Mal3 masks catastrophe events in Schizosaccharomyces pombe microtubules by inhibiting shrinkage and promoting rescue.

Schizosaccharomyces pombe Mal3 is a member of the EB family of proteins, which are proposed to be core elements in a tip-tracking network that regulates microtubule dynamics in cells. How Mal3 itself influences microtubule dynamics is unclear. We tested the effects of full-length recombinant Mal3 on dynamic microtubules assembled in vitro from purified S. pombe tubulin, using dark field video microscopy to avoid fluorescent tagging and data-averaging techniques to improve spatiotemporal resolution. We find that catastrophe occurs stochastically as a fast (<2.2 s) transition from constant speed growth to constant speed shrinkage with a constant probability that is independent of the Mal3 concentration. This implies that Mal3 neither stabilizes nor destabilizes microtubule tips. Mal3 does, however, stabilize the main part of the microtubule lattice, inhibiting shrinkage and increasing the frequency of rescues, consistent with recent models in which Mal3 on the lattice forms stabilizing lateral links between neighboring protofilaments. At high concentrations, Mal3 can entirely block shrinkage and induce very rapid rescue, making catastrophes impossible to detect, which may account for the apparent suppression of catastrophe by Mal3 and other EBs in vivo. Overall, we find that Mal3 stabilizes microtubules not by preventing catastrophe at the microtubule tip but by inhibiting lattice depolymerization and enhancing rescue. We argue that this implies that Mal3 binds microtubules in different modes at the tip and on the lattice.

Nucleotide- and Mal3-dependent changes in fission yeast microtubules suggest a structural plasticity view of dynamics.

Using cryo-electron microscopy, we characterize the architecture of microtubules assembled from Schizosaccharomyces pombe tubulin, in the presence and absence of their regulatory partner Mal3. Cryo-electron tomography reveals that microtubules assembled from S. pombe tubulin have predominantly B-lattice interprotofilament contacts, with protofilaments skewed around the microtubule axis. Copolymerization with Mal3 favors 13 protofilament microtubules with reduced protofilament skew, indicating that Mal3 adjusts interprotofilament interfaces. A 4.6-Å resolution structure of microtubule-bound Mal3 shows that Mal3 makes a distinctive footprint on the S. pombe microtubule lattice and that unlike mammalian microtubules, S. pombe microtubules do not show the longitudinal lattice compaction associated with EB protein binding and GTP hydrolysis. Our results firmly support a structural plasticity view of microtubule dynamics in which microtubule lattice conformation is sensitive to a variety of effectors and differently so for different tubulins.

Identification of a 250 kDa putative microtubule-associated protein as bovine ferritin. Evidence for a ferritin-microtubule interaction.

We reported previously on the purification and partial characterization of a putative microtubule-associated protein (MAP) from bovine adrenal cortex with an approximate molecular mass of 250 kDa. The protein was expressed ubiquitously in mammalian tissues, and bound to microtubules in vitro and in vivo, but failed to promote tubulin polymerization into microtubules. In the present study, partial amino acid sequencing revealed that the protein shares an identical primary structure with the widely distributed iron storage protein, ferritin. We also found that the putative MAP and ferritin are indistinguishable from each other by electrophoretic mobility, immunological properties and morphological appearance. Moreover, the putative MAP conserves the iron storage and incorporation properties of ferritin, confirming that the two are structurally and functionally the same protein. This fact led us to investigate the interaction of ferritin with microtubules by direct electron microscopic observations. Ferritin was bound to microtubules either singly or in the form of large intermolecular aggregates. We suggest that the formation of intermolecular aggregates contributes to the intracellular stability of ferritin. The interactions between ferritin and microtubules observed in this study, in conjunction with the previous report that the administration of microtubule depolymerizing drugs increases the serum release of ferritin in rats [Ramm GA, Powell LW & Halliday JW (1996) J Gastroenterol Hepatol11, 1072-1078], support the probable role of microtubules in regulating the intracellular concentration and release of ferritin under different physiological circumstances.

Ectopic A-lattice seams destabilize microtubules.

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

Preparation of dual-color polarity-marked fluorescent microtubule seeds.

Assaying microtubule dynamics in vitro requires stabilized nucleation centers, a method to immobilize individual microtubules onto a surface, and a specialized microscope to image the microtubule. Microtubules are polar structures with different dynamic properties at the plus and minus ends. However, the dynamics of the two ends can be modified by the addition of other proteins, such as microtubule plus-end-tracking proteins (+TIPs), so that it becomes impossible to distinguish the microtubule polarity by measuring the differences in the dynamic properties of the ends alone. In this chapter, we describe a method for labeling tubulin protein with N-hydroxysuccinimide ester fluorescent dyes, enabling the formation of dual-color polarity-marked stable microtubule seeds that can be immobilized onto a microscopic cover glass for imaging by fluorescence microscopy. These seeds create functional nucleation centers for the growth of dynamic microtubules.

