extfieldsofstudy: Chemistry Medicine

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Materials Science

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XMAP215 is a microtubule nucleation factor that functions synergistically with the gamma-tubulin ring complex

How microtubules (MT) are generated in the cell is a major question in understanding how the cytoskeleton is assembled. For several decades, γ-tubulin has been accepted as the cell’s universal MT nucleator. Although there is evidence that γ-tubulin complexes are not the sole MT nucleators, identification of other nucleation factors has proven difficult. Here, we report that the well-characterized MT polymerase XMAP215 (chTOG/Msps/Stu2p/Alp14/Dis1 homologue) is essential for MT nucleation in Xenopus egg extracts. The concentration of XMAP215 determines the extent of MT nucleation. Even though XMAP215 and γ-tubulin ring complex (γ-TuRC) possess minimal nucleation activity individually, together these factors synergistically stimulate MT nucleation in vitro. The N-terminal TOG domains 1–5 of XMAP215 bind αβ-tubulin and promote MT polymerization, while the conserved C-terminus is required for efficient MT nucleation and directly binds γ-tubulin. In sum, XMAP215 and γ-TuRC together function as the principal nucleation module that generates MTs in cells.

There is growing evidence that γ-TuRC alone does not account for all the MT nucleation activity in the cell. In the absence of γ-TuRC, MTs still form, albeit with significantly reduced numbers and kinetics [11][12][13] . Additionally, at least one factor of unknown identity besides γ-TuRC is needed to promote nucleation from centrosomes 14 . Furthermore, purified γ-tubulin complexes display low nucleation potential in vitro 5,7,9,[14][15][16][17][18] that does not match the nucleation activity observed from cellular MTOCs. Together, these data suggest that the current γ-TuRC-centered model of MT nucleation is incomplete. Thus, the search for factors that generate MTs in the cell is ongoing and how MT nucleation occurs remains to be elucidated.
After their nucleation, growth of MTs is necessary for building cytoskeletal structures. The major MT polymerase in the cell, XMAP215, increases polymerization rates up to 10-fold in vitro and in vivo [19][20][21][22][23][24][25][26][27][28] . XMAP215 is a multi-domain protein consisting of five TOG domains at the N-terminus and a C-terminal domain 23 . The TOG domains bind to soluble αβ-tubulin dimers 22,29,30 and are essential for the polymerase activity of XMAP215 24 . Recently, TOG domains 4 and 5 have been suggested to interact with αβ-tubulin incorporated in the MT lattice 31 . While primarily characterized as a polymerase, XMAP215, similar to a number of other MT-associated proteins, promotes spontaneous MT assembly 27,29,32 and re-growth from MT templates in vitro 33 . However, mass balance model of the mitotic spindle suggested that XMAP215 only enhances growth of individual MTs and does not influence their nucleation 26 . Therefore, it remains unclear whether XMAP215 is involved in nucleating MTs in the cytoplasm, because assays to visualize nucleation events in the cell are lacking. Furthermore, if XMAP215 were to function as a MT nucleator, its relationship with the universal nucleator γ-TuRC needs to be established and their mechanism determined 27,34,35 .
In this study, we demonstrate that XMAP215 is a bona-fide MT nucleation factor that cooperates with γ-TuRC. Using Xenopus egg extracts to resolve individual nucleation events, we show that XMAP215 determines the extent of MT nucleation in the cytoplasm. XMAP215 and γ-TuRC operate synergistically to generate MTs in vitro, and XMAP215's C-terminus is required for this cooperation. We further demonstrate that XMAP215 directly interacts with γ-tubulin via its C-terminus. Thus, XMAP215 is a major MT nucleation factor that functions with γ-TuRC to give rise to the MT cytoskeleton.
To assess XMAP215's role, we first added recombinant XMAP215 to Xenopus egg extracts, which contains approximately 120 nM endogenous XMAP215 26 . We expected the branched structures to appear larger due to longer and faster growing MTs, but less dense due to unaltered level of MT nucleation 26 . We quantified the number of MTs by detecting each plus-tip as the readout for MT nucleation. Surprisingly, XMAP215 increased the number of MTs in a concentration-dependent manner ( Supplementary Fig. 1A-B, Supplementary Video 1). Addition of 480 nM XMAP215 increased MT numbers up to 30-fold, while the growth speed of MTs remained nearly constant ( Supplementary Fig. 1B-C). These results show that XMAP215 has a dramatic effect on MT generation. XMAP215 increases nucleation events, but is it required to generate MTs? To assess this, we immunodepleted XMAP215 from Xenopus extracts (Fig. 1A). Remarkably, MT nucleation was completely abolished in the absence of XMAP215 (Fig. 1B). This was confirmed by observing the reaction up to an hour, throughout the entire sample (2000 fields of view), and across tens of extract preparations. This phenotype has previously been observed only by immunodepletion of γ-tubulin, the universal MT nucleator 36 . To verify the specificity of this effect, we added back purified XMAP215, which rescued the branched structures with comparable MT count to the control-depleted extracts ( Fig. 1A-B, Supplementary Fig. 1D). Proteins such as TACC3, α-tubulin, augmin, TPX2 and γ-TuRC were not depleted ( Supplementary Fig. 1E). In summary, XMAP215 is required for generating MTs in Xenopus egg extracts.

Characterization of XMAP215's microtubule nucleation activity
To further characterize its role, we investigated the effect of XMAP215 concentration in MT nucleation and polymerization processes. We added increasing concentrations of XMAP215 to immunodepleted extracts and evaluated a range of XMAP215 concentrations: from subendogenous levels (15 nM) to 6-fold the endogenous concentration (720 nM). No MTs formed at 15 nM XMAP215 and only a few MTs were visible at 30 nM ( Fig. 1C-D, Supplementary Video 2). From 60 to 720 nM XMAP215, MT nucleation increased dramatically and the measured rate of MT nucleation varied linearly with XMAP215 concentration, without reaching saturation (Fig. 1E). We simultaneously measured the rate of polymerization or growth speed of MTs, which saturated at twice the endogenous concentration (240 nM), as previously reported 26 .
In summary, MT nucleation and polymerization by XMAP215 demonstrates different characteristics. First, no saturation limit for MT nucleation was observed for the range of XMAP215 concentrations tested (Supplementary Fig. 1A-B, Fig. 1E), yet the MT polymerization rate saturates near the endogenous concentration ( Supplementary Fig. 1C,  Fig. 1E). Second, a minimum concentration of XMAP215 (30 nM) is required for nucleating MTs, while XMAP215 promotes MT growth even at low concentrations 24,26 . These results support that XMAP215 not only functions as a polymerase, but also promotes MT nucleation in the cytoplasm.

XMAP215 is a general microtubule nucleation factor
These findings led us to investigate whether XMAP215 increases only branching MT nucleation events, or generates MTs independently, which we define as de novo nucleation events. The number of de novo nucleation events and consequently, the branched MT networks formed, increased linearly with XMAP215 concentration (Fig. 1F), suggesting that XMAP215 functions as a general MT nucleation factor. To directly characterize this, XMAP215-depleted extracts were used in the absence of RanQ69L, thereby not inducing branching MT nucleation, and increasing concentration of XMAP215 was added ( Fig. 2A,  Supplementary Video 3). Remarkably, de novo MT nucleation events increased by 150-fold between 60 nM and 720 nM of XMAP215 (Fig. 2B). The occurrence of branching nucleation events was negligible (Fig. 2B). As before, no MTs formed at XMAP215 concentration below 30 nM and the polymerization rate saturated at 240 nM (Supplementary Fig. 2A). Additionally, we verified that XMAP215 stimulates de novo nucleation in the absence of the branching factors TPX2 and augmin 36 (Fig. 2C-E, Supplementary Video 4). Thus, XMAP215 functions as a general MT nucleation factor in the cytoplasm. Notably, MT nucleation by XMAP215 was indistinguishable in extracts depleted of TPX2 or augmin, excluding a specific synergy between TPX2 and XMAP215 in this assay that was previously observed in vitro 27 .
To further understand the relationship between XMAP215's nucleation and polymerization activities, we designed an experiment to specifically observe MT polymerization by XMAP215 in Xenopus extracts. Instead of observing endogenous MT nucleation, we added pre-formed MT seeds stabilized by GMPCPP to XMAP215-depleted extracts. Subsequently, wild-type XMAP215 was added back to initiate MT growth ( Supplementary Fig. 2B-E). In absence of XMAP215, polymerization was suppressed, resulting in infrequent EB1 blinking events on one end of MT seeds (roughly 20% seeds; Supplementary Fig. 2C,E). When 7.5 nM of XMAP215 was added back, MT growth immediately occurred from a large proportion of seeds (nearly 70%). At 15 nM XMAP215, MTs polymerized from all seeds. In contrast, no MT nucleation events were observed in the absence of seeds at XMAP215 concentrations below 30 nM (Figs. 1, 2A-B). Therefore, by titrating XMAP215 in Xenopus egg extracts, we find regimes where polymerization from MT plus-end occurs while nucleation does not (Figs. 1

γ-TuRC is required for microtubule nucleation by XMAP215
Our results show that XMAP215 is essential for MT generation, similar to the universal nucleator γ-TuRC, yet is γ-TuRC required for nucleation by XMAP215? To address this, we depleted γ-TuRC from Xenopus extracts and induced MT nucleation by RanQ69L, while XMAP215 and proteins required for branching MT nucleation were not depleted ( Supplementary Fig. 3A). Almost no MTs formed with endogenous XMAP215 level in the absence of γ-TuRC (Fig. 3A). Surprisingly, both de novo and branching nucleation events were severely reduced even upon addition of excess XMAP215 to γ-TuRC-depleted extracts ( Fig. 3A-B, Supplementary Fig. 3B-C; Supplementary Video 5). By specifically assaying de novo MT nucleation in Xenopus extracts as before ( Fig. 2A), we further verified that  3D-E). These results clearly demonstrate that both XMAP215 and γ-TuRC are required for all MT nucleation events in Xenopus extracts.

