Characterization of a 60S complex of the adenomatous polyposis coli tumor suppressor protein

The tumor suppressor protein adenomatous polyposis coli (APC) is a multifunctional protein with a well characterized role in the Wnt signal transduction pathway and roles in cytoskeletal regulation and cell polarity. The soluble pool of APC protein in colon epithelial tumor cells exists in two distinct complexes fractionating at approximately 20S and approximately 60S in size. The 20S complex contains components of the beta-catenin destruction complex and probably functions in the Wnt pathway. In this study, we characterized the molecular nature of the 60S APC- containing complex by examining known potential binding partners of APC. 60S APC did not contain EB1 or diaphanous, proteins that have been reported to interact with APC and are implicated in microtubule plus end stabilization. Nor did the two other microtubule associated proteins, MAP4 or KAP3, which is thought to link APC to kinesin motor proteins, associate with the 60S complex. Minor fractions of alpha-tubulin, gamma-tubulin and IQGAP1, a Rac1 and CDC42 effector that interacts with APC, specifically associated with APC in the 60S fraction. We propose that 60S APC is a discrete high molecular weight complex with a novel function in cytoskeletal regulation in epithelial cells apart from its well established role in targeting catenin destruction or its proposed role in microtubule plus end stabilization.     

A role for the Adenomatous Polyposis Coli protein in chromosome segregation

Mutations in the Adenomatous Polyposis Coli (APC) gene are responsible for familial colon cancer and also occur in the early stages of sporadic colon cancer. APC functions in the Wnt signalling pathway to regulate the degradation of beta-catenin (reviewed in refs 1-3). APC also binds to and stabilizes microtubules in vivo and in vitro, localizes to clusters at the ends of microtubules near the plasma membrane of interphase cells, and is an important regulator of cytoskeletal function. Here we show that cells carrying a truncated APC gene (Min) are defective in chromosome segregation. Moreover, during mitosis, APC localizes to the ends of microtubules embedded in kinetochores and forms a complex with the checkpoint proteins Bub1 and Bub3. In vitro, APC is a high-affinity substrate for Bub kinases. Our data are consistent with a role for APC in kinetochore-microtubule attachment and suggest that truncations in APC that eliminate microtubule binding may contribute to chromosomal instability in cancer cells.     

Adenomatous Polyposis Coli as a Scaffold for Microtubule End-Binding Proteins

End-binding proteins (EBs), referred to as the core components of the microtubule plus-end tracking protein network, interact with the C-terminus of the adenomatous polyposis coli (APC) tumor suppressor. This interaction is disrupted in colon cancers expressing truncated APC. APC and EBs act in synergy to regulate microtubule dynamics during spindle formation, chromosome segregation and cell migration. Since EBs autonomously end-track microtubules and partially co-localize with APC at microtubule tips in cells, EBs have been proposed to direct APC to microtubule ends. However, the interdependency of EB and APC localization on microtubules remains elusive. Here, using in vitro reconstitution and single-molecule imaging, we have investigated the interplay between EBs and the C-terminal domain of APC (APC-C) on dynamic microtubules. Our results show that APC-C binds along the microtubule wall but does not accumulate at microtubule tips, even when EB proteins are present. APC-C was also found to enhance EB binding at the extremity of growing microtubules and on the microtubule lattice: APC-C promotes EB end-tracking properties by increasing the time EBs spend at microtubule growing ends, whereas a pool of EBs with a fast turnover accumulates along the microtubule surface. Overall, our results suggest that APC is a promoter of EB interaction with microtubules, providing molecular determinants to reassess the relationship between APC and EBs.     

Membrane localization of adenomatous polyposis coli protein at cellular protrusions: targeting sequences and regulation by beta-catenin

Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo- (binds KAP3A, B56alpha, and ASEF) and beta-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of beta-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of beta-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of beta-catenin reduced APC at membrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with beta-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only beta-catenin stimulated APC anchorage at the membrane. Our findings identify beta-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.     

Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules

The positioning of growth sites in fission yeast cells is mediated by spatially controlled microtubule dynamics brought about by tip1p, a CLIP-170-like protein, which is localized at the microtubule tips and guides them to the cell ends. The kinesin tea2p is also located at microtubule tips and affects microtubule dynamics. Here we show that tea2p interacts with tip1p and that the two proteins move with high velocity along the microtubules toward their growing tips. There, tea2p and tip1p accumulate in larger particles. Particle formation requires the EB1 homolog, mal3p. Our results suggest a model in which kinesins regulate microtubule growth by transporting regulatory factors such as tip1p to the growing microtubule tips.     

