Stephen J. Royle

Clathrin is required for the function of the mitotic spindle.

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.

Endocytosis at the synaptic terminal

Exocytosis of neurotransmitter from a synaptic vesicle is followed by efficient retrieval of its constituent membrane and proteins. Real‐time measurements indicate that fast and slow modes of retrieval operate in parallel at a number of presynaptic terminals. Two mechanisms can be distinguished by electron microscopy: clathrin‐mediated retrieval of small vesicles and bulk retrieval of large cisternae. Methods that investigate the behaviour of individual vesicles have recently demonstrated a third route of retrieval: the rapid reversal of a pore‐like connection between the vesicle and surface (‘kiss‐and‐run’). Key aims for the future are to identify the molecules underlying different mechanisms of endocytosis at the synapse and the signals that select between them.

Behavioural analysis and susceptibility to CNS injury of four inbred strains of mice

Interpretation of data from gene targeting studies can be confounded by the inherent traits of the background inbred strains used in the generation of transgenic and null mutant mice. We have therefore compared the behaviour and response to CNS injury of four inbred strains commonly used in molecular genetic studies to produce models of neurological disease. Adult, male 129/Ola, BALB/c, C57BL/6 and FVB/N mice (2-4 months) were initially subjected to behavioural tests that comprised a neurological examination, determination of motor function and cognitive testing in the Morris water maze. Also the response to CNS injury following an acute kainic acid (KA) challenge (30 mg kg-1, i.p.) was determined. The 129/Ola and BALB/c strains showed significant motor deficits when compared with the C57BL/6 and FVB/N strains. In contrast, only the FVB/N strain showed evidence of apparent cognitive impairments in the water maze as evidenced by increased pathlengths to locate the escape platforms and impaired performance in a probe trial. In addition, the FVB/N strain showed the most severe seizure response and mortality rate (62%) following administration of KA (30 mg kg-1, i.p.). These behavioural changes were also associated with a greater degree of cell body and synaptophysin loss in the pyramidal CA3 hippocampal cell layer and astrogliosis 72-h post-dose. These data suggest that the FVB/N strain may not be the most suitable background strain for the development of new transgenic mice for the study of genes implicated in the learning and memory process.

A TACC3/ch-TOG/clathrin complex stabilises kinetochore fibres by inter-microtubule bridging.

Kinetochore fibres (K‐fibres) of the spindle apparatus move chromosomes during mitosis. These fibres are discrete bundles of parallel microtubules (MTs) that are crosslinked by inter‐MT ‘bridges’ that are thought to improve fibre stability during chromosomal movement. The identity of these bridges is unknown. Clathrin is a multimeric protein that has been shown to stabilise K‐fibres during early mitosis by a mechanism independent of its role in membrane trafficking. In this study, we show that clathrin at the mitotic spindle is in a transforming acidic colied‐coil protein 3 (TACC3)/colonic, hepatic tumour overexpressed gene (ch‐TOG)/clathrin complex. The complex is anchored to the spindle by TACC3 and ch‐TOG. Ultrastructural analysis of clathrin‐depleted K‐fibres revealed a selective loss of a population of short inter‐MT bridges and a general loss of MTs. A similar loss of short inter‐MT bridges was observed in TACC3‐depleted K‐fibres. Finally, immunogold labelling confirmed that inter‐MT bridges in K‐fibres contain clathrin. Our results suggest that the TACC3/ch‐TOG/clathrin complex is an inter‐MT bridge that stabilises K‐fibres by physical crosslinking and by reducing rates of MT catastrophe.

Identification of a Non-canonical Tyrosine-based Endocytic Motif in an Ionotropic Receptor

Rapid modulation of the surface number of certain ionotropic receptors is achieved by altering the relative rates of insertion and internalization. These receptors are internalized by a clathrin-mediated pathway; however, a motif that is necessary for endocytosis of ionotropic receptors has not yet been identified. Here, we identified a motif that is required for constitutive and agonist-regulated internalization of the ionotropic P2X4 receptor. Three amino acids in the C terminus of P2X4 (Tyr378, Gly381, and Leu382) compose a non-canonical tyrosine-based sorting signal of the form YXXGL. We found that P2X4 protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathioneS-transferase fusion of the P2X4 C terminus pulled down the adaptor protein-2 complex from brain extract. Mutation of either the tyrosine-binding pocket of the μ2 subunit of adaptor protein-2 or the YXXGL motif in the receptor C terminus caused a decrease in receptor internalization and a dramatic increase in the surface expression of P2X4 receptors. The YXXGL motif represents a non-canonical tyrosine-based sorting signal that is necessary for efficient endocytosis of the P2X4 receptor. Similar motifs are present in other receptors and may be important for the control of their functional expression.

Non-canonical YXXGPhi endocytic motifs: recognition by AP2 and preferential utilization in P2X4 receptors

During clathrin-mediated endocytosis, proteins on the cell surface are selected for inclusion in clathrin-coated vesicles by clathrin adaptors, mainly the adaptor complex AP2. The P2X4 subtype of ATP-gated ion channel has in its C-terminus two putative endocytic motifs: a canonical YXXΦ motif and a non-canonical YXXGΦ motif (YEQGL). We demonstrate that endocytosis of P2X4 receptors is mediated preferentially by the YXXGΦ motif because the YXXΦ motif is inaccessible to AP2 owing to the structure of the channel. The crystal structure of a complex between residues 160-435 of the μ2 subunit of AP2 and a P2X4 C-terminal peptide showed that the YEQGL motif binds to μ2 at the same site as YXXΦ motifs. Y and Φ residues are accommodated in the same hydrophobic pockets in μ2 with the extra residue between them being accommodated by changes in the peptides backbone configuration, when compared to YXXΦ motifs. These data demonstrate that the family of potential tyrosine-based endocytic signals must be expanded to include motifs with an additional glycine at Y+3 (YXXGΦ).

The cellular functions of clathrin.

Abstract.Membranes and proteins are moved around the cell in small vesicles. A protein coat aids the budding of such vesicles from donor membranes. The major type of coat used by the cell is composed of clathrin, a three-legged protein that can form lattice-like coats on membranes destined for trafficking. In this review, I outline what we know about clathrin and discuss some recent advances in understanding the basic biology of this fascinating molecule, which include building a molecular model of a clathrin lattice and discovery of a new function for clathrin that occurs during mitosis.

Constitutive clathrin-mediated endocytosis of CTLA-4 persists during T cell activation

Background: CTLA-4 is an essential regulator of T cell immune responses with unusual intracellular trafficking. Results: Endocytosis of CTLA-4 is continuous with subsequent recycling and degradation. Conclusion: Clathrin-mediated endocytosis of CTLA-4 persists in activated T cells. Significance: This alters our understanding of CTLA-4 behavior and, therefore, how it might function. CTLA-4 is one of the most important negative regulators of the T cell immune response. However, the subcellular distribution of CTLA-4 is unusual for a receptor that interacts with cell surface transmembrane ligands in that CTLA-4 is rapidly internalized from the plasma membrane. It has been proposed that T cell activation can lead to stabilization of CTLA-4 expression at the cell surface. Here we have analyzed in detail the internalization, recycling, and degradation of CTLA-4. We demonstrate that CTLA-4 is rapidly internalized from the plasma membrane in a clathrin- and dynamin-dependent manner driven by the well characterized YVKM trafficking motif. Furthermore, we show that once internalized, CTLA-4 co-localizes with markers of recycling endosomes and is recycled to the plasma membrane. Although we observed limited co-localization of CTLA-4 with lysosomal markers, CTLA-4 was nonetheless degraded in a manner inhibited by lysosomal blockade. T cell activation stimulated mobilization of CTLA-4, as judged by an increase in cell surface expression; however, this pool of CTLA-4 continued to endocytose and was not stably retained at the cell surface. These data support a model of trafficking whereby CTLA-4 is constitutively internalized in a ligand-independent manner undergoing both recycling and degradation. Stimulation of T cells increases CTLA-4 turnover at the plasma membrane; however, CTLA-4 endocytosis continues and is not stabilized during activation of human T cells. These findings emphasize the importance of clathrin-mediated endocytosis in regulating CTLA-4 trafficking throughout T cell activation.

Coordination of adjacent domains mediates TACC3–ch-TOG–clathrin assembly and mitotic spindle binding

Aurora A phosphorylation-induced interaction of TACC3 and clathrin coordinates adjacent domains in each protein to create a microtubule-binding interface, whereas a distinct site in TACC3 recruits ch-TOG to mitotic spindles.

The role of clathrin in mitotic spindle organisation.

Clathrin, a protein best known for its role in membrane trafficking, has been recognised for many years as localising to the spindle apparatus during mitosis, but its function at the spindle remained unclear. Recent work has better defined the role of clathrin in the function of the mitotic spindle and proposed that clathrin crosslinks the microtubules (MTs) comprising the kinetochore fibres (K-fibres) in the mitotic spindle. This mitotic function is unrelated to the role of clathrin in membrane trafficking and occurs in partnership with two other spindle proteins: transforming acidic coiled-coil protein 3 (TACC3) and colonic hepatic tumour overexpressed gene (ch-TOG; also known as cytoskeleton-associated protein 5, CKAP5). This review summarises the role of clathrin in mitotic spindle organisation with an emphasis on the recent discovery of the TACC3–ch-TOG–clathrin complex.