XMAP215 and γ-TuRC synergistically nucleate microtubules in vitro
Because we found that XMAP215 and γ-TuRC are both required for nucleation in Xenopus extract, we investigated their effect on MT nucleation in vitro. We first purified γ-TuRC via affinity purification from Xenopus egg extracts. All components of γ-TuRC were present as confirmed by mass spectrometry (Supplementary Fig. 4A), while proteins such as XMAP215, TPX2, α-tubulin, or TACC3 were not detected ( Supplementary Fig. 4B). Furthermore, the characteristic ring shape of γ-TuRC was observed by electron microscopy (Fig. 4A). Affinity-purified γ-TuRC was further purified by sucrose gradient centrifugation, where γ-TuRC fractionated at the expected size (Fig. 4B).
MT nucleation was analyzed in vitro by incubating soluble αβ-tubulin with purified γ-TuRC and XMAP215 independently or together (Fig. 4B). At minimal αβ-tubulin concentration of 10-12 μM, γ-TuRC (250-400 pM) or XMAP215 alone nucleated few MTs above the buffer control. Addition of γ-TuRC to increasing concentrations of XMAP215 (5-130 nM) resulted in a continuous increase in the amount of MTs observed (Fig. 4C). Remarkably, low concentration of XMAP215 (5-30 nM) together with γ-TuRC resulted in 6 to 10-fold increase in the number of MTs generated when compared to the sum of MTs generated by XMAP215 and γ-TuRC individually ( Supplementary Fig. 4C). With higher XMAP215 concentrations, even more prominent increase in MT generation was observed when γ-TuRC was present (Fig. 4C). However, due to emergence of long, bundled MTs, individual MTs could not be counted and total MT fluorescence intensity across all reactions was measured instead (Fig. 4D). These measurements demonstrate that XMAP215 and γ-TuRC together induce more MT generation than the sum of MT mass produced by either component individually across the range of XMAP215 concentration ( Fig. 4C-D, Supplementary Fig. 4D). Similar results were obtained with the gradient-fractionated γ-TuRC ( Supplementary Fig. 4E). This in vitro reconstitution agrees with our results in Xenopus extracts ( Fig. 1-2). Thus, XMAP215 and γ-TuRC synergistically promote MT nucleation both in vitro and ex vivo.

TOG domains of XMAP215 are essential for microtubule nucleation
We next asked what regions of XMAP215 are required for MT nucleation activity in the cytoplasm. We generated versions of XMAP215 lacking TOG domains from the N-terminus or missing its C-terminus. All constructs were tested for their nucleation activity in Xenopus extracts following depletion of endogenous XMAP215. Branching MT nucleation was stimulated by TPX2's C-terminal half in addition to RanQ69L as described previously 36 , which allows for robust measurements of low nucleation levels by some XMAP215 constructs.
Deleting the TOG1 domain (TOG2-CT) caused significant reduction in MT nucleation ( Smaller branched structures were generated when the C-terminal domain of XMAP215 was removed (TOG1-5), while further truncation from the C-terminus preserving TOG regions 1-4 additionally reduced the nucleation activity of the protein ( Fig. 5B-C, Supplementary Video 7). These results indicate that the entire protein is needed for full MT nucleation activity, with the TOG domains being essential for retaining a minimal level of nucleation.
XMAP215's TOG domains 1 and 2 linked to a MT-binding region has been reported to recapitulate the minimal polymerase activity 24 . This protein construct (TOG12-Kloop) did not have any discernable nucleation activity, suggesting that the minimal polymerization domains are not sufficient to promote MT nucleation.
Because TOG domains interact with αβ-tubulin to promote polymerization 30 , we tested whether disrupting αβ-tubulin binding to TOGs by mutating two residues in each TOG domain 24  Mutating residues in TOGs 1-3 individually dramatically reduced the nucleation activity of TOG1-5, with the more N-terminally situated TOGs having a greater effect, matching their described function in polymerization 24 . In contrast, mutation of TOG4 had a minor effect, while mutating TOG5 did not significantly impair MT nucleation. Interestingly, the loss of TOG5 domain caused significant reduction in MT nucleation ( Fig. 5B-C, compare TOG1-5 with TOG1-4) while mutating TOG5 did not, suggesting that TOG5's role may not involve its interaction with αβ-tubulin dimers. Lastly, maintaining the αβ-tubulin binding of TOGs 1 and 2 (TOG1-5 3-5AA) did not restore nucleation, demonstrating that TOG1 and TOG2 alone are not able to promote MT nucleation. Taken together, the interaction between αβ-tubulin and XMAP215's TOG domains, in particular by the N-terminally situated TOGs, is important for MT nucleation activity.

C-terminus of XMAP215 is required for efficient microtubule nucleation
To compare how XMAP215's domains contribute to MT polymerization versus MT nucleation, we chose three constructs, namely the N-terminal TOG domains 1-5 (TOG1-5), the construct containing TOG5 and the C-terminus (TOG5-CT) and the minimal polymerase (TOG12-Kloop). First, we performed in vitro MT polymerization from stabilized MT seeds in the presence of these constructs. Wild-type XMAP215 promoted MT plus-end growth in a concentration-dependent manner (Fig. 6A, Supplementary Fig. 6A), as described previously 23,24,26,37 . TOG1-5 strongly promoted MT polymerization to an extent similar to the full-length protein. The maximum MT polymerization rate by the minimal polymerase TOG12-Kloop was 2-3 fold lower than wild-type XMAP215, as previously reported 24 . Most importantly, the C-terminal construct TOG5-CT did not increase MT polymerization rate even at high protein concentrations. In summary, XMAP215's N-terminal TOG domains constitute the MT polymerizing region of the protein, and its C-terminus is neither required for nor affects the growth rate of MT plus-ends.
To thoroughly compare the nucleation activity to polymerization by these domains, we repeated the branching MT nucleation assay in Xenopus extracts as a function of XMAP215 concentration. As before, upon depletion of endogenous XMAP215 followed by add-back of increasing concentrations of wild-type XMAP215, an increase in the rate of MT nucleation was observed (Fig. 6B, Supplementary Fig. 6B; Supplementary Video 9). Although addback of TOG1-5 forms MT networks, the rate of MT nucleation was significantly reduced. Even at high TOG1-5 concentrations (500 nM -1 μM), the rate of MT nucleation remained lower than at endogenous concentrations of full-length XMAP215 (120 nM). Neither addition of up to 1 μM of the C-terminal construct (TOG5-CT) nor the minimal polymerase (TOG12-Kloop) restored MT nucleation. The side-by-side comparison of these constructs in MT polymerization and nucleation assays ( Fig. 6A-B) demonstrates that XMAP215's Cterminus does not directly promote polymerization, yet is required for rapid MT nucleation in Xenopus extracts.
To further analyze the requirement of XMAP215's C-terminus in nucleating MTs, we performed in vitro MT nucleation assay with purified γ-TuRC and increasing concentrations of TOG1-5 protein (Fig. 6C). Similar to full-length XMAP215, TOG1-5 alone displays low nucleation activity. Combining γ-TuRC and TOG1-5 resulted in slightly increased MT nucleation when compared to γ-TuRC alone (Fig. 6C, Supplementary Fig. 6C), yet TOG1-5 induced lower MT nucleation with γ-TuRC than the wild-type XMAP215. At higher protein concentrations, the difference in nucleation activity of full-length XMAP215 and TOG1-5 was more pronounced and TOG1-5 shows a dramatically reduced effect on MT generation when combined with γ-TuRC (Fig. 6D). Together, these results demonstrate that the polymerizing region of XMAP215, the N-terminal TOG1-5, is not sufficient to promote MT nucleation similar to the full-length XMAP215 and γ-TuRC, and that the conserved Cterminus is required for robust MT nucleation.

XMAP215 and γ-TuRC components interact in Xenopus egg extract
Because γ-TuRC and XMAP215 cooperate to generate MTs, we hypothesized that these components may interact in Xenopus egg extracts. Indeed, components of γ-TuRC coimmunoprecipitate with XMAP215 ( Supplementary Fig. 7A) and reciprocally, endogenous XMAP215 was co-precipitated with γ-TuRC ( Supplementary Fig. 7B), confirming an interaction between these components in egg extracts. Notably, the branching factor TPX2 did not co-precipitate in either experiments, while augmin did not co-precipitate with XMAP215, verifying the specificity of this interaction. We note that in Drosophila S2 cells, XMAP215 and augmin were co-immunoprecipitated 38 . This could be due to system-specific differences, such as the species, cell-cycle state, or presence of MTOCs that are absent from Xenopus extracts.
It is noteworthy that αβ-tubulin was not co-immunoprecipitated with endogenous XMAP215 (Supplementary Fig. 7A). This was surprising because XMAP215 and αβtubulin directly interact in vitro 23,24 . We determined that XMAP215 and αβ-tubulin interact in vitro using size exclusion chromatography only under low salt buffer 23,24 ( Supplementary Fig. 7C, 75 mM NaCl). This interaction was not maintained near the physiological salt concentration (150 mM NaCl), suggesting that XMAP215's binding to αβ-tubulin is transient in the cytoplasmic environment of Xenopus extracts.

