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

Abstract

A microtubule network on the basal cortex of polarized epithelial cells consists of noncentrosomal 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 p150 Glued -although considered to be microtubule plus-end-binding proteins -are localized along the entire length of microtubules within the network, and at Tjunctions 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.Microtubule interactions with the cell cortex are thought to be important in a wide variety of cell functions, including cyokinesis, cell migration, membrane retraction during cell motility, and vesicle and protein delivery to, and retrieval from, the plasma membrane. However, molecular mechanisms that are involved in specifying microtubule attachment to, and organization at, the cell cortex are poorly understood.Proteins that associate with microtubule plus ends (+Tip proteins) might act as one point of regulation for microtubule-cortex interactions, because the plus ends are localized to the cell periphery in close apposition to the plasma membrane 1 . Several +Tip proteins have been identified, including APC, CLIP-170, EB1 and p150 Glued (reviewed in refs 2, 3). However, many questions remain unanswered regarding the regulation and function of +Tip proteins.Although +Tip proteins have been highlighted as regulators of microtubule dynamics and stability [4][5][6][7][8][9] , their role in microtubule binding and organization at the cell cortex remains poorly understood. Overexpression studies indicate that +Tip proteins are delivered to the cortex in association with microtubules 10,11 , and that they colocalize on the membrane cortex, perhaps in interacting complexes 8,12 . Examination of endogenous EB1 and APC showed that these +Tip proteins generally co-distribute in the same areas of the cell, but that their subcellular localization is not identical 13 .© 2005 Nature Publishing Group 2 Correspondence should be addressed to W.J.N. (wjnelson@stanford.edu).Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. To address questions concerning the organization of microtubules and the functions of +Tip proteins at the cell cortex, we isolated intact basal plasma membranes with the associated cytoskeleton (basal patches) from polarized ...

Cytoskeleton Regulation of Glycine Receptor Number at Synapses and Diffusion in the Plasma Membrane

“…Direct contacts between the cytoskeleton and the plasma membrane were observed at extrasynaptic locations (Gulley and Reese, 1981). Moreover, the major effect of nocodazole on GlyR lateral diffusion in the extrasynaptic compartment supports the idea that the submembrane microtubule network is crucial in the control of the "apparent viscosity" of the neuronal membrane and might be quite dynamic, as seen in polarized epithelial cells (Reilein and Nelson, 2005).…”

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

“…MACF directly binds to EB1, microtubules and F-actin, and is required for tethering EB1-tagged microtubules to the submembranous F-actin network at specialized junctions (Kodama et al, 2003;Subramanian et al, 2003;Slep et al, 2005). Microtubule binding to all three proteins, APC, EB1 and MACF, promotes their stabilization and leads to more frequent and longer lasting attachments (Dikovskaya et al, 2001;Tirnauer et al, 2002;Kodama et al, 2003;Reilein and Nelson, 2005). Further, IQGAP1 plays a key role in regulating actin cytoskeleton dynamics at intercellular contact sites.…”

“…Thus, IQGAP1 further links APC and EB1-tagged microtubules to the submembranous F-actin network. Perturbing the function of APC, EB1, MACF or IQGAP1 (by heterozygous mutation, RNAi knockdown or function-blocking antibodies) reduces the accumulation of microtubules and F-actin at intercellular contact sites (Mogensen et al, 2002;Kodama et al, 2003;Noritake et al, 2004;Reilein and Nelson, 2005;Shaw et al, 2007) . Similarly, the interactions of APC, EB1 and shot/kakapo (Drosophila ortholog of MACF) are essential for organizing and maintaining a unique microtubule-rich and F-actin-rich domain at the Drosophila muscle-tendon junction, and for restricting the localization of specific components to this site (Prokop et al, 1998;Strumpf and Volk, 1998;Subramanian et al, 2003).…”

“…APC is a known negative regulator in the canonical Wnt gene transcription pathway (Polakis, 1997). In addition, APC organizes and stabilizes the polarized microtubule cytoskeleton in epithelial cells by binding to the microtubule plus-end binding protein EB1, a member of the family of microtubule tracking proteins (Akhmanova and Hoogenraad, 2005;Reilein and Nelson, 2005). Recent studies show that APC also carries out microtubule-organizing functions in neurons.…”

NAD + and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

“…These polymers exhibit increased curvature, particularly near the cell periphery. Microtubule curvature is influenced by microtubule-associated proteins, such as adenomatous polyposis coli protein among other +TiP proteins (45). Thus, the effects of NAD + may reflect an alteration in microtubule-bound proteins, especially those that bind the plus-end of microtubules.…”