GTP Cap Research Articles

Stable GDP-tubulin islands rescue dynamic microtubules.

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.

Alternative Approaches to Understand Microtubule Cap Morphology and Function.

Microtubules (MTs) are essential cellular machines built from concatenated αβ-tubulin heterodimers. They are responsible for two central and opposite functions from the dynamic point of view: scaffolding (static filaments) and force generation (dynamic MTs). These roles engage multiple physiological processes, including cell shape, polarization, division and movement, and intracellular long-distance transport. At the most basic level, the MT regulation is chemical because GTP binding and hydrolysis have the ability to promote assembly and disassembly in the absence of any other constraint. Due to the stochastic GTP hydrolysis, a chemical gradient from GTP-bound to GDP-bound tubulin is created at the MT growing end (GTP cap), which is translated into a cascade of structural regulatory changes known as MT maturation. This is an area of intense research, and several models have been proposed based on information mostly gathered from macromolecular crystallography and cryo-electron microscopy studies. However, these classical structural biology methods lack temporal resolution and can be complemented, as shown in this mini-review, by other approaches such as time-resolved fiber diffraction and computational modeling. Together with studies on structurally similar tubulins from the prokaryotic world, these inputs can provide novel insights on MT assembly, dynamics, and the GTP cap.

Covalent Catalytic Strategies for Enzymes That Modify RNA Molecules on their Tripartite Building Blocks.

The tripartite structures of the four 5'-nucleotide monophosphate (NMP) building blocks in all RNAs enable enzyme-catalyzed chemical modifications to three types of sites: the heterocyclic bases via N- and C-methylations and other alkylations, conversion of the N-glycoside linkages of the uridine moiety to the C-C glycoside link in pseudouridines, and the phosphodiester-mediated processes of 5'-capping, splicing, and 3'-tailing of premRNAs. We examine known cases for enzymatic covalent catalytic strategies that entail transient formation and breakdown of covalent enzyme-RNA adducts in each catalytic cycle. One case involves generation of the required carbon nucleophile during C5 methylation of cytosine residues in RNAs. A second examines the mechanism proposed for pseudouridine synthases and for replacement of a guanine residue in tRNAs by queuosine. The third category involves phosphoric anhydride and phosphodiester chemistry by which viral RNAs encode enzymes for making their own mRNA 5'-caps. This strategy includes the recent finding that the SARS-CoV2 proteins assemble a canonical 5',5'-GTP cap on their 28 900 nucleotide genomic RNA to enable its translation as an mRNA by host translational machinery by way of a covalent RNA-viral enzyme intermediate.

Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo. We find that in mitotic cells HURP accumulates on the kinetochore-proximal region of depolymerising kinetochore-fibres, whilst avoiding recruitment to nascent polymerising K-fibres, giving rise to a growing “HURP-gap”. The absence of end-binding proteins in the HURP-gaps leads us to postulate that they reflect a mixed-nucleotide zone. We generate a minimal quantitative model based on the preferential binding of HURP to GDP-tubulin to show that such a mixed-nucleotide zone is sufficient to recapitulate the observed in vivo dynamics of HURP-gaps.

Phosphomimetic Mutation at Ser165 of α-Tubulin Promotes the Persistence of GTP Caps in Microtubules.

Protein kinase C (PKC)-mediated phosphorylation of α-tubulin at Ser165 or expression of phosphomimetic (S165D)-α-tubulin stimulates microtubule (MT) polymerization (Cytoskeleton 2014, 71, 257-272). Ser165 lies near the interface between adjacent αβ-tubulin heterodimers and helix H8, which contains Glu254, the catalytic residue in α-tubulin that hydrolyzes the exchangeable GTP in β-tubulin (β:GTP) and triggers MT depolymerization. It was hypothesized that S165D, a phosphomimetic variant of α-tubulin, perturbs the alignment of α:Glu254 with respect to β:GTP, thereby impairing its hydrolysis. Molecular simulations were performed with cryoEM structures of MTs (PDB ID: 3J6E) in which phosphomimetic S165D or control S165N had been substituted. Unlike native and S165N structures, the distance between S165D and α:Glu254 increased by 0.6 Å, while the distance between α:Glu254 and β:GTP decreased by 0.4 Å. Rotation of β:GTP by 4 Å occurred in the S165D variant, whereas β:GTP in the S165N control was unchanged from the native structure. Additionally, the S165D variant exhibited an altered pattern of H-bonding to β:GTP, including the loss of three H-bonds. The significance of these findings to β:GTP hydrolysis was analyzed in MCF-10A human breast cells treated with an antibody that detects GTP-bound tubulin. Compared with controls, GTP-tubulin signals were at higher levels in cells that ectopically expressed S165D-α-tubulin (TUBA1C) or had been treated with PKC activator DAG-lactone to induce phosphorylation of Ser165 in native α-tubulin. These findings support a model whereby conformational changes induced by Ser165 phosphorylation alter the spatial relationship between β:GTP and α:Glu254, thereby slowing GTP hydrolysis and promoting GTP caps.

Counting fluorescently labeled proteins in tissues in the spinning-disk microscope using single-molecule calibrations.

Quantification of molecular numbers and concentrations in living cells is critical for testing models of complex biological phenomena. Counting molecules in cells requires estimation of the fluorescence intensity of single molecules, which is generally limited to imaging near cell surfaces, in isolated cells, or where motions are diffusive. To circumvent this difficulty, we have devised a calibration technique for spinning–disk confocal microscopy, commonly used for imaging in tissues, that uses single–step bleaching kinetics to estimate the single–fluorophore intensity. To cross–check our calibrations, we compared the brightness of fluorophores in the SDC microscope to those in the total internal reflection and epifluorescence microscopes. We applied this calibration method to quantify the number of end–binding protein 1 (EB1)–eGFP in the comets of growing microtubule ends and to measure the cytoplasmic concentration of EB1–eGFP in sensory neurons in fly larvae. These measurements allowed us to estimate the dissociation constant of EB1–eGFP from the microtubules as well as the GTP–tubulin cap size. Our results show the unexplored potential of single–molecule imaging using spinning–disk confocal microscopy and provide a straightforward method to count the absolute number of fluorophores in tissues that can be applied to a wide range of biological systems and imaging techniques.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

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 invivo 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 invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

Regulation of microtubule dynamics, mechanics and function through the growing tip.

Microtubule dynamics and their control are essential for the normal function and division of all eukaryotic cells. This plethora of functions is, in large part, supported by dynamic microtubule tips, which can bind to various intracellular targets, generate mechanical forces and couple with actin microfilaments. Here, we review progress in the understanding of microtubule assembly and dynamics, focusing on new information about the structure of microtubule tips. First, we discuss evidence for the widely accepted GTP cap model of microtubule dynamics. Next, we address microtubule dynamic instability in the context of structural information about assembly intermediates at microtubule tips. Three currently discussed models of microtubule assembly and dynamics are reviewed. These are considered in the context of established facts and recent data, which suggest that some long-held views must be re-evaluated. Finally, we review structural observations about the tips of microtubules in cells and describe their implications for understanding the mechanisms of microtubule regulation by associated proteins, by mechanical forces and by microtubule-targeting drugs, prominently including cancer chemotherapeutics.

Microtubule Growth Is Slowed through Transient Exposure of GDP Tubulin

Microtubule plus-end growth dynamics are determined by a kinetic race dictated by tubulin on- and off-rates at the microtubule tip. The apparent tubulin on- and off-rates can be estimated by measuring the mean plus-end growth rate across a range of free tubulin concentrations. However, when growth phases are studied at higher temporal resolution, fluctuations around the average growth rate are observed, implying more complex dynamics. To understand growth dynamics at the microtubule plus-end, we used Interference Reflection Microscopy (IRM) to quantify time-dependent variations in microtubule growth using GMPCPP, a non-hydrolyzable GTP analog. We found an expected linear dependence of the average growth rate on tubulin concentration. However, there was also a linear relationship between growth variance and tubulin concentration. Utilizing these measured values, we were able to constrain a biochemical model of microtubule growth with an on-rate of 0.7/ μM/pf/s. To determine the degree to which growth in GMPCPP mimics the GTP cap, we repeated the measurements in GTP. We again found a linear dependence of the average growth rate on tubulin; but found a much higher tubulin-dependent growth variance than expected from the GMPCPP experiments. When the growth variance was normalized to growth rate for each condition, there was a 5-fold increase in the relative growth variance in GTP compared to GMPCPP. This result suggests that GTP hydrolysis impacts tubulin binding kinetics at the microtubule plus-end. Consistent with this hypothesis, we then artificially varied the hydrolysis rates from 0 to 0.15 s−1 in the biochemical model. Increasing the hydrolysis rate increased the growth variance, and transient suppression of growth coincided with increasing numbers of exposed GDP tubulin at the plus-end. This work suggests that during growth, exposure of terminal GDP tubulin slows growth until a terminal GTP is restored.

Evidence for Conformational Change-Induced Hydrolysis of β-Tubulin-GTP

Microtubules, protein polymers of α/β-tubulin dimers, form the structural framework for many essential cellular processes including cell shape formation, intracellular transport, and segregation of chromosomes during cell division. It is known that tubulin-GTP hydrolysis is closely associated with microtubule polymerization dynamics. However, the precise roles of GTP hydrolysis in tubulin polymerization and microtubule depolymerization, and how it is initiated are still not clearly defined. We report here that tubulin-GTP hydrolysis can be triggered by conformational change induced by the depolymerizing kinesin-13 proteins or by the stabilizing chemical agent paclitaxel. We provide biochemical evidence that conformational change precedes tubulin-GTP hydrolysis, confirming this process is mechanically driven and structurally directional. Furthermore, we quantitatively measure the average size of the presumptive stabilizing “GTP cap” at growing microtubule ends. Together, our findings provide the molecular basis for tubulin-GTP hydrolysis and its role in microtubule polymerization and depolymerization.

Microtubule Simulations Provide Insight into the Molecular Mechanism Underlying Dynamic Instability

The dynamic instability of microtubules (MTs), which refers to their ability to switch between polymerization and depolymerization states, is crucial for their function. It has been proposed that the growing MT ends are protected by a “GTP cap” that consists of GTP-bound tubulin dimers. When the speed of GTP hydrolysis is faster than dimer recruitment, the loss of this GTP cap will lead the MT to undergo rapid disassembly. However, the underlying atomistic mechanistic details of the dynamic instability remains unclear. In this study, we have performed long-time atomistic molecular dynamics simulations (1 μs for each system) for MT patches as well as a short segment of a closed MT in both GTP- and GDP-bound states. Our results confirmed that MTs in the GDP state generally have weaker lateral interactions between neighboring protofilaments (PFs) and less cooperative outward bending conformational change, where the difference between bending angles of neighboring PFs tends to be larger compared with GTP ones. As a result, when the GDP state tubulin dimer is exposed at the growing MT end, these factors will be more likely to cause the MT to undergo rapid disassembly. We also compared simulation results between the special MT seam region and the remaining material and found that the lateral interactions between MT PFs at the seam region were comparatively much weaker. This finding is consistent with the experimental suggestion that the seam region tends to separate during the disassembly process of an MT.

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability.

Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length determines microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region.

Effect of Nucleotide State on the Protofilament Conformation of Tubulin Octamers.

At the molecular level, the dynamic instability (random growth and shrinkage) of the microtubule (MT) is driven by the nucleotide state (GTP vs GDP) in the β subunit of the tubulin dimers at the MT cap. Here, we use large-scale molecular dynamics (MD) simulations and normal-mode analysis (NMA) to characterize the effect of a single GTP cap layer on tubulin octamers composed of two neighboring protofilaments (PFs). We utilize recently reported high-resolution structures of dynamic MTs to simulate a GDP octamer both with and without a single GTP cap layer. We perform multiple replicas of long-time atomistic MD simulations (3 replicas, 0.3 μs for each replica, 0.9 μs for each octamer system, and 1.8 μs total) of both octamers. We observe that a single GTP cap layer induces structural differences in neighboring PFs, finding that one PF possesses a gradual curvature, compared to the second PF which possesses a kinked conformation. This results in either curling or splaying between these PFs. We suggest that this is due to asymmetric strengths of longitudinal contacts between the two PFs. Furthermore, using NMA, we calculate mechanical properties of these octamer systems and find that octamer system with a single GTP cap layer possesses a lower flexural rigidity.

Tubulin isoform composition tunes microtubule dynamics.

Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility, and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2-Å cryo-electron microscopy (cryo-EM) structure and in vitro dynamics parameters of α1B/βI+βIVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared with brain microtubules. Cryo-EM reveals that the dynamic ends of α1B/βI+βIVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Last, we show that the addition of recombinant α1A/βIII tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of α1B/βI+βIVb microtubules. Our study is an important step toward understanding how tubulin isoform composition tunes microtubule dynamics.

Mechanisms of kinetic stabilization by the drugs paclitaxel and vinblastine.

Microtubule-targeting agents (MTAs), widely used as biological probes and chemotherapeutic drugs, bind directly to tubulin subunits and "kinetically stabilize" microtubules, suppressing the characteristic self-assembly process of dynamic instability. However, the molecular-level mechanisms of kinetic stabilization are unclear, and the fundamental thermodynamic and kinetic requirements for dynamic instability and its elimination by MTAs have yet to be defined. Here we integrate a computational model for microtubule assembly with nanometer-scale fluorescence microscopy measurements to identify the kinetic and thermodynamic basis of kinetic stabilization by the MTAs paclitaxel, an assembly promoter, and vinblastine, a disassembly promoter. We identify two distinct modes of kinetic stabilization in live cells, one that truly suppresses on-off kinetics, characteristic of vinblastine, and the other a "pseudo" kinetic stabilization, characteristic of paclitaxel, that nearly eliminates the energy difference between the GTP- and GDP-tubulin thermodynamic states. By either mechanism, the main effect of both MTAs is to effectively stabilize the microtubule against disassembly in the absence of a robust GTP cap.

TubZ filament assembly dynamics requires the flexible C-terminal tail

Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY.

Biophysical Studies of Liposome Encapsulated Pokeweed Antiviral Protein and its use as a HIV Therapeutic

Human Immunodeficiency Virus (HIV) is a virus that attacks the human immune system, compromising its effect in regards to disease prevention. HIV results in the destruction of CD4 cells, which are vital in the defense against human immune responses. HIV can severely damage the immune system and lead to Acquired Immunodeficiency Syndrome (AIDS). There is presently no cure or effective HIV vaccine and as a result it is important to seek alternative measures in the defense against HIV/AIDS. Pokeweed Antiviral Protein (PAP), a protein isolated from pokeweed plant, Phytolacca americana, provides a new and profoundly promising direction in the field of HIV/AIDS research. PAP plays a vital role in the immune system of pokeweed and is a ribosome inactivating protein (RIP), inhibiting viral protein production. PAP possesses antiviral properties, and reduces the infectivity of many plant and animal viruses, including HIV-1. My project focuses on mechanisms using liposomal encapsulated PAP, targeted to infected cells as a therapeutic for HIV infection. Steady state fluorescence was used to identify the thermodynamic parameters of interaction between PAP and HIV RNA. PAP binds to the m7 GTP cap and the 5′ UTR region of HIV RNA. Various isoforms of PAP were tested and the optimal affinity was identified as a Km of 20.09 nM. Fluorescence observed V.S [m7 GTP] was plotted through nonlinear regression using the Prism 6.0 program. Varying liposomes were prepared in order to identify the optimal characteristics for PAP encapsulation. Liposomes were lyophilized through rotary evaporation and later extruded to ensure homogeneity. Fluorescent titration enables the testing of liposomal PAP encapsulation efficiency. It was concluded that a 1/1 v% mixture of liposomes DOPE-DOTAP is most efficient for PAP encapsulation. These accomplishments will enable further modification of the liposomes by covalently conjugating HIV-specific monoclonal antibodies (anti-pg120 and/or anti-pg41), thus facilitating the selectivity of the liposomal PAP to the target tissues (HIV-infected CD4+ lymphocytes). With the aforementioned completed the basis for PAP-liposome interactions can be established and also the modulation of these interactions in order to target HIV-infected cells.

Structure of the NS5 methyltransferase from Zika virus and implications in inhibitor design

Recent outbreak of flavivirus Zika virus (ZIKV) in America has urged the basic as well as translational studies of this important human pathogen. The nonstructural protein 5 (NS5) of the flavivirus has an N-terminal methyltransferase (MTase) domain that plays critical roles in viral RNA genome capping. The null mutant of NS5 MTase is lethal for virus. Therefore, NS5 is a potential drug target for the treatment of Zika virus infection. In this study, we determined crystal structures of the ZIKV MTase in complex with GTP and RNA cap analogue 7meGpppA. Structural analyses revealed highly conserved GTP/cap-binding pocket and S-adenosylmethionine (SAM)-binding pocket. Two conformations of the second base of the cap were identified, which suggests the flexibility of RNA conformation. In addition, the ligand-binding pockets identified a continuous region of hotspots suitable for drug design. Docking calculation shows that the Dengue virus inhibitor compound 10 may bind to the ZIKV MTase.

GDP-to-GTP exchange on the microtubule end can contribute to the frequency of catastrophe.

Microtubules are dynamic polymers of αβ-tubulin that have essential roles in chromosome segregation and organization of the cytoplasm. Catastrophe-the switch from growing to shrinking-occurs when a microtubule loses its stabilizing GTP cap. Recent evidence indicates that the nucleotide on the microtubule end controls how tightly an incoming subunit will be bound (trans-acting GTP), but most current models do not incorporate this information. We implemented trans-acting GTP into a computational model for microtubule dynamics. In simulations, growing microtubules often exposed terminal GDP-bound subunits without undergoing catastrophe. Transient GDP exposure on the growing plus end slowed elongation by reducing the number of favorable binding sites on the microtubule end. Slower elongation led to erosion of the GTP cap and an increase in the frequency of catastrophe. Allowing GDP-to-GTP exchange on terminal subunits in simulations mitigated these effects. Using mutant αβ-tubulin or modified GTP, we showed experimentally that a more readily exchangeable nucleotide led to less frequent catastrophe. Current models for microtubule dynamics do not account for GDP-to-GTP exchange on the growing microtubule end, so our findings provide a new way of thinking about the molecular events that initiate catastrophe.