Microtubules are capital intracellular polymers, congenital from tubulin subunits, that authorize corpuscle shape, move organelles, and choose chromosomes during corpuscle division. Vemu et al. appearance that microtubule-severing enzymes abstract tubulin subunits forth the microtubule shaft. This nanoscale accident is repaired by the assimilation of chargeless tubulin, which stabilizes the microtubule adjoin depolymerization. Back abstraction outpaces repair, microtubules are severed, arising with counterbalanced ends composed of beginning tubulin. The burst microtubules act as templates for new microtubule growth, arch to accession of microtubule cardinal and mass. Thus, acutely paradoxically, disengagement enzymes can access microtubule accumulation in processes such as neurogenesis and mitotic arbor assembly.
Science, this affair p. eaau1504
The microtubule cytoskeleton is continuously sculpted by polymerization, depolymerization, cross-linking, and microtubule severing. Microtubule disengagement generates centralized break in microtubules through a ailing accepted mechanism. It is advised by the AAA [adenosine triphosphatases (ATPases) associated with assorted cellular activities] ATPases katanin, spastin, and fidgetin. Paradoxically, admitting the annihilative activity of disengagement enzymes, accident of disengagement agitator activity leads to a abatement rather than an access in microtubule mass. It was accepted that this disengagement enzyme–dependent access in microtubule accumulation after-effects from templated nucleation from the burst ends. This is an adorable antecedent for a apparatus to accomplish microtubule mass, abnormally in the absence of centrosome-based nucleation as in neurons or meiotic spindles. However, for this accession to operate, the guanosine diphosphate (GDP)–tubulin filigree apparent through disengagement would accept to be stabilized. The GDP-microtubule filigree is the artefact of guanosine triphosphate (GTP)–tubulin polymerization and depolymerizes spontaneously back apparent in the absence of a stabilizing GTP cap. We advised this absurdity by analytical the furnishings of the disengagement enzymes spastin and katanin on microtubule anatomy and dynamics in vitro.
Because ablaze microscopy–based disengagement assays abort to abduction ultrastructural appearance of disengagement intermediates due to resolution limitations, we acclimated negative-stain chiral electron microscopy (TEM) to abduction and angel spastin- and katanin-mediated microtubule disengagement in vitro. We accumulated these abstracts with quantitative analyses of tubulin and microtubule polymer dynamics by application absolute centralized absorption fluorescence (TIRF) microscopy to accept the furnishings of disengagement on microtubule networks.
Our electron microscopy analyses accompanying with TIRF microscopy appear that spastin and katanin actively abstract tubulin dimers out of the microtubule, introducing nanoscale accident forth the microtubule, and that this activity is counteracted by spontaneous, de novo assimilation of GTP-tubulin dimers from the acrid pool. Depending on the bounded antithesis amid the ante of alive tubulin abstraction and acquiescent repair, there are two non–mutually absolute consequences: The microtubule is ambulatory with GTP-tubulin islands that antithesis it adjoin depolymerization, or disengagement gain to achievement and the anew burst microtubule ends appear with a aerial body of stabilizing GTP-tubulin. Constant with this, we begin that spastin and katanin activities access ante of microtubule accomplishment and that rescues activity preferentially at sites of enzyme-dependent GTP-tubulin incorporation. Lastly and unexpectedly, we begin that the assimilation of GTP-tubulin at disengagement sites ensures that the anew burst additional ends are abiding because they appear with a aerial body of GTP-tubulin that protects them adjoin ad-lib depolymerization and promotes elongation. The synergy amid the added accomplishment ante and the stabilization of the anew burst ends leads to microtubule amplification.
Our abstraction identifies the microtubule-severing enzymes spastin and katanin as biological agents that acquaint GTP-tubulin islands aural microtubules and demonstrates that microtubule-severing enzymes abandoned can amplify microtubule cardinal and accumulation by announcement GTP-tubulin assimilation into the microtubule shaft, abroad from the activating ends continued anticipation to be the sole locus of tubulin exchange. This microtubule-based accession apparatus in the absence of a nucleating agency helps explain why the accident of spastin and katanin after-effects in the accident of microtubule accumulation in systems that are abased on noncentrosomal microtubule generation.
Spastin and katanin bisect and destabilize microtubules. Paradoxically, admitting their annihilative activity they access microtubule accumulation in vivo. We accumulated single-molecule absolute centralized absorption fluorescence microscopy and electron microscopy to appearance that the basal footfall in microtubule disengagement is the bearing of nanoscale accident throughout the microtubule by alive abstraction of tubulin heterodimers. These accident sites are repaired spontaneously by guanosine triphosphate (GTP)–tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin access microtubule accomplishment rates. Furthermore, anew burst ends appear with a aerial body of GTP-tubulin that protects them adjoin depolymerization. The stabilization of the anew burst additional ends and the college accomplishment abundance synergize to amplify microtubule cardinal and mass. Thus, disengagement enzymes adapt microtubule architectonics and dynamics by announcement GTP-tubulin assimilation aural the microtubule shaft.
The bendability of the microtubule cytoskeleton follows from assorted levels of adjustment through microtubule-end polymerization and depolymerization, cross-linking, and microtubule severing. Microtubule disengagement generates centralized break in microtubules. It is advised by three enzymes of the AAA [adenosine triphosphatases (ATPases) associated with assorted cellular activities] ATPase family—katanin, spastin, and fidgetin [reviewed in (1)]—that are broadly conserved in animals and plants. They are analytical for the bearing and aliment of circuitous noncentrosomal microtubule arrays in neurons (2–5) and the bulb case (6–8) and adapt meiotic and mitotic arbor appraisal and breadth (9–12), cilial biogenesis (13, 14), axis duplication (14, 15), cytokinesis (16, 17), axonal advance (18), anguish healing (19), and bulb phototropism (7, 8). Both spastin and katanin are associated with debilitating diseases. Spastin is mutated in ancestral spastic paraplegias, neurodegenerative disorders characterized by lower-extremity weakness due to axonopathy [reviewed in (1)]. Katanin mutations account microcephaly, seizures, and astringent adorning defects (14, 15, 20). Ache mutations blemish microtubule disengagement (21, 22).
Paradoxically, in abounding of these systems, the accident of the microtubule-severing agitator leads to a abatement in microtubule accumulation [reviewed in (1)]. Spastin accident causes dispersed chaotic microtubule arrays at Drosophila synaptic boutons (2) and broken axonal bulge and dispersed microtubule arrays in adjudicator angle axons (23). Similarly, katanin accident leads to dispersed cortical microtubule arrays in Arabidopsis (8, 24), admitting in Caenorhabditis elegans meiotic spindles, it after-effects in the accident of microtubule accumulation and cardinal (25). It was accepted that the empiric access in microtubule cardinal and accumulation after-effects from templated nucleation from the burst ends (26, 27). This is an adorable apparatus for rapidly breeding microtubule mass, abnormally in the absence of centrosome-based nucleation as in neurons or meiotic spindles. This severing-dependent microtubule accession has been anon empiric in bulb cortical microtubule arrays (8). However, for this accession to operate, the guanosine diphosphate (GDP)–tubulin filigree apparent through disengagement would accept to be counterbalanced because GDP-microtubules depolymerize spontaneously in the absence of a stabilizing guanosine triphosphate (GTP) cap (28–31). To abstraction this paradox, we accumulated time-resolved chiral electron microscopy (TEM) and absolute centralized absorption fluorescence (TIRF) microscopy to anon beam the furnishings of the disengagement enzymes spastin and katanin on microtubule anatomy and dynamics in vitro.
Because ablaze microscopy–based disengagement assays abort to abduction ultrastructural appearance of disengagement intermediates due to resolution limitations, we acclimated negative-stain TEM to abduction and angel spastin-mediated microtubule-severing intermediates in vitro with purified, recombinant spastin. To abbreviate severing-intermediate breakage, we performed disengagement reactions anon on electron microscopy (EM) grids. These on-grid reactions appear a aerial body of “bites” into the protofilament anatomy (Fig. 1) that resulted in the abatement of tubulin dimers. Disengagement reactions performed in a appraisal tube with Taxol-stabilized microtubules that were afresh transferred to EM grids by pipetting produced abounding abbreviate microtubules with edgeless ends (fig. S1A), agnate to those ahead appear in vitro with katanin (32), advertence that the brittle nanoscale-damaged disengagement intermediates are absent during pipetting. Thus, in our on-grid disengagement setup, we were able to abduction intermediates that were contrarily disrupted by microburst armament alien by pipetting. Aloft abiding evolution (>5 min), disengagement was apprenticed to achievement on the EM grid, with astringent aition of the microtubule anatomy advertence that the intermediates empiric were on alleyway (fig. S1B). The nanoscale accident sites were empiric with GDP-microtubules behindhand of whether they were nonstabilized or counterbalanced with Taxol (Fig. 1, A and B). Agnate observations were fabricated with microtubules polymerized with the nonhydrolyzable analog guanylyl (α,β)-methylene diphosphonate (GMPCPP) (Fig. 1C). The nanoscale accident we empiric in vitro is evocative of that empiric by electron tomography in freeze-substituted C. elegans meiotic spindles (25). The aforementioned abstraction of tubulin dimers and protofilament fraying were empiric if reactions were performed in band-aid and afresh microtubules were deposited on an EM filigree afterwards pipetting to abstain microburst (Fig. 1D and Abstracts and methods). In ascendancy reactions afterwards the enzyme, the candor of the filigree was preserved (fig. S1, C and D), admitting in the spastin-treated samples, nanoscale accident sites were detected every ~2.2 μm (fig. S1D). Time-course abstracts appear a bit-by-bit access in nanoscale damage, as able-bodied as in the cardinal of beneath microtubules (Fig. 1E). We continued our TEM analyses to the microtubule-severing agitator katanin (Fig. 1, F and G, and fig. S1, E to H). As with spastin, TEM appear that katanin microtubule disengagement gain through accelerating abstraction of tubulin dimers out of the microtubule.
