function in mitosis, we designed a panel of clathrin constructs based on structural (Fotin et al., 2004) and biochemical information (Liu et al., 1995; Nathke et al., 1992; Ybe et al., 2003). These various constructs were expressed in HEK293 cells in which levels of endogenous CHC were reduced by more than 90% using RNA interference (RNAi). Constructs used in this study The CHC constructs used in this study are illustrated in Fig. 1B. The first two constructs have the trimerisation domain and should be able to form trimers: full-length CHC (1-1675) and Fig. 1. Overview of the organisation of clathrin and of the constructs used in this study. (A) Model of a clathrin triskelion proposed by Fotin et al. (Fotin et al., 2004). The triskelion is viewed looking down onto the vertex. Coloured regions show the features of a CHC molecule (see key, right). (B) Schematic representations of each CHC construct used in the study. Variable region (residues 1631-1675) is shown in grey, GFP has been omitted for clarity. Short names used in the paper are in black and full descriptive names are in grey. Trimerisation was predicted based on previous publications (Fotin et al., 2004; Liu et al., 1995; Nathke et al., 1992; Ybe et al., 2003). X22 epitope is between residues 1109-1128 of CHC (Liu et al., 1995). Constructs were compared with GFP, GFP expressed on a CHC RNAi background and with Control, GFP expressed on a control RNAi background. the major splice variant (1-1639). Four other constructs are all predicted to be unable to trimerise: three truncations (1-479, 11516, 1-1597) and a point mutant (C1573S). We also included a construct that is predicted to trimerise but lacks the Nterminal domain (331-1639) in order to test the role of the propeller interaction domain. Note that our earlier analysis was limited to 1-1639 and 1-479 only (Royle et al., 2005). All CHC constructs were GFP tagged at the N-terminus and any that included CHC residues 60-66 (the region targeted for RNAi) were rendered resistant to knockdown (see Materials and Methods). For comparison we expressed GFP alone on a CHC-depleted background (GFP) or as a control we expressed GFP alone on an endogenous clathrin background (control). As the constructs were expressed on a CHC RNAi background, we first assessed the level of CHC in these cells by immunocytochemistry using the monoclonal antibody, X22 (Fig. 2). We found that in GFP cells the level of endogenous clathrin was ~10% of that in the control (Royle et al., 2005). Knockdown occurred to a similar extent in cells expressing 1479. In cells expressing 1-1675, 1-1639, 1-1516, 1-1597, C1573S and 331-1639, X22 recognised the expressed protein (Fig. 2, supplementary material Fig. S1). This is consistent with previous stu (...truncated)