The microtubule plus-end tracking protein Bik1 is required for chromosome congression

During mitosis, sister chromatids congress on both sides of the spindle equator to facilitate the correct partitioning of the genomic material. Chromosome congression requires a finely tuned control of microtubule dynamics by the kinesin motor proteins. In Saccharomyces cerevisiae, the kinesin proteins Cin8, Kip1, and Kip3 have a pivotal role in chromosome congression. It has been hypothesized that additional proteins that modulate microtubule dynamics are involved. Here, we show that the microtubule plus-end tracking protein Bik1—the budding yeast ortholog of CLIP-170—is essential for chromosome congression. We find that nuclear Bik1 localizes to the kinetochores in a cell cycle–dependent manner. Disrupting the nuclear pool of Bik1 with a nuclear export signal (Bik1-NES) leads to slower cell-cycle progression characterized by a delayed metaphase–anaphase transition. Bik1-NES cells have mispositioned kinetochores along the spindle in metaphase. Furthermore, using proximity-dependent methods, we identify Cin8 as an interaction partner of Bik1. Deleting CIN8 reduces the amount of Bik1 at the spindle. In contrast, Cin8 retains its typical bilobed distribution in the Bik1-NES mutant and does not localize to the unclustered kinetochores. We propose that Bik1 functions with Cin8 to regulate kinetochore–microtubule dynamics for correct kinetochore positioning and chromosome congression.     

Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking

Visualization of specific molecules and their interactions in real time and space is essential to delineate how cellular dynamics and the signaling circuit are orchestrated. Spatial regulation of conformational dynamics and structural plasticity of protein interactions is required to rewire signaling circuitry in response to extracellular cues. We introduce a method for optically imaging intracellular protein interactions at nanometer spatial resolution in live cells, using photoactivatable complementary fluorescent (PACF) proteins. Subsets of complementary fluorescent protein molecules were activated, localized, and then bleached; this was followed by the assembly of superresolution images from aggregate position of sum interactive molecules. Using PACF, we obtained precise localization of dynamic microtubule plus-end hub protein EB1 dimers and their distinct distributions at the leading edges and in the cell bodies of migrating cells. We further delineated the structure–function relationship of EB1 by generating EB1-PACF dimers (EB1^wt:EB1^wt, EB1^wt:EB1^mt, and EB1^mt:EB1^mt) and imaging their precise localizations in culture cells. Surprisingly, our analyses revealed critical role of a previously uncharacterized EB1 linker region in tracking microtubule plus ends in live cells. Thus PACF provides a unique approach to delineating spatial dynamics of homo- or heterodimerized proteins at the nanometer scale and establishes a platform to report the precise regulation of protein interactions in space and time in live cells.     

The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150(Glued) subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150(Glued) in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150(Glued) on microtubule polymerization, and they show that p150(Glued) has a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.     

The microtubule plus-end binding protein EB1 functions in root responses to touch and gravity signals in Arabidopsis

Microtubules function in concert with associated proteins that modify microtubule behavior and/or transmit signals that effect changes in growth. To better understand how microtubules and their associated proteins influence growth, we analyzed one family of microtubule-associated proteins, the END BINDING1 (EB1) proteins, in Arabidopsis thaliana (EB1a, EB1b, and EB1c). We find that antibodies directed against EB1 proteins colocalize with microtubules in roots, an observation that confirms previous reports using EB1-GFP fusions. We also find that T-DNA insertion mutants with reduced expression from EB1 genes have roots that deviate toward the left on vertical or inclined plates. Mutant roots also exhibit extended horizontal growth before they bend downward after tracking around an obstacle or after a 90 degrees clockwise reorientation of the root. These observations suggest that leftward deviations in root growth may be the result of delayed responses to touch and/or gravity signals. Root lengths and widths are normal, indicating that the delay in bend formation is not due to changes in the overall rate of growth. In addition, the genotype with the most severe defects responds to low doses of microtubule inhibitors in a manner indistinguishable from the wild type, indicating that microtubule integrity is not a major contributor to the leftward deviations in mutant root growth.     