(A to C) Microtubules in the absence or attendance of 33 nM spastin. The acknowledgment proceeded on an EM filigree for 1 min and was beheld by application negative-stain TEM (Materials and methods). Boxed regions are apparent at 2× deepening in insets. Microtubules were beheld at 30,000× magnification. Arrows announce nanoscale accident sites. Calibration bar, 50 nm. (D) Fields of GMPCPP-microtubules incubated with absorber or 25 nM spastin. Disengagement proceeded in solution, and acknowledgment mixtures were irenic deposited assimilate EM grids, abnormally stained, and visualized by TEM (Materials and methods). Arrows announce nanoscale damage. Microtubules were beheld at 13,000× magnification; boxed regions are apparent at 30,000× deepening in insets. Calibration bar, 50 nm. (E) Microtubule breadth administration afterwards evolution with spastin. (F) Fields of GMPCPP-microtubules incubated with absorber or 100 nM katanin and beheld as in (D). Calibration bar, 50 nm. (G) Microtubule breadth administration afterwards evolution with katanin.
Our TEM appraisal showed that GMPCPP-microtubules, Taxol-stabilized microtubules, or nonstabilized microtubules do not bisect alike back brindled with spastin- and katanin-induced nanoscale accident and do not catastrophically depolymerize aloft abatement of the antecedent tubulin subunits. This aloft the achievability that this accident could be repaired by assimilation of tubulin subunits from the acrid pool, as afresh empiric with mechanically damaged or photodamaged microtubules in vitro (33, 34). To appraisal this hypothesis, we preassembled GMPCPP-microtubules fluorescently labeled with HiLyte 647 and incubated them with spastin (or katanin) and adenosine triphosphate (ATP) to admit disengagement (Materials and methods). Beneath these conditions, we empiric attenuate disengagement contest (Fig. 2). Aloft perfusion with acrid HiLyte 488–labeled tubulin and GTP, we empiric tubulin assimilation in detached patches forth microtubules. These patches were numerous, far beyond the cardinal of disengagement events. Mock-treated microtubules showed no assimilation of tubulin into microtubules (Fig. 2, A to C). The tubulin absorption acclimated was beneath the analytical absorption for tubulin polymerization. Agnate after-effects were acquired with Taxol-stabilized microtubules (fig. S2). Because photodamage can abet filigree defects in fluorescently labeled microtubules (34), we additionally performed abstracts with unlabeled microtubules visualized by cogwheel arrest adverse (DIC) microscopy and additionally empiric assimilation of tubulin into spastin-treated microtubules but not into controls (Fig. 2D).
(A and B) HiLyte 647–labeled GMPCPP-microtubules (MT) incubated with absorber (A) or 10 nM spastin (B) for 35 s and afresh incubated with 1 μM HiLyte 488–labeled GTP-tubulin and done to aish antithesis tubulin (Materials and methods). Arrowheads announce disengagement events. Calibration bar, 5 μm. (C) HiLyte 647–labeled GMPCPP-microtubules incubated with 2 nM katanin for 90 s and afresh incubated with 1 μM HiLyte 488–labeled GTP-tubulin and done to aish antithesis tubulin (Materials and methods). Arrowheads announce disengagement events. (D) DIC-imaged unlabeled GMPCPP-microtubules incubated with 10 nM spastin and afresh with 1 μM HiLyte 488–labeled GTP-tubulin (cyan) and done to aish antithesis tubulin (Materials and methods). Insets accord to boxed areas and appearance the progression to a disengagement event. Arrowheads announce the disengagement site.
In time-course experiments, both the cardinal of repaired nanoscale accident sites and the beggarly fluorescence forth repaired microtubules added over time (figs. S3, A and B, and S4, A and B). The admeasurement of the adjustment sites [full amplitude at bisected best (FWHM)] (figs. S3C and S4C) was initially diffraction apprenticed and confused adjoin beyond ethics at best evolution times, advertence an accession of the accident as detected by acrid GTP-tubulin incorporation. Frequent nanoscale accident contest were arresting back disengagement contest were acutely sparse: As aboriginal as 35 s, the body of spastin-induced nanoscale accident sites was 0.35 ± 0.01 μm−1, compared with 0.0008 ± 0.0004 μm−1 for disengagement contest (fig. S3, A and D). Thus, best nanoscale accident contest did not advance to arresting disengagement events. Once a acceptable cardinal of tubulin dimers was removed from the lattice, the microtubule unraveled and a arresting disengagement accident was visible. Constant with this, we empiric an brusque access in mesoscale disengagement at 120 and 90 s for spastin and katanin, appropriately (figs. S3D and S4D).
Next, we probed the aftereffect of acrid tubulin on spastin microtubule disengagement by assuming disengagement assays in the attendance of fluorescently labeled acrid tubulin (fig. S5). This accustomed us to ascertain microtubule nanoscale accident and disengagement simultaneously. Spastin-induced disengagement was not decidedly afflicted with 100 nM tubulin, alike admitting we empiric assimilation of HiLyte 488–labeled tubulin into microtubules (Fig. 3A, fig. S5, and Abstracts and methods). However, disengagement was appreciably bargain in the attendance of 2 μM acrid tubulin (Fig. 3A), and in this case, tubulin fluorescence acuteness at adjustment sites was additionally decidedly college (Fig. 3B). Thus, the tubulin abstraction activity of the agitator was not decidedly inhibited by acrid tubulin as proposed ahead for katanin (35), but the amount of tubulin assimilation at nanoscale accident sites added with tubulin concentration. This college amount of tubulin assimilation at accident sites delays (and can alike prevent) the achievement of a disengagement event. Constant with this, the time amid the assimilation of HiLyte 488–tubulin at a nanoscale accident armpit and the achievement of a disengagement accident was best in the attendance of 2 μM tubulin than in the attendance of 100 nM tubulin (Fig. 3C). Thus, admitting about all nanoscale accident sites apparent beneath our beginning altitude proceeded to complete disengagement aural 65 s afterwards tubulin assimilation in the attendance of 100 nM acrid tubulin, alone 47% did so at 2 μM tubulin (Fig. 3D). We additionally monitored alive the accession of audible fluorescently labeled tubulin dimers by TIRF microscopy (Fig. 3E and Abstracts and methods). Fluorescence acuteness analyses appear that adjustment proceeded mainly through the assimilation of tubulin heterodimers and not through the accession of beyond tubulin polymers or aggregates because the fluorescence acuteness administration of congenital tubulin was agnate to that of audible tubulin subunits anchored to bottle (Fig. 3F).
(A) Disengagement ante in the attendance of acrid tubulin (n = 31, 28, and 36 microtubules from assorted accommodation for no tubulin, 100 nM tubulin, and 2 μM tubulin, respectively). Thin curve announce SEM. (B) Acuteness administration of beaming tubulin puncta congenital at spastin-induced nanoscale accident sites (n = 50 and 49 puncta from assorted accommodation for 100 nM and 2 μM tubulin, respectively). Confined announce the agency and SD. A.U., approximate units. (C) Adjustment at accident sites delays disengagement (n = 81 and 83 disengagement contest from assorted accommodation for 100 nM and 2μM tubulin, respectively). Tsevering and Tincorporation, time appropriate for a disengagement accident and for antecedent assimilation of tubulin, respectively. (D) Fraction of GMPCPP-microtubules burst by 20 nM spastin aural 65 s of antecedent tubulin assimilation in the attendance of 100 nM and 2 μM HiLyte 488–labeled acrid tubulin. Error confined announce SEM in (C) and (D). (E) Alive imaging of Alexa 488–labeled GTP-tubulin (cyan) assimilation into HiLyte 647–labeled GMPCPP-microtubules (magenta) afterwards spastin-induced damage. Calibration bar, 1.5 μm. (F) Fluorescence acuteness administration of Alexa 488–labeled tubulin (labeling ratio, ~1.0) anchored on bottle or congenital into spastin-induced nanoscale accident sites (n = 188 and 398 for glass-immobilized and microtubule-incorporated particles, respectively). (G) Spastin-induced nanoscale accident and ad-lib tubulin adjustment of GDP-microtubules (magenta) developed from axonemes and counterbalanced with a GMPCPP cap (bright cyan) in the attendance of spastin (5 nM) and 5 μM acrid HiLyte 488–labeled GTP-tubulin (cyan). Images were achromatize corrected. White arrowheads, tubulin assimilation sites; chicken arrowheads, disengagement events. Calibration bar, 5 μm. (H) Average achievement time of a disengagement accident afterwards spastin perfusion. Brown, GMPCPP-microtubules; gray, GMPCPP-capped GDP-microtubules in the absence or attendance of acrid tubulin (n = 36, 63, 34, and 27 microtubules from assorted accommodation for GMPCPP-microtubules and GMPCPP-capped GDP-microtubules with 0, 2 μM, and 5 μM acrid GTP-tubulin, respectively). Confined announce the agency and SD. ****P < 0.0001, bent by a two-tailed t test, for (B), (C), (D), and (H).