Purification of tubulin from the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an attractive source of tubulin for biochemical experiments as it contains few tubulin isoforms and is amenable to genetic manipulation. We describe the preparation of milligram quantities of highly purified native tubulin from S. pombe suitable for use in microtubule dynamics assays as well as structural and other biochemical studies. S. pombe cells are grown in bulk in a fermenter and then lysed using a bead mill. The soluble protein fraction is bound to anion-exchange chromatography resin by batch binding, packed in a -chromatography column and eluted by a salt gradient. The tubulin-containing fraction is ammonium sulphate precipitated to further concentrate and purify the protein. A round of high-resolution anion-exchange chromatography is carried out before a cycle of polymerisation and depolymerisation to select functional tubulin. Gel filtration is used to remove residual contaminants before a final desalting step. The purified tubulin is concentrated, and then frozen and stored in liquid nitrogen.

Mal3, the Schizosaccharomyces pombe homolog of EB1, changes the microtubule lattice.

In vitro studies of pure tubulin have suggested that tubulin heterodimers in cells assemble into B-lattice microtubules, where the 8-nm dimers in adjacent protofilaments are staggered by 0.9 nm. This arrangement requires the tube to close by forming a seam with an A-lattice, in which the protofilaments are staggered by 4.9 nm. Here we show that Mal3, an EB1 family tip-tracking protein, drives tubulin to assemble in vitro into exclusively 13-protofilament microtubules with a high proportion of A-lattice protofilament contacts. We present a three-dimensional cryo-EM reconstruction of a purely A-lattice microtubule decorated with Mal3, in which Mal3 occupies the groove between protofilaments and associates closely with one tubulin monomer. We propose that Mal3 promotes assembly by binding to freshly formed tubulin polymer and particularly favors any with A-lattice arrangement. These results reopen the question of microtubule structure in cells.

Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin.

The kinesin-binding site on the microtubule has not been identified because of the technical difficulties involved in the mutant analyses of tubulin. Exploiting the budding yeast expression system, we succeeded in replacing the negatively charged residues in the alpha-helix 12 of beta-tubulin with alanine and analyzed their effect on kinesin-microtubule interaction in vitro. The microtubule gliding assay showed that the affinity of the microtubules for kinesin was significantly reduced in E410A, D417A, and E421A, but not in E412A mutant. The unbinding force measurement revealed that in the former three mutants, the kinesin-microtubule interaction in the adenosine 5'-[beta,gamma-imido]triphosphate state (AMP-PNP state) became less stable when a load was imposed towards the microtubule minus end. In parallel with this decreased stability, the stall force of kinesin was reduced. Our results implicate residues E410, D417, and E421 as crucial for the kinesin-microtubule interaction in the strong binding state, thereby governing the size of kinesin stall force.

The number of repeat sequences in microtubule-associated protein 4 affects the microtubule surface properties.

The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a Repeat region with tandemly organized repeat sequences. In this study, we focused on the variations of the Repeat region, and searched for MAP4 isoforms with diverse Repeat region organizations. We successfully isolated four types of MAP4 cDNAs, which differed from each other in both the number and the arrangement of the repeat sequences, from a single source (bovine adrenal gland). To examine the functional differences among the isoforms, we prepared the microtubule-binding domain polypeptides of three of the four isoforms, and examined their activities. The isoform fragments showed similar degrees of microtubule assembly promoting activity and microtubule binding affinity. This result suggested that the Repeat region variation is not important for the control of microtubule dynamics, which is believed to be the main function of MAPs. On the other hand, the microtubule bundle-forming activity differed among the isoform fragments. The bundle formation was augmented by increasing the number of repeat sequences in the fragments. Based on these results, we propose the hypothesis that the role of the MAP4 isoforms is to regulate the surface charge of microtubules.

A novel LIM and SH3 protein (lasp-2) highly expressing in chicken brain.

From eluates of F-actin affinity chromatography of chicken brain, we identified a novel actin-binding protein (lasp-2) whose gene was predicted in silico. We cloned cDNA of chicken lasp-2 and analyzed its structure, expression, activity, and localization with lasp-1 (LIM and SH3 protein 1), a previously identified actin-binding protein closely related to lasp-2. Chicken lasp-2 showed high homology to mammalian putative lasp-2. Both chicken lasp-1 and chicken lasp-2 have N-terminal LIM domains, C-terminal SH3 domains, and internal nebulin repeats. However, lasp-2 is greatly different from lasp-1 in the sequence between the second nebulin repeat and a SH3 domain, and the region is conserved in chicken, mouse, and human. As expected from its structural similarity to lasp-1, lasp-2 possessed actin-binding activity and localized with actin filament in filopodia of neuroblastoma. In contrast to lasp-1, which is widely distributed in non-muscle tissues, lasp-2 was highly expressed in brain.