Clathrin is required for the function of the mitotic spindle

Abstract

Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell [1][2][3][4] . The formation of clathrin-coated vesicles occurs continuously in nondividing cells 5 , but is shut down during mitosis 6 , when clathrin concentrates at the spindle apparatus 7,8 . Here we show that clathrin stabilises fibres of the mitotic spindle to aid congression of chromsomes. Clathrin bound the spindle directlyvia the N-terminal domain of clathrin heavy chain (CHC). Depletion of CHC using RNA interference prolonged mitosis; kinetochore fibres were destabilised leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia but not the N-terminal domain of CHC indicating that stabilisation of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain. Keywordsclathrin; mitosis; mitotic spindle; endocytosis; cancer; cell divisionThe subcellular distribution of clathrin depended on the phase of the cell cycle [7][8][9] ( Supplementary Fig. S1). During interphase, GFP-tagged clathrin light chain a (GFP-LCa) in NRK cells was associated with the Golgi apparatus and numerous puncta representing clathrin-coated pits and vesicles 5 (Fig. 1a). But during metaphase, clathrin localised to kinetochore fibres of the mitotic spindle 10 and possibly interpolar microtubules, but not astral microtubules (Fig. 1a,b). Localisation of clathrin to kinetochore fibres was confirmed by chilling cells for 10 min at 4 °C to selectively disassemble microtubules not associated with kinetochores 11 ; after this treatment, clathrin in metaphase cells remained bound to the kinetochore fibres, indicating that these microtubules were a potential site of clathrin function (Fig. 1b). Similar changes in the distribution of clathrin were observed using other variants of the light chain tagged by GFP or by immunocytochemistry using a monoclonal antibody specific for CHC ( Supplementary Figs. S2,3).Two observations indicated that clathrin bound the mitotic spindle rather than membrane localised to this region. First, none of the major adaptor proteins which allow clathrin to coat membranes (AP-1, AP-2 and AP-3) 2,3 were found at the spindle apparatus ( Supplementary Fig. S4a-c). To test whether clathrin at the spindle was associated with membranes at all, we indiscriminately labelled intracellular membranes by incubating cells with the styryl dyeCorrespondence and requests for materials should be addressed to S.J. R. (sjr51@mrc-lmb.cam.ac.uk Fig. S4d). In cells at metaphase, none of these membranes were found at the spindle (Fig. 1c).The localisation of clathrin to the mitotic spindle was examined at higher resolution using immunoelectron microscopy. CHC and α-tubulin were immunolabelled with 15 nm and 10 nm colloidal gold-conjugated reagents, re...

Monomeric but not trimeric clathrin heavy chain regulates p53-mediated transcription

“…Polyhedral clathrin is recruited to the plasma membrane and promotes the internalization of certain receptors to take nutrients or to transduce correct intracellular signaling from receptors (McPherson et al, 2001). In addition, more recently, it has been reported that CHC plays a role in mitosis (Royle et al, 2005). CHC consists of an Nterminal b-propeller domain that is necessary for binding to adapter proteins for the internalization of various molecules, followed by seven a-helical repeat structures named 'clathrin repeats' (Kirchhausen, 2000).…”

Novel clathrin activity: developments in health and disease

“…At the onset of mitosis, membrane trafficking is inhibited (20,21) and a proportion of CHC17 clathrin is recruited to the kinetochore fibers (K-fibers) of mitotic spindles to enhance integrity (22)(23)(24). In the M phase, microtubule-stabilizing protein TACC3 is phosphorylated by Aurora kinase A (25,26).…”

“…For example, the depletion of CHC17 clathrin causes spindle morphology defects (28) and leads to chromosome dysfunction (24).…”

Novel functions of endocytic player clathrin in mitosis

“…During the process of spindle assembly, the endocytic protein clathrin has been supposed to be involved [12]. It was found that clathrin is localized on spindle during cell division [10][11][12][13][14].…”

“…Clathrin was reported to be localized on mitotic spindles and function in mitosis a few years ago [10][11][12][13][14]. More recently, the role of clathrin in mitosis has been widely recognized.…”