Microtubules are dynamic polymers of αβ-tubulin that are essential for intracellular organization, organelle trafficking and chromosome segregation. Microtubule growth and shrinkage occur via ...addition and loss of αβ-tubulin subunits, which are biochemical processes. Dynamic microtubules can also engage in mechanical processes, such as exerting forces by pushing or pulling against a load. Recent advances at the intersection of biochemistry and mechanics have revealed the existence of multiple conformations of αβ-tubulin subunits and their central role in dictating the mechanisms of microtubule dynamics and force generation. It has become apparent that microtubule-associated proteins (MAPs) selectively target specific tubulin conformations to regulate microtubule dynamics, and mechanical forces can also influence microtubule dynamics by altering the balance of tubulin conformations. Importantly, the conformational states of tubulin dimers are likely to be coupled throughout the lattice: the conformation of one dimer can influence the conformation of its nearest neighbours, and this effect can propagate over longer distances. This coupling provides a long-range mechanism by which MAPs and forces can modulate microtubule growth and shrinkage. These findings provide evidence that the interplay between biochemistry and mechanics is essential for the cellular functions of microtubules.
Microtubules are long, slender polymers of αβ-tubulin found in all eukaryotic cells. Tubulins associate longitudinally to form protofilaments, and adjacent protofilaments associate laterally to form ...the microtubule. In the textbook view, microtubules are 1) composed of 13 protofilaments, 2) arranged in a radial array by the centrosome, and 3) built into the 9+2 axoneme. Although these canonical structures predominate in eukaryotes, microtubules with divergent protofilament numbers and higher-order microtubule assemblies have been discovered throughout the last century. Here we survey these noncanonical structures, from the 4-protofilament microtubules of
to the 40-protofilament accessory microtubules of mantidfly sperm. We review the variety of protofilament numbers observed in different species, in different cells within the same species, and in different stages within the same cell. We describe the determinants of protofilament number, namely nucleation factors, tubulin isoforms, and posttranslational modifications. Finally, we speculate on the functional significance of these diverse polymers. Equipped with novel tubulin-purification tools, the field is now prepared to tackle the long-standing question of the evolutionary basis of microtubule structure.
Microtubules are not like other polymers. Whereas polymers such as F-actin will grow continuously as long as the subunit concentration is high enough, a steadily growing microtubule can suddenly ...shrink even when there is ample αβ-tubulin around. This remarkable behavior was discovered in 1984 when Tim Mitchison and Marc Kirschner deduced that microtubules switch from growth to shrinkage when they lose their GTP caps. Here, I review the canonical explanation of dynamic instability that was fleshed out in the years after its discovery. Many aspects of this explanation have been recently subverted, particularly those related to how GTP-tubulin forms polymers and why GTP hydrolysis disrupts them. I describe these developments and speculate on how our explanation of dynamic instability can be changed to accommodate them.
Neurons, like all cells, face the problem that tubulin forms microtubules with too many or too few protofilaments (pfs). Cells overcome this heterogeneity with the γ-tubulin ring complex, which ...provides a nucleation template for 13-pf microtubules. Doublecortin (DCX), a protein that stabilizes microtubules in developing neurons, also nucleates 13-pf microtubules in vitro. Using fluorescence microscopy assays, we show that the binding of DCX to microtubules is optimized for the lateral curvature of the 13-pf lattice. This sensitivity depends on a cooperative interaction wherein DCX molecules decrease the dissociation rate of their neighbors. Mutations in DCX found in patients with subcortical band heterotopia weaken these cooperative interactions. Using assays with dynamic microtubules, we discovered that DCX binds to polymerization intermediates at growing microtubule ends. These results support a mechanism for stabilizing 13-pf microtubules that allows DCX to template new 13-pf microtubules through associations with the sides of the microtubule lattice.
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► DCX “measures” microtubule thickness by a cooperative mechanism ► DCX tracks microtubule ends like the canonical end-tracking protein EB1 ► Single-molecule experiments define the biochemical basis of double-cortex syndrome ► DCX-GFP is a fluorescence-based marker for microtubule structure
Doublecortin (DCX) nucleates uniform 13-protofilament (pf) microtubules by a mechanism distinct from γ-TuRC. Bechstedt and Brouhard show that DCX binds optimally to the 13-pf lattice by cooperative interactions and tracks microtubule ends. DCX mutations in patients with subcortical band heterotopia weaken DCX cooperativity, providing a biochemical basis for the disease.
Microtubule ends have distinct biochemical and structural features from those of the lattice. Several proteins that control microtubule behavior can distinguish the end of a microtubule from the ...lattice. The end-binding protein EB1, for example, recognizes the nucleotide state of microtubule ends, which are enriched in GTP-tubulin. EB1 shares its binding site with Doublecortin (DCX), a protein expressed in developing neurons. We showed recently that DCX binds with highest affinity to microtubule ends.
Here we show that DCX recognizes microtubule ends by a novel mechanism based on lattice curvature. Using single-molecule microscopy, we show that DCX “comets” do not elongate at faster microtubule growth rates and DCX does not recognize two out of three GTP analogs. We demonstrate that DCX binds with higher affinity to curved microtubule lattices than to straight ones. We find that curvature recognition is a property of single DCX molecules. Straightening of protofilaments (pfs) at microtubule ends with paclitaxel significantly attenuates end-recognition by DCX, but not EB1. Mutations in DCX found in patients with double cortex syndrome disrupted curvature recognition.
We propose a model in which DCX recognizes microtubule ends through specific interactions with their structure. We conclude that microtubule ends have two distinct features that proteins can recognize independently, namely a structural feature related to curvature and nucleotide state.
•DCX recognizes a structural feature of microtubule ends, namely their curvature•DCX binds with higher affinity to curved microtubule lattices than to straight ones•Mutations found in double cortex syndrome patients disrupt curvature recognition
Microtubule ends have distinct properties from those of the lattice. Several proteins can distinguish the end of a microtubule from the lattice. Bechstedt et al. show that Doublecortin (DCX), a protein expressed in developing neurons, recognizes microtubule ends by a novel mechanism based on lattice curvature.
The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular ...functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.
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•Osmotic shifts modulate microtubule assembly and disassembly proportionally in cells•Effects correlate with changes in cytoplasmic concentration, viscosity, and diffusion•Effects are conserved across eukaryotes (yeast, plant, mammalian cells)•Cytoplasmic dampening of microtubules is recapitulated by a viscous agent in vitro
How dynamic cellular processes operate in the complex environment of the cytoplasm remains poorly understood. Molines et al. show that the cytoplasm physically dampens microtubule assembly and disassembly through its viscous properties. These findings demonstrate the importance of cytoplasmic viscosity to biochemical reaction rates within the living cell.
Microtubules are dynamic polymers of αβ-tubulin that form diverse cellular structures, such as the mitotic spindle for cell division, the backbone of neurons, and axonemes. To control the ...architecture of microtubule networks, microtubule-associated proteins (MAPs) and motor proteins regulate microtubule growth, shrinkage, and the transitions between these states. Recent evidence shows that many MAPs exert their effects by selectively binding to distinct conformations of polymerized or unpolymerized αβ-tubulin. The ability of αβ-tubulin to adopt distinct conformations contributes to the intrinsic polymerization dynamics of microtubules. αβ-Tubulin conformation is a fundamental property that MAPs monitor and control to build proper microtubule networks.
Microtubules are born and reborn continuously, even during quiescence. These polymers are nucleated from templates, namely γ-tubulin ring complexes (γ-TuRCs) and severed microtubule ends. Using ...single-molecule biophysics, we show that nucleation from γ-TuRCs, axonemes and seed microtubules requires tubulin concentrations that lie well above the critical concentration. We measured considerable time lags between the arrival of tubulin and the onset of steady-state elongation. Microtubule-associated proteins (MAPs) alter these time lags. Catastrophe factors (MCAK and EB1) inhibited nucleation, whereas a polymerase (XMAP215) and an anti-catastrophe factor (TPX2) promoted nucleation. We observed similar phenomena in cells. We conclude that GTP hydrolysis inhibits microtubule nucleation by destabilizing the nascent plus ends required for persistent elongation. Our results explain how MAPs establish the spatial and temporal profile of microtubule nucleation.
Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. ...Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a "reduction in dimensionality." We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates.
The dynamic instability of microtubules is a conserved and fundamental mechanism in eukaryotes. Yet microtubules from different species diverge in their growth rates, lattice structures, and ...responses to GTP hydrolysis. Therefore, we do not know what limits microtubule growth, what determines microtubule structure, or whether the mechanisms of dynamic instability are universal. Here, we studied microtubules from the nematode C. elegans, which have strikingly fast growth rates and non-canonical lattices in vivo. Using a reconstitution approach, we discovered that C. elegans microtubules combine intrinsically fast growth with very frequent catastrophes. We solved the structure of C. elegans microtubules to 4.8 Å and discovered sequence divergence in the lateral contact loops, one of which is ordered in C. elegans but unresolved in other species. We provide direct evidence that C. elegans tubulin has a higher free energy in solution and propose a model wherein the ordering of lateral contact loops activates tubulin for growth.
•C. elegans microtubules reconstituted in vitro are fast growing and short lived•Lateral contact loops are ordered in the C. elegans free dimer and in the lattice•C. elegans tubulin has a higher free energy in solution than B. taurus tubulin•Structuring of lateral contacts is a limiting step in tubulin activation and microtubule growth
Microtubule polymerization involves complex structural transitions that are not fully understood. Chaaban et al. study microtubules from the nematode C. elegans, which have unique structures and dynamics in cells. They uncover a key limiting step in microtubule polymerization that involves the structuring of tubulin-tubulin interacting residues.
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