Unconjugated
The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network.
EBs and CLIPs are evolutionarily conserved proteins, which associate with the tips of growing microtubules, and regulate microtubule dynamics and their interactions with intracellular structures. In this study we investigated the functional relationship of CLIP-170 and CLIP-115 with the three EB family members, EB1, EB2(RP1), and EB3 in mammalian cells. We showed that both CLIPs bind to EB proteins directly. The C-terminal tyrosine residue of EB proteins is important for this interaction. When EB1 and EB3 or all three EBs were significantly depleted using RNA interference, CLIPs accumulated at the MT tips at a reduced level, because CLIP dissociation from the tips was accelerated. Normal CLIP localization was restored by expression of EB1 but not of EB2. An EB1 mutant lacking the C-terminal tail could also fully rescue CLIP dissociation kinetics, but could only partially restore CLIP accumulation at the tips, suggesting that the interaction of CLIPs with the EB tails contributes to CLIP localization. When EB1 was distributed evenly along the microtubules because of overexpression, it slowed down CLIP dissociation but did not abolish its preferential plus-end localization, indicating that CLIPs possess an intrinsic affinity for growing microtubule ends, which is enhanced by an interaction with the EBs.