Dynamics of the Kv1.2 voltage-gated K+ channel in a membrane environment

All-atom molecular dynamics simulations are used to better understand the dynamic environment experienced by the Kv1.2 channel in a lipid membrane. The structure of the channel is stable during the trajectories. The pore domain keeps a well-defined conformation, whereas the voltage-sensing domains undergo important lateral fluctuations, consistent with their modular nature. A channel-like region at the center of the S1-S4 helical bundle fills rapidly with water, reminiscent of the concept of high-dielectric aqueous crevices. The first two arginines along S4 (R294 and R297) adopt an interfacial position where they interact favorably with water and the lipid headgroups. The following two arginines (R300 and R303) interact predominantly with water and E226 in S2. Despite the absence of a structurally permanent gating pore formed by protein residues and surrounding the S4 helix, as traditionally pictured, the charged residues are located in a favorable environment and are not extensively exposed to the membrane nonpolar region. Continuum electrostatic computations indicate that the transmembrane potential sensed by the charged residues in the voltage sensor varies abruptly over the outer half of the membrane in the arginine-rich region of S4; thus, the voltage gradient or membrane electric field is "focused". Interactions of basic residues with the lipid headgroups at the intracellular membrane-solution interface reduce the membrane thickness near the channel, resulting in an increased transmembrane field.     

Targeting of the F-actin-binding protein drebrin by the microtubule plus-tip protein EB3 is required for neuritogenesis

Interactions between dynamic microtubules and actin filaments (F-actin) underlie a range of cellular processes including cell polarity and motility. In growth cones, dynamic microtubules are continually extending into selected filopodia, aligning alongside the proximal ends of the F-actin bundles. This interaction is essential for neuritogenesis and growth-cone pathfinding. However, the molecular components mediating the interaction between microtubules and filopodial F-actin have yet to be determined. Here we show that drebrin, an F-actin-associated protein, binds directly to the microtubule-binding protein EB3. In growth cones, this interaction occurs specifically when drebrin is located on F-actin in the proximal region of filopodia and when EB3 is located at the tips of microtubules invading filopodia. When this interaction is disrupted, the formation of growth cones and the extension of neurites are impaired. We conclude that drebrin targets EB3 to coordinate F-actin-microtubule interactions that underlie neuritogenesis.     

An inwardly rectifying K+ channel is required for patterning

Mutations that disrupt function of the human inwardly rectifying potassium channel KIR2.1 are associated with the craniofacial and digital defects of Andersen-Tawil Syndrome, but the contribution of Kir channels to development is undefined. Deletion of mouse Kir2.1 also causes cleft palate and digital defects. These defects are strikingly similar to phenotypes that result from disrupted TGFβ/BMP signaling. We use Drosophila melanogaster to show that a Kir2.1 homolog, Irk2, affects development by disrupting BMP signaling. Phenotypes of irk2 deficient lines, a mutant irk2 allele, irk2 siRNA and expression of a dominant-negative Irk2 subunit (Irk2DN) all demonstrate that Irk2 function is necessary for development of the adult wing. Compromised Irk2 function causes wing-patterning defects similar to those found when signaling through a Drosophila BMP homolog, Decapentaplegic (Dpp), is disrupted. To determine whether Irk2 plays a role in the Dpp pathway, we generated flies in which both Irk2 and Dpp functions are reduced. Irk2DN phenotypes are enhanced by decreased Dpp signaling. In wild-type flies, Dpp signaling can be detected in stripes along the anterior/posterior boundary of the larval imaginal wing disc. Reducing function of Irk2 with siRNA, an irk2 deletion, or expression of Irk2DN reduces the Dpp signal in the wing disc. As Irk channels contribute to Dpp signaling in flies, a similar role for Kir2.1 in BMP signaling may explain the morphological defects of Andersen-Tawil Syndrome and the Kir2.1 knockout mouse.     

Molecular basis for Kv1.5 channel block: conservation of drug binding sites among voltage-gated K+ channels

Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) that contributes to action potential repolarization of human atrial myocytes. Block of these channels has been proposed as a treatment for atrial arrhythmias. Here we report a novel and potent inhibitor of Kv1.5 potassium channels, N-benzyl-N-pyridin-3-yl-methyl-2-(toluene-4-sulfonylamino)-benzamide hydrochloride (S0100176), which exhibits features consistent with preferential block of the open state. The IC50 of S0100176 for Kv1.5 expressed in Xenopus oocytes was 0.7 microm. Ala-scanning mutagenesis within the pore helix and the S6 segment, regions that form the walls of the central cavity, was combined with voltage clamp analysis to identify point mutations that altered drug affinity. This approach identified Thr-479, Thr-480, Val-505, Ile-508, and Val-512 as the most important residues for block by S0100176. Mutations of these key residues to Ala or other amino acids caused marked changes in the IC50 of S0100176 (p<0.01). For example, the IC50 of S0100176 increased 362-fold for T480A, 26-fold for V505A, 150-fold for I508A, and 99-fold for V512A. We used modeling to dock S0100176 into the inner cavity of a Kv1.5 pore homology model that was generated based on the crystal structure of KcsA. The docking predicted that the five residues identified by the Ala scan were positioned less than 4.5 A from the compound. Based on the homology models, the positions of the five amino acids identified to interact with S0100176 face toward the central cavity and overlap with putative binding sites for other blockers and voltage-gated potassium channels.     

