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. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus. Here, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Clathrin bound to the spindle directly by the amino-terminal domain of clathrin heavy chain. Depletion of clathrin heavy chain using RNA interference prolonged mitosis; kinetochore fibres were destabilized, 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 clathrin heavy chain, indicating that stabilization 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.

Figure 1

Clathrin was targeted to the mitotic spindle of NRK cells. a, Confocal micrographs showing the subcellular distribution of clathrin at interphase and metaphase. GFP-LCa (left, green), α-tubulin (centre, red) and nucleic acids (blue). b, Cells expressing GFP-LCa fixed before (left) or after (right) depolymerisation of non-kinetochore microtubules. c-d, The association of clathrin with microtubules was not via coated membranes. c, Example images of live cells expressing either GFP-α-tubulin (left) or GFP-LCa (right) imaged following 24-28 h incubation with FM4-64 (red). d, Association of clathrin with microtubules visualised by immunogold EM. CHC (15 nm) and α-tubulin (10 nm gold) in mitotic NRK cells. Chromosomes are denoted by asterisks. A morphologically distinct CCV (ii) is indicated by an arrow. Arrowheads denote CHC labelling associated with microtubules. Scale bars, 10 μm (a-c) 250 nm (d).

Figure 2

Clathrin was targeted to the mitotic spindle via the N-terminal domain of the heavy chain. a, Example images of GFP and GFP-LCa in cells at metaphase. b, Histogram of spindle recruitment of GFP or GFP-LCa. A value of one represents no specific recruitment (see Methods). c, Schematic diagram of a clathrin triskelion and the CHC fragments used in d-e (right). CHC N-terminal domain (residues 1-330) is yellow. d, Example images of GFP-tagged CHC fragments in cells at metaphase. e, Histogram of spindle recruitment of GFP or GFP-tagged CHC fragments. Results are mean ± s.e.m, **, p < 0.01. Scale bars, 10 μm.

Figure 3

Inhibition of CME did not disrupt mitosis. a, Effect of clathrin-depletion on mitotic index (black bars) and transferrin uptake (red bars) 24, 48 and 72 hours after transfection with CHC siRNA. Open bars show mitotic index in control siRNA-transfected cells. Transferrin uptake normalised to control values (dotted line). Within 48 hours CME was reduced by 70% and the mitotic index increased four-fold. b, Effects of inhibiting CME by over-expression of GFP-CHC(1-479), measured 48, 72 and 96 hours after transfection. Transferrin uptake was inhibited by 60-70%, without any change in the mitotic index. Control (open bars) was GFP alone. Results are mean ± s.e.m., **, p < 0.01.

Figure 4

Depletion of clathrin resulted in destabilised kinetochore fibres, defective congression of chromosomes and prolonged activation of the spindle checkpoint. a, Clathrin depletion increased the frequency of misaligned chromosomes and caused thicker metaphase plates. b, Clathrin-depleted metaphase-like plates were more disorganised. Cells were marked by GFP (green) stained for nucleic acids (blue) and CENP-B (red) to visualise centromeres. Schematic drawings illustrate centromere arrangement (right). c, Cells expressing GFP-α-tubulin (green) after depolymerisation of non-kinetochore fibres. Close-up of boxed centromere pairs (right). d, Misaligned chromosomes in CHC RNAi cells were pairs of sister chromatids. e, Representative images of one control (left) and two CHC RNAi (middle and right) cells expressing very low levels of GFP-hMad2. Mad2-positive kinetochores are indicated by arrows. Scale bars, 5 μm.

Figure 5

“Full-length” CHC, but not CHC N-terminal domain, was sufficient to rescue the mitotic defects found in cells depleted of endogenous CHC. a, Representative images of GFP-tagged knockdown-proof CHC (GFP-CHC(1-1639)) expressed in HEK293 cells that were depleted of endogenous CHC. Right panel shows the normal uptake of transferrin (red) in these cells, GFP-CHC(1-1639) (green) and DNA (blue). Quantification of transferrin uptake at interphase (b), mitotic index (c) and the frequency of metaphase-like cells with misaligned chromosomes (d) in cells expressing GFP, GFP-CHC(1-479) or GFP-CHC(1-1639) in control cells (open bars) or cells depleted of endogenous CHC (closed bars), 72 h post-transfection.