Peter Bieling

Reconstitution of a microtubule plus-end tracking system in vitro

The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites

The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end–binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated α-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.

A Minimal Midzone Protein Module Controls Formation and Length of Antiparallel Microtubule Overlaps

During cell division, microtubules are arranged in a large bipolar structure, the mitotic spindle, to segregate the duplicated chromosomes. Antiparallel microtubule overlaps in the spindle center are essential for establishing bipolarity and maintaining spindle stability throughout mitosis. In anaphase, this antiparallel microtubule array is tightly bundled forming the midzone, which serves as a hub for the recruitment of proteins essential for late mitotic events. The molecular mechanism of midzone formation and the control of its size are not understood. Using an in vitro reconstitution approach, we show here that PRC1 autonomously bundles antiparallel microtubules and recruits Xklp1, a kinesin-4, selectively to overlapping antiparallel microtubules. The processive motor Xklp1 controls overlap size by overlap length-dependent microtubule growth inhibition. Our results mechanistically explain how the two conserved, essential midzone proteins PRC1 and Xklp1 cooperate to constitute a minimal protein module capable of dynamically organizing the core structure of the central anaphase spindle.

GTPgammaS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs)

Microtubule plus-end-tracking proteins (+TIPs) localize to growing microtubule plus ends to regulate a multitude of essential microtubule functions. End-binding proteins (EBs) form the core of this network by recognizing a distinct structural feature transiently existing in an extended region at growing microtubule ends and by recruiting other +TIPs to this region. The nature of the conformational difference allowing EBs to discriminate between tubulins in this region and other potential tubulin binding sites farther away from the microtubule end is unknown. By combining in vitro reconstitution, multicolor total internal reflection fluorescence microscopy, and electron microscopy, we demonstrate here that a closed microtubule B lattice with incorporated GTPγS, a slowly hydrolyzable GTP analog, can mimic the natural EB protein binding site. Our findings indicate that the guanine nucleotide γ-phosphate binding site is crucial for determining the affinity of EBs for lattice-incorporated tubulin. This defines the molecular mechanism by which EBs recognize growing microtubule ends.

Peptide bond formation does not involve acid-base catalysis by ribosomal residues

Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNAfMet with a modified A-site tRNA substrate, Phelac-tRNAPhe, in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3′ termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNAPhe is estimated to be >300 s−1.

Directional Switching of the Kinesin Cin8 Through Motor Coupling

A molecular motor switches direction upon interacting with individual microtubules or antiparallel microtubules. Kinesin motor proteins are thought to move exclusively in either one or the other direction along microtubules. Proteins of the kinesin-5 family are tetrameric microtubule cross-linking motors important for cell division and differentiation in various organisms. Kinesin-5 motors are considered to be plus-end–directed. However, here we found that purified kinesin-5 Cin8 from budding yeast could behave as a bidirectional kinesin. On individual microtubules, single Cin8 motors were minus-end–directed motors, whereas they switched to plus-end–directed motility when working in a team of motors sliding antiparallel microtubules apart. This kinesin can thus change directionality of movement depending on whether it acts alone or in an ensemble.

Phosphorylation Relieves Autoinhibition of the Kinetochore Motor Cenp-E

During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore.

Processive kinesins require loose mechanical coupling for efficient collective motility

Processive motor proteins are stochastic steppers that perform actual mechanical steps for only a minor fraction of the time they are bound to the filament track. Motors usually work in teams and therefore the question arises whether the stochasticity of stepping can cause mutual interference when motors are mechanically coupled. We used biocompatible surfaces to immobilize processive kinesin‐1 motors at controlled surface densities in a mechanically well‐defined way. This helped us to study quantitatively how mechanical coupling between motors affects the efficiency of collective microtubule transport. We found that kinesin‐1 constructs that lack most of the non‐motor sequence slow each other down when collectively transporting a microtubule, depending on the number of interacting motors. This negative interference observed for a motor ensemble can be explained quantitatively by a mathematical model using the known physical properties of individual molecules of kinesin‐1. The non‐motor extension of kinesin‐1 reduces this mutual interference, indicating that loose mechanical coupling between motors is required for efficient transport by ensembles of processive motors.

Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles.

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein-dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.

Obstacles on the Microtubule Reduce the Processivity of Kinesin-1 in a Minimal In Vitro System and in Cell Extract

Inside cells, a multitude of molecular motors and other microtubule-associated proteins are expected to compete for binding to a limited number of binding sites available on microtubules. Little is known about how competition for binding sites affects the processivity of molecular motors and, therefore, cargo transport, organelle positioning, and microtubule organization, processes that all depend on the activity of more or less processive motors. Very few studies have been performed in the past to address this question directly. Most studies reported only minor effects of crowding on the velocity of motors. However, a controversy appears to exist regarding the effect of crowding on motor processivity. Here, we use single-molecule imaging of mGFP-labeled minimal dimeric kinesin-1 constructs in vitro to study the effects of competition on kinesins processivity. For competitors, we use kinesin rigor mutants as static roadblocks, minimal wild-type kinesins as motile obstacles, and a cell extract as a complex mixture of microtubule-associated proteins. We find that mGFP-labeled kinesin-1 detaches prematurely from microtubules when it encounters obstacles, leading to a strong reduction of its processivity, a behavior that is largely independent of the type of obstacle used here. Kinesin has a low probability to wait briefly when encountering roadblocks. Our data suggest, furthermore, that kinesin can occasionally pass obstacles on the protofilament track.