XMAP215's C-terminus directly binds to γ-tubulin
To further characterize the interaction between XMAP215 and γ-TuRC, we specifically tested binding between XMAP215 and γ-tubulin, the most abundant component of γ-TuRC. We performed size exclusion chromatography with purified human γ-tubulin and XMAP215. To prevent formation of γ-tubulin filaments at several hundred nM concentration 39 , we used a low molar ratio of γ-tubulin to XMAP215. In this setup, γtubulin alone elutes in fractions H-K (Fig. 7A) and partially oligomerizes in the chromatography buffer (compare to Supplementary Fig. 8A). Interestingly, in the presence of XMAP215, γ-tubulin shifts toward an earlier elution volume corresponding to XMAP215's elution, demonstrating its direct interaction with γ-tubulin. This binding was verified using in vitro immunoprecipitations ( Supplementary Fig. 8B). Notably, this interaction occurs even at the low γ-tubulin concentrations used in these experiments (140 nM in Fig. 7A, 80 nM in Supplementary Fig. 8B), which are within the cytoplasmic γtubulin concentrations 40 . Mutating the residues essential for αβ-tubulin interaction in TOG domains did not abolish γ-tubulin binding ( Supplementary Fig. 8C). Next, we identified the XMAP215 domain responsible for interacting with γ-tubulin. The N-terminal truncation containing TOGs 1-4, or TOGs 1-5, or the protein containing TOGs 1 and 2 repeated twice did not interact with γ-tubulin (Fig. 7A, Supplementary Fig. 8C). Surprisingly, the Cterminal construct containing TOG5 and the C-terminal domain (TOG5-CT) strongly shifted γ-tubulin to an earlier elution volume, similar to full-length XMAP215 (Fig. 7A). These results demonstrate that XMAP215 directly binds to γ-tubulin, potentially in an oligomeric form, via its C-terminal domain.
We next validated the region of XMAP215 that interacts with αβ-tubulin using size exclusion chromatography. The N-terminal TOG1-4 protein formed a complex with αβtubulin similar to wild-type XMAP215, while the C-terminal construct TOG5-CT did not bind αβ-tubulin (Fig. 8A, Supplementary Fig. 8D). Altogether, these results show that XMAP215's N-terminus binds to αβ-tubulin, while its C-terminus interacts with γ-tubulin.

Discussion
How MT nucleation occurs is critical for understanding how MT cytoskeletal structures that enable cell function are assembled. γ-TuRC has been thought to play a prominent role in MT nucleation since its discovery in 1989 41 , but evidence suggested other factors might exist as well. In this study, we uncover that the major polymerase of the cell, XMAP215, is a principal MT nucleation factor that directly interacts with γ-tubulin and functions synergistically with γ-TuRC to generate MTs in the cytoplasm.
We propose several reasons why XMAP215's role in MT nucleation has been underappreciated until now. Of primary importance, assays to resolve MT nucleation in the cytoplasm and to distinguish it from plus-end growth were previously unavailable. Additionally, compensatory nucleation mechanisms in the mitotic spindle 11,26,42 , introduction of MTOCs such as centrosomes 32 that contain XMAP215 43 , or incomplete loss of XMAP215 20,44 could have masked its function in MT nucleation. Here, by developing a high-resolution assay and analyses system, stimulating nucleation without MTOCs, and via complete XMAP215-depletion, we demonstrate that XMAP215, along with γ-TuRC, is necessary for all nucleation events in Xenopus egg extracts and determines the rate of MT generation in the cytoplasm. One widely accepted model for MT nucleation 7 proposes that γ-tubulin complexes perform the entire nucleation process. In this scenario, XMAP215, acting solely as a polymerase, catalyzes the addition of αβ-tubulin dimers to the plus-end after MT nucleation has occurred 26 . In contrast, our study clearly demonstrates that XMAP215 and γ-TuRC function cooperatively in the nucleation process, while either factor independently supports low MT nucleation (Fig. 8B, left). What is the molecular mechanism by which γ-TuRC and XMAP215 nucleate MTs? We propose a synergistic nucleation model in which both XMAP215 and γ-TuRC are directly involved in assembling MT precursors. The C-terminal region of XMAP215 binds to γ-TuRC where the TOG domains recruit αβ-tubulin dimers and both factors together form the MT nucleation intermediates (Fig. 8B, right). The domain organization of the conserved C-terminus still remains to be characterized, but the existence of a sixth TOG domain has been speculated 45,46 , which could be a potential γ-tubulin interaction site. In the N-terminus, TOG domains 1 and 2 could primarily recruit αβ-tubulin dimers, whereas the later TOG domains stabilize the newly forming MT. This is supported by recent work proposing TOG5's interaction with lattice-incorporated αβ-tubulin 31,47 . Following MT nucleation, XMAP215 could translate with the growing MT plus-end to catalyze MT polymerization. In the future, it will be important to further investigate the mechanism of how XMAP215 and γ-TuRC cooperate to nucleate a MT using single molecule techniques and structural studies.
Because MT nucleation is the rate-limiting process in constructing the cytoskeleton, activity of the principal nucleation module consisting of XMAP215 and γ-TuRC must be critically regulated in space and time to rapidly assemble complex MT structures. While γ-TuRC has been proposed to be regulated by activator proteins such as CDK5RAP2 15 , it is feasible that XMAP215 is also regulated. For example, the localization of XMAP215 at the centrosome by TACC3 48 could function to concentrate XMAP215 near γ-TuRCs, or post-translational modifications 19,49 could temporally regulate its nucleation activity. With the discovery of XMAP215 and γ-TuRC's cooperation in the MT nucleation process, we can now begin evaluating additional factors that regulate MT generation.

Antibodies
See Supplementary Table 1 for information on antibodies used in this study.

Xenopus egg extracts, immunodepletion and immunoprecipitation experiments
CSF extracts were prepared from Xenopus laevis oocytes as described 53,54 and either used immediately or immunodepleted 51,53 . When working with Xenopus laevis, all relevant ethical regulations were followed, and all procedures were approved by Princeton IACUC. For immunodepletions, XMAP215-, TPX2-, HAUS1-, or γ-tubulinand IgG(control)antibody were conjugated to Protein A Dynabeads (Life Technologies #10002D) overnight. 50-150 μl extracts were subjected to two sequential rounds of immunodepletion by incubating with roughly equal volume of antibody-conjugated bead slurry for 30-40 minutes in each round, as described previously 51,53 . See Supplementary Table 1 for antibody concentrations. Control immunodepletion was always performed and depletion efficiency was assessed using western blots and functional assays.
The immunoprecipitation (IP) experiment of XMAP215 and reciprocal IP of γ-TuRC (Supplemental Fig. 7A-B) was performed by coupling either XMAP215, γ-tubulin or Mzt1 antibody to Dynabeads. For IP of XMAP215, 75μl of egg extract was incubated with 125μl of Dynabeads slurry for 1 hour on ice. Beads were washed thrice with 5 volumes of TBS buffer + 0.1% Tween20. For IP of γ-TuRC, 50μl of extract was incubated with 50μl of Dynabeads slurry for 1 hour at 4°C, then washed twice with 5 volumes of CSF-XB. Beads were boiled and protein content analyzed using immunoblot (chemiluminescence), imaged via X-ray film or iBright imaging system (ThermoFisher Scientific).

Microtubule nucleation assays in Xenopus egg extracts
Microtubule (MT) nucleation was assayed in Xenopus egg extracts as previously described 36,55 . Briefly, all reactions were performed with 0.5mM vanadate (sodium orthovanadate, NEB) to avoid sliding of MTs. 5.5μM RanQ69L was added for branching nucleation experiments. For de novo nucleation reactions, an equal volume of buffer was added. MTs were labeled with 0.89μM Cy5-labeled porcine brain tubulin and 200nM EB1-mCherry (plus-tips). For testing activity of XMAP215 constructs (Fig. 5, 6), 1 μM GSTtagged X.l. TPX2 α3-7 36 was also added to decrease the time lag until MT nucleation. For all experiments, either untreated extracts were used, or endogenous XMAP215, TPX2, augmin or γ-tubulin was immunodepleted prior to use, and purified wild-type XMAP215 or XMAP215 constructs were added to the extract mixture at specified final concentrations. For Fig. 6, freshly purified proteins (TOG1-5, TOG12-Kloop, TOG5-CT) were used for comparing MT nucleation in extracts to in vitro polymerization (Fig. 6A), and the same protein purification was used in both assays. The reaction mixture was prepared, incubated on ice for 2-3 minutes, 6μl mixture was pipetted into a flow cell to start the reaction (timed as 0 seconds), and imaged for 15-20 minutes at 18-20°C in a temperature-controlled room. All reagents added to extracts were stored in CSF-XB buffer, no more than 25% volumetric dilution of extracts was performed, and reaction composition was kept constant for each experimental set. All reactions shown in individual figure panels were performed with one Xenopus extract preparation. TIRF imaging was performed with Nikon TiE microscope using 100x 1.49 NA objective. Andor Zyla sCMOS camera was used for acquisition, with the field of view of [165.1x139.3 μm] or [132.1x132.1 μm]. 2x2 binned, dual color images were acquired every 2 seconds using NIS-Elements software (Nikon). Brightness and contrast were optimized individually for display.

Analysis of microtubule nucleation in Xenopus egg extracts
Number of MTs and their growth speed in extracts was measured using a combination of custom-built and existing MATLAB software. EB1 intensity in the image sequence was homogenized by dividing a blank background image that was pre-blurred with Gaussian kernel of 20 pixels. For experiments in Figs. 1-3, immotile features were first discarded by applying temporal median filter of 6-8 frames. EB1 comets, were detected by comet detection and tracked by plus-end tracking module of uTrack 56,57 . An alternative preprocessing method was devised to analyze nucleation by XMAP215 constructs (Fig. 5-6) that was insensitive to growth speed or EB1 comet size, which varied between constructs. After background correction, EB1 comets were enhanced by applying 2 pixels wide, Laplacian of Gaussian filter, followed by uTrack's comet detection. MT nucleation curves were generated by plotting the number of EB1 comets detected or the number of EB1 tracks for each frame over time, as specified in each figure legend. For growth speed calculation, all EB1 tracks were classified into growth and forward gaps using uTrack's plus-end dynamics classification. Growth speed was measured from consecutive frames of growth events for all tracks.
De novo nucleation events and branched MT networks (Figs. 1F, 2B) were counted manually for the entire experimental duration or until a fixed time specified in figure legends. Lifetime of MTs was measured manually for Supplementary Fig. 1D. Data regression to Michaelis-Menten kinetics or straight line was performed using curve fit function in MATLAB.