The voltage-gated potassium channel subunit, Kv1.3, is expressed in epithelia

The Shaker-type voltage-gated potassium channel, Kv1.3, is believed to be restricted in distribution to lymphocytes and neurons. In lymphocytes, this channel has gained intense attention since it has been proven that inhibition of Kv1.3 channels compromise T lymphocyte activation. To investigate possible expression of Kv1.3 channels in other types of tissue, such as epithelia, binding experiments, immunoprecipitation studies and immunohistochemical studies were performed. The double-mutated, radiolabeled peptidyl ligand, ¹²⁵I-HgTX₁-A19Y/Y37F, which selectively binds Kv1.1, Kv1.2, Kv1.3 and Kv1.6 channels, was used to perform binding studies in epithelia isolated from rabbit kidney and colon. The equilibrium dissociation constant for this ligand was found to be in the sub-picomolar range and the maximal receptor concentration (in fmol/mg protein) 1.68 for colon and 0.61-0.75 for kidney epithelium. To determine the subtype of Kv1 channels, immunoprecipitation studies with ¹²⁵I-HgTX₁-A19Y/Y37F labeled epithelial membranes were performed with specific antibodies against Kv1.1, Kv1.2, Kv1.3, Kv1.4 or Kv1.6 subunits. These studies demonstrated that Kv1.3 subunits constituted more than 50% of the entire Kv1 subunit population. The precise localization of Kv1.3 subunits in epithelia was determined by immunohistochemical studies.     

Folding of the voltage-gated K+ channel T1 recognition domain

Voltage-gated K(+) channels (Kv) are tetramers whose assembly is coordinated in part by a conserved T1 recognition domain. Although T1 achieves its quaternary structure in the ER, nothing is known about its acquisition of tertiary structure. We developed a new folding assay that relies on intramolecular cross-linking of pairs of cysteines engineered at the folded T1 monomer interface. Using this assay, we show directly that the T1 domain is largely folded while the Kv protein is still attached to membrane-bound ribosomes. The ER membrane facilitates both folding and oligomerization of Kv proteins. We show that folding and oligomerization assays can be used to study coupling between these two biogenic events and diagnose defects in assembly of Kv channels.     

The voltage-gated potassium channel subunit, Kv1.3, is expressed in epithelia

The Shaker-type voltage-gated potassium channel, Kv1.3, is believed to be restricted in distribution to lymphocytes and neurons. In lymphocytes, this channel has gained intense attention since it has been proven that inhibition of Kv1.3 channels compromise T lymphocyte activation. To investigate possible expression of Kv1.3 channels in other types of tissue, such as epithelia, binding experiments, immunoprecipitation studies and immunohistochemical studies were performed. The double-mutated, radiolabeled peptidyl ligand, (125)I-HgTX(1)-A19Y/Y37F, which selectively binds Kv1.1, Kv1.2, Kv1.3 and Kv1.6 channels, was used to perform binding studies in epithelia isolated from rabbit kidney and colon. The equilibrium dissociation constant for this ligand was found to be in the sub-picomolar range and the maximal receptor concentration (in fM/mg protein) 1.68 for colon and 0.61-0.75 for kidney epithelium. To determine the subtype of Kv1 channels, immunoprecipitation studies with (125)I-HgTX(1)-A19Y/Y37F labeled epithelial membranes were performed with specific antibodies against Kv1.1, Kv1.2, Kv1.3, Kv1.4 or Kv1.6 subunits. These studies demonstrated that Kv1.3 subunits constituted more than 50% of the entire Kv1 subunit population. The precise localization of Kv1.3 subunits in epithelia was determined by immunohistochemical studies.     

A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons

The discrete localization of ion channels is a critical determinant of neuronal excitability. We show here that the dendritic K+ channels Kv2.1 and Kv2.2 were differentially targeted in cultured hippocampal neurons. Kv2.1 was found in high-density clusters on the soma and proximal dendrites, while Kv2.2 was uniformly distributed throughout the soma and dendrites. Chimeras revealed a proximal restriction and clustering domain on the cytoplasmic tail of Kv2.1. Truncations and internal deletions revealed a 26-amino acid targeting signal within which four residues were critical for localization. This signal is not related to other known sequences for neuronal and epithelial membrane protein targeting and represents a novel cytoplasmic signal responsible for proximal restriction and clustering.     

Cortactin is required for N-cadherin regulation of Kv1.5 channel function

The intercalated disc serves as an organizing center for various cell surface components at the termini of the cardiomyocyte, thus ensuring proper mechanoelectrical coupling throughout the myocardium. The cell adhesion molecule, N-cadherin, is an essential component of the intercalated disc. Cardiac-specific deletion of N-cadherin leads to abnormal electrical conduction and sudden arrhythmic death in mice. The mechanisms linking the loss of N-cadherin in the heart and spontaneous malignant ventricular arrhythmias are poorly understood. To investigate whether ion channel remodeling contributes to arrhythmogenesis in N-cadherin conditional knock-out (N-cad CKO) mice, cardiac myocyte excitability and voltage-gated potassium channel (Kv), as well as inwardly rectifying K(+) channel remodeling, were investigated in N-cad CKO cardiomyocytes by whole cell patch clamp recordings. Action potential duration was prolonged in N-cad CKO ventricle myocytes compared with wild type. Relative to wild type, I(K,slow) density was significantly reduced consistent with decreased expression of Kv1.5 and Kv accessory protein, Kcne2, in the N-cad CKO myocytes. The decreased Kv1.5/Kcne2 expression correlated with disruption of the actin cytoskeleton and reduced cortactin at the sarcolemma. Biochemical experiments revealed that cortactin co-immunoprecipitates with Kv1.5. Finally, cortactin was required for N-cadherin-mediated enhancement of Kv1.5 channel activity in a heterologous expression system. Our results demonstrate a novel mechanistic link among the cell adhesion molecule, N-cadherin, the actin-binding scaffold protein, cortactin, and Kv channel remodeling in the heart. These data suggest that in addition to gap junction remodeling, aberrant Kv1.5 channel function contributes to the arrhythmogenic phenotype in N-cad CKO mice.     

Mutant analysis of the Shal (Kv4) voltage-gated fast transient K+ channel in Caenorhabditis elegans

Shal (Kv4) alpha-subunits are the most conserved among the family of voltage-gated potassium channels. Previous work has shown that the Shal potassium channel subfamily underlies the predominant fast transient outward current in Drosophila neurons (Tsunoda, S., and Salkoff, L. (1995) J. Neurosci. 15, 1741-1754) and the fast transient outward current in mouse heart muscle (Guo, W., Jung, W. E., Marionneau, C., Aimond, F., Xu, H., Yamada, K. A., Schwarz, T. L., Demolombe, S., and Nerbonne, J. M. (2005) Circ. Res. 97, 1342-1350). We show that Shal channels also play a role as the predominant transient outward current in Caenorhabditis elegans muscle. Green fluorescent protein promoter experiments also revealed SHL-1 expression in a subset of neurons as well as in C. elegans body wall muscle and in male-specific diagonal muscles. The shl-1 (ok1168) null mutant removed all fast transient outward current from muscle cells. SHL-1 currents strongly resembled Shal currents in other species except that they were active in a more depolarized voltage range. We also determined that the remaining delayed-rectifier current in cultured myocytes was carried by the Shaker ortholog SHK-1. In shl-1 (ok1168) mutants there was a significant compensatory increase in the SHK-1 current. Male shl-1 (ok1168) animals exhibited reduced mating efficiency resulting from an apparent difficulty in locating the hermaphrodite vulva. SHL-1 channels are apparently important in fine-tuning complex behaviors, such as mating, that play a crucial role in the survival and propagation of the species.     

The potential role of Kv4.3 K+ channel in heart hypertrophy

Transient outward K⁺ current (I_to) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K⁺ channel is an important component of I_to. The function and expression of Kv4.3 K⁺ channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. In this review, we summarized the changes of cardiac Kv4.3 K⁺ channel in heart diseases and discussed the potential role of Kv4.3 K⁺ channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, downregulation of Kv4.3 K⁺ channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca²⁺]_i, activation of calcineurin and heart hypertrophy/heart failure. However, in canine and human, Kv4.3 K⁺ channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K⁺ channel/APD/[Ca²⁺]_i pathway, there exits another mechanism of Kv4.3 K⁺ channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K⁺ channels leads to CaMKII dissociation from Kv4.3–CaMKII complex and subsequent activation of the dissociated CaMKII, which induces heart hypertrophy/heart failure. Upregulation of Kv4.3 K⁺ channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K⁺ channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K⁺ channel might be potentially harmful or beneficial to hearts through CaMKII.     