To aphorism out adjustment as an antiquity of alive with counterbalanced microtubules (either Taxol or GMPCPP stabilized), we continued our abstracts to nonstabilized GDP-microtubules. We polymerized GDP-microtubules from axonemes and counterbalanced their ends with a GMPCPP cap to abstain ad-lib depolymerization (Materials and methods). We afresh alien spastin in the absence or attendance of fluorescently labeled acrid GTP-tubulin. Aural 50 s of introducing 5 nM spastin and 5 μM acrid tubulin [tubulin concentrations in vivo are 5 to 20 μM (36, 37)], we empiric the assimilation of tubulin as puncta forth microtubules (Fig. 3G and cine S1). At these agitator and tubulin concentrations, best tubulin assimilation sites did not advance to a disengagement event, and the disengagement amount was appreciably lower than in the absence of acrid tubulin (Fig. 3H). However, tubulin assimilation consistently preceded microtubule severing. No adjustment sites were empiric in the absence of spastin. Thus, the bounded antithesis amid alive tubulin abatement catalyzed by the agitator and acquiescent tubulin assimilation determines whether a nanoscale accident armpit progresses to a mesoscale disengagement accident or fails to do so because of the adjustment with GTP-tubulin from the acrid pool.
We additionally visualized the lattice-incorporated tubulin at a college resolution by application TEM. We generated recombinant animal α1AβIII tubulin with an engineered FLAG tag at the β-tubulin C aals (38). We afresh acclimated this recombinant tubulin to adjustment academician microtubules damaged at the nanoscale by spastin. The attendance of the FLAG tag on the recombinant tubulin accustomed specific apprehension of recombinant tubulin both in fluorescence and TEM images with the use of beaming or gold-conjugated accessory antibodies adjoin FLAG antibodies (Materials and methods). Fluorescence microscopy appear that the recombinant tubulin robustly incorporates forth microtubules with nanoscale accident by spastin with ATP. No assimilation was detected with spastin and adenosine 5′-O-(3-thiotriphosphate) (ATP-γ-S) (fig. S6). TEM showed the discrete, advantageous assimilation of recombinant α1AβIII tubulin in islands forth microtubules and the absence of tubulin aggregates at nanoscale accident sites (fig. S7). The FLAG primary and accessory gold-conjugated antibodies are specific for the recombinant tubulin, as academician microtubules showed alone accomplishments antibiotic adornment (fig. S7, C and D). In the absence of recombinant acrid tubulin in the reaction, microtubules were robustly damaged at the nanoscale beneath these altitude (fig. S7E). Moreover, neither recombinant tubulin assimilation nor affiliation with the microtubule filigree was empiric by fluorescence and TEM assays with the slow-hydrolyzing analog ATP-γ-S (figs. S6 and S7, A and C). Thus, acrid tubulin was congenital productively into the microtubule filigree at nanoscale accident sites created by spastin in an ATP hydrolysis–dependent manner.
Because spastin and katanin activate GTP-tubulin assimilation forth microtubules, we aing advised their furnishings on microtubule dynamics. It has been accustomed for 30 years that tubulin assimilation into a growing microtubule stimulates hydrolysis of the apprenticed GTP. The constant GDP-tubulin filigree is ambiguous but is adequate from depolymerization by a bandage of GTP-tubulin. This GTP cap at the microtubule end after-effects from a lag amid the GTP hydrolysis amount on the congenital tubulin and the microtubule advance acceleration (30, 31, 39–42). Added recently, islands of GTP-tubulin were detected forth microtubules in beef and were activated with accomplishment (34, 43)—the alteration from depolymerization to growth, one of the ambit of microtubule activating instability. As in counterbalanced GMPCPP-microtubules and GMPCPP-capped GDP-microtubules, the anew perfused GTP-tubulin was rapidly congenital forth the GDP-microtubule filigree of activating microtubules in the attendance of spastin and katanin with ATP; by contrast, in the ascendancy afterwards ATP, accession was arresting alone at microtubule ends (Fig. 4, A to D, and movies S2 and S3). We afresh characterized microtubule dynamics in the attendance of spastin or katanin at physiological concentrations [25 nM; spastin and katanin concentrations in HeLa beef are 46 and 28 nM, appropriately (37)]. At these agitator concentrations, we empiric able-bodied microtubule disengagement and centralized GTP-tubulin incorporation. Spastin and katanin added accomplishment frequencies by factors of ~13- and 9, appropriately (with 0.5 ± 0.2 min−1 for the ascendancy adjoin 6.6 ± 1.6 min−1 and 4.5 ± 0.7 min−1 for spastin and katanin, respectively) (Fig. 4E). Admitting alone 13% of the depolymerization contest were rescued in the control, 61% were rescued in the attendance of spastin or katanin (Fig. 4F). Constant with their advance of tubulin barter forth the microtubule shaft, spastin and katanin had no cogent aftereffect on ante of microtubule advance and accident (Fig. 4, G and H). This is in adverse to added rescue-promoting factors, such as cytoplasmic linker-associated proteins, which advance accomplishment by accretion the on amount of tubulin dimers at microtubule ends and appropriately abatement accident and access advance ante (44), or accepted microtubule-associated proteins (MAPs) such as MAP2, which advance accomplishment by stabilizing the microtubule filigree (45).
(A and B) Time advance of a activating 10% HiLyte 647–labeled microtubule at 12 μM tubulin in the attendance of 25 nM spastin afterwards (A) or with (B) ATP assuming HiLyte 488–labeled tubulin assimilation at the microtubule tip (A) or assimilation (arrowheads) forth the microtubule in accession to the tip (B). The aboriginal micrograph for anniversary activity was recorded aloof afore the perfusion of the alcove with 12 μM 10% HiLyte 488–labeled tubulin. Calibration bar, 2 μm. (C and D) Time advance of a activating 10% HiLyte 647–labeled microtubule at 12 μM tubulin in the attendance of 25 nM katanin afterwards (C) or with (D) ATP assuming HiLyte 488–labeled tubulin assimilation at the microtubule tip (C) or assimilation (arrowheads) forth the microtubule in accession to the tip (D). The aboriginal micrograph for anniversary activity was recorded aloof afore the perfusion of the alcove with 12 μM 10% HiLyte 488–labeled tubulin. (E) Accomplishment abundance at 10 μM tubulin in the absence or attendance of 25 nM spastin and 25 nM katanin with ATP (n = 47, 45, and 61 microtubules from assorted accommodation for the ascendancy afterwards enzyme, spastin, and katanin, respectively). ****P < 0.0001, bent by the Mann-Whitney test. (F) Anticipation of accomplishment of a depolymerizing microtubule in the absence or attendance of spastin and katanin with ATP (n = 68, 57, and 78 depolymerization contest for the control, spastin, and katanin, respectively). ****P < 0.0001, bent by a two-tailed t test. (G and H) Advance ante (G) and accident abundance (H) in the absence or attendance of spastin and katanin with ATP [n = 56, 37, and 34 advance contest for the control, spastin, and katanin, respectively, in (G) and n = 62, 70, and 71 microtubules for the control, spastin, and katanin, respectively, in (H)]. Additional signs in (G) announce the means. ns, not significant. Error confined announce SEM throughout.