Association of the testis-specific TRIM/RBCC protein RNF33/TRIM60 with the cytoplasmic motor proteins KIF3A and KIF3B

The Rnf33/Trim60 gene is temporally transcribed in the preimplantation embryo before being silenced at the blastocyst stage but Rnf33 expression is detected in adult testis of the mouse. The putative RNF33 protein is a tripartite motif (TRIM)/RBCC protein composed of a typical RING zinc finger, a B-box 2, two α-helical coiled-coil segments, and a B30.2 domain. As a first step towards the elucidation of the biologic function of RNF33, we aimed in this study to elucidate proteins that associate with RNF33. RNF33-interacting proteins were first derived by the yeast two-hybrid system followed by co-immunoprecipitation assays. Interacting domains were determined by deletion mapping in genetic and biochemical analyzes. RNF33 was shown to interact with the kinesin-2 family members 3A (KIF3A) and 3B (KIF3B) motor proteins in the heterodimeric form known to transport cargos along the microtubule. Domain mapping showed that the RB and B30.2 domains of RNF33 interacted with the respective carboxyl non-motor domains of KIF3A and KIF3B. Since RNF33 interacted with the carboxyl-terminal tail of the KIF3A-KIF3B heterodimer, the motor head section of KIF3A-KIF3B was free and available for association with designated cargo(s) and movement along the microtubule. Data also suggest that RNF33 most likely interacted with KIF3A-KIF3B independent of the adaptor kinesin-associated protein KAP3. This study is a first demonstration of a TRIM protein, namely RNF33, that interacts with the kinesin molecular motors possibly contributing to kinesin-dependent mobilization of specific cargo(s) along the microtubule in the testis of the mouse.     

The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules

Adenomatous polyposis coli protein (APC) is a well-characterized tumor suppressor protein [1] [2] [3]. We previously showed that APC tagged with green fluorescent protein (GFP) in Xenopus A6 epithelial cells moves along a subset of microtubules and accumulates at their growing plus ends in cell extensions [4]. EB1, which was identified as an APC-binding protein by yeast two-hybrid analysis [5], was also reported to be associated with microtubules [6] [7] [8]. To examine the interaction between APC and EB1 within cells, we compared the dynamic behavior of EB1-GFP with that of APC-GFP in A6 transfectants. Time-lapse microscopy of live cells at interphase revealed that EB1-GFP was concentrated at all of the growing microtubule ends throughout the cytoplasm and abruptly disappeared from the ends when microtubules began to shorten. Therefore, EB1 appeared to be co-localized and interact with APC on the growing ends of a subset of microtubules. When APC-GFP was overexpressed, endogenous EB1 was recruited to APC-GFP, which accumulated in large amounts on microtubules. On the other hand, when microtubules were disassembled by nocodazole, EB1 was not co-localized with APC-GFP, which was concentrated along the basal plasma membrane. During mitosis, APC appeared to be dissociated from microtubules, whereas EB1-GFP continued to concentrate at microtubule growing ends. These findings showed that the APC-EB1 interaction is regulated within cells and is allowed near the ends of microtubules only under restricted conditions.     

APC is a component of an organizing template for cortical microtubule networks

A microtubule network on the basal cortex of polarized epithelial cells consists of non-centrosomal microtubules of mixed polarity. Here, we investigate the proteins that are involved in organizing this network, and we show that end-binding protein 1 (EB1), adenomatous polyposis coli protein (APC) and p150Glued - although considered to be microtubule plus-end-binding proteins - are localized along the entire length of microtubules within the network, and at T-junctions between microtubules. The network shows microtubule behaviours that arise from physical interactions between microtubules, including microtubule plus-end stabilization on the sides of other microtubules, and sliding of microtubule ends along other microtubules. APC also localizes to the basal cortex. Microtubules grew over and paused at APC puncta; an in vitro reconstituted microtubule network overlaid APC puncta; and microtubule network reconstitution was inhibited by function-blocking APC antibodies. Thus, APC is a component of a cortical template that guides microtubule network formation.     

Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.     