Purification of γ-TuRC from Xenopus egg extracts
γ-TuRC purification was based on previous reports 5, 17, 58, 59 . 10 ml of Xenopus egg extract was diluted with 5 volumes of CSF-XB+ buffer (CSF-XB buffer + 1mM GTP, 1mM DTT and protease inhibitors) supplemented with 10% w/v sucrose and spun at 3500 rpm for 10 minutes to pellet large particles. 1mM GTP was added to all buffers and all steps were performed at 4°C. The supernatant was diluted two-fold with CSF-XB+ buffer, and filtered through a 0.22μm filter. γ-TuRC was precipitated by addition of 30% polyethylene glycol (PEG) solution to final PEG concentration of 6.5%, incubated for 30 minutes, then centrifuged for 20 minutes at 17,500xg. The PEG pellet was resuspended in 20ml CSF-XB+ buffer with 0.05% NP-40, sample was centrifuged at 136,000xg for 7 minutes, and supernatant was pre-cleared with Protein A Sepharose beads (GE LifeSciences #17127901) for 20 minutes. Beads were removed, 1ml of γ-tubulin antibody (1mg/ml) was added, and sample was incubated for 2 hours with rotation. 1ml of washed Protein A Sepharose beads were then added and incubated for 2 hours. Beads containing bound γ-TuRC were centrifuged and the flow-through removed. Beads were washed thrice with 10ml of CSF-XB + with 0.05% NP-40, with 30ml of CSF-XB+ containing 250mM KCl, 10ml of CSF-XB+ with 1mM ATP to remove heat-shock proteins, and finally with 10ml of CSF-XB+ buffer. 1ml of γ-tubulin peptide (residues 413-451) at 0.4mg/ml in CSF-XB+ buffer incubated with beads overnight. After 10 hours, additional CSF-XB+ buffer was added and fractions 1-3 (1ml each) were collected. For sucrose gradient fractionation, CSF-XB+ buffer with 10% sucrose and CSF-XB+ with 50% sucrose was layered in an ultra-clear 2.2ml tube (11x34 mm, Beckman Coulter) and continuous 10-50% sucrose gradient was made using two-step program in Gradient Master 108 machine. Peptide-eluted fractions were combined, concentrated, layered on top of the gradient and subjected to centrifugation at 200,000xg in TLS55 rotor for 4 hours. The gradient was fractionated from the top in 10-11 fractions and immunoblotted against GCP4, GCP5 and γ-tubulin to determine γ-TuRC fractions.

Negative stain electron microscopy
5μl of γ-TuRC sample was pipetted onto carbon grids and incubated at room temperature for 5 minutes. The grid was rinsed briefly in deionized water, and 1% uranyl acetate was flowed over. Uranyl acetate was wicked away and the grid was air-dried. Images were taken on CM100 TEM microscope.

Mass Spectrometry
Peptide-eluted γ-TuRC obtained was precipitated with Trichloroacetic acid (TCA), resuspended in SDS buffer, separated by PAGE and stained with Coomassie dye. Gel bands were excised and analyzed by the Mass Spectrometry Core facility at Princeton University. Gel bands were trypsin-digested and peptides analyzed on Thermo Orbitrap Elite in data dependent mode with 120,000 MS1 resolution and up to 20 MS/MS scans in ion trap. Raw files were searched using Proteome Discoverer 2.1 (Thermo Scientific) with 10ppm MS1 and 0.5Da MS2 tolerances. Caramidomethylation of cysteine was set as fixed modification, while oxidation of methionine, deamination of asparagine and conversion glutamine to pyro-glutamate (at N-termini) as dynamic modifications. Data were searched against Xenopus protein database defined in ref. 40 supplemented with common contaminant proteins. Scaffold 4.7.5 (Proteome Software) was used to validate MS/MS identifications. 1% protein FDR and at least 2 peptides were accepted as confident protein identifications.

Nucleation assay with purified γ-TuRC
Peptide-eluted γ-TuRC was diluted two-fold in BRB80 buffer (80mM K-PIPES, 1mM MgCl 2 , 1mM EGTA, pH 6.8) to final concentration of 3-6nM GCP4 subunit, determined by quantitative immunoblot against purified, recombinant GCP4. Gradient fractionated γ-TuRC was concentrated and exchanged into BRB80 using Amicon Ultra-4 ml 10,000 NMWL centrifugal filter unit. Bovine brain αβ-tubulin (Pursolutions) +10% Alexa-568 labeled porcine brain tubulin, BRB80 buffer and 1.5mM GTP were combined and clarified by centrifugation at 80,000 rpm (TLA100.1 rotor, Beckman Coulter) for 20 minutes at 2°C. The final tubulin concentration for each reaction ranged within 10-12μM. Microtubule nucleation was performed as described previously 5,17,58,60 . Clarified tubulin mixture was combined with γ-TuRC with or without recombinant XMAP215 or TOG1-5 at described concentrations to 20μl final volume on ice. The solution was incubated at 37°C for 5 minutes, diluted with 80μl warm BRB80, and the reaction terminated with addition of 100μl of 2% glutaraldehyde in BRB80 buffer, then incubated at room temperature for 5 minutes. Samples were diluted 1:10 in BRB80, layered on top of 5ml cushion of 20% glycerol in BRB80 buffer in 15ml Corex tube containing custom inserts to support round, poly-lysine coated coverslips. The sample was centrifuged for 45 minutes at 25,000xg in HB-6 rotor at 4°C. Following centrifugation, MTs were fixed with ice-cold methanol, coverslip mounted in Prolong Diamond and imaged via TIRF microscopy as described above. All images from each experimental set were taken in the same imaging session with constant TIRF angle, laser power and exposure.
Number of MTs in the entire field of view was counted manually, and their total intensity was determined using a MATLAB script. Images were thresholded via Otsu method, eroded to eliminate tubulin aggregates having aspect ratio of one and a mask for MT signal was generated. Average intensity from reverse mask was subtracted from the image as background. The residual fluorescent intensity per image was summed and reported.

In vitro microtubule polymerization
Coverslips and glass slides were coated with dichlorodimethylsilane 61 . Biotin-labeled, Alexa-594, GMPCPP MTs were made as described 61 , flash frozen and stored at −80°C. Aliquots were thawed at 37°C and diluted 2000-fold in BRB80 buffer. TIRF objective was warmed to 35°C using objective heater (Bioptechs, model 150819-13) prior to experiments.
Polymerization reactions were performed similar to previous reports 62 . Briefly, the flow chamber was incubated with anti-biotin antibody for 5 minutes, rinsed with BRB80 buffer, incubated with 1% Pluronic F127 in BRB80 for 5-10 minutes, rinsed again with BRB80, and incubated with diluted MT seeds for 10 minutes. The slide was protected from light. Unattached seeds were remove with BRB80 buffer + Trolox mix (2.5mM PCA, 25nM PCD, 2mM Trolox) 63 . Attached seeds were visualized on the microscope. Polymerization mix (7.5μM tubulin, 1mg/ml κ-casein, 14.3mM BME, 1mM GTP in BRB80) was flowed in at room temperature to prime the channel, followed by XMAP215 reaction mix (XMAP215 protein at indicated concentration, 6.75μM unlabeled bovine tubulin, 0.75μM Cy5-labeled tubulin, 1mg/ml κ-casein, 14.3mM BME, 1mM GTP and Trolox mix in BRB80). The chamber was immediately placed on the objective, and imaged using the TIRF setup described above with the field of view of [132.1x132.1μm]. Single color images were obtained every 6 seconds, using 647nm excitation (Cy5-dye), for 15 minutes, starting 2 minutes after the slide placement.
MT polymerization was analyzed using ImageJ software. Maximum intensity projection of movie stacks was produced and all polymerized MTs were marked using ROI generator. The stacks were resliced using multi-kymograph plugin. Growth speed of plus-end was obtained as the slope of each kymograph.

Microtubule polymerization in Xenopus egg extracts
Coverslips were passivated with dichlorodimethylsilane 61 . 20μM biotin and Alexa-488labeled, GMPCPP seeds were prepared fresh as described 23 , digested with 0.2mg/ml subtilisin A protease (P5380, Sigma) for 20 minutes at 37°C. The digestion was stopped with 10μM PMSF. MTs were pelleted for 20 minutes and resuspended in BRB80. Flow chambers were treated sequentially with anti-biotin antibody (10 minutes), BRB80 buffer, 500-1000-fold dilution of subtilisin-digested seeds (10 minutes), and finally with CSF-XB buffer to remove unattached seeds. XMAP215 was depleted from egg extracts, and low concentrations of recombinant XMAP215-GFP was added back, along with 0.5mM vanadate, 0.89μM Cy5-tubulin and 200nM EB1-mCherry. Extract mixture was added to chamber containing subtilisin-digested seeds (timed as 0 seconds). Growth from most MT seeds was observed immediately (within 30-60 seconds). Imaging was completed within 300 seconds due to disappearance of seeds by motors and slow degradation in extracts. TIRF imaging was performed as described above. 2x2 binned, triple channel images of Alexa-488 GMPCPP seeds (pseudocolored as blue), EB1-mCherry (pseudocolored as green), Cy5tubulin (red) were acquired every 3.5 seconds at 18-20°C.
Growth speed was obtained by analyzing composite kymographs of EB1 and tubulin intensity, as described above. Central, most-illuminated part of movie stack was cropped [103.08x103.08μm], GMPCPP seeds were observed roughly between 90-140 seconds from the start of reaction, and composite EB1 and tubulin signal was used to visually assess if the observed GMPCPP seed polymerized. For buffer addback (0nM XMAP215), tubulin signal was not observed on all seeds, and this quantification was based on seeds where one end clearly showed EB1 spots associating, translating and dissociating in the movie stack (or EB1 blinking).