The potential role of Kv4.3 K+ channel in heart hypertrophy

Transient outward K⁺ current (I_to) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K⁺ channel is an important component of I_to. The function and expression of Kv4.3 K⁺ channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. In this review, we summarized the changes of cardiac Kv4.3 K⁺ channel in heart diseases and discussed the potential role of Kv4.3 K⁺ channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, downregulation of Kv4.3 K⁺ channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca²⁺]_i, activation of calcineurin and heart hypertrophy/heart failure. However, in canine and human, Kv4.3 K⁺ channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K⁺ channel/APD/[Ca²⁺]_i pathway, there exits another mechanism of Kv4.3 K⁺ channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K⁺ channels leads to CaMKII dissociation from Kv4.3–CaMKII complex and subsequent activation of the dissociated CaMKII, which induces heart hypertrophy/heart failure. Upregulation of Kv4.3 K⁺ channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K⁺ channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K⁺ channel might be potentially harmful or beneficial to hearts through CaMKII.     

Interaction between EB1 and p150glued is required for anaphase astral microtubule elongation and stimulation of cytokinesis

In animal cells, microtubules (MTs) of the mitotic apparatus (MA) communicate with the cell cortex to stimulate cytokinesis; however, the molecular nature of this stimulus remains elusive . A signal for cytokinesis likely involves the MT plus end binding family of proteins, which includes EB1, p150glued, APC, LIS1, and CLIP-170. These proteins modulate MT dynamics and facilitate interactions between growing MTs and their intracellular targets, including kinetochores, organelles, and the cell cortex . The dynein-dynactin complex mediates many of these microtubule capture events . We report that EB1 and p150glued interactions are required for stimulation of cytokinesis in dividing sea urchin eggs. Injected antibodies against EB1 or p150glued suppressed furrow ingression but did not prevent elongation of anaphase astral MTs toward the cortex, suggesting that EB1 and dynactin are both required for communication between the MA and the cortex. Targeted disruption of the interaction between EB1 and p150glued suppressed anaphase astral MT elongation and resulted in a delay of cytokinesis that could not be overcome by manipulation of the asters toward the cortex. We conclude that EB1 and dynactin participate in stimulation of the cleavage furrow, and their interaction promotes elongation of astral MTs at anaphase onset.     

The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules

Sertoli cells of testis belong to a unique type of polarized epithelial cells and are essential for spermatogenesis. They form the blood-testis barrier at the base of seminiferous tubule. Their numerous long, microtubule-rich processes extend inward and associate with developing germ cells to sustain germ cell growth and differentiation. How Sertoli cells develop and maintain their elaborate processes has been an intriguing question. Here we showed that, by microinjecting lentiviral preparations into mouse testes of 29 days postpartum, we were able to specifically label individual Sertoli cells with GFP, thus achieving a clear view of their natural configurations together with associated germ cells in situ. Moreover, compared to other microtubule plus end-tracking proteins such as CLIP-170 and p150(Glued), EB1 was highly expressed in Sertoli cells and located along microtubule bundles in Sertoli cell processes. Stable overexpression of a GFP-tagged dominant-negative EB1 mutant disrupted microtubule organizations in cultured Sertoli cells. Furthermore, its overexpression in testis Sertoli cells altered their shapes. Sertoli cells in situ became rod-like, with decreased basal and lateral cell processes. Seminiferous tubule circularity and germ cell number were also reduced. These data indicate a requirement of proper microtubule arrays for Sertoli cell plasticity and function in testis.     

EB1 is required for spindle symmetry in mammalian mitosis

Most information about the roles of the adenomatous polyposis coli protein (APC) and its binding partner EB1 in mitotic cells has come from siRNA studies. These suggest functions in chromosomal segregation and spindle positioning whose loss might contribute to tumourigenesis in cancers initiated by APC mutation. However, siRNA-based approaches have drawbacks associated with the time taken to achieve significant expression knockdown and the pleiotropic effects of EB1 and APC gene knockdown. Here we describe the effects of microinjecting APC- or EB1- specific monoclonal antibodies and a dominant-negative EB1 protein fragment into mammalian mitotic cells. The phenotypes observed were consistent with the roles proposed for EB1 and APC in chromosomal segregation in previous work. However, EB1 antibody injection also revealed two novel mitotic phenotypes, anaphase-specific cortical blebbing and asymmetric spindle pole movement. The daughters of microinjected cells displayed inequalities in microtubule content, with the greatest differences seen in the products of mitoses that showed the severest asymmetry in spindle pole movement. Daughters that inherited the least mobile pole contained the fewest microtubules, consistent with a role for EB1 in processes that promote equality of astral microtubule function at both poles in a spindle. We propose that these novel phenotypes represent APC-independent roles for EB1 in spindle pole function and the regulation of cortical contractility in the later stages of mitosis. Our work confirms that EB1 and APC have important mitotic roles, the loss of which could contribute to CIN in colorectal tumour cells.