Identification of a phragmoplast-associated kinesin-related protein in higher plants

The phragmoplast executes cytokinesis in higher plants. The major components of the phragmoplast are microtubules, which are arranged in two mirror-image arrays perpendicular to the division plane [1]. The plus ends of these microtubules are located near the site of the future cell plate. Golgi-derived vesicles are transported along microtubules towards the plus ends to deliver materials bound for the cell plate [2] [3]. During cell division, rapid microtubule reorganization in the phragmoplast requires the orchestrated activities of microtubule motor proteins such as kinesins. We isolated an Arabidopsis cDNA clone of a gene encoding an amino-terminal motor kinesin, AtPAKRP1, and have determined the partial sequence of its rice homolog. Immunofluorescence experiments with two sets of specific antibodies revealed consistent localization of AtPAKRP1 and its homolog in Arabidopsis and rice cells undergoing anaphase, telophase and cytokinesis. AtPAKRP1 started to accumulate along microtubules towards the spindle midzone during late anaphase. Once the phragmoplast microtubule array was established, AtPAKRP1 conspicuously localized to microtubules near the future cell plate. Our results provide evidence that AtPAKRP1 is a hitherto unknown motor that may take part in the establishment and/or maintenance of the phragmoplast microtubule array.     

Mutated APC and Asef are involved in the migration of colorectal tumour cells

The tumour suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC binds to beta-catenin, a key component of the Wnt signalling pathway, and induces its degradation. APC interacts with microtubules and accumulates at their plus ends in membrane protrusions, and associates with the plasma membrane in an actin-dependent manner. In addition, APC interacts with the Rac-specific guanine nucleotide exchange factor Asef and stimulates its activity, thereby regulating the actin cytoskeletal network and cell morphology. Here we show that overexpression of Asef decreases E-cadherin-mediated cell-cell adhesion and promotes the migration of epithelial Madin-Darby canine kidney cells. Both of these activities are stimulated by truncated APC proteins expressed in colorectal tumour cells. Experiments based on RNA interference and dominant-negative mutants show that both Asef and mutated APC are required for the migration of colorectal tumour cells expressing truncated APC. These results suggest that the APC-Asef complex functions in cell migration as well as in E-cadherin-mediated cell-cell adhesion, and that truncated APC present in colorectal tumour cells contributes to their aberrant migratory properties.     

A balance of KIF1A-like kinesin and dynein organizes early endosomes in the fungus Ustilago maydis

In Ustilago maydis, bidirectional transport of early endosomes is microtubule dependent and supports growth and cell separation. During early budding, endosomes accumulate at putative microtubule organizers within the bud, whereas in medium-budded cells, endosome clusters appear at the growing ends of microtubules at the distal cell pole. This suggests that motors of opposing transport direction organize endosomes in budding cells. Here we set out to identify these motors and elucidate the molecular mechanism of endosome reorganization. By PCR we isolated kin3, which encodes an UNC-104/KIF1-like kinesin from U.maydis. Recombinant Kin3 binds microtubules and has ATPase activity. Kin3-green fluorescent protein moves along microtubules in vivo, accumulates at sites of growth and localizes to endosomes. Deletion of kin3 reduces endosome motility to approximately 33%, and abolishes endosome clustering at the distal cell pole and at septa. This results in a transition from bipolar to monopolar budding and cell separation defects. Double mutant analysis indicates that the remaining motility in Deltakin3-mutants depends on dynein, and that dynein and Kin3 counteract on the endosomes to arrange them at opposing cell poles.     

Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends.

Mutants of the yeast Kar3 protein are defective in nuclear fusion, or karyogamy, during mating and show slow mitotic growth, indicating a requirement for the protein both during mating and in mitosis. DNA sequence analysis predicts that Kar3 is a microtubule motor protein related to kinesin, but with the motor domain at the C-terminus of the protein rather than the N-terminus as in kinesin heavy chain. We have expressed Kar3 as a fusion protein with glutathione S-transferase (GST) and determined the in vitro motility properties of the bacterially expressed protein. The GST-Kar3 fusion protein bound to a coverslip translocates microtubules in gliding assays with a velocity of 1-2 microns/min and moves towards microtubule minus ends, unlike kinesin but like kinesin-related Drosophila ncd. Taxol-stabilized microtubules bound to GST-Kar3 on a coverslip shorten as they glide, resulting in faster lagging end, than leading end, velocities. Comparison of lagging and leading end velocities with velocities of asymmetrical axoneme-microtubule complexes indicates that microtubules shorten preferentially from the lagging or minus ends. The minus end-directed translocation and microtubule bundling of GST-Kar3 is consistent with models in which the Kar3 protein crosslinks internuclear microtubules and mediates nuclear fusion by moving towards microtubule minus ends, pulling the two nuclei together. In mitotic cells, the minus end motility of Kar3 could move chromosomes polewards, either by attaching to kinetochores and moving them polewards along microtubules, or by attaching to kinetochore microtubules and pulling them polewards along other polar microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the kinesin-2 family protein, KIF3, during mitosis

During mitosis, kinesin and dynein motor proteins play critical roles in the equal segregation of chromosomes between two daughter cells. Kinesin-2 is composed of two microtubule-based motor subunits, KIF3A/3B, and a kinesin-associated protein known as KAP3, which links KIF3A/3B to cargo that is carried to cellular organelles along microtubules in interphase cells. We have shown here that the kinesin-2 complex is localized with components of the mitotic apparatus such as spindle microtubules and centrosomes. Furthermore, we found that expression of a mutant KIF3B, which is able to associate with KIF3A but not KAP3 in NIH3T3 cells, caused chromosomal aneuploidy and abnormal spindle formation. Our data suggested that the kinesin-2 complex plays an important role not only in interphase but also in mitosis.     

The minus end-directed motor Kar3 is required for coupling dynamic microtubule plus ends to the cortical shmoo tip in budding yeast

The budding yeast shmoo tip is a model system for analyzing mechanisms coupling force production to microtubule plus-end polymerization/depolymerization. Dynamic plus ends of astral microtubules interact with the shmoo tip in mating yeast cells, positioning nuclei for karyogamy. We have used live-cell imaging of GFP fusions to identify proteins that couple dynamic microtubule plus ends to the shmoo tip. We find that Kar3p, a minus end-directed kinesin motor protein, is required, whereas the other cytoplasmic motors, dynein and the kinesins Kip2p and Kip3p, are not. In the absence of Kar3p, attached microtubule plus ends released from the shmoo tip when they switched to depolymerization. Furthermore, microtubules in cells expressing kar3-1, a mutant that results in rigor binding to microtubules [2], were stabilized specifically at shmoo tips. Imaging of Kar3p-GFP during mating revealed that fluorescence at the shmoo tip increased during periods of microtubule depolymerization. These data are the first to localize the activity of a minus end-directed kinesin at the plus ends of microtubules. We propose a model in which Kar3p couples depolymerizing microtubule plus ends to the cell cortex and the Bim1p-Kar9p protein complex maintains attachment during microtubule polymerization. In support of this model, analysis of Bim1p-GFP at the shmoo tip results in a localization pattern complementary to that of Kar3p-GFP.     

Yeast Bim1p Promotes the G1-specific Dynamics of Microtubules

Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro. Proteins that promote dynamic instability are therefore central to microtubule behavior in living cells. Here, we report that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cytoplasmic microtubule dynamics specifically during G1. During G1, microtubules in cells lacking BIM1 showed reduced dynamicity due to a slower shrinkage rate, fewer rescues and catastrophes, and more time spent in an attenuated/paused state. Human EB1 was identified as an interacting partner for the adenomatous polyposis coli (APC) tumor suppressor protein. Like human EB1, Bim1p localizes to dots at the distal ends of cytoplasmic microtubules. This localization, together with data from electron microscopy and a synthetic interaction with the gene encoding the kinesin Kar3p, suggests that Bim1p acts at the microtubule plus end. Our in vivo data provide evidence of a cell cycle–specific microtubule-binding protein that promotes microtubule dynamicity.     

Single Molecule Imaging Reveals Differences in Microtubule Track Selection Between Kinesin Motors

Cells generate diverse microtubule populations by polymerization of a common α/β-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.     

Colocalization of APC and DLG at the tips of cellular protrusions in cultured epithelial cells and its dependency on cytoskeletons

Adenomatous polyposis coli gene product (APC) is a tumor suppressor linked to familial adenomatous polyposis and is thought to be involved in cellular polarization and migration in moving epithelial cells. APC interacts with the mammalian homolog of Discs large (DLG). DLG is a member of the membrane-associated guanylate kinase superfamily and is thought to function as a scaffolding protein that coordinates the assembly of a lateral plasma membrane-localized protein complex in epithelial cells. We confirmed the suitability of several anti-APC antibodies for immunocytochemical analysis. Using these antibodies, we showed that APC clusters were colocalized with DLG protein at cellular protrusions of subconfluent MDCK cells. A portion of the clusters was found at the tips of microtubules extending into the cellular protrusions. In addition, actin stress fibers converged near the clusters. When microtubules were disrupted by nocodazole, the colocalization of APC and DLG was lost due to the disappearance of APC clusters. However, the coclusters remained after depolymerization of actin filaments with latrunculin A. This is the first report showing colocalization of APC and DLG in non-polarized epithelial cells. This colocalization suggests that DLG functions not only at the lateral cell–cell contact sites of polarized epithelial cells but also at the protrusions of non-polarized epithelial cells through the interaction with APC protein.