Structural heterogeneity of the microtubule lattice

Microtubules are polymers assembled from tubulin α-β-heterodimers. They typically display lateral α-α and β-β-homotypic interactions, except at one region, called the seam, where heterotypic α-β and β-α interactions occur. Here, we decorated microtubules assembled in vitro or in cytoplasmic Xenopus egg extracts with kinesin-motor domains, and analyzed their lattice organization using dual axis cryo-electron tomography followed by segmented sub-tomogram averaging. In both conditions, microtubules incorporated variable protofilament and/or tubulin subunit helix start numbers. While microtubules assembled in vitro displayed variable numbers of seams, those assembled in extracts displayed preferentially one seam. The seam location varied within individual microtubules implying the presence of lattice holes. Thus, the formation of discontinuous microtubule lattices is an intrinsic property of tubulin assembly, a process that is controlled in cells.

Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, creating a link between the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, to link the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

Mechanics and kinetics of dynamic instability

During dynamic instability, self-assembling microtubules (MTs) stochastically alternate between phases of growth and shrinkage. This process is driven by the presence of two distinct states of MT subunits, GTP- and GDP-bound tubulin dimers, that have different structural properties. Here, we use a combination of analysis and computer simulations to study the mechanical and kinetic regulation of dynamic instability in three-dimensional (3D) self-assembling MTs. Our model quantifies how the 3D structure and kinetics of the distinct states of tubulin dimers determine the mechanical stability of MTs. We further show that dynamic instability is influenced by the presence of quenched disorder in the state of the tubulin subunit as reflected in the fraction of non-hydrolysed tubulin. Our results connect the 3D geometry, kinetics and statistical mechanics of these tubular assemblies within a single framework, and may be applicable to other self-assembled systems where these same processes are at play.

The growing landscape of tubulin acetylation: lysine 40 and many more

Tubulin heterodimers are the building block of microtubules, which are major elements of the cytoskeleton. Several types of post-translational modifications are found on tubulin subunits as well as on the microtubule polymer to regulate the multiple roles of microtubules. Acetylation of lysine 40 (K40) of the α-tubulin subunit is one of these post-translational modifications which has been extensively studied. We summarize the current knowledge about the structural aspects of K40 acetylation, the functional consequences, the enzymes involved and their regulation. Most importantly, we discuss the potential importance of the recently discovered additional acetylation acceptor lysines in tubulin subunits and highlight the urgent need to study tubulin acetylation in a more integrated perspective.

The feasibility of coherent energy transfer in microtubules

It was once purported that biological systems were far too ‘warm and wet’ to support quantum phenomena mainly owing to thermal effects disrupting quantum coherence. However, recent experimental results and theoretical analyses have shown that thermal energy may assist, rather than disrupt, quantum coherent transport, especially in the ‘dry’ hydrophobic interiors of biomolecules. Specifically, evidence has been accumulating for the necessary involvement of quantum coherent energy transfer between uniquely arranged chromophores in light harvesting photosynthetic complexes. The ‘tubulin’ subunit proteins, which comprise microtubules, also possess a distinct architecture of chromophores, namely aromatic amino acids, including tryptophan. The geometry and dipolar properties of these aromatics are similar to those found in photosynthetic units indicating that tubulin may support coherent energy transfer. Tubulin aggregated into microtubule geometric lattices may support such energy transfer, which could be important for biological signalling and communication essential to living processes. Here, we perform a computational investigation of energy transfer between chromophoric amino acids in tubulin via dipole excitations coupled to the surrounding thermal environment. We present the spatial structure and energetic properties of the tryptophan residues in the microtubule constituent protein tubulin. Plausibility arguments for the conditions favouring a quantum mechanism of signal propagation along a microtubule are provided. Overall, we find that coherent energy transfer in tubulin and microtubules is biologically feasible.

First Report of Neofusicoccum australe Associated with Botryosphaeria Canker of Grapevine in Chile

A survey of trunk diseases was conducted in 2010 in vineyards (n = 14) in central Chile (latitude 33°51′ to 36°30′), specifically of Vitis vinifera ‘Cabernet Sauvignon,’ which is the main wine-grape cultivar (38,806 ha) in Chile. The following symptoms of trunk disease were observed in 5- to 19-year-old grapevines: short internodes, dead spurs, dead cordons (arms), and shoot dieback. Upon cutting into cordons and trunks of symptomatic vines, brown, V-shaped cankers of hard consistency were observed. A total of 56 wood cankers were collected, and small pieces of symptomatic wood (approximately 4 mm in diameter) taken from the canker margin were surface disinfected (75% ethanol, 30 s) and placed on acidified PDA (0.5 ml of 96% lactic acid per liter; APDA), which was incubated for 4 to 7 days at 24°C. Colonies, tentatively identified as a species within the Botryosphaeriaceae based on the presence of whitish-to-gray aerial mycelium and exhibiting rapid growth (4 to 5 cm colony diameter in 48 h), were hyphal-tip purified to APDA for identification. Colonies produced globose, black pycnidia with unicellular, hyaline, ellipsoidal, densely granulate, externally smooth, and thin-walled conidia of 17.0 ± 0.7 ± 6.7 ± 0.4 μm (n = 20). A yellow pigmentation was observed at the center of 48-h colonies on APDA. Morphologically, these isolates were identified as Neofusicoccum australe (Slippers, Crous & M.J. Wingfield) Crous, Slippers & A.J.L. Phillips (2,3). BLASTn searches of the ITS rDNA region, amplified with PCR primers ITS4/ITS5 (532 bp), and a 400-bp section of the beta-tubulin subunit 2 gene amplified with primers Bt2a and Bt2b of N. australe (GenBank Accession No. JX290091 and JX679868, respectively) revealed 99% similarity with the ITS and beta-tubulin sequences of N. australe reference strains EF638778 and HQ392761, respectively. Pathogenicity tests were conducted using N. australe isolate Vid1559 on 2-year-old Cabernet Sauvignon plants (n = 4), which were inoculated by wounding the woody stem with a scalpel approximately 1 cm below the most basal bud, placing an 8-mm mycelial plug taken from a 7-day culture into the wound, and then sealing the wound with Parafilm. Non-inoculated controls (n = 4) were ‘mock’ inoculated with sterile agar plugs. After 3 months under field conditions, during spring and summer, the woody stems were examined for vascular discoloration (VD), characteristic of a wood canker. Inoculated plants had stems with light-brown, necrotic VD with a mean length of 15.2 cm, measured from the inoculation point. No VD was observed on the controls. N. australe was reisolated from 100% of the inoculated plants, completing Koch's postulates. Of 14 vineyards surveyed, 8% were infected with N. australe. N. australe is known as a trunk pathogen of grape (4), and other species of Botryosphaeriaceae have been associated with grapevine trunk disease in Chile (1). To our knowledge, this is the first report of N. australe causing Botryosphaeria canker of grape in Chile, where the pathogen is previously reported on blueberry (2). References: (1) G. A. Díaz et al. Plant Dis. 95:1032, 2011. (2) J. G. Espinoza et al. Plant Dis. 92:1407, 2008. (3) Slippers et al. Mycologia 96:1030, 2004. (4) J. R. Úrbez-Torres Phytopathol. Mediterr. 50:S5, 2011.

INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells

T cell antigen receptor–proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor–induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cells.

The dynamic behaviour of microtubules and their contributions to hyphal tip growth in Aspergillus nidulans

Creating and maintaining cell polarity are complex processes that are not fully understood. Fungal hyphal tip growth is a highly polarized and dynamic process involving both F-actin and microtubules (MTs), but the behaviour and roles of the latter are unclear. To address this issue, MT dynamics and subunit distribution were analysed in a strain of Aspergillus nidulans expressing GFP–α-tubulin. Apical MTs are the most dynamic, the bulk of which move tipwards from multiple subapical spindle pole bodies, the only clear region of microtubule nucleation detected. MTs populate the apex predominantly by elongation at rates about three times faster than tip extension. This polymerization was facilitated by the tipward migration of MT subunits, which generated a tip-high gradient. Subapical regions of apical cells showed variable tubulin subunit distributions, without tipward flow, while subapical cells showed even tubulin subunit distribution and low MT dynamics. Short MTs, of a similar size to those reported in axons, also occasionally slid into the apex. During mitosis in apical cells, MT populations at the tip varied. Cells with less distance between the tip and the first nucleus were more likely to loose normal MT populations and dynamics. Reduced MTs in the tip, during mitosis or after exposure to the MT inhibitor carbendazim (MBC), generally correlated with reduced, but continuing growth and near-normal tip morphology. In contrast, the actin-disrupting agent latrunculin B reduced growth rates much more severely and dramatically distorted tip morphology. These results suggest substantial independence between MTs and hyphal tip growth and a more essential role for F-actin. Among MT-dependent processes possibly contributing to tip growth is the transportation of vesicles. However, preliminary ultrastructural data indicated a lack of direct MT–organelle interactions. It is suggested that the population of dynamic apical MTs enhance migration of the ‘cytomatrix’, thus ensuring that organelles and proteins maintain proximity to the constantly elongating tip.