Size exclusion chromatography experiments
Size exclusion chromatography (SEC) with γ-tubulin was performed as follows. All steps were performed at 4°C. Purified, human γ-tubulin was generous gift from Michelle Moritz in γ-TB buffer [defined as 50mM K-MES pH6.6, 5mM MgCl2, 1mM EGTA, 10mM thioglycerol, 10μM GDP] with additional 500mM KCl and 10% glycerol. At high protein concentration, γ-tubulin forms large oligomers or filaments below physiological salt amounts 39 , therefore the salt concentration was reduced only when γ-tubulin concentration was simultaneously decreased. Small volumes (100-300μl) of XMAP215 proteins were dialyzed (69590, Life Technologies) into γ-TB buffer without salt or GDP for 2 hours. After dialysis, protein aggregates were pelleted and concentration was estimated using Bradford Reagent (Bio-Rad). γ-tubulin was diluted to 280nM in γ-TB buffer containing 220mM KCl. γ-tubulin and XMAP215 proteins were individually clarified by ultracentrifugation at 80,000 rpm in TLA 100 (Beckman Coulter) for 15 minutes, then mixed in 1:1 volume ratio and incubated on ice for 10 minutes. 500μl of the mixture was loaded onto Superdex 200 Increase 10/300 column (GE Healthcare). The final concentration loaded to the column was 140nM for γ-tubulin and between 1-1.4μM for most XMAP215 proteins, except XMAP215 FL 1-5AA at 0.7μM. The column was equilibrated with γ-TB buffer containing 85 mM KCl and chromatography was performed in this buffer.
For all control chromatography runs, equal volume of corresponding buffer was used. Absorbance at 280 nm was recorded. High molecular weight gel filtration standards (Thyroglobulin, Aldolase and Ovalbumin) were purchased from GE Healthcare (Catalog #28403842) and used to estimate the Stoke's radii of eluted proteins in the same buffer as used for corresponding SEC run 64 . For γ-tubulin SEC experiments, 0.3ml fractions were collected and alternate fractions eluted between 8.5ml and 16.6ml were analyzed via immunoblot against γ-tubulin and GFP to detect XMAP215-GFP constructs. Secondary antibody conjugated to 800nm IRDye (LI-COR) was used and imaged with Odyssey CLx imaging station (LI-COR). For αβ-tubulin SEC experiments, 0.2ml fractions were collected and alternate fractions between 8.5ml and 13.9ml were analyzed via SDS-PAGE, stained with Coomassie dye.

In vitro immunoprecipitation with γ-tubulin
Immunoprecipitation (IP) was performed using GFP tag on XMAP215 as the bait and γtubulin as the prey. Anti-GFP antibody was coupled to Protein A Dynabeads overnight. All proteins were pre-clarified via ultracentrifugation. 500nM XMAP215-GFP or GFP-tag was each mixed with 80nM γ-tubulin in pulldown buffer (γ-TB buffer from above with final 90mM KCl and 0.04% Tween20). Empty beads (buffer) were used as additional control without any GFP-tagged protein. One volume of Dynabeads was suspended in one volume each IP mixture, incubated on rotisserie for 1 hour at 4°C, then washed twice with 5 volumes of pulldown buffer. Protein content on the beads was analyzed by immunoblot against γtubulin and GFP.

Statistics and reproducibility
All nucleation or polymerization experiments with Xenopus egg extracts were reproduced with at least three independent experiments, unless stated otherwise, with extracts freshlyprepared from eggs laid by different animals on different days. Representative results are displayed, or all data was reported, as specified in individual figure legends. Similar results were seen in all replicates that were performed. For in vitro polymerization, at least two independent set of experiments were performed on different days with different protein purifications, where at least two replicates were completed for each purification. All results were pooled and reported, and no difference was observed between different experimental sets. Nucleation experiments with purified γ-TuRC were repeated at least thrice, unless stated otherwise. Each replicate consisted of an independent preparation of γ-TuRC from different extract preparations. All results from the replicates showed similar results, and were pooled and reported. All chromatography runs with γ-tubulin were repeated at least twice on different days with freshly prepared buffers and multiple protein purifications. Similar results were seen with all runs performed. Chromatography runs with αβ-tubulin were performed once at the exact buffer and concentration specified, with supporting runs performed in slightly varied conditions on different days with freshly prepared buffers and multiple protein purifications, as specified in figure legends. Immunoprecipitation experiments in Xenopus extracts or in vitro were performed two-four times as indicated in respective figure legends. All replicates showed similar results and representative experiment was reported. No statistical tests were performed or reported.

Life Sciences Reporting Summary
Further information on experimental design is available in Life Sciences Reporting Summary.

Code availability
MATLAB-based custom-built software in conjunction with open source software (uTrack, Danuser lab) was used for detecting and tracking EB1 particles. For other measurements and data analysis, MATLAB-based custom-built scripts were used. All codes are available from the authors upon request.

Data availability
Source data for figs. 1E, 1F, 2B, 6B, 6D, and supplemental figs. 2D, 2E, 4C, 4E and 6C have been provided as Supplemental Table 2. All data supporting the findings of this study are available from the corresponding author upon request.   (B) Total number of MT-independent (de novo) and MT-dependent nucleation events that occurred until 600 seconds of the reaction were counted and plotted against XMAP215 concentration. Data for MT-independent nucleation events versus XMAP215 concentration was fitted to a linear curve (solid green). XMAP215 concentration is plotted on log scale. Few MT-dependent nucleation events were also observed (red circles). The analysis was repeated twice with independent extract preparations. See Supplementary Table 2 for source data.
(C) Immunoblot for TPX2 and augmin depletion corresponding to (D-E). Augmin was immunodepleted using anti-HAUS1 antibodies, and depletion was assessed by western blotting for two subunits -HAUS1 and HAUS6. The experiments in (C-E) were repeated twice with independent extract preparations, along with two supporting experiments performed. See Supplementary Fig. 9 for unprocessed blot.
(D) XMAP215-GFP was added to TPX2 and augmin immunodepletion at 360 nM in addition to endogenous protein in the presence of 5.5 μM RanQ69L. Equal volume of buffer was added for control reactions. Representative images at 20 minutes at displayed. Scale bar, 10 μm. See Supplementary Video 4.
(E) EB1 comets in the entire field of view were tracked using the analysis procedure described in Methods. Number of EB1 tracks were counted and plotted over time. The experiment was repeated with three independent extract preparations, including verification with XenC antibody for γ-TuRC immunodepletion 5,11 . (B) Number of MT-independent and MT-dependent nucleation events observed was tabulated with excess XMAP215 concentration. Nucleation events were counted until 800 seconds for each reaction. The few MTs that emerged in γ-tubulin depleted extracts were counted manually (both MT-independent and MT-dependent nucleation events). For IgG depletion, total number of MTs (EB1 comets) in the field of view was counted using image analysis, while de novo nucleation events were counted manually. The number of MTdependent nucleation events was calculated by subtracting MT-independent nucleation events from the total number of MTs. See Supplementary Fig. 3. The analyses were repeated twice with experiments performed on independent extract preparations, with one additional supporting set of results with anti-XenC immunodepletion. Fractions were analyzed by Silver-stained SDS-PAGE (top) and immunoblot (bottom) with antibodies against γ-tubulin, GCP5 and GCP4. These components were observed to peak in fractions 5 and 6, at the expected size for intact γ-TuRC. The bands for GCP6, 5, 4, 3, 2, γtubulin, NEDD1 and Mzt2 can clearly be seen in fractions 5 and 6 using silver staining. Representative image is displayed, and the experiment was repeated more than three times. See Supplementary Fig. 9 for unprocessed scans.
(C) Combination of purified γ-TuRC and XMAP215 promotes MT nucleation in vitro.
Purified γ-TuRC at 250-400 pM added to αβ-tubulin with GTP promotes MT nucleation as compared to control (elution from IgG beads, upper left panel). Addition of recombinant XMAP215, at concentrations from 5 nM to 130 nM, promotes low levels of MT nucleation, with bundling and increased MT length seen at high concentrations. Addition of both XMAP215 and γ-TuRC together causes a significant increase in the number of MTs nucleated, greater than simply adding the MTs generated by each component independently.
Representative fields of MTs are shown, nucleation assays were repeated with at least 3 independent γ-TuRC purifications. Scale bar, 10 μm. (D) Fluorescent intensity was quantified as the readout of amount of polymerized tubulin for 10 fields of view for each reaction condition. Data from three independent γ-TuRC preparations was pooled and displayed as mean±s.d. n=30 fields of view analyzed per reaction. The XMAP215+γ-TuRC reactions had greater fluorescent intensity as compared to control. A significant increase between microtubule mass can be seen starting from 35nM XMAP215 when combined with γ-TuRC. See Supplementary Fig. 4.   Supplementary Fig. 6B). Rate of nucleation by 120nM wild-type XMAP215 was normalized to 1 in each experimental set with Xenopus extracts. Where more than one reaction at 120nM wild-type XMAP215 was performed, their average was set to 1. Data was pooled from the following independent extract preparations: wild-type XMAP215 (8) (A) Size exclusion chromatography was performed with 140 nM of human γ-tubulin alone and with 1-1.4 μM XMAP215 protein constructs. 500 μl sample volume was injected into the column, 300 μl fractions were collected and alternate fractions eluted between 8.5 ml to 16.6 ml were analyzed via SDS-PAGE followed by immunoblot with γ-tubulin antibodies and GFP antibodies to detect the elution profile of γ-tubulin and GFP labeled XMAP215 proteins, respectively. Shift in γ-tubulin signal was observed to assess the region of XMAP215 that γ-tubulin interacts with. Stoke's radii of reference proteins are marked at their peak elution: Thyroglobulin (8.6 nm), Aldolase (4.6 nm) and Ovalbumin (2.8 nm). The contrast of each image was adjusted to display the entire elution profile clearly. Each chromatography run was repeated at least twice on different days at the specified concentration, and at least one additional supporting experiment for each displayed run (more than nine supporting runs in total) was performed at slightly different protein concentrations. Void volume of the column was measured as 8.7-8.8 ml. See Supplementary Fig. 8A-C. See also Supplementary Fig. 9 for unprocessed scans. and bovine αβ-tubulin (6.2 μM) were mixed and applied to size exclusion chromatography. 100 μl sample volume was injected into the column. Each chromatography run was performed once at the specified concentration. More than three additional supporting experiments were performed either with slightly different protein concentrations or using alternative protein constructs. Void volume of the column was measured as 8.7-8.8 ml. See Supplementary Fig. 8D. See also Supplementary Fig. 9 for unprocessed scans. (B) Schematic representation for how XMAP215 and γ-TuRC could together promote MT nucleation. While γ-TuRC independently induces low MT nucleation (left), its cooperation with XMAP215 promotes efficient nucleation of MTs. Synergistic microtubule nucleation model (right): XMAP215 binds to γ-TuRC with its C-terminus and promotes the assembly of αβ-tubulin dimers onto γ-TuRC via its N-terminus.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Mast cells are essential intermediaries in regulating IL-33/ST2 signaling for an immune network favorable to mucosal healing in experimentally inflamed colons

Mast cells (MCs) are potent tissue-resident immune cells that are distributed in the intraepithelial space of the intestine and have been implicated in regulating immune homeostasis and coordinating epithelial responses in inflamed mucosa of inflammatory bowel disease (IBD). IL-33 functions as an endogenous danger signal or alarmin in inflamed intestine segments. MCs highly express the IL-33 receptor ST2. However, the mechanisms underlying the immune regulation of MC-dependent IL-33/ST2 signaling at the barrier surface of the intestine remain largely unknown. We confirmed that MCs are required for the effective resolution of tissue damage using an experimental colitis model that allows for conditional ablation of MCs. After elucidating the IL-33 signaling involved in MC activity in the context of intestinal inflammation, we found that the function of restricted IL-33/ST2 signaling by MCs was consistent with an MC deficiency in response to the breakdown of the epithelial barrier. We observed that a tissue environment with a spectrum of protective cytokines was orchestrated by MC-dependent IL-33/ST2 signaling. Given the significant downregulation of IL-22 and IL-13 due to the loss of MC-dependent IL-33/ST2 signaling and their protective functions in inflammation settings, induction of IL-22 and IL-13 may be responsible for an immune network favorable to mucosal repair. Collectively, our data showed an important feedback loop in which cytokine cues from damaged epithelia activate MCs to regulate tissue environments essential for MC-dependent restoration of epithelial barrier function and maintenance of tissue homeostasis.

Introduction
To function properly, the intestine immune system detects a wide variety of agents over time, maintaining tolerance of commensal microbiota and dietary antigens while retaining the ability to launch an effective immune response against invading pathogens. 1 Disorders of the immune system manifest as inflammatory bowel disease (IBD), in which continual activation of the mucosal immune response in the gastrointestinal tract leads to chronic remittent and progressive inflammation. 2,3 Colitis appears to be involved in chronic engagement of the stress response of epithelial barrier and dysregulation of a carefully balanced immune system, which represents the underlying pathogenesis. 4,5 Studies implicate crosstalk between the epithelial barrier and the mammalian immune system in which damaged epithelial cells release cytokine signals that activate sentinel immune cell populations that may result in mucosal healing with epithelial regeneration and immune network regulation. [6][7][8][9] Understanding the details of regulatory mechanisms of the intestinal immune system that direct tissue protection and remodeling is key to identifying new therapeutic targets for IBD.
Mast cells (MCs) are tissue-resident immune cells that are preferentially distributed throughout barrier tissues such as the skin and mucosa, including the intestinal intraepithelial space. 10 MCs participate in several physiological processes and are important in initiation and regulation of immune reactions to environmental stimuli that occur in their homing tissues. The functions of MCs during colitis development has drawn substantial interest in recent years. [11][12][13] Although some evidence exists on a pathogenic role for intestinal MC activation in experimental colitis, conclusive data associated with colon inflammation are lacking. 14,15 An article on immunity found that factors weakening Treg cell function most likely prevail during the acute phase. However, when inflammation subsides, MCs may have more prominent functions, preventing excessive tissue damage and development of chronic inflammation. 16 This hypothesis has been confirmed by evidence. 12,13,17 The hallmarks of IBD are a dysregulated intestinal immune response in which MCs accumulate in the inflamed gut of IBD patients. However, the specific functions of MCs in IBD development remain to be elucidated, especially the aberrant interactions between MCs and epithelial cells or other underlying resident cells, which are fundamental for immunopathological regulation.
The importance of events involved in stress-triggered inflammation and tissue remodeling at mucosal tissues in orchestrating immune response is highlighted by the effects of tissue-derived cytokine signals in development of the underlying immune network of IBD. Studies have advanced considerable interest in interleukin-33 (IL-33) as a central instigator and candidate for therapeutic intervention of colitis. [18][19][20] IL-33 is released as one of the earliest signaling molecules following epithelial damage or stress. Abundant IL-33 receptors (ST2) are expressed on MCs and coordinate spatially and temporally with IL-33 signaling, which may trigger a key regulatory amplification loop involved in immune homeostasis. 13,21,22 As key effector cells to IL-33 signaling, MCs are recognized as driving production of high amounts of the (T helper) Th2 cytokines IL-5, IL-9, and IL-13. 23,24 In colitis, the interaction of MCs with epithelial cells depends on the inflammatory stage and activation of the tissue repair program. However, how the IL-33/ST2-dependent MC response contributes to IBD development remains largely unknown.
We previously showed that MCs participate in the tight regulation of multiple immune responses. [25][26][27] Our interest in MC function and adaptation to the inflammatory environment developed with our investigation of the infiltration of intestinal mucosal MCs in the remission phase of dextran sodium sulfate (DSS)-induced colitis. Based on studies that implicate MCs in the IL-33/ST2 axis, we investigated and characterized the unknown function of MCs in this setting when perturbed in IBD tissue environments. We confirmed that MCs are required for the effective resolution of DSS-induced colitis and are involved in the tissue environment that favors mucosal healing. In addition, this beneficial effect from MCs seemed to be facilitated by IL-33/ST2 signaling that increased the release of IL-13 and IL-22.

MC infiltration increased during the DSS-induced colitis remission phase
Colitis was induced with 2.5% DSS in drinking water for 7 days. The activating phase was from day 3 to day 7 and the remission phase from day 10 to day 14 after DSS withdrawal (Fig. 1a). Histology change of colon by H&E staining during DSS-induced colitis was showed in Figure S1. We found MC numbers dramatically increased during the remission phase (Fig. 1b). The proportion without MCs was as high as 60% under basal conditions (Fig. 1c), and distributed throughout the outer layers of the muscularis propria at day 0. As colitis progressed, MCs tended to move from the outer to the inner intestinal layers, closer to the sites of mucosal regeneration (Fig. 1d). We found SCF mRNA increased in tissues mainly at days 3, 7, and 10 after DSS administration compared with sham-operated mice (Fig. 1e). In addition, expression level of c-KIT and mMCP7 increased only during remission phase (Fig. 1f, g). These results suggested that MCs develop in an SCF-dependent manner in a pathological process before mucosal repair. The percentage of FcεRIα + CD117 + MCs among infiltrating cells during the activation phase from day 3 to 7 was similar to the percentage at day 0. During the remission phase after DSS withdrawal (from 10 to day 14), we detected more than two-fold more infiltrating MCs (Fig. 1h, i).
MC deficiency in KIT Wsh mice results in tissue repair program destruction in colitis models DSS administration caused significant body weight loss with a similar progression in WT and KIT Wsh mice (Fig. 2a). In the activation phase, there are no differences in survival rate, colon length, or disease score between the WT and KIT Wsh groups ( Fig. 2b-d). MC deficiency mainly affect the colitis recovery (Fig. 2a). Survival rates were also significantly decreased in KIT Wsh mice during a 15-day observation and deaths occurred mainly around the days of DSS withdrawal (Fig. 2b). An analysis of intestines in the remission phase showed that KIT Wsh mice has delayed resolution of inflammation and impaired repair of damaged mucosal tissue, which led to a continuing severe colon inflammation condition characterized by shorter colon length and higher disease scores and pathological evaluations ( Fig. 2c-f). Intestinal permeability was examined using FITC-labeled dextran assays. We found that KIT Wsh mice had significantly high intestinal permeability even in the recovery phase (Fig. 2g).
Expression of Ki67 in both WT and KIT Wsh mice was significantly higher in WT mice and was associated with a better recovery (Fig. 2h, i). Expression of zonula  c Whole colon lengths from sham, activation phase, and remission phase. d Disease score evaluated as weight loss, feces, rectal bleeding, and general appearance as indicated. e Representative H&E staining images of transverse colon. f Pathological scores from colonic sections, calculated as indicated. g Serum FITC-dextran quantified as a measure of intestinal permeability. h Percentage of positive cells for Ki-67 immunostaining. i Representative stain of Ki-67. j Heat map of relative transcriptional level of selected mRNA involved in mucosal recovery and homeostasis maintenance. Data are mean ± SD. Student t-test, *P < 0.05; **P < 0.01 occludens-1 (ZO-1) was decreased in tissue from KIT Wsh mice compared to WT mice, suggesting that MCs may benefit tight junctions and signal transduction between epithelial cells. The expression of basic fibroblast growth factor (FGF-2, bFGF, or FGF-β) and angiopoietin-related protein 2 (ANGPTL2) was significantly lower in KIT Wsh tissue. They are important for maintaining tissue homeostasis and promoting tissue repair. MC deficiency in KIT Wsh mice also influenced expression of the innate immune genes Reg3γ and Reg3β, which were expressed by diverging epithelial cell types and important for defense peptides in regulating intestinal inflammation 28 (Fig. 2j). These diverse effects were largely consistent with the function of intestinal MCs. Although we did not detect differences between WT and KIT Wsh mice in the remaining tested genes associated with tissue protection and intestine homeostasis, the results suggested a limited range of meaningful effects from MCs.

BMMC-reconstituted KIT Wsh mice exhibit a tissue repair activity in DSS-induced colitis similar to the WT counterpart
To further confirm that MCs are required for the effective resolution of DSS-induced colitis in mice, i.p. injections of BMMCs into KIT Wsh mice (Kit Wsh + MCs) have been used in this study. The purity of MCs, evaluated as a percentage of c-Kit+ and FcεRIα+ cells, was >95% (Fig. 3a). After 5 weeks of MC injections, we detected the MC populations in the gut tissue (Fig. 3b). Body mass change in reconstituted mice was consistent with that of the WT counterpart during colitis, whereas impaired recovery occurred after the activate phase in Kit Wsh mice, suggesting that MCs are required for the effective resolution of DSS-induced inflammation (Fig. 3c). In addition, reconstituted mice obtain a similar survival rate as that of the WT (Fig. 3d). Corresponding to the above results, we detected changes in intestinal length and disease score similar to WT in reconstituted KIT Wsh mice (Fig. 3e, f). During the remission phase of colitis, the crypt architecture and pathological score of reconstituted mice was more similar to WT than to KIT Wsh mice at the same time point (Fig. 3g, h).

IL-33/ST2 signaling is involved in MC activity during colitis
Similar to previous research on human IBD 29 and mouse models, 30 we found that mRNA/protein level of IL-33 was significantly elevated in inflamed colon (Fig. 4a, b). Immunostaining for IL-33 further confirmed these findings (Fig. 4c, d). IL-33 expression in WT mice decreased in the remission phase at days 10 and 14, but not in KIT Wsh mice ( Fig. 4a-d). The percentage of T1/ST2 + MCs increased significantly at days 10 and 14 compared to day 0. Some immune cells including ILC2s, Th2 cells, and Treg cells may also express T1/ST2. However, we detected only a slightly increase in ST2 expression from non-MCs (Fig. 5a). Significant positive correlations were seen between T1/ST2 expression and expression of MC-specific markers Kit and mMCP7 (Fig. 5b, c). We detected significantly increased T1/ST2 expression in WT but not KIT Wsh mice during the remission phase of DSS-induced colitis (Fig. 5d). We focused on whether MC degranulation affected T1/ST2 expression. In colitis development, MCs appeared to degranulate in the activation phase and primarily in the first 2 days (Fig. 5e). In addition, MC degranulation in the colon primarily occurred at the early stage of colitis ( Figure S2a, b). MC degranulation was also negatively correlated with T1/ST2 expression in MCs ( Fig. 5f, g). Consistent with the results from the colitis model, degranulation contributed to decreased T1/ST2 expression in the activated cell population compared to the naïve MC population (Fig. 5i). By reviewing and analyzing data obtained from a database (GSE10246), we confirmed that T1/ST2 was primarily expressed on MCs. Consistent with our results in vitro, IgE and IgE + antigen treatments resulted in lower T1/ST2 expression compared with the normal condition (Fig. 5h). Recovery after degranulation of MCs promoted T1/ST2 expression (Fig. 5i). Therefore, we hypothesized that some MCs in the inflamed colon went through the degranulation-restore process to promote T1/ ST2 expression.

MC-dependent IL-33/ST2 signals promote mucosal healing
To formally assess the role of MC-dependent IL-33/ ST2 signals in intestinal homeostasis, we explored if absence of the IL-33-mediated signaling pathway promoted colitis recovery in MC-deficient KIT Wsh or WT mouse models. All DSS-treated mice had significant progressive weight loss over the acute phase (Fig. 6A). Body weight loss with WT mice treated with blocking anti-ST2 antibody was comparable to KIT Wsh mice in the acute and recovery phases of colitis. This result was confirmed by FITC-dextran assays, colon length, histological analyses, and disease scores (Fig. 6b-f). No significant difference in tissue recovery was seen in KIT Wsh mice treated with isotype or anti-ST2 antibodies, suggesting MCs were essential for a redundant IL-33 signal to promote mucosal healing. We also demonstrated that an IL-33/ST2 signaling pathway in MCs was required for expression of ZO-1, ANPTL2, Reg3β, and Reg3γ, which were associated with signal transduction and immune regulation ( Fig. 6g-j).

MC-dependent IL-33/ST2 signal orchestrates an immune network favorable to mucosal healing
To identify potential tissue-specific cytokines of immune cells, we compared protein expression profiles of colon tissue. We identified IL-13 and IL-22 as important cytokines from colon tissue (Fig. 7a, b). To further assess and confirm the effect of MC-dependent IL33/ ST2 signaling on the colon tissue immune network during the remission phase, we used enzyme-linked immunosorbent assay (ELISA) to quantified the expression of potentially important cytokines from T cells. Only decreased expression of IL-13 and IL-22 was largely and tightly restricted to blocking the MC-dependent IL33/ST2 pathway (Fig. 7c).

IL-33 signal facilitates MCs survival and function in vitro
We assessed the impact of IL-33 on MCs in vitro. In line with previous findings, we showed that IL-33 attenuated apoptosis of murine BMMCs, promoting their survival (Fig. 8a-c). MC degranulation was not promoted by IL-33 alone (Fig. 8d). Given that T cells are prominent in the colon, we postulated that IL-33/ST2 signaling in MCs may modulate in vitro-induced T cell differentiation and function. To test this, we sort-purified naïve CD4+T cells from WT mice and cocultured them with or without BMMCs. Consistent with in vivo results, both IL-13 and IL-22 secretion were promoted by IL-33 in the co-culture system. IL-13 was secreted by MCs alone, confirming that MCs were an important source of Th2 cytokines (Fig. 8e,  f). The presence of IL-33 in MCs cultures significantly promoted IL-13-producing and IL-22-producing T cell percentage in the proliferation phase, suggesting that IL-33 preferentially depended on intermediary MCs to regulate T cell differentiation and function (Fig. 8g).

Discussion
Animal and human clinical studies that have implicated the participation of MCs in IBD [11][12][13]31,32 suggest that the contribution of these cells includes regulating epithelium permeability, immune signal transmittance, maintenance and resolution of inflammatory responses, and subsequent tissue remodeling. 33 Our data provide evidence for a tissue-protective function for MCs in the intestinal tract and implicate the IL-33/ST2 signaling pathway as a critical mechanism by which MCs mediate the production of IL-13 and IL-22 to orchestrate the immune network to limit inflammation and promote epithelial repair. This study identified a critical feedback loop in which cytokine signals from damaged epithelia mediate innate immune cell responses and promote expression of a spectrum of cytokines essential for restoration of epithelial barrier function and tissue homeostasis. Strategies designed to regulate this feedback loop may provide novel therapeutic agents for IBD management.
Accumulating evidence indicates that the function of MCs in IBD is more complicated than originally thought, for example, the composition of MCs and how their phenotypes can be altered under various conditions. However, this evidence also raises the opportunity to explore information about MCs and their associated signaling pathways in IBD. In this study, we confirmed the dynamic changes of MC infiltration in inflamed colons and MC functions in repairing DSS-induced colon damage. Consistent with previous studies, 13 the increase in MC number in the lamina propria of WT mice after withdrawal parallels the delayed recovery of weight loss occurring in KIT Wsh mice. Although we have no evidence that MCs have a special function in the activation phase of colitis, signaling such as SCF released from damaged tissue in this phase was essential for facilitating MC development and survival in the subsequent remission phase. Our findings highlighted MC functions in colonic epithelial regeneration and intestinal homeostasis suggested Relative mRNA for g ZO-1, h ANGPTL2, i Reg3β, and j Reg3γ from real-time-quantification PCR. Data were pooled from three repeated experiments. Data are mean ± SD. Student t-test, *P < 0.05; **P < 0.01 by indicators for detecting colitis and by Ki-67 immunostaining. The increase of MC infiltration near the epithelium resulted in a favorable microenvironment that expressed more beneficial proteins including ZO-1, FGF-2, ANGPTL2, REG3γ, and REG3β, which are involved in signal transduction, cell growth, tissue repair, and homeostasis maintenance. Thus, we have added new insights about a novel innate immune component to the pathway that involves these factors and suggest that intestinal tissue protection requires a progressive MC response that is initiated by rapid damage signals. 33 IL-1 family member IL-33 is constitutively expressed in epithelial cells at barrier sites where it functions as an endogenous danger signal or alarmin following tissue damage. 34,35 Although high levels of IL-33 are found in inflamed intestinal segments, how IL-33 functions in colitis has remained a formidable challenge due to diverse conclusions from previous studies. 19,20,30,[36][37][38] Our data  suggested that delayed resolution of inflammation in the KIT Wsh mice was accompanied with a failure to reduce IL-33. MC granules contain a wide range of proteases and mMCP-4 degrades several alarmins, including IL-33, both in vitro and in vivo. 39 Based on our findings and previous studies [40][41][42][43] implicating MC with IL-33/ST2, we investigated the relationship of MC activity and ST2 expression during colitis development. Our data identified that MCs acted as the primary cell type that expressed ST2 among the CD45 + CD11b− lamina propria mononuclear cells (LPMCs) during colitis development and among immune cells and important functional cells. In addition, recovery after MC degranulation promoted ST2 expression. Taken together, these results suggested the development and function of MCs was involved in the IL-33/ST2 signaling pathway.
From an immune perspective, MCs appear to be cellular sensors and effectors for IL-33, in keeping with their functional relationship with other systems, where they lead to different biological endpoints. 10,21,24,[44][45][46][47][48] However, further assessing if IL-33 is required for MC response and function during colitis is still necessary. We identified MC-dependent IL-33/ST2 signaling as one pathway for orchestrating intestinal tissue protection, consistent with IL-33 function in previous studies. However, we did not observe significant differences in colitis degree between groups with or without ST2 blocking in KIT Wsh mice. IL-33 is a potent in vivo stimulus that activates different resident immune cell populations in multiple inflammatory settings. 49 We remain confident that IL-33 has a key function in activating other cell populations but in colitis, this process in largely thought to act via MC-dependent signaling. Supporting our view is the decreased expression of ZO-1, ANGPTL2, Reg3β, and Reg3γ in ST2-blocked groups. Consistent with our findings, a study suggests that the IL-33-MC axis suppresses tissue inflammation with an immunosuppressive negative-feedback role in airways, 50 suggesting that downstream signals from MCs involving IL-33 orchestrate a specific immune microenvironment.
The cellular and molecular mechanisms acting downstream of IL-33 to regulate disease are poorly defined. To some extent, we demonstrated that IL-33 signaling activated MCs to form a tissue microenvironment favorable to tissue repair. Given the known influence of IL-33 on signals from T cells, 35 exploring if intestinal tissue protection requires a cooperative response from both MCs and T cells to expand downstream signals to orchestrate a special immune network to facilitate tissue repair is worthwhile. We identified IL-22 and IL-13 as significantly downregulated cytokines preferentially involved in blocking MCdependent IL-33/ST2 signaling using protein array and ELISA assays. IL-33 is a well-known regulator of mucin responses in multiple inflammatory settings, but our colitis model largely found IL-13-and IL-22-dependent signaling.
MCs are long-lived cells that accumulate in chronically inflamed tissues, 51 consistent with the long process of colon epithelium repair during colitis. Despite the gradual decline in IL-33 levels with increasing repair of intestinal mucosal damage, IL-33 also contributes to MC survival and function and possibly until damaged colon tissue recovers the normal structure. In line with the results of a previous study, our data revealed a cell-intrinsic role for IL-33 signal in the regulation of MC apoptosis. 52 Considering that we did not examine directly whether MC-derived ST2 plays a role, for instance, using mice that are deficient of ST2 only in the MC population, the potential role of other possible confounding factors cannot be completely ruled out during the both the acute and recovery phases of colitis. Nevertheless, MCs are tissue-based stationary effector cells that present a first-line defense against challenges and are closely related to the inflammation process. 51 For these reasons, MCs are more prominent in the remission phase.
IL-13 was largely released from MCs treated with IL-33, with more production in an MC-T cell co-culture system. Most biological effects of IL-13, similar to those of IL-4, are linked to a single transcription factor, signal transducer and activator of transcription 6 (STAT6). 53 IL-13 is responsible for promoting the survival and migration of epithelial cells, 54 consistent with our findings. Similarly, IL-22R is specifically expressed in epithelial cells and IL-22 acts directly on epithelium, which is thought to be beneficial for the intestinal epithelial barrier by enhancing mucus production and promoting the proliferation and migration of epithelial cells. 55,56 Therefore, a powerful proliferative stimulus linking IL-33, MCs, IL-13, and IL-22 to neighboring epithelia in vivo maybe sufficient for tissue repair with increased cytokine expression including ANGPTL2, Reg3β, and Reg3γ.
In summary, our data indicated that MCs, as important tissue-resident effector cells with a long life in inflamed colons, were essential intermediaries in regulating IL-33/ ST2 signaling to orchestrate an immune network favorable to mucosal healing. Furthermore, production of IL-13 and IL-22 mediated by an MC-dependent IL-33/ST2 pathway, as a previously unrecognized multiple mechanism, was responsible for the beneficial effects of tissue homeostasis and immune regulation. However, an open area of investigation remains if human intestinal MCs have a consistent function as murine MCs for resolution of colon inflammation, and long term, if the repair capacity of MCs is also adjusted by genetic alterations in tumor-associated modification.

Animals and treatments
Mice were maintained under pathogen-free conditions and housed in filter-top cages. All animal experiments were conducted with the approval of the Local Ethics Committee responsible for regulating animal research in Tongji University. Animal care was strictly according to the guidelines for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Wild-type (WT) C57BL/6 mice were from Shanghai Laboratory Animal Co Ltd. (Shanghai, China), and MC-deficient mice C57BL/6-Kit W-sh/W-sh (Kit Wsh ) were from Jackson Laboratories (Bar Harbor, ME, USA). Reconstitution of KIT Wsh mice with murine bone marrow-derived MCs (BMMCs), termed the "mast cell knock-in" approach, was performed by intraperitoneal (i.p.) injection of 5 × 10 6 cells per mouse. Wait 5 weeks after i.p. engraftment before performing in vivo experiments. DSS-induced colitis models were established using a method described previously. 57 WT and Kit Wsh mice were give 2.5% DSS (molecular mass, 36,000-50,000; MP Biomedicals) in drinking water for 7 days continuously, then the recovery phase was evaluated followed with regular water. Characteristics of acute colitis were observed daily, including diarrhea, rectal bleeding, body weight, and survival. Disease severity was scored as in a previous study. 58 ST2 neutralization was by i.p. administration of 20 μg anti-ST2 blocking antibody or rat anti-mouse IgG2b antibodies as the isotype control (R&D Systems).

Cell preparation
BMMCs were differentiated and cultured. Bone marrow cells were isolated from femurs of C57BL/6 mice, differentiated in complete RPMI (Gibco), and supplemented with 10% FBS (Gibco), 100 U/mL penicillin/streptomycin (Gibco), 10 ng/mL recombinant murine IL-3 (R&D Systems), and 10 ng/mL recombinant murine SCF (R&D Systems) at 37°C under 5% CO 2 . After 7 days, nonadherent cells were carefully removed and replaced with fresh culture medium to increase the purity of the MCs. This step was repeated every 7 days until adherent cells disappeared (after 5-6 passages). After 5 weeks of culture, BMMC purity was evaluated as the percentage of FcεRIα and c-kit (CD117) cells. BMMCs were used at a purity more than 90%. CD4 + T cells were enriched using an isolation kit (Miltenyi Biotech) according to the manufacturer's instructions. To analyze immune cells infiltrating the colon, collagenase IV (0.05% w/v; Roche) was used to digest the tiny colon tissue (0.1-0.5 cm) for 1 h. Singlecell suspensions were collected and further purified via density gradient centrifugation with 40% (v/v) and 70% (v/ v) Percoll-RPMI. LPMCs were collected from the interface and suspended in RPMI medium. 59

Histopathology
Colons isolated from euthanized animals were embedded in paraffin and 5-μm sections were deparaffinized in xylene and hydrated through a graded series of alcohol to water. For toluidine blue staining (Sigma) to assess MCs in the colon, slides were rinsed with PBS for 10 min and stained with 0.5% w/v toluidine blue (Sigma) in 0.5 N HCl (Baker, Philipsburg) for 30 min. Staining with H&E (Sigma) or H&E plus Alcian Blue was under standard conditions. Slides were dehydrated and mounted in Richard-Allan Scientific Cytoseal XYL (Invitrogen). Histology was scored blindly by lab technicians from the Department of Pathology using previously described criteria: 0, no signs of inflammation; 1, very low level of inflammation; 2, low level of leukocyte infiltration; 3, high level of leukocyte infiltration, high vascular density, thickening of the colon wall; and 4, transmural infiltration, loss of globet cells, high vascular density, thickening of the colon wall. For immunofluorescence or immunohistochemistry, antigen retrieval was performed by microwave irradiation in citrate buffer for 20 min and cooling to room temperature. Sections were incubated with 3% H 2 O 2 in distilled water for 15 min to quench endogenous peroxidase activity. After rinsing three times with PBS, sections were incubated with Ki-67 IgG antibody (2.5 μg/mL; 12202, CST) and IL-33 IgG antibody (2 μg/mL, ab229698; Abcam) overnight at 4°C, and washed in PBS. Sections were incubated with secondary antibody for 1 h at room temperature. Negative controls were processed in the same manner but without primary antibody.

FITC-labeled dextran intestinal permeability assays
Intestinal permeability was examined using a FITClabeled dextran method. Mice were gavaged with 60 mg/ 100 g FITC dextran (MW 4000 at 80 mg/mL; Sigma) 4 h before sacrifice. Sera were obtained to detect FITCdextran levels using fluorescence microplates and an excitation maximum of 490 nm and emission maximum of 520 nm (Fluorimeter Pharos FX; BioRad). Standard curves were obtained by diluting serial concentrations of FITC-dextran in mouse serum.

Real-time quantitative PCR
We extracted RNA using the TRIzol method (Invitrogen) and purified it using RNeasy columns (Qiagen). Real-time PCR was with Light Cycler RNA Master SYBR Green kits (Roche) in a LightCycler instrument (Roche) using primers specific for detected genes (Table S1). On completion of amplification, DNA melting curve analysis was carried out to confirm the presence of a single amplicon. GAPDH was the internal reference gene for normalizing transcript levels. Relative mRNA levels (2−ΔΔCt) were determined by comparing PCR cycle thresholds (Ct) for the gene of interest and GAPDH (ΔCt) between WