Rapid functional analysis of protein-protein interactions by fluorescent C-terminal labeling and single-molecule imaging.

Detection of protein-protein interactions is a fundamental step to understanding gene function. Here we report a sensitive and rapid method for assaying protein-protein interactions at the single-molecule level. Protein molecules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The interaction of proteins thus prepared was visualized using a single-molecule imaging technique. As a demonstration of this method, a motor protein, kinesin, was labeled with Cy5-puro at an efficiency of about 90%, and the processive movement of kinesin along microtubules was observed by using total internal reflection microscopy. It took only 2 h from the synthesis of proteins to the functional analysis. This method is applicable to the functional analysis of various kinds of proteins.

Where is APC going?

Adenomatous polyposis coli (APC) protein has been thought to function as a tumor suppressor through its involvement in the Wnt/beta-catenin signaling pathway. However, its connections to the cytoskeleton and microtubules in particular are becoming apparent, and the discovery of these new functions for APC is leading to a reevaluation of its role not only in tumorigenesis, but also in normal physiology.

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.

Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells.

Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.

Role of the outermost subdomain of Salmonella flagellin in the filament structure revealed by electron cryomicroscopy.

A mutant strain of Salmonella typhimurium, SJW46, has flagellar filaments supercoiled in the same form as the wild-type strain, SJW1103, and swims normally. However, its flagellar filaments are mechanically unstable and show anomalous behaviors of polymorphism. Flagellin from SJW46 has a large central deletion from Ala204 to Lys292 of SJW1103 flagellin, which has been thought to be located in the outer surface of the filament. Since the filament structure is determined by intersubunit interactions of the terminal regions in the densely packed core of the filament, no serious involvement of the deleted portion was expected in the filament stability and polymorphism. In order to locate the deleted portion and to understand the underlying mechanism of these anomalous characteristics, we carried out structure analysis of the L-type straight filament reconstituted from a mutant flagellin of SJW46 (SJW46S) and compared the structure with that of the SJW1660 filament, which is also the L-type but composed of flagellin with no deletion. The deleted portion was identified as the outermost subdomain, and the structure in the core region showed no appreciable differences. The structure revealed the previously identified folding of flagellin in further detail, and the significance of intersubunit interactions between outer domains, which are present in the SJW1660 filament but absent in the SJW46 filament. This suggests that these contacts have a significant contribution to the filament stability and polymorphic behavior, despite the fact that the contacting surface area occupies only a minor portion of the whole intersubunit interactions.

Structure and switching of bacterial flagellar filaments studied by X-ray fiber diffraction.

Bacterial motility involves switching between the left and right supercoiled states of the flagellar filament. The polymorphism of this assembly of identical flagellin molecules has presented a structural puzzle. Supercoiling has been attributed to coexistence of two conformational states of the 11 nearly axially aligned protofilament strands of subunits. The helical parameters of straight filaments in the left (L) and right (R) lattice states have now been accurately determined by X-ray fiber diffraction. The 9 A resolution electron density map of the R-type filament, refined from the X-ray data, reveals the interlocked alpha-helical segments of the core portion, which constitute the inner and outer tubes. While the inner-tube domain interactions remain invariant, the strand joints in the outer tube can switch between the L- and R-state by 2-3 A axial shifts, which change the strand periodicity of approximately 50 A by 0.8 A. This bi-stable quaternary switching results in supercoiling. Based on the measured helical parameters of the L and R lattices and the switching model, the twist and curvature calculated for the ten possible supercoils are in quantitative accord with observed supercoiled forms of flagellar filaments.

Locations of terminal segments of flagellin in the filament structure and their roles in polymerization and polymorphism.

Terminal regions of flagellin, about 180 NH2 and 100 COOH-terminal residues, are well conserved and play important roles in polymerization and polymorphism of bacterial flagellar filaments. About 65 NH2 and 45 COOH-terminal residues are disordered in the monomeric form, but become folded upon filament formation. Taking advantage of the facts that relatively small segments can be cleaved off these disordered termini by limited proteolysis, and isolated fragments still form straight filaments, locations of those terminal segments have been mapped out in the filament structure by electron cryomicroscopy and helical image reconstruction. The fragments studied are F(1-486), F(20-494), F(1-461), F(30-461) and F(30-452). Regardless of the size and terminal side of truncation, the structures of the filaments reconstituted from the truncated fragments all have identical subunit packing arrangements of the Lt-type symmetry. Structural differences compared to the filament reconstituted from intact flagellin are found only around the filament axis, namely in the inner-tube region, and no obvious changes are observed in the outer-tube or the outer part of the filament. Truncation of only a few terminal residues results in misfolding of the inner-tube domains and their aggregation around the filament axis; further truncation reduces the densities of different parts of the aggregate. The filament reconstituted from F(30-461) fragment shows complete disappearance of the density corresponding to the inner-tube. When a further nine residues are removed, the spoke-like features left on the inner wall of the outer-tube become significantly smaller. Based on the structures and radial mass distributions of the filaments obtained, the previous amino acid sequence assignment to the morphological domains has been confirmed and further refined. The roles of terminal segments in the assembly regulation, and those of the double-tubular structure in the polymorphic mechanism are discussed.

Direct interaction of flagellin termini essential for polymorphic ability of flagellar filament.

We report the structures of flagellar filaments reconstituted from various flagellins with small terminal truncations. Flagellins from Salmonella typhimurium strains SJW1103 (wild type), SJW1660, and SJW1655 were used, which form a left-handed supercoil, the L- and R-type straight forms, respectively. Structure analyses were done by electron cryomicroscopy and helical image reconstruction with a help of x-ray fiber diffraction for determining precise helical symmetries. Truncation of either terminal region, irrespective of the original flagellin species, results in a straight filament having a helical symmetry distinct either from the L- or R-type. This filament structure is named Lt-type. Although the local subunit packing is similar in all three types, a close comparison shows that the Lt-type packing is almost identical to the R-type but distinct from the L-type, which demonstrates the strong two-state preference of the subunit interactions. The structure clearly suggests that both termini are located in the inner tube of the concentric double-tubular structure of the filament core, and their proper interaction is responsible for the correct folding of fairly large terminal regions that form the inner tube. The double tubular structure appears to be essential for the polymorphic ability of flagellar filaments, which is required for the swimming-tumbling of bacterial taxis.