Microtubule Elongation Research Articles

Collective effect of Vigna sp. (mung) tubulin GTP hydrolysis rate divergence on microtubule filament assembly.

Microtubules (MTs) are dynamic cytoskeletal filaments with highly conserved sequences across evolution, polymerizing by the GTP-dependent assembly of tubulin subunits. Despite the sequence conservation, MT polymerization kinetics diverge quantitatively between vertebrate brain, the model plant Arabidopsis and the protozoan Plasmodium. Previously, tubulin purified from seedlings of the plant Vigna sp. (mung) by temperature cycling was found to have a very low critical concentration. However, the lengths of MTs were sub-micron, much shorter than brain tubulin filaments. This was explained in simulations to be the result of the collective effect of high nucleation and GTP hydrolysis rates. Here, we test the effect of GTPase rates of affinity-purified Vigna sp. tubulin on microtubule polymerization and elongation. Affinity-purified mung tubulin is active and has a critical concentration of .37 μM. The GTP-dependent polymerization kinetics are transient, consistent with previous results. Polymerization is stabilized in the presence of either GTP analog GMPPNP (non-hydrolyzable) or GMPCPP (slow-hydrolyzable). Using interference reflection microscopy (IRM) we find polymerization with the non-hydrolysable analog significantly increases filament numbers, while lengths are unaffected for both GTP analogs. However, prolonged incubation with slow-hydrolyzable GMPCPP results in long filaments, pointing to GTP hydrolysis as a key factor determining MT length. We find the average GTPase turnover number of mung tubulin is 22.8 min-1, compared to 2.04 min-1 for goat brain tubulin. Thus modulating GTPase rates affects both nucleation and elongation. This quantitative divergence in kinetics despite high sequence conservation in the GTPase domains of α- and β-tubulin could help better understand the roles of selective pressure and function in the diverse organisms.

Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends.

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The 'self-acting' (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the 'interface-acting' (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with a self-acting nucleotide, plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide, plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and 'poisoning' plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics.

Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The ‘self-acting’ (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the ‘interface-acting’ (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with a self-acting nucleotide, plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide, plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and ‘poisoning’ plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics.

CCDC183 is essential for cytoplasmic invagination around the flagellum during spermiogenesis and male fertility.

Sperm flagellum plays a crucial role in male fertility. Here, we generated Ccdc183 knockout mice using the CRISPR/Cas9 system to reveal the protein function of the testis-specific protein CCDC183 in spermiogenesis. We demonstrated that the absence of CCDC183 causes male infertility with morphological and motility defects in spermatozoa. Owing to the lack of CCDC183, centrioles after elongation of axonemal microtubules do not connect the cell surface and nucleus during spermiogenesis, which causes subsequent loss of cytoplasmic invagination around the flagellum. As a result, the flagellar compartment does not form properly and cytosol-exposed axonemal microtubules collapse during spermiogenesis. In addition, ectopic localization of accessory structures, such as the fibrous sheath and outer dense fibers, and abnormal head shape as a result of abnormal sculpting by the manchette are observed in Ccdc183 knockout spermatids. Our results indicate that CCDC183 plays an essential role in cytoplasmic invagination around the flagellum to form functional spermatozoa during spermiogenesis.

In vivo impact of tubulin polymerization promoting protein (Tppp) knockout to the airway inflammatory response

Microtubule dysfunction has been implicated as a mediator of inflammation in multiple diseases such as disorders of the cardiovascular and neurologic systems. Tubulin polymerization promoting protein (Tppp) facilitates microtubule elongation and regulates tubulin acetylation through inhibition of cytosolic deacetylase enzymes. Pathologic alterations in microtubule structure and dynamics have been described in cystic fibrosis (CF) and associated with inflammation, however the causality and mechanism remain unclear. Likewise, Tppp has been identified as a potential modifier of CF airway disease severity. Here we directly assess the impact of microtubule dysfunction on infection and inflammation by interrogating wild type and a Tppp knockout mouse model (Tppp − / −). Mice are challenged with a clinical isolate of Pseudomonas aeruginosa-laden agarose beads and assessed for bacterial clearance and inflammatory markers. Tppp − / − mouse model demonstrate impaired bacterial clearance and an elevated inflammatory response compared to control mice. These data are consistent with the hypothesis microtubule dysregulation is sufficient to lead to CF-like airway responses in mice.

Manipulation of the Tubulin Code Alters Directional Cell Migration and Ciliogenesis.

Conjunction of epithelial cells into monolayer sheets implies the ability to migrate and to undergo apicobasal polarization. Both processes comprise reorganization of cytoskeletal elements and rearrangements of structural protein interactions. We modulated expression of tubulin tyrosin ligase (TTL), the enzyme that adds tyrosine to the carboxy terminus of detyrosinated α-tubulin, to study the role of tubulin detyrosination/-tyrosination in the orientation of cell motility and in epithelial morphogenesis. Oriented cell migration and the organization of focal adhesions significantly lose directionality with diminishing amounts of microtubules enriched in detyrosinated tubulin. On the other hand, increasing quantities of detyrosinated tubulin results in faster plus end elongation of microtubules in migrating and in polarized epithelial cells. These plus ends are decorated by the plus end binding protein 1 (EB1), which mediates interaction between microtubules enriched in detyrosinated tubulin and the integrin-ILK complex at focal adhesions. EB1 accumulates at the apical cell pole at the base of the primary cilium following apicobasal polarization. Polarized cells almost devoid of detyrosinated tubulin form stunted primary cilia and multiluminal cysts in 3D-matrices. We conclude that the balance between detyrosinated and tyrosinated tubulin alters microtubule dynamics, affects the orientation of focal adhesions and determines the organization of primary cilia on epithelial cells.

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end.

Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αβ-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently 'poisoned' growth, yielding a wider range of growth rates compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP-tubulin at the growing microtubule end may be an important early event determining catastrophe.

Mechanisms of microtubule organization in differentiated animal cells.

Microtubules are polarized cytoskeletal filaments that serve as tracks for intracellular transport and form a scaffold that positions organelles and other cellular components and modulates cell shape and mechanics. In animal cells, the geometry, density and directionality of microtubule networks are major determinants of cellular architecture, polarity and proliferation. In dividing cells, microtubules form bipolar spindles that pull chromosomes apart, whereas in interphase cells, microtubules are organized in a cell type-specific fashion, which strongly correlates with cell physiology. In motile cells, such as fibroblasts and immune cells, microtubules are organized as radial asters, whereas in immotile epithelial and neuronal cells and in muscles, microtubules form parallel or antiparallel arrays and cortical meshworks. Here, we review recent work addressing how the formation of such microtubule networks is driven by the plethora of microtubule regulatory proteins. These include proteins that nucleate or anchor microtubule ends at different cellular structures and those that sever or move microtubules, as well as regulators of microtubule elongation, stability, bundling or modifications. The emerging picture, although still very incomplete, shows a remarkable diversity of cell-specific mechanisms that employ conserved building blocks to adjust microtubule organization in order to facilitate different cellular functions.

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end

In the present study, we used interference reflection microscopy (IRM) to observe the in vitro growth rates and fluctuations of bovine brain microtubules at high temporal resolution. We performed measurements in the presence of GTP and in the presence of the hydrolysis-resistant GTP analog GMPCPP. We fit a computational to the resulting measurements to provide insight into the molecular interactions governing microtubule elongation. Microtubules growing with GMPCPP showed a very low apparent critical concentration, reflecting high affinity interactions between αβ-tubulin subunits and the microtubule end.

Multiplication of Motor-Driven Microtubules for Nanotechnological Applications.

Microtubules gliding on motor-functionalized surfaces have been explored for various nanotechnological applications. However, when moving over large distances (several millimeters) and long times (tens of minutes), microtubules are lost due to surface detachment. Here, we demonstrate the multiplication of kinesin-1-driven microtubules that comprises two concurrent processes: (i) severing of microtubules by the enzyme spastin and (ii) elongation of microtubules by self-assembly of tubulin dimers at the microtubule ends. We managed to balance the individual processes such that the average length of the microtubules stayed roughly constant over time while their number increased. Moreover, we show microtubule multiplication in physical networks with topographical channel structures. Our method is expected to broaden the toolbox for microtubule-based in vitro applications by counteracting the microtubule loss from substrate surfaces. Among others, this will enable upscaling of network-based biocomputation, where it is vital to increase the number of microtubules during operation.

The drebrin/EB3 pathway regulates cytoskeletal dynamics to drive neuritogenesis in embryonic cortical neurons.

Co-ordinating the dynamic behaviour of actin filaments (F-actin) and microtubules in filopodia is an important underlying process in neuritogenesis, but the molecular pathways involved are ill-defined. The drebrin/end-binding protein 3 (EB3) pathway is a candidate pathway for linking F-actin to microtubules in filopodia. Drebrin binds F-actin and, simultaneously, the microtubule-binding protein EB3 when bound to microtubule plus-ends. We assessed the effect on neuritogenesis of gain- or loss-of-function of proteins in the drebrin/EB3 pathway in rat embryonic cortical neurons in culture. Loss-of-function of drebrin by gene editing or pharmacological inhibition of drebrin binding to F-actin reduced the number of dynamic microtubules in the cell periphery and simultaneously delayed the initiation of neuritogenesis, whereas over-expression of drebrin induced supernumerary neurites. Similarly, loss of EB3 inhibited neuritogenesis, whereas loss of end-binding protein 1 (EB1), a related protein that does not bind to drebrin, did not affect neuritogenesis. Over-expression of EB3, but not EB1, induced supernumerary neurites. We discovered that EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia allowing for continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F-actin in filopodia. Finally, we showed that preventing the entry of dynamic microtubules into filopodia using a pharmacological inhibitor of microtubule dynamics is associated with a loss of EB3, but not EB1, from microtubule plus-ends and a concurrent attenuation of neuritogenesis. Collectively, these findings support the idea that neuritogenesis depends on microtubule/F-actin zippering in filopodia orchestrated by the drebrin/EB3 pathway.

Differential regulation of single microtubules and bundles by a three-protein module.

A remarkable feature of the microtubule cytoskeleton is co-existence of sub-populations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: microtubule-crosslinker PRC1, and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find their collective activity promotes elongation of single microtubules, while simultaneously stalling polymerization of crosslinked bundles. This differentiation arises from (i) Strong rescue activity of CLASP1, which overcomes weaker effects of Kif4A on single microtubules, (ii) Lower microtubule and PRC1-binding affinity of CLASP1, which permit dominance of Kif4A at overlaps. In addition to canonical mechanisms where antagonistic regulators set microtubule lengths, our findings illuminate design principles by which collective regulator activity creates microenvironments of arrays with distinct dynamic properties.

Microtubule-associated proteins and motors required for ectopic microtubule array formation in Saccharomyces cerevisiae.

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here, we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules (MTs), and the organization of MTs relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Furthermore, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

Differential Regulation of Single Microtubules and Cross-Linked Bundles by a Minimal Three-Protein Module

A remarkable feature of the microtubule cytoskeleton is co-existence of distinct populations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: the microtubule-cross-linker PRC1, and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find their collective activity promotes elongation of single microtubules, while simultaneously stalling polymerization of cross-linked bundles. This striking differentiation arises from (i) higher activity of CLASP1 despite lower microtubule occupancy than Kif4A, (ii) amplification of Kif4A activity on microtubule bundles through stronger PRC1-affinity. In contrast to canonical mechanisms where antagonistic regulators set microtubule lengths of single arrays, our findings illuminate design principles by which their collective activity instead creates microenvironments comprised of arrays with distinct dynamic properties.

Microtubule Nucleation Properties of Single Human γTurcs Investigated by TIRF Microscopy and Cryo-Electron Microscopy

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a TIRF microscopy-based real-time nucleation assay. We find that single γTuRC complexes nucleate microtubules is a cooperative and stochastic manner. They stably cap microtubule minus ends. Nucleation is inefficient compared to microtubule elongation. A 4 Å resolution cryo-EM structure of γTuRC, combined with crosslinking-mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a ‘closed’ conformation matching the microtubule geometry. The opposite side of γTuRC is in an ‘open’, nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure. [published in Dev. Cell 53, 603-617, 2020; https://doi.org/10.1016/j.devcel.2020.04.019].

The actin regulator profilin 1 is functionally associated with the mammalian centrosome.

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction

While intracytoplasmic sperm injection (ICSI) is an asset in human Assisted Reproduction Technologies (ART), its outcomes, in terms of blastocyst, is still unacceptably low in ruminants. The picture typically found in ICSI derived bovine and ovine embryos is an asymmetry between a high activation rate, marked by a pronuclear development, and a low first cleavage rate. Abnormal centriole function has been indicated as a possible factor which undermines embryonic development following ICSI, especially when Freeze Dried spermatozoa (FD) are used. In order to verify the hypothesis that centriole dysfunction might be responsible for low ICSI outcomes in sheep, we have investigated micro-tubular dynamics, markedly aster nucleation, in fertilized sheep zygotes by ICSI with frozen/thawed (FT) and FD spermatozoa; In Vitro Fertilized (IVF) sheep oocytes were used as control. The spermatozoa aster nucleation was assessed at different time points following ICSI and IVF by immune-detection of α-tubulin. Pronuclear stage, syngamy and embryo development were assessed. No difference was noticed in the timing of aster nucleation and microtubule elongation in ICSI-FT derived embryos with control IVF ones, while a delay was recorded in ICSI-FD ones. The proportion of 2-pronuclear stage zygotes was similar in ICSI-FT and ICSI-FD (47% and 53%, respectively), both much lower comparing the IVF ones (73%). Likewise, syngamy was observed in a minority of both ICSI groups (28.5% vs 12.5% in ICSI-FT/FD respectively) comparing to IVF controls (50%), with a high number of zygotes blocked at the 2-pronuclear stage (71.5% vs 87.5% respectively). While no significant differences were noticed in the cleavage rate between ICSI-FD, ICSI-FT and IVF groups (31%, 34% and 44%) respectively, development to blastocyst stage was markedly compromised in both ICSI groups, especially with FD spermatozoa (10% in ICIS-FD and 19% in ICSI-FT vs 33% in IVF (P < 0.005, ICSI-FD vs IVF and P < 0.05, IVF vs ICSI-FT, respectively). Hence, here we have demonstrated that the reduced cleavage, and the ensuing impaired development to blastocysts stage of ICSI derived sheep embryos is not related to centriole dysfunction, as suggested by other authors. The major recorded problem is the lack of syngamy in ICSI derived zygotes, an issue that should be addressed in further studies to improve ICSI procedure in sheep embryos.

Fear Deficits in HypomyelinatedTpppKnock-Out Mice

Oligodendrocytes in the central nervous system (CNS) produce myelin sheaths that insulate axons to facilitate efficient electrical conduction. These myelin sheaths contain lamellar microtubules that enable vesicular transport into the inner sheath. Mechanistically, oligodendrocytes rely on Golgi outpost organelles and the associated protein tubulin polymerization promoting protein (TPPP) to nucleate or form new microtubules outside of the cell body. Consequently, elongation of lamellar microtubules is defective in Tppp knock-out (KO) mice, which have thinner and shorter myelin sheaths. We now explore the behavioral phenotypes of Tppp KO mice using a number of different assays. In open-field assays, Tppp KO mice display similar activity levels and movement patterns as wild-type mice, indicating that they do not display anxiety behavior. However, Tppp KO mice lack fear responses by two types of assays, traditional fear-conditioning assays and looming fear assays, which test for innate fear responses. Deficits in fear conditioning, which is a memory-dependent task, as well as in spatial memory tests, support possible short-term memory defects in Tppp KO mice. Together, our experiments indicate a connection between CNS myelination and behavioral deficits.

Microtubule organization defects in Arabidopsis thaliana.

Microtubules (MT) are critical cytoskeletal filaments that have several functions in cell morphogenesis, cell division, vesicle transport and cytoplasmic separation in the spatiotemporal regulation of eukaryotic cells. Formation of MT requires the co-interaction of MT nucleation and α-β-tubulins, as well as MT-associated proteins (MAP). Many key MAP contributing to MT nucleation and elongation are essential for MT nucleation and regulation of MT dynamics, and are conserved in the plant kingdom. Therefore, the deletion or decrease of γ-tubulin ring complex (γTuRC) components and related MAP, such as the augmin complex, NEDD1, MZT1, EB1, MAP65, etc., in Arabidopsis thaliana results in MT organizational defects in the spindle and phragmoplast MT, as well as in chromosome defects. In addition, similar defects in MT organization and chromosome structure have been observed in plants under abiotic stress conditions, such as under high UV-B radiation. The MT can sense the signal from UV-B radiation, resulting in abnormal MT arrangement. Further studies are required to determine whether the abnormal chromosomes induced by UV-B radiation can be attributed to the involvement of abnormal MT arrays in chromosome migration after DNA damage.

AKAP350 enables p150glued /EB1 interaction at the spindle poles

A-kinase anchoring protein 350 (AKAP350) is a centrosomal/Golgi scaffold protein, critical for the regulation of microtubule dynamics. AKAP350 recruits end-binding protein 1 (EB1) to the centrosome in mitotic cells, ensuring proper spindle orientation in epithelial cells. AKAP350 also interacts with p150glued, the main component of the dynactin complex. In the present work, we found that AKAP350 localized p150glued to the spindle poles, facilitating p150glued/EB1 interaction at these structures. Our results further showed that the decrease in AKAP350 expression reduced p150glued localization at astral microtubules and impaired the elongation of astral microtubules during anaphase. Overall, this study provides mechanistic data on how microtubule regulatory proteins gather to define microtubule dynamics in mitotic cells.