Abstract. The role of the epithelial adhesion molecule uvomorulin in the formation of the epithelial junctional complex in the Madin-Darby canine kidney (MDCK) cell line was investigated. Experiments were carried out to determine whether specific inhibition of uvomorulin function would interfere selectively with the formation, stability, or function of the apical zonula adherens (ZA) and zonula occludens (ZO), or whether it would interfere with all forms of intercellular contact including the desmosomes. The effects of blocking antibodies and Fab fragments to uvomorulin on the formation of the junctional complex was examined with a Ca 2+ switch assay for de novo junction assembly. The formation of the ZO, the ZA, and the desmosomes was assayed by fluorescence staining with an antibody to the tight junction-specific protein ZO-1, with rhodamine-phalloidin for ZA-associated actin filaments, and with an anti-desmoplakin antibody, respectively. Under different conditions and times of antibody treatment the extent of inhibition of the formation of each of the junctional elements was very similar. The ability of the cells to eventually overcome the inhibitory effect of the antibodies and form junctions correlated with the reappearance of uvomorulin at the regions of cell-cell contact. Therefore uvomorulin seems to mediate an early adhesion event between epithelial cells that is a prerequisite for the assembly of all elements of the junctional complex. In contrast, the transepithelial electrical resistance of confluent, well-established monolayers of MDCK cells grown on filters was not greatly affected by treatment with the various antibodies or Fab fragments. A small transient decrease in resistance observed with the polyclonal ct-uvomorulin IgG may be due to a more subtle modulation of the junctional complex.

Using computational modeling and laser microsurgery, we establish that neither the centrosomal microtubule array nor the Golgi-derived array is solely sufficient for correct Golgi assembly. Only the concerted effort of both MT arrays results in the integral, polarized Golgi complex necessary for polarized trafficking and cell motility.

The kinesin KIF1C is known to regulate podosomes, actin-rich adhesion structures, which remodel the extracellular matrix during physiological processes. Here we show that KIF1C is a player in the podosome-inducing signaling cascade. Upon induction of podosome formation by protein kinase C, KIF1C translocation to the cell periphery intensifies and KIF1C accumulates in the proximity of peripheral microtubules enriched with plus tip-associated proteins CLASPs and around podosomes. Importantly, without CLASPs, both KIF1C trafficking and podosome formation are suppressed. Moreover, chimeric mitochondria-targeted CLASP2 recruits KIF1C, suggesting a transient CLASP-KIF1C association. We propose that CLASP creates preferred microtubule tracks for KIF1C to promote podosome induction downstream of PKC.

CLIP-associated proteins CLASPs are mammalian microtubule (MT) plus-end tracking proteins (+TIPs) that promote MT rescue in vivo. Their plus-end localization is dependent on other +TIPs, EB1 and CLIP-170, but in the leading edge of the cell, CLASPs display lattice-binding activity. MT association of CLASPs is suggested to be regulated by multiple TOG (tumor overexpressed gene) domains and by the serine-arginine (SR)-rich region, which contains binding sites for EB1. Here, we report the crystal structures of the two TOG domains of CLASP2. Both domains consist of six HEAT repeats, which are similar to the canonical paddle-like tubulin-binding TOG domains, but have arched conformations. The degrees and directions of curvature are different between the two TOG domains, implying that they have distinct roles in MT binding. Using biochemical, molecular modeling and cell biological analyses, we have investigated the interactions between the TOG domains and αβ-tubulin and found that each domain associates differently with αβ-tubulin. Our findings suggest that, by varying the degrees of domain curvature, the TOG domains may distinguish the structural conformation of the tubulin dimer, discriminate between different states of MT dynamic instability and thereby function differentially as stabilizers of MTs.

Summary Microtubule (MT) plus-end tracking proteins (+TIPs) preferentially localize to MT plus-ends. End-binding proteins (EBs) are master regulators of the +TIP complex; however, it is unknown whether EBs are regulated by other +TIPs. Here, we show that Cytoplasmic linker associated proteins (CLASPs) modulate EB localization at MTs. In CLASP-depleted cells, EBs localized along the MT lattice in addition to plus-ends. The MT-binding region of CLASP was sufficient for restoring normal EB localization, while neither EB-CLASP interactions nor EB tail-binding proteins are involved. In vitro assays revealed that CLASP directly functions to remove EB from MTs. Importantly, this effect occurs specifically during MT polymerization, but not at pre-formed MTs. Increased GTP-tubulin content within MTs in CLASP-depleted cells suggests that CLASPs facilitate GTP-hydrolysis to reduce EB lattice binding. Together, these findings suggest that CLASPs influence the MT lattice itself to regulate EB and determine exclusive plus-end localization of EBs in cells.