In our dynamics assays, tubulin was always extracted by the enzyme, while at the aforementioned time the filigree was healed with anew congenital GTP-tubulin that was gradually adapted into GDP-tubulin. To decouple these processes and authorize anon whether the GTP-tubulin islands alien by these enzymes can act as microtubule accomplishment sites, we alien nonhydrolyzable GTP-tubulin islands into the microtubule. We induced nanoscale accident to a GMPCPP-capped GDP-microtubule with spastin or katanin and healed it with GMPCPP-tubulin, removed the agitator and GMPCPP-tubulin from the chamber, and accomplished microtubule depolymerization through laser ablation aing to the GMPCPP cap (Fig. 5; fig. S8, A to C; and Abstracts and methods). No GMPCPP-tubulin assimilation was detected in the ascendancy performed in the attendance of agitator afterwards ATP. These microtubules depolymerized all the way to the berry aloft ablation (Fig. 5B and fig. S8A). In contrast, microtubules with GMPCPP-tubulin islands congenital forth their lengths through the ATP hydrolysis–dependent activity of spastin or katanin were counterbalanced adjoin depolymerization at the breadth of the island (Fig. 5C and fig. S8B), admitting the absence of acrid tubulin in the chamber: 75% and 76% paused back they encountered a GMPCPP island alien by spastin and katanin, appropriately (Fig. 5D; fig. S8, A to C; and cine S4). Those that depolymerized through the island showed a abatement in the depolymerization acceleration (Fig. 5E and fig. S8D). Moreover, fluorescence acuteness appraisal appear that GMPCPP islands that paused depolymerization were statistically decidedly brighter than those that did not (Fig. 5F and fig. S8E). Next, we capital to authorize whether these enzyme-generated GMPCPP islands were competent to abutment microtubule regrowth. We afresh performed the above-described experiment, but during the aftermost footfall we alien 7 μM acrid GTP-tubulin into the alcove (Fig. 5, A, G, and H, and cine S5). Admitting at these tubulin concentrations accomplishment contest were actual attenuate in the control, we saw a college anticipation of accomplishment of microtubules with spastin-incorporated GMPCPP islands (Fig. 5I). Back the GMPCPP island did not abutment a rescue, it did apathetic bottomward depolymerization (Fig. 5J). Moreover, fluorescence acuteness appraisal appear that GMPCPP islands that accurate rescues were decidedly brighter than those that did not (Fig. 5K). Thus, microtubule dynamics abstracts and abstracts with GMPCPP-tubulin islands announce that GTP islands alien in a microtubule disengagement enzyme–dependent address advance microtubule accomplishment and that there is a basal bounded GTP-tubulin body appropriate to robustly abutment accomplishment at that site. Because the microtubule is rescued back the antithesis accouterment from net tubulin accident to net tubulin addition, it is acceptable that the alternation amid the admeasurement of the GTP-tubulin island and accomplishment anticipation will alter with the tubulin absorption or the attendance of MAPs. Thus, abate GTP-tubulin islands may still be able as accomplishment sites at college tubulin concentrations or in the attendance of MAPs that access the tubulin on rate.
(A) Agreement schematic. GDP-microtubules (solid magenta) were polymerized from seeds and capped with GMPCPP-tubulin (magenta outline). Spastin, ATP, and GMPCPP-tubulin (green) were added and done out of the chamber. Microtubules were laser ablated in the absence (B to F) or attendance (G to K) of GTP-tubulin (Materials and methods). (B) Kymograph of a depolymerizing laser-ablated microtubule (magenta) preincubated with spastin and no ATP. Horizontal calibration bar, 5 μm; vertical bar, 10 s. (C) Kymographs of depolymerizing laser-ablated microtubules pausing at GMPCPP-tubulin islands (green) alien by spastin with 1 mM ATP. Arrowheads, pauses. (D) Pie blueprint assuming the admeasurement of depolymerization contest that paused at GMPCPP islands (white) or did not (gray) (n = 44 events). (E) Depolymerization ante of microtubules afterwards GMPCPP islands preincubated with spastin and no ATP or of microtubules depolymerized through GMPCPP islands alien by spastin with 1 mM ATP (n = 17 and 7 microtubules for no ATP and ATP, respectively). (F) Fluorescence acuteness of GMPCPP islands through which microtubules depolymerized or paused (n = 9 and 14 islands, respectively). (G and H) Kymographs of laser-ablated microtubules in the attendance of 7 μM acrid GTP-tubulin afterwards preincubation with spastin and no ATP assuming complete depolymerization (G) or accomplishment (arrowheads) at a GMPCPP island alien by spastin with ATP (H). Horizontal calibration bar, 5 μm; vertical bar, 20 s. (I) Accomplishment abundance for laser-ablated microtubules incubated with spastin with or afterwards ATP (n = 23 and 24 microtubules with and afterwards ATP, respectively). (J) Depolymerization ante in the attendance of 7 μM GTP-tubulin for microtubules preincubated with spastin and no ATP or for microtubules that depolymerized through GMPCPP islands alien by spastin with ATP (n = 9 and 6 microtubules afterwards and with ATP, respectively). (K) Fluorescence acuteness of GMPCPP islands that did not stop depolymerization (n = 6) or at which microtubules were rescued in the attendance of spastin and ATP (n = 9). **P < 0.01, ***P < 0.001, bent by the Mann-Whitney test. Additional signs in (E), (F), (J), and (K) announce means. Error confined announce SEM throughout.
The GTP accompaniment of tubulin is accustomed by MAPs acceptance to the end-binding (EB) protein family. EB1 preferentially binds to growing microtubule ends by appraisal the GTP [or GDP–inorganic phosphate (Pi)] accompaniment of tubulin (46, 47). Constant with the conception of GTP-tubulin islands, in the attendance of spastin or katanin and ATP we empiric EB1 not alone at the growing ends as in the ascendancy but additionally as audible puncta forth microtubules (Fig. 6, A to D). These puncta are evocative of the EB3 puncta empiric at sites of tubulin adjustment afterwards laser-induced accident (34). Of the anew congenital GTP-tubulin islands, 89% colocalized with EB1 (Fig. 6, E and F). These EB1 puncta were transient, constant with the activating abatement and assimilation of new tubulin into the filigree and the bit-by-bit GTP hydrolysis of the congenital tubulin (Fig. 6, A and C; fig. S9; and cine S6). Constant with a careful aftereffect of the GTP islands, microtubule dynamics assays in the attendance of spastin and EB1 appear that 74% of rescues were associated with the attendance of EB1 at the accomplishment armpit (fig. S10A). This cardinal is decidedly college than the anticipation accustomed by the accidental superposition of EB1 puncta and accomplishment contest (74% adjoin 14%; P < 0.0001 by Fisher’s exact test) (Materials and methods). Similarly, 63% of rescues in the attendance of katanin occurred at the armpit of an EB1 atom (fig. S10B), compared with 0% back the administration was randomized (P < 0.00001 by Fisher’s exact test) (Materials and methods). Laser ablation of microtubules brindled with EB1 puncta additionally appear a apparent access in accomplishment frequency. Admitting microtubules were rescued from 100% of ablation-induced depolymerization contest aural 4 s, they were rescued from alone 15% of contest in the attendance of spastin and ATP-γ-S (Fig. 6, G and H). Agnate after-effects were acquired with katanin (Fig. 6I and cine S7). Thus, the ATP-dependent activity of the agitator that promotes tubulin barter aural the filigree is appropriate for the empiric access in accomplishment frequency.
(A) Time advance of EB1-GFP on a activating microtubule in the attendance of 25 nM spastin afterwards or with ATP. Calibration bar, 2 μm. Bandage scans on the appropriate appearance EB1-GFP acuteness profiles forth the microtubule at the adumbrated times. Acuteness profiles alpha on the microtubule filigree and end at the microtubule tip. Arrowheads appearance filigree EB1 puncta. (B) Body of EB1-GFP puncta on microtubules incubated afterwards spastin or with spastin afterwards and with ATP. Error confined announce SEM. ****P < 0.0001. (C) Time advance of EB1-GFP on a activating microtubule in the attendance of 25 nM katanin afterwards and with ATP. Acuteness profiles are as in (A). Arrowheads appearance filigree EB1 puncta. (D) Body of EB1-GFP puncta on microtubules incubated afterwards katanin or with katanin afterwards or with ATP. Error confined announce SEM. ****P < 0.0001. (E) Colocalization of anew congenital GTP-tubulin (top) and EB1-GFP (middle) in the attendance of spastin and ATP. (Bottom) Overlay. Images were acquired anon afterwards the perfusion of the alcove with agitator and EB1-GFP. Calibration bar, 2 μm. (F) Fluorescence acuteness of congenital tubulin (magenta) and EB1-GFP (green) forth the microtubule filigree in (E) assuming their colocalization. Eighty-nine percent of tubulin islands colocalize with EB1-GFP (n = 38 puncta from 22 microtubules from assorted accommodation abstinent anon afterwards perfusion with 10% HyLite 647–tubulin). (G) Time advance of laser-ablated activating microtubules (magenta) incubated with 25 nM spastin with ATP-γ-S or spastin with ATP in the attendance of 50 nM EB1-GFP (green) (Materials and methods). The dotted bandage marks the ablated arena and the alpha of depolymerization. Calibration bar, 2 μm. (H and I) Pie archive appearance the fates of additional ends generated through laser ablation of microtubules incubated with spastin (H) or katanin (I) with ATP-γ-S or ATP. The allotment of additional ends that depolymerized (gray) or were rescued (white) aural 4 s afterwards ablation is apparent (n = 13 and 13 microtubules from assorted accommodation for spastin with ATP-γ-S and ATP, respectively; n = 54 and 9 microtubules from assorted accommodation for katanin with ATP-γ-S and ATP, respectively).
The GDP-tubulin filigree is ambiguous and, back apparent by laser ablation, is rapidly depolymerized at the additional ends, alike in the attendance of acrid tubulin (Fig. 7A), a aftereffect constant with those of archetypal abstracts performed with laser-ablated or mechanically cut microtubules (28, 29, 48–51). Surprisingly, at 12 μM tubulin, the majority of new additional ends generated by spastin or katanin were abiding and rapidly reinitiated advance (Fig. 7, B and C). By contrast, in the absence of either agitator or in the attendance of spastin or katanin and ATP-γ-S, new additional ends generated through laser ablation rapidly depolymerized (Figs. 6, H and I, and 7A). This indicates that it is not the acquiescent bounden of the protein that stabilizes the new additional ends adjoin ad-lib depolymerization but the ATP-dependent assimilation of GTP-tubulin at disengagement sites. The bare ends were abiding behindhand of whether they were generated through agitator activity or laser ablation, constant with after-effects of beforehand abstracts application laser ablation (28, 29). Thus, back bounded tubulin abstraction by spastin or katanin outpaces the amount of tubulin incorporation, a disengagement accident occurs and the anew burst microtubule ends appear with a aerial body of GTP-tubulin that is careful (Fig. 7, B and C). Moreover, the additional ends that depolymerize anon afterwards disengagement resume advance afterwards a lower net accident of polymer accumulation (fig. S8, F and G). Thus, the access in microtubule cardinal with anniversary disengagement accident (Fig. 7, D and E) synergizes with the college accomplishment abundance to aftermath a accelerated accession of absolute microtubule cardinal and accumulation (Fig. 7, F to J).
(A) Additional ends generated through laser ablation depolymerize. The pie blueprint shows the percentages of additional ends that are abiding (white) or depolymerize (gray) (n = 32 microtubules from assorted chambers). Calibration bar, 5 μm. The abject bandage marks the ablated arena and the alpha of depolymerization. (B and C) Spastin (B)- or katanin (C)-severed ends appear with anew congenital GTP-tubulin and are stable. The pie archive appearance the percentages of additional ends that are abiding (white) or depolymerize (gray) (n = 96 and 94 microtubules from assorted accommodation for spastin and katanin, respectively). White and chicken arrowheads announce tubulin assimilation and a disengagement event, respectively. Calibration bars, 2 μm. (D and E) Time-lapse images assuming afterwards spastin (D)- or katanin (E)-induced disengagement contest on a microtubule. Curve and numbers announce alone microtubules afterwards severing. and − mark microtubule ends. Magenta, microtubule; green, congenital tubulin. Calibration bars, 2 μm. (F) Time-lapse images assuming microtubule dynamics at 12 μM tubulin in the absence of a disengagement enzyme. Green, anew congenital tubulin at the growing ends. The aftermost two frames are achromatize corrected. Calibration bar, 5 μm. (G and H) Time-lapse images assuming microtubule cardinal and accumulation accession through spastin (G) and katanin (H) severing. Green, anew congenital HiLyte 488–tubulin perfused into the accommodation calm with the disengagement enzymes. (I and J) Microtubule accumulation as a activity of time (n = 4, 5, and 4 accommodation for the control, spastin, and katanin, respectively). Error confined announce SEM.
The classical appearance of microtubule dynamics has been that tubulin dimer barter occurs alone at microtubule ends through polymerization and depolymerization (30, 52). By visualizing a disengagement acknowledgment at the ultrastructural level, we accept apparent that spastin and katanin abstract tubulin subunits from the microtubule (Fig. 1) and that this ATP hydrolysis–dependent tubulin abatement is counteracted by ad-lib filigree assimilation of acrid GTP-tubulin (Figs. 2 to 4 and figs. S5 to S7). The nanoscale-damaged microtubules do not anon break but are abiding abundant to accept a adventitious to alleviate through the advantageous assimilation of tubulin into the lattice. Because longitudinal filigree contacts are stronger than crabbed ones (42), we brainstorm that tubulin dimer accident from the microtubule bank has a slight longitudinal bent that gain forth the protofilament. This would accord the microtubule a adventitious to alleviate afore it is burst beyond and accomplish GTP-tubulin islands that abide of several tubulin dimers in the longitudinal direction. The geometry of the nanoscale accident sites and the apparatus of tubulin assimilation and conformational changes at these sites will be agitative and axiological areas for approaching exploration.
This apparatus of filigree adjustment can explain the beforehand ascertainment of the inhibition of katanin disengagement by acrid tubulin (35, 53). The ragged, Swiss cheese attributes of the nanoscale-damaged microtubules is accessory to healing, as the admission tubulin dimers can accomplish stabilizing crabbed interactions. Thus, depending on the bounded ante of the disengagement enzyme–catalyzed tubulin abatement and the ad-lib assimilation of new GTP-tubulin into the lattice, the activity of a microtubule-severing agitator after-effects in a disengagement accident breadth the anew arising ends accept a aerial body of GTP-tubulin or a microtubule that preserves candor but acquires a GTP island at the armpit of agitator action. The college GTP body at the anew burst ends can additionally act to bound recruit atomic motors and MAPs that can attune the fate of the anew generated end.
Although in vitro microtubule adjustment afterwards the accession of defects through laser-induced photodamage (34) or automated accent (33, 54) has been appear previously, our abstraction identifies a ancestors of enzymes as biological agents that advance the ATP-dependent assimilation of GTP-tubulin islands into microtubules. Microtubule adjustment has a aerial accident in vivo at microtubule crossovers or bundles (34), breadth microtubule-severing enzymes accept been apparent to act (7, 8, 17, 55). Our allegation appropriately advance that the aerial accident of adjustment at these sites is due not alone to automated accident (34) but additionally to the activity of microtubule-severing enzymes. As spastin and katanin preferentially ambition glutamylated microtubules (13, 56, 57), they may additionally selectively rejuvenate crumbling microtubules with accumulated glutamylation marks through GTP-tubulin incorporation. GTP-tubulin islands accept been articular forth axonal microtubules (58), a neuronal alcove breadth disengagement enzymes act. This award raises the achievability that disengagement enzymes are additionally acclimated as affection ascendancy and aliment factors in hyperstable microtubule arrays, such as those in axons, centrioles, and cilia, breadth spastin and katanin are important for biogenesis and aliment (2, 5, 13, 14) and breadth spastin and katanin may serve to aish and alter old, possibly damaged tubulin subunits afterwards affecting all-embracing microtubule organization. Approaching assignment should authorize how broken filigree adjustment contributes to the ache phenotypes apparent in patients with spastin and katanin mutations.
Our abstraction shows that the disengagement enzyme–catalyzed assimilation of GTP-tubulin forth microtubules has two physiological consequences: It increases the abundance at which microtubules are rescued (Figs. 4 to 6), and it stabilizes anew burst additional ends that appear adjoin depolymerization with a aerial body of GTP-tubulin (Fig. 7). Thus, microtubule dynamics can be articulate not alone by factors that affect tubulin assimilation at microtubule ends but additionally by disengagement enzymes that advance the barter of tubulin subunits aural the microtubule shaft. The synergy amid the added accomplishment ante and the stabilization of the anew burst ends leads to microtubule accession in the absence of a nucleating factor, answer why, paradoxically, the accident of spastin and katanin after-effects in the accident of microtubule accumulation in abounding systems (2, 23, 25, 27). Such a apparatus of polymer accession has parallels to the actin cytoskeleton, breadth burst filaments are acclimated for templated actin polymerization [(26, 59); advised in (60)]. Back disengagement enzymes are bidding at aerial levels or are absolutely regulated, tubulin abstraction outpaces adjustment and the microtubule arrangement disassembles. Beef acceptable attune disengagement activity and the amount of tubulin filigree assimilation through the activity of MAPs to arm-twist these two altered outcomes. This adjustment will be a notable breadth of approaching exploration.
Drosophila melanogaster feature spastin was antiseptic by affection chromatography and ion barter as ahead declared (61). Caenorhabditis elegans MBP-tagged katanin Mei1/Mei2 (12) was antiseptic on amylose resin. The affection tag was removed by tobacco compose virus protease, and the protein was added antiseptic on an ion barter MonoS cavalcade (GE Healthcare) as ahead declared (62). Peak fractions were concentrated, absorber was exchanged into 20 mM HEPES (pH 7.0), 300 mM KCl, 10 mM MgCl2, and 1 mM TCEP, and fractions were beam arctic in baby aliquots in aqueous nitrogen. Homo sapiens EB1–green beaming protein (GFP) was bidding and antiseptic as ahead declared (63). Animal α1AβIII tubulin with an engineered FLAG tag at the β-tubulin C aals was bidding by application baculovirus and antiseptic as declared ahead (38).
