+TIPs: SxIPping along microtubule ends

Abstract

+TIPs are a heterogeneous class of proteins that specifically bind to growing microtubule ends. Because dynamic microtubules are essential for many intracellular processes, +TIPs play important roles in regulating microtubule dynamics and microtubule interactions with other intracellular structures. End-binding proteins (EBs) recognize a structural cap at growing microtubule ends, and have emerged as central adaptors that mediate microtubule plus-end tracking of potentially all other +TIPs. The majority of these +TIPs bind to EBs through a short hydrophobic (S/T)x(I/L)P sequence motif (SxIP) and surrounding electrostatic interactions. These recent discoveries have resulted in a rapid expansion of the number of possible +TIPs. In this review, we outline our current understanding of the molecular mechanism of plus-end tracking and provide an overview of SxIP-recruited +TIPs.

Figure 1

EB proteins recognize an intermediate GTP hydrolysis state at growing microtubule ends. (a) Contrast-inverted image of a tissue culture cell expressing EB1-EGFP illustrating the characteristic +TIP comet shape at growing microtubule ends. Scale bar is 10 μm. The inset shows the indicated region at the cell edge at higher magnification, and the graph below is the fluorescence intensity profile of the topmost EB1 comet in the inset. The dashed line is an exponentially modified Gaussian fit that takes into account the blur introduced by the microscope point spread function [31]. The green line is a first order exponential fit of only the decay phase. (b) Diagram highlighting central features of microtubule structure and dynamic instability. The structural cap at growing microtubule ends likely consists of a layer of GTP-loaded tubulin dimers at the very end that is followed by a longer section of tubulin dimers in an intermediate GTP hydrolysis state, possibly GDP/P_i. High affinity EB binding sites at the interface of four tubulin dimers in a GDP/P_i state near the growing microtubule end are indicated in blue. These sites do not exist along the microtubule seam. The first order decay of these binding sites determines the exponential fluorescence profile of +TIPs at growing microtubule ends. The remainder of the lattice is mostly GDP-tubulin. (c) High resolution model of the microtubule-bound Mal3 calponin homology (CH) domain reconstructed from cryo-electron microscopy data. The CH domain binds at the interface of four tubulin dimers in the B-type microtubule lattice. Image reproduced with permission from [15].

Figure 2

EB protein structure and interactions. (a) Model of the EB1 homodimer, reproduced with permission from [25]. Crystal structures of the calponin homology (CH) and EB homology (EBH) domains were manually fitted into the envelope shape calculated from SAXS data (blue mesh). (b) Diagram of interactions between EB1 and SxIP- or CAP-Gly-containing +TIPs. Dashed arrows indicate interactions of SxIP motif containing +TIPs with other proteins. Some SxIP motif +TIPs such as CLASPs and SLAINs interact with CLIP-170, but the functional significance of these interaction networks is incompletely understood. (c) Interaction of the EBH domains with SxIP motif containing CLASP2 peptides. The structure was derived from homology modeling and molecular dynamics simulations [31]. Only the backbone is shown for CLASP2 peptides except for the SxIP motif (green) and nearby arginine residues. The inset shows electrostatic surface coloring of the intrinsically disordered C-terminal part of the EBH domain highlighting the interaction of positively charged arginine residues with the negatively charged EBH C-terminus.