Fiona E. Hood

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.

Phosphorylation Regulates the Dynamic Interaction of RCC1 with Chromosomes during Mitosis

The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.

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.

RCC1 isoforms differ in their affinity for chromatin, molecular interactions and regulation by phosphorylation

RCC1 is the guanine nucleotide exchange factor for Ran GTPase. Generation of Ran-GTP by RCC1 on chromatin provides a spatial signal that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. We show that RCC1 is expressed in human cells as at least three isoforms, named RCC1α, RCC1β and RCC1γ, which are expressed at different levels in specific tissues. The β and γ isoforms contain short inserts in their N-terminal regions (NTRs) that are not present in RCC1α. This region mediates interaction with chromatin, binds importin α3 and/or importin β, and contains regulatory phosphorylation sites. RCC1γ is predominantly localised to the nucleus and mitotic chromosomes like RCC1α. However, compared to RCC1α, RCC1γ has a greatly reduced interaction with an importin α3-β and a stronger interaction with chromatin that is mediated by the extended NTR. RCC1γ is also the isoform that is most highly phosphorylated at serine 11 in mitosis. Unlike RCC1α, RCC1γ supports cell proliferation in tsBN2 cells more efficiently when serine 11 is mutated to non-phosphorylatable alanine. Phosphorylation of RCC1γ therefore specifically controls its function during mitosis. These results show that human RCC1 isoforms have distinct chromatin binding properties, different molecular interactions, and are selectively regulated by phosphorylation, as determined by their different NTRs.

Aurora A kinase activity is required for localization of TACC3/ch-TOG/clathrin inter-microtubule bridges

Accurate chromosome segregation during mitosis is achieved by the kinetochore fibers (K-fibers) of the spindle apparatus. These fibers are bundles of microtubules (MTs) connected by non-motor bridges. We recently identified a TACC3/ch-TOG/clathrin complex that constitutes the shortest class of inter-MT bridge in K-fibers. TACC3 anchors the complex to MTs and this is dependent on phosphorylation by Aurora A kinase. Here we show that inhibition of Aurora A kinase using MLN8237 results in (1) loss of clathrin and TACC3 from spindles, (2) destabilization of K-fibers and (3) loss of inter-MT bridges. These results are similar to those in cells depleted of clathrin or TACC3; suggesting that TACC3/ch-TOG/clathrin bridges are the major class of bridge that is regulated by this kinase.

Pulling it together: The mitotic function of TACC3

Transforming acidic coiled coil 3 (TACC3) is a non-motor microtubule-associated protein (MAP) that is important for mitotic spindle stability and organisation. The exact mechanism by which TACC3 acts at microtubules to stabilise the spindle has been unclear. However, several recent studies identified that the TACC3 complex at microtubules contains clathrin in addition to its previously identified binding partner, colonic and hepatic tumour over-expressed gene (ch-TOG). In this complex, phosphorylated TACC3 interacts directly with both ch-TOG and clathrin heavy chain, promoting accumulation of all complex members at the mitotic spindle. This complex stabilises kinetochore fibres within the spindle by forming cross-bridges that link adjacent microtubules in these bundles. So, TACC3 is an adaptor that recruits ch-TOG and clathrin to mitotic microtubules, in an Aurora A kinase-regulated manner. In this mini-review we will describe the recent advances in the understanding of TACC3 function and present a model that pulls together these new data with previous observations.

Functional equivalence of the clathrin heavy chains CHC17 and CHC22 in endocytosis and mitosis.

Clathrin is crucial for endocytosis and plays a recently described role in mitosis. Two clathrin heavy chains (CHCs) are found in humans: the ubiquitous CHC17, and CHC22, a CHC that is enriched in skeletal muscle. Functional differences have been proposed for these clathrins despite high sequence similarity. Here, we compared each paralogue in functional assays of endocytosis and mitosis. We find that CHC17 and CHC22 are functionally equivalent. We also describe how previous work on CHC22 has involved a splice variant that is not usually expressed in cells.

Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression

Hsp72 is a novel mitotic substrate of Nek6, and, together, these proteins play an essential role in assembly of robust mitotic spindles capable of efficient chromosome congression through K-fiber recruitment of the ch-TOG and TACC3 complex.

The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells

BackgroundRegulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA.ResultsWe have investigated the mechanism of the dynamic interaction of the α isoform of human RCC1 (RCC1α) with chromatin in live cells using fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) fusions. We show that the N-terminal tail stabilises the interaction of RCC1α with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1α with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1α. The interaction of RCC1α with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes α-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, α-N-terminal methylation of RCC1α is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1α does not require the N-terminal tail of RCC1α or its methylation. The mobility of RCC1α on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1αD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N.ConclusionsThese results show that the stabilisation of the dynamic interaction of RCC1α with chromatin by Ran in live cells requires the N-terminal tail of RCC1α. α-N-methylation is not regulated by formation of the binary complex with Ran, but it promotes chromatin binding through the tail. This work supports a model in which the association of RCC1α with chromatin is promoted by a conformational change in the α-N-terminal methylated tail that is induced allosterically in the binary complex with Ran.

The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle

Kinetochore fibers (K-fibers) of the mitotic spindle are force-generating units that power chromosome movement during mitosis. K-fibers are composed of many microtubules that are held together throughout their length. Here, we show, using 3D electron microscopy, that K-fiber microtubules (MTs) are connected by a network of MT connectors. We term this network ‘the mesh’. The K-fiber mesh is made of linked multipolar connectors. Each connector has up to four struts, so that a single connector can link up to four MTs. Molecular manipulation of the mesh by overexpression of TACC3 causes disorganization of the K-fiber MTs. Optimal stabilization of K-fibers by the mesh is required for normal progression through mitosis. We propose that the mesh stabilizes K-fibers by pulling MTs together and thereby maintaining the integrity of the fiber. Our work thus identifies the K-fiber meshwork of linked multipolar connectors as a key integrator and determinant of K-fiber structure and function. DOI: http://dx.doi.org/10.7554/eLife.07635.001