Cold-induced Disassembly Research Articles

Optimal Preservation of PFFs in Glycerol Enhances Suitability for Modeling Parkinson's Disease.

Injecting α-synuclein pre-formed fibrils (αSyn PFFs) into various tissues and organs involves converting monomeric αSyn into a fibrillar form, inducing extensive αSyn pathology that effectively models Parkinson's disease (PD). However, the distinct physicochemical properties of αSyn amyloid fibrils can potentially reduce their seeding activity, especially during storage. In this study, it is demonstrated that αSyn PFFs exhibit significant sensitivity to low temperatures, with notable denaturation occurring between -20 and 4°C, and gradual disassembly persisted even under storage conditions at -80°C. To mitigate this issue, a commonly used protein stabilizer, glycerol is introduced, which significantly reverses the cold-induced disassembly of PFFs. Remarkably, storing PFFs with 20% glycerol at -80°C for a month preserved their morphology and seeding activity as freshly prepared PFFs. Glycerol-stabilized αSyn PFFs resulted in compromised neuronal survival, with the extent of these impairments correlating with the formation of αSyn pathology both in vivo and in vitro, indistinguishable from freshly prepared PFFs. Storing sonicated PFFs with 20% glycerol at -80°C provides an optimal storage method, as sonication is necessary for activating their seeding potential. This approach reduces the frequency of sonication, simplifies handling, and ultimately lowers the overall workload, enhancing the practicality of using PFFs.

Tau isoform–specific stabilization of intermediate states during microtubule assembly and disassembly

The microtubule (MT)-associated protein tau regulates the critical growing and shortening behaviors of MTs, and its normal activity is essential for neuronal development and maintenance. Accordingly, aberrant tau action is tightly associated with Alzheimer's disease and is genetically linked to several additional neurodegenerative diseases known as tauopathies. Although tau is known to promote net MT growth and stability, the precise mechanistic details governing its regulation of MT dynamics remain unclear. Here, we have used the slowly-hydrolyzable GTP analog, guanylyl-(α,β)-methylene-diphosphonate (GMPCPP), to examine the structural effects of tau at MT ends that may otherwise be too transient to observe. The addition of both four-repeat (4R) and three-repeat (3R) tau isoforms to pre-formed GMPCPP MTs resulted in the formation of extended, multiprotofilament-wide projections at MT ends. Furthermore, at temperatures too low for assembly of bona fide MTs, both tau isoforms promoted the formation of long spiral ribbons from GMPCPP tubulin heterodimers. In addition, GMPCPP MTs undergoing cold-induced disassembly in the presence of 4R tau (and to a much lesser extent 3R tau) also formed spirals. Finally, three pathological tau mutations known to cause neurodegeneration and dementia were differentially compromised in their abilities to stabilize MT disassembly intermediates. Taken together, we propose that tau promotes the formation/stabilization of intermediate states in MT assembly and disassembly by promoting both longitudinal and lateral tubulin-tubulin contacts. We hypothesize that these activities represent fundamental aspects of tau action that normally occur at the GTP-rich ends of GTP/GDP MTs and that may be compromised in neurodegeneration-causing tau variants.

Safranal Inhibits HeLa Cell Viability by Perturbing the Reassembly Potential of Microtubules.

Saffron, a spice from Crocus sativus, has been known for its health benefits and medicinal properties. Safranal is a component of saffron and is known for its antioxidant and anticancer properties. In this study, we elucidated a possible tubulin-targeted antiproliferative mechanism of action of safranal. In vitro, the compound perturbed secondary structure of tubulin without altering net microtubule polymer mass. It inhibited HeLa cell viability in a concentration-dependent manner, with minimal damage to cellular microtubules. However, it strongly inhibited recovery of microtubule network after cold-induced disassembly, indicating its ability to interfere with the nucleation potential of tubulin. Further, as the acetylation pattern of the safranal-treated microtubules revealed, unlike many tubulin-targeted agents, the compound did not appear to induce persistent stabilization of microtubules. Our data shows an unusual, tubulin-targeted antiproliferative mechanism of safranal. Copyright © 2017 John Wiley & Sons, Ltd.

Thermal Memory in Self-Assembled Collagen Fibril Networks

Collagen fibrils form extracellular networks that regulate cell functions and provide mechanical strength to tissues. Collagen fibrillogenesis is an entropy-driven process promoted by warming and reversed by cooling. Here, we investigate the influence of noncovalent interactions mediated by the collagen triple helix on fibril stability. We measure the kinetics of cold-induced disassembly of fibrils formed from purified collagen I using turbimetry, probe the fibril morphology by atomic force microscopy, and measure the network connectivity by confocal microscopy and rheometry. We demonstrate that collagen fibrils disassemble by subunit release from their sides as well as their ends, with complex kinetics involving an initial fast release followed by a slow release. Surprisingly, the fibrils are gradually stabilized over time, leading to thermal memory. This dynamic stabilization may reflect structural plasticity of the collagen fibrils arising from their complex structure. In addition, we propose that the polymeric nature of collagen monomers may lead to slow kinetics of subunit desorption from the fibril surface. Dynamic stabilization of fibrils may be relevant in the initial stages of collagen assembly during embryogenesis, fibrosis, and wound healing. Moreover, our results are relevant for tissue repair and drug delivery applications, where it is crucial to control fibril stability.

Aurora B Regulates Formin mDia3 in Achieving Metaphase Chromosome Alignment

Proper bipolar attachment of sister kinetochores to the mitotic spindle is critical for accurate chromosome segregation in mitosis. Here we show an essential role of the formin mDia3 in achieving metaphase chromosome alignment. This function is independent of mDia3 actin nucleation activity, but is attributable to EB1-binding by mDia3. Furthermore, the microtubule binding FH2 domain of mDia3 is phosphorylated by Aurora B kinase invitro, and cells expressing the nonphosphorylatable mDia3 mutant cannot position chromosomes at the metaphase plate. Purified recombinant mDia3 phosphorylated by Aurora B exhibits reduced ability to bind microtubules and stabilize microtubules against cold-induced disassembly invitro. Cells expressing the phosphomimetic mDia3 mutant do not form stable kinetochore microtubule fibers; despite they are able to congress chromosomes to the metaphase plate. These findings reveal a key role for mDia3 and its regulation by Aurora B phosphorylation in achieving proper stable kinetochore microtubule attachment.

Microtubule Association of the Neuronal p35 Activator of Cdk5

Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to alpha/beta-tubulin and microtubules. Microtubule polymers but not the alpha/beta-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to cold-induced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.

ARP2 and ARP3 are localized to sites of actin filament nucleation in tobacco BY-2 cells

Complete depolymerization of actin filaments (AFs) at low temperature (0 degrees C) is followed by the formation of transient actin structures at 25 degrees C in tobacco BY-2 cells (Nicotiana tabacum L.). Using antibodies against fission yeast actin-related proteins (ARP2 and ARP3), we show here that transient actin structures (dots, dotted filaments, rods) colocalize with epitopes stained by these antibodies and thus are likely to represent sites of actin filament nucleation (SANs). In contrast to the cold-induced disassembly of AFs, no transient actin structures were detectable during recovery of AFs from latrunculin B-induced depolymerization. However, the staining pattern obtained with ARP antibodies in latrunculin B-treated cells was similar to that in controls and cold-treated cells. This suggests that, in addition to the complete depolymerization of AFs, disruption of other cellular structures is needed for the formation of transient actin structures during the early phase of recovery from cold treatment.

The centrosome-nucleus complex and microtubule organization in theDrosophilaoocyte

Molecular motors transport the axis-determining mRNAs oskar, bicoid and gurken along microtubules (MTs) in the Drosophila oocyte. However, it remains unclear how the underlying MT network is organized and how this transport takes place. We have identified a centriole-containing centrosome close to the oocyte nucleus. Remarkably, the centrosomal components, gamma-tubulin and Drosophila pericentrin-like protein also strongly accumulate at the periphery of this nucleus. MT polymerization after cold-induced disassembly in wild type and in gurken mutants suggests that in the oocyte the centrosome-nucleus complex is an active center of MT polymerization. We further report that the MT network comprises two perpendicular MT subsets that undergo dynamic rearrangements during oogenesis. This MT reorganization parallels the successive steps in localization of gurken and oskar mRNAs. We propose that in addition to a highly polarized microtubule scaffold specified by the cortex oocyte, the repositioning of the nucleus and its tightly associated centrosome could control MT reorganization and, hence, oocyte polarization.

Microtubule organization and distribution of gamma-tubulin in male meiosis of lepidoptera

Meiotic spindles in males of higher Lepidotera are unusual in that the bulk of the spindle microtubules (MTs) ends about halfway between the equatorial plate and the centrosomes in metaphase. It appears worthwhile to determine how the MTs are nucleated, while their pole proximal ends are distant from the centrosomes. To this end, spermatocytes of Phragmatobia fuliginosa (Arctiidae), collected in the field, were double-labeled with antibodies to beta- and gamma-tubulin. The former antibody reveals the entire microtubular cytoskeleton, and the latter is directed against a newly-discovered tublin isoform that is prevalent in microtubule-organizing centers (MTOCs). The immunocytochemical work was supplemented by a fine structural analysis of MTOCs and spindles. Gamma-tubulin was clearly detected at the spindle poles, and prominent microtubular asters originated from these sites. Additionally, MT arrays at both sides of the equatorial plate in metaphase spermatocytes contained gamma-tubulin. The staining persisted in late anaphase, when kinetochore MTs are depolymerized. This indicates that at least nonkinetochore MTs contain gamma-tubulin. The analysis of ultrathin sections through spindles revealed large amounts of pericentriolar material at the spindles poles, in prometaphase through anaphase. The spindle MTs appeared as regular, straight elements in longitudinal sections. We assume that gamma-tubulin is located at the pole proximal ends of the MTs and/or is associated with the spindle MTs throughout their lengths. In order to distinguish between these possibilities, testes of Ephestia kuehniella (Pyralidae), a laboratory species, were cold-treated prior to double-labeling with antibodies to beta- and gamma-tubulin. The treatment was expected to depolymerize MTs. Astral MTs, which were nucleated end-on by gamma-tubulin-containing material, indeed depolymerized. In contrast, the gamma-tubulin-containing spindle MTs persisted. It is, therefore, conceivable that gamma-tubulin is associated with MTs throughout their lengths in male meiosis of Lepidoptera species. It is plausible that this association stabilizes the MTs against cold-induced disassembly.

Orientation, assembly, and stability of microtubule bundles induced by a fragment of tau protein.

The neuronal microtubule-associated protein tau has been implicated in the development of axonal morphology including the organization of microtubules into a uniformly oriented array of microtubules commonly referred to as "bundle." Determination of the functional organization of tau has revealed that regions of tau protein which flank the microtubule-binding domain affect the bundling of microtubules in vitro with a microtubule-binding fragment of tau being most effective [Brandt and Lee, 1993: J. Biol. Chem. 268:3414-3419]. In order to study the relation of microtubule bundles that form in vitro to those observed in the axon, we determined the orientation of individual microtubules in bundles and the effects of bundling on microtubule assembly and stability in cell-free assembly reactions. Here we report that bundles induced by a microtubule-binding fragment of tau contain randomly oriented microtubules as determined by using the difference in growth rates at microtubule plus and minus ends. We demonstrate that in vitro bundling increases microtubule growth (about 30%), stabilizes microtubules against dilution- and cold-induced disassembly, and allows microtubule nucleation despite the absence of a tau region which has previously been shown to be required for tau-dependent microtubule nucleation. We conclude that conditions that stabilize microtubules can lead to bundle formation and allow microtubule assembly by a mechanism different from that employed by microtubule-associated proteins. The data also support the view that additional mechanisms besides the action of tau and tubulin exist in order to organize microtubules in the axon.

Rhazinilam mimics the cellular effects of taxol by different mechanisms of action

We have investigated the effects of the microtubule poison rhazinilam on microtubule assembly in vivo and in vitro. In mammalian cells, rhazinilam mimics the effects of taxol and leads to microtubule bundles, multiple asters, and microtubule cold stability. In vitro, rhazinilam protected preassembled microtubules from cold-induced disassembly, but not from calcium ion-induced disassembly. Moreover, both at 0 degrees C and at 37 degrees C, rhazinilam induced the formation of anomalous tubulin assemblies (spirals). This process was prevented by maytansine and vinblastine, but not by colchicine. Preferential saturable and stoichiometric binding of radioactive rhazinilam to tubulin in spirals was observed with a dissociation constant of 5 microM. This binding was abolished in the presence of vinblastine and maytansine. In contrast, specific binding of radioactive rhazinilam to tubulin assembled in microtubules was undetectable. These results demonstrate that rhazinilam alters microtubule stability differently than taxol, and that the overall similar effects of rhazinilam and taxol on the cellular cytoskeleton are the consequence of two distinct mechanisms of action at the molecular level.

Differences in the abilities of human tau isoforms to promote microtubule assembly

Three isoforms of human tau protein were compared for their abilities to induce microtubule assembly. The three isoforms, tau 3 (tau containing three microtubule-binding domains), tau 4 (tau containing four microtubule-binding domains) and tau 4L (tau containing four microtubule binding domains plus a 58-amino-acid insert near the N-terminus) were expressed in E. coli and purified using ammonium sulfate precipitation, ion exchange, and size exclusion chromatography. All three isoforms induced microtubule assembly at micromolar concentrations and showed similar critical concentrations for assembly of 0.4-0.45 microM. However, tau 4 induced microtubule formation at a rate five- to tenfold faster than either tau 3 or tau 4L. The rate of microtubule elongation seen with tau 4 was twofold greater than with tau 3 or tau 4L, suggesting that the faster rate of microtubule assembly seen with tau 4 was due, at least in part, to faster elongation. Tau 4 induced a greater number of microtubules to form at steady state than did tau 3 or tau 4L. The microtubules generated with each tau isoform had similar steady-state length distributions and were equally susceptible to cold-induced disassembly. These results indicate that the additional microtubule-binding domain in tau 4 enhances microtubule assembly, while the 58-amino-acid insert negates the stimulatory effect of the fourth microtubule-binding domain.

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

Tubulin, but not microtubules, is the substrate for tubulin:tyrosine ligase in mature avian erythrocytes.

Chicken erythrocytes, which contain a marginal band of microtubules, were used to study the influence of the aggregation state of tubulin on the post-translational incorporation of tyrosine into the alpha-tubulin subunit. We found that the incorporation of [14C]tyrosine occurs almost exclusively into the nonassembled tubulin pool. The marginal band was practically not labeled. The low incorporation into microtubules was not due to the lack of tubulin with acceptor capacity since after cold-induced disassembly, an additional amount of [14C]tyrosine could be incorporated. 14C-Tyrosinated tubulin of the nonassembled pool could not be incorporated into microtubules of the marginal band after prolonged incubation at 37 degrees C or when the marginal band was regenerated after cold-induced depolymerization. In erythrocytes, tubulin:tyrosine ligase behaved as a soluble entity when the cells were lysed under microtubule-preserving conditions.

Cytoplasmic microtubule assembly-disassembly from endogenous tubulin in a Brij-lysed cell model.

We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-lysed 3T3 cell system. Cells that were pretreated in vivo with colcemid retain endogenous tubulin in the depolymerized state after lysis. When lysed cells were removed from colcemid block and incubated in GTP-PIPES reassembly buffer at pH 6.9, microtubules repolymerized randomly throughout the cytoplasm, appeared to be free-ended and were generally not associated with the centrosomes. However, tubulin could be induced to polymerize in an organized manner from the centrosomes by increasing the pH to 7.6 in the presence of ATP and cAMP. Microtubules polymerized in ATP had significantly longer lengths than those assembled in GTP or UTP. When cells not treated with colcemid were lysed, the integrity of the cytoplasmic microtubule complex (CMTC) was maintained during subsequent incubation in reassembly buffer. However, in contrast to unlysed, living cells, microtubules of lysed cells were stable to colchicine. A significant fraction of the CMTC was stable to cold-induced disassembly whereas microtubules reassembled after lysis were extremely cold-sensitive. When cells not treated with colcemid were lysed and incubated in millimolar Ca++, microtubules depolymerized from their distal ends and a much reduced CMTC was observed. Ca++ reversal with EGTA rapidly resulted in a reformation of the CMTC apparently by elongation of Ca++ resistant microtubules.

Microtubule interactions with GDP provide evidence that assembly-disassembly properties depend on the method of brain microtubule protein isolation

Microtubule stability in the presence of GDP was found to depend on the microtubule protein purification method used, even though all are variations on the use of warm-induced assembly and cold-induced disassembly with intervening centrifugation steps. Substantial differences in the assembly-disassembly properties were observed with protein prepared in the absence or presence of glycerol under hypotonic extraction conditions as compared to the microtubule protein from a sucrose extraction protocol which minimizes contamination resulting from osmotic shock of cellular organelles. Assembled protein from either hypotonic preparation partially maintained its state of assembly after GDP addition. With protein from the sucrose method, assembled tubules are almost completely depolymerized upon enzymatic conversion of GTP to GDP. The characteristics of the elongation reaction following addition of GDP to assembling microtubule protein also depend on the isolation method. We discuss the conflicting claims in the literature on microtubule interactions with GDP in the light of these new findings.

3′,5′-cyclic AMP binds to and promotes polymerisation on platelet tubulin

IN spite of considerable research, the regulatory mechanism of the reversible assembly–disassembly of microtubules remains obscure. Of the various processes and agents which have been examined as potential regulators probably none has received as much attention as cyclic nucleotides. Indeed, the number of reports which indicate that adenosine 3′,5′-cyclic monophosphate (cyclic AMP) exerts a stimulatory effect in the formation of microtubules, or maintains the latter in the presence of dissociative influences is steadily growing1–13. Most of these studies used intact cells, however, leaving unclear whether the cyclic nucleotide was directly or indirectly involved in this process. To try to resolve this problem, I have used tubulin extracted from platelets: addition of exogenous cyclic AMP has been reported to prevent cold-induced disassembly of microtubules in about 50% of the platelets14. I show here that cyclic AMP stimulates the assembly of microtubules and binds to platelet tubulin, primarily to the α-subunit.