Taxol-stabilized GDP-microtubules were able by polymerizing 10 μl of 100 μM glycerol-free beefy tubulin (Cytoskeleton, Denver, CO) in 80 mM K-PIPES (pH 6.8), 1 mM MgCl2, 1 mM EGTA, 10% DMSO, and 1 mM GTP for 1 hour in a 37°C baptize bath. Taxol was added to 20 μM final concentration, and the acknowledgment was incubated on the bank top for 1 to 2 hours. Microtubules were loaded assimilate a 60% glycerol beanbag [BRB80, 60% (v/v) glycerol, and 20 μM Taxol] at 37°C by application a pipette tip with the tip cut off. Nonpolymerized tubulin was removed by centrifugation in a TLA100 rotor at 35,000 rpm for 15 min at 37°C. The pellet was acclaim resuspended to 2.5 μM tubulin in BRB80 supplemented with 20 μM Taxol and 1 mM GTP at 37°C by application a pipette tip with the tip cut off.
For GDP-microtubules, all polymerization and disengagement reactions were performed at 37°C. Twenty microliters of 100 μM glycerol-free beefy tubulin (Cytoskeleton) was polymerized in 10% DMSO, 1 mM GTP, and 10 mM MgCl2 for 1 hour at 37°C in a baptize bath. The microtubules were anesthetized through a 60% glycerol beanbag [BRB80, 60% (v/v) glycerol, and 1mM GTP] by application a TLA100 rotor at 53,000 × g for 15 min to aish nonpolymerized tubulin. The pellet was done alert application 50 μl of absorber (BRB80, 10% DMSO, 1 mM GTP) and acclaim resuspended to 30 μM in the aforementioned absorber by application a pipette tip with the tip cut off.
GMPCPP-microtubules were able by polymerizing 20 μl of 100 μM glycerol-free beefy tubulin (Cytoskeleton) in 1 mM GMPCPP in BRB80 [80 mM PIPES-KOH (pH 6.8), 1mM MgCl2, 1mM EGTA, 1mM DTT] on ice for 5 min and afresh in a baptize ablution at 37°C for 1 hour. Nonpolymerized tubulin was removed by centrifugation in a TLA100 rotor at 126,000 × g for 5 min at 37°C. The pellet was done alert with 50 μl of BRB80 at 37°C and resuspended in 50 μl of ice-cold BRB80. The acknowledgment admixture was kept on ice for 30 min and periodically alloyed up and bottomward to absolutely depolymerize microtubules. GMPCPP was added to 1 mM, and the polymerization acknowledgment admixture was kept on ice for 10 min and afresh transferred to 37°C for 2 to 4 hours or overnight. Nonpolymerized tubulin was removed by centrifugation and done as declared above. The microtubule pellet was acclaim resuspended to 2.5 μM tubulin in BRB80 by application a pipette tip with the tip cut off.
We begin that assuming disengagement reactions in the tube followed by pipetting assimilate EM grids resulted in microtubule breakage. We accordingly aboriginal agitated out disengagement reactions on the EM grid. Briefly, 2 μl of microtubule band-aid (at 1 to 3 μM) in BRB80 [80 mM PIPES (pH 6.8), 1 mM MgCl2, 1 mM EGTA] was activated to a glow-discharged Cu grid, followed by pipetting of 2 μl of ATP band-aid (10 mM ATP in BRB80 supplemented with 20 mM Taxol for Taxol-stabilized microtubules) and 2 μl of spastin (at 100 nM). The acknowledgment was accustomed to advance on the filigree for 1 min or as specified, afterwards which the aqueous was abandoned off with calcium-free clarify cardboard and the filigree was decrepit with 0.75% (w/v) uranyl formate and air-dried. Images were calm on a FEI Morgagni 286 electron microscope operated at 80 kV and able with an AMT lens-coupled 1k × 1k CCD camera. For the band-aid disengagement acknowledgment time courses, 20 μl of GMPCPP or Taxol-stabilized microtubules in BRB80 absorber at 2.5 and 1.0 μM was activated to parafilm, followed by the accession of 20 μl of 50 nM spastin or 200 nM katanin in 20 mM HEPES (pH 7.5), 300 mM KCl, 10 mM MgCl2, 1mM TCEP, and 1mM ATP to a final absorption of 25 nM spastin and 100 nM katanin. For the band-aid disengagement acknowledgment time courses of nonstabilized GDP-microtubules, 20 μl of 30 μM GDP-microtubules in the attendance of 10% DMSO was incubated with 2 μl of 20 nM katanin. Absorber afterwards disengagement enzymes was added to microtubules as a abrogating control. The disengagement acknowledgment mixtures were incubated for 30 s or 2 or 5 min, and carbon-coated grids (carbon blur alone on 400 mesh; Ted Pella) were biconcave into the acknowledgment mixtures. Antithesis aqueous was blotted with clarify paper. Grids were done three times with 40 μl BRB80, decrepit with 0.75% (w/v) uranyl formate, and air-dried. Images were calm on a T12 Technai electron microscope (FEI) able with a 2k × 2k Gatan US1000 CCD camera. Images were calm at nominal magnifications of 550×, 13,000×, or 30,000×, agnate to pixel sizes of 84 Å/pix, 3.55 Å/pix, or 1.54 Å/pix, respectively.
Double-cycled, GMPCPP-stabilized microtubules (64) were polymerized from 2-mg/ml beefy academician tubulin (Cytoskeleton). The aboriginal polymerization was 1 hour, and the additional polymerization footfall was at atomic 4 hours to access continued microtubules. Afresh microtubules were centrifuged, resuspended in balmy BRB80 [80 mM K-PIPES (pH 6.8), 1 mM MgCl2, 1 mM EGTA], and stored at 37°C or allowance temperature (RT) afore use. The aforementioned after-effects were acquired behindhand of whether the accumulator temperature was 37°C or RT. Taxol-stabilized microtubules (61) were polymerized from 5-mg/ml beefy academician tubulin absolute 1% biotinylated and 20% HiLyte 647–labeled tubulin (Cytoskeleton) in BRB80 with 10% DMSO, 0.5 mM GTP, and 10 mM MgCl2. Afterwards 1 hour of evolution at 37°C, 20 μM Taxol was added and the admixture was added incubated overnight. Microtubules were afresh centrifuged through a 60% glycerol beanbag for 12 min at 109,000 × g at 35°C. The microtubule pellet was done with balmy BRB80 supplemented with 14.3 mM 2-mercaptoethanol and 20 μM Taxol and was resuspended acclaim in the aforementioned buffer.
Chambers for TIRF microscopy were accumulated as ahead declared (61). Double-cycled GMPCPP-microtubules absolute 1% biotinylated tubulin and 20% HiLyte 647–labeled tubulin (or unlabeled tubulin for the DIC assays) accumulated as declared aloft were anchored in the alcove with 2-mg/ml NeutrAvidin (Thermo Fisher Scientific) and beheld by TIRF or DIC microscopy in disengagement absorber [BRB80 absorber with 2-mg/ml casein, 14.3 mM 2-mercaptoethanol, 2.5% glycerol, 50 mM KCl, 2.5 mM MgCl2, 1 mM ATP, 1% Pluronic F127 (Life Technologies), and oxygen scavengers]. To acquaint and ascertain nanoscale accident in microtubules (Fig. 2), anchored microtubules were afresh incubated with 10 nM spastin or 2 nM katanin in disengagement absorber for 35 or 90 s, respectively. Microtubules in ascendancy abstracts were incubated afterwards disengagement enzyme. The agitator admixture was afresh replaced with 1 μM HiLyte 488–labeled tubulin (Cytoskeleton), 1 mM ADP, 0.5 mM GTP, 1% Pluronic F127, and 2.5-mg/ml casein in BRB80 and larboard to bear for 5 min. The tubulin-containing band-aid was afresh done out with 45 μl of BRB80 supplemented with oxygen scavengers, 1.5-mg/ml casein, 10 mM 2-mercaptoethanol, and 1% Pluronic F127. Microtubules and HiLyte 488–labeled tubulin were beheld by TIRF. Assorted fields of appearance were imaged. The aforementioned appraisal was performed for Taxol-stabilized microtubules, but in this case the adjustment footfall was performed with 0.1 μM acrid tubulin to anticipate microtubule nucleation in the attendance of Taxol. For time-course experiments, the aforementioned agreement was acclimated except that microtubules were incubated with 2 nM spastin (fig. S3) or 2 nM katanin (fig. S4) for 35 to 120 s. Ascendancy microtubules were incubated afterwards disengagement agitator for 120 s. HiLyte 488–labeled tubulin (1 μM) was acclimated for the adjustment step. For adjustment with 1 μM recombinant animal tubulin (fig. S6), nanoscale-damaged microtubules were incubated for 5 min with recombinant tubulin. Unincorporated tubulin was done away, and tubulin congenital into microtubules was detected by anti-FLAG M2 antibodies (Sigma-Aldrich; adulterated 1:500) and dupe anti-mouse antibodies conjugated with Alexa Fluor 488 (Invitrogen; adulterated 1:1000). All assays were performed at RT. Details apropos angel accretion and appraisal are declared in the annex below.
Images were acquired by application a Nikon Ti-E microscope able with a 100× 1.49 NA oil cold and a TI-TIRF adapter (Nikon). The 488 action laser (Coherent) was set at 20 mW, and the 647 nm laser (Coherent) was set to 2 mW afore actuality accompanying into the Ti-TIRF optical cilia (Nikon). Two-color accompanying imaging was performed by application a TuCAM (Andor) accessory that splits the discharge assimilate two abstracted EMCCD cameras (Andor iXon 897). The action and discharge were breach by a cloister bandage dichroic (Semrock), and the discharge was added breach by an FF640 clarify (Semrock) and added filtered with an FF01-550/88 (Semrock) for the 488 approach and an FF01-642/LP (Semrock) for the 640 channel. The TuCAM imaging arrangement introduces an added 2× magnification, acquiescent a final pixel admeasurement of 77 nm. The images from the two cameras were accumbent by aboriginal imaging a filigree of spots (Nanogrid MiralomaTech) on anniversary camera and application the GridAligner constituent for ImageJ.
DIC beam was provided by a SOLA-SE-II (Lumencor) accompanying to the microscope by a aqueous ablaze guide. A accepted set of polarizer and analyzer (Nikon 100 X-II Aerial NA/Oil) prisms was used, and the angel was captured on a CoolSNAP (Photometrics) camera. The final pixel admeasurement for DIC images was 65 nm. Raw DIC images were candy application an FFT band-pass filter. DIC images were scaled and adapted to bury with beaming images by imaging beaming microtubules in both channels for angel registration. The absolute imaging bureaucracy was controlled by Micro-Manager (65).
For abstracts apparent in figs. S3 and S4, images were analyzed application scripts in ImageJ and MATLAB. First, the account amid 640 and 488 channels was adapted with the GridAligner plug-in. Afresh microtubules were called with 7 px-wide bandage selection, and bandage scans were generated. These bandage scans were alien into a MATLAB calligraphy that articular the peaks in the 488 approach and recorded the number, intensity, and FWHM of the adjustment sites. The FWHM for a diffraction-limited atom was acquired by application 100-nm TetraSpeck chaplet (Thermo Fischer Scientific). Abstracts were exported to Prism software for graphing.
GMPCPP-microtubules at 1 μM absorption in 1× BRB80 were activated to parafilm in a clamminess alcove and incubated with 20 nM spastin in agitator absorber [20 mM HEPES (pH 7.5), 300 mM KCl, 10 mM MgCl2, 1 mM TCEP, and 0.5 mM ATP]. Absorber absolute 0.5 mM ATP-γ-S instead of ATP was acclimated as a control. Disengagement was accustomed to advance for 30 s, followed by the accession of 0.6 μM acrid FLAG-tagged single-isoform recombinant neuronal animal α1AβIII tubulin to adjustment the microtubule filigree in the attendance of 1 mM GTP and 5 mM ADP to inactivate the enzyme. The adjustment acknowledgment was agitated out for 5 min. Microtubules were afresh counterbalanced by the accession of 5 volumes of 0.2% glutaraldehyde in 1× BRB80 (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA). Afterwards 3 min, cross-linking was quenched by the accession of Tris-HCl (pH 7.5) to a 20 mM final absorption and cross-linked microtubules were transferred into a 10-ml centrifuge tube (Beckman Coulter). The microtubule disengagement and healing action was again three added times, acknowledgment mixtures were affiliated into the aforementioned centrifuge tube, and microtubules were afresh spun bottomward in an MLA-80 rotor at 100,000 × g for 15 min at 30°C. The microtubule pellet was acclaim done with 200 μl of 1× BRB80 at 37°C alert and resuspended in 50 μl of balmy 1× BRB80. Five microliters of 6.7 μM monoclonal mouse-raised anti-FLAG M2 antibiotic (Sigma-Aldrich) and 5 μl of 11.45 μM dupe anti-mouse antibiotic conjugated to 4-nm all-around gold nanoparticles, C11-4-TGAMG-50 (Nanopartz), were added to microtubules to characterization repaired sites. Antibiotic labeling was accustomed to advance for 5 min, and the acknowledgment was alloyed with 10 volumes of 30% glycerol in 1× BRB80. Microtubules in 30% glycerol were loaded assimilate a 1× BRB80 beanbag absolute 40% glycerol and spun bottomward assimilate glow-discharged carbon-coated grids (carbon blur alone on 400 mesh; Ted Pella) at 4200 × g for 20 min at 30°C. Antithesis aqueous was blotted with clarify paper. Grids were done three times with 30 μl of BRB80, decrepit with 0.75% (w/v) uranyl formate, and air-dried. Images were calm on a T12 Technai electron microscope (FEI) able with a 2k × 2k Gatan US1000 CCD camera. Images were calm at nominal magnifications of 6800× and 18,500×, agnate to pixel sizes of 6.8 Å/pix and 2.5 Å/pix, respectively. Images in fig. S7F were calm on a TF20 electron microscope (FEI) able with a K2 camera (Gatan). Images were calm at 50,000× and 9600× magnifications, agnate to pixel sizes of 0.73 Å/pix and 3.65 Å/pix, respectively.
To beam microtubule disengagement and tubulin assimilation at accident sites accompanying (Fig. 3 and fig. S5, A to C), GMPCPP-stabilized double-cycled microtubules labeled with 1% biotin and 20% HiLyte 647–tubulin were anchored in imaging chambers. Angel accretion was started by application 100-ms connected acknowledgment in the 647 and 488 channels simultaneously, and the alcove was perfused with disengagement absorber absolute 0.5 mM GTP, 20 nM spastin, and 0, 0.1, or 2 μM HiLyte 488–labeled tubulin. Disengagement ante were affected by chiral counting of disengagement contest (microtubule breaks) as a activity of time. Tubulin assimilation sites were readily arresting in the 488 channel. To beam the alive assimilation of audible tubulin dimers into microtubules damaged by spastin (Fig. 3, F and G), double-cycled GMPCPP-microtubules composed of 20% HiLyte 647–labeled and 1% biotinylated tubulin were anchored in imaging accommodation as declared above. The alcove was afresh perfused with disengagement buffer, and images of microtubules were acquired. Microtubules were afresh incubated for 30 s with 20 nM spastin in disengagement buffer. Angel accretion was started during the spastin evolution step, and a band-aid absolute fluorescently labeled tubulin [50 nM Alexa 488–labeled tubulin (PurSolutions) in BRB80 with 2-mg/ml casein, 14.3 mM 2-mercaptoethanol, 50 mM KCl, 2.5 mM MgCl2, 1 mM ADP, 0.5 mM GTP, 1% Pluronic F127, and oxygen scavengers] was ablaze in. Images were acquired for 5 min at 10 Hz in the 488-nm channel. Afterwards tubulin perfusion, the 640 laser was angry off to anticipate photobleaching and microtubule photodamage. Images of beaming tubulin molecules landing on the microtubule were analyzed by application a 7 × 7 pixel box, and the acuteness of tubulin molecules congenital into the microtubule was calibrated adjoin the acuteness of audible tubulin dimers acquired by immobilizing 0.5 nM Alexa 488–tubulin on bottle with an anti–β-tubulin antibiotic (SAP.4G5; Sigma-Aldrich) and imaging beneath the aforementioned conditions.
For imaging of nonstabilized GDP-microtubules with a GMPCPP cap, sea brat axonemes antiseptic as declared ahead (66) were nonspecifically adhered to the coverslip, and 15 μM tubulin absolute 20% HiLyte 647–tubulin and 1 mM GTP were added to alpha microtubule advance from the axonemes. Afterwards the adapted microtubule breadth (10 to 20 μm) was achieved, the band-aid was exchanged bound to acquaint HiLyte 488–tubulin (20%) and 0.5 mM GMPCPP. Afterwards the advance of the GMPCPP cap, tubulin and nucleotide were done out and spastin (5 nM) was alien into the alcove with 1 mM ATP in the absence or attendance of acrid tubulin at 2 μM (500 nM HiLyte 488–tubulin 1.5 μM unlabeled tubulin) or 5 μM (500 nM HiLyte 488–tubulin 4.5 μM unlabeled tubulin) and 0.5 mM GTP. Polymerization and imaging were performed at 30°C.
TIRF microscopy accommodation were able as declared above. HiLyte 647 (10%)–labeled microtubules were polymerized at 30°C at 10 μM tubulin. The alcove was perfused with 25 nM spastin or katanin and 10 μM beefy academician tubulin absolute 10% HyLite 647–labeled tubulin in disengagement appraisal absorber (50 mM KCl, 1% Pluronic F127, 0.2-mg/ml casein, 6.2 mM 2-mercaptoethanol, 1.5% glycerol, 0.1% methylcellulose 4000cP, and oxygen scavengers in 1× BRB80) with 1 mM GTP and 1 mM ATP calm with 50 nM EB1-GFP. Images were acquired in the 647 and 488 channels accompanying at 2 Hz. Microtubule rescues are authentic as the alteration of microtubules from abbreviating to growth. Accomplishment abundance was affected as the cardinal of rescues disconnected by the time spent depolymerizing. Catastrophes are authentic as the alteration of microtubules from advance to shrinkage. Accident abundance was affected as the cardinal of catastrophes disconnected by the time spent in the polymerization state. The EB1 puncta and the microtubule accomplishment armpit were advised colocalized back the ambit amid the EB1 atom and the end of the depolymerizing microtubule was beneath than two pixels. The blow for an EB1 punctum was authentic as accepting a beggarly acuteness in a 5 × 5 pixel box that is at atomic 3 accepted deviations aloft the beggarly accomplishments EB1 filigree intensity. Accomplishments EB1 filigree acuteness was bent from ascendancy accommodation afterwards disengagement enzymes. Accomplishments EB1 filigree acuteness was the aforementioned in the absence of disengagement enzymes or the attendance of disengagement enzymes but in the absence of ATP. For statistical acceptation calculation, accomplishment armpit appraisal was additionally performed by application constructed abstracts generated by alive the position of the EB1 spots by 7 pixels on the microtubule (alternatively, both adjoin the additional and the bare ends).
For the GTP-tubulin and EB1-GFP colocalization abstracts apparent in Fig. 6E, microtubule extensions were developed in the absence of beaming tubulin for 8 min at 30°C at 12 μM beefy academician tubulin (Cytoskeleton) in disengagement appraisal buffer. The alcove was perfused with 20 nM spastin, 50 nM EB1-GFP, and 12 μM beefy academician tubulin absolute 10% HyLite 647–labeled tubulin in disengagement appraisal buffer. Angel accretion was started during perfusion in the 640 and 488 channels accompanying at 5 Hz. The account amid the 640 and 488 channels was adapted by application a nanogrid (Nanogrid Miraloma Tech) and the GridAligner constituent in ImageJ.
GMPCPP-stabilized blunt microtubule seeds were anchored on glass. To pregrow microtubules, 16 μM tubulin absolute 12.5% HiLyte 647–labeled tubulin with 1 mM GTP was perfused into the alcove and incubated for 10 min at 30°C. Microtubules were afresh capped application 6 μM tubulin with 10% HiLyte 647 and 0.5 mM GMPCPP. The alcove was done afterwards 2 min with disengagement appraisal absorber afterwards GTP and afresh incubated with 4 nM spastin and 6 μM tubulin absolute 25% HiLyte 488–labeled tubulin in the attendance of 200 μM GMPCPP in disengagement appraisal absorber (50 mM KCl, 1% Pluronic F127, 0.2-mg/ml casein, 6.2 mM 2-mercaptoethanol, 2.5% glycerol, 0.1% methylcellulose 4000cP, and oxygen scavengers in 1× BRB80) with or afterwards 1 mM ATP for 3 min. The alcove was done with absorber absolute disengagement appraisal buffer. Microtubules were ablated with a 405-nm laser at 40% ability application the iLas laser illuminator (BioVision). Images in the 488 and 647 channels were acquired sequentially with 100-ms exposure. For the accomplishment abundance measurements, 15% HiLyte 647–labeled tubulin at 7 μM in disengagement appraisal absorber absolute 1 mM GTP was perfused into the chamber. For the katanin experiments, the alcove was done afterwards microtubule capping with disengagement appraisal absorber afterwards GTP and afresh incubated with 20 nM katanin and 8 μM tubulin absolute 25% HiLyte 488–labeled tubulin in the attendance of 200 μM GMPCPP in disengagement appraisal absorber with or afterwards ATP for 45 s. Microtubule depolymerization ante through the GMPCPP islands were bent by adding the breadth of the island by the time it takes to depolymerize through it.
TIRF microscopy accommodation were able as declared above. HiLyte 647–labeled microtubule extensions were polymerized for 8 min at 30°C at 12 μM beefy academician tubulin (Cytoskeleton) absolute 20% HiLyte 647–labeled tubulin in disengagement appraisal buffer. The alcove was perfused with 25 nM spastin or katanin, 50 nM EB1-GFP, and 12 μM beefy academician tubulin absolute 20% HyLite 647–labeled tubulin in disengagement appraisal absorber with ATP or ATP-γ-S. Microtubules were ablated by application a DeltaVision OMX with the 405-nm laser at 100% ability for 1 s or with a 405-nm laser at 40% ability application an iLas laser illuminator (BioVision). Images were acquired in the 647 and 488 channels at 5 Hz on the DeltaVision OMX and 2.9 Hz on the iLas system.
Chambers for TIRF microscopy were able as declared above. GMPCPP-stabilized, blunt microtubules absolute 2% biotinylated tubulin were anchored with 0.1-mg/ml NeutrAvidin (Thermo Fisher Scientific). Microtubule extensions were polymerized for 12 min at 30°C at 10 or 12 μM beefy academician tubulin (Cytoskeleton) absolute 10% HiLyte 647–tubulin in disengagement appraisal absorber (50 mM KCl, 1% Pluronic F127, 1 mM ATP, 1 mM GTP, 0.2-mg/ml casein, 6.2 mM 2-mercaptoethanol, 1.5% glycerol, 0.1% methylcellulose 4000cP, and oxygen scavengers in 1× BRB80). Then, 25 nM katanin or spastin with 12 μM beefy academician tubulin absolute 10% HyLite 488–labeled tubulin was perfused into the alcove in disengagement appraisal buffer. Images were acquired with 488 and 640 lasers accompanying at 2 Hz at 100-ms exposure. The assimilation of the HiLyte 488–tubulin was anon arresting aloft perfusion alone at microtubule tips in the ascendancy and forth the microtubules and the activating tips in the agitator and ATP conditions. Absolute polymer accumulation was acquired by barometer the background-corrected absolute chip fluorescence in both the 488 and 640 channels. The laser ablation controls were performed at the aforementioned agitator and tubulin concentrations but with 1 mM ATP-γ-S. Microtubules were ablated with a 405-nm laser at 40% ability application an iLas laser illuminator (BioVision) for the katanin abstracts and the DeltaVision OMX for spastin.
n numbers and statistical tests are appear for all abstracts in the amount legends. All abstracts were performed assorted times, and alone adumbrative images are shown. ImageJ was acclimated for angel analysis. Prism (GraphPad) was acclimated for graphing and statistical analysis.
C. Gell, C. T. Friel, B. Borgonovo, D. N. Drechsel, A. A. Hyman, J. Howard, “Purification of tubulin from beefy brain,” in Microtubule Dynamics: Methods and Protocols, A. Straube, Ed. (Springer, 2011), pp. 15–28.
Acknowledgments: We acknowledge A. Szyk, National Institute of Neurological Disorders and Stroke (NINDS), for antiseptic spastin and katanin, and X. Wu, National Heart, Lung, and Blood Institute (NHLBI), for advice in the Ablaze Microscopy Core. Funding: N.G. is a Howard Hughes Medical Institute investigator. A.M.D. is accurate by NIH admission R01GM121975. A.R.-M. is accurate by the civil programs of NINDS and NHLBI. Columnist contributions: A.V. performed TIRF abstracts with activating microtubules and laser ablation. E.S. performed TIRF abstracts with counterbalanced and activating microtubules. A.V., E.S., and J.O.S. performed angel analysis. E.A.Z. performed EM time courses with spastin and all EM abstracts with katanin and recombinant tubulin. A.M.D. performed antecedent in-the-tube and on-grid EM abstracts with spastin. A.M.D. and N.G. discussed EM experiments. A.R.-M. wrote the arrangement with ascribe from A.M.D., E.S, A.V., and E.A.Z. All authors planned abstracts and advised the manuscript. A.R.-M. conceived of the project, conceptualized the manuscript, and supervised research. Aggressive interests: The authors acknowledge no aggressive banking interests. Abstracts and actual availability: All abstracts bare to accept and appraise the abstracts of this analysis are accessible in the capital argument and added materials. Information requests and requests for reagents should be directed to the agnate author.
Correction (13 September 2018): The advertence “M. E. Bailey, M. M. Morelli, J. D. Diaz, J. L. Ross, Biophys. J. 102, 701a (2012)” was afield included as advertence 54. This advertence has been removed, and the argument has been adapted to adduce advertence 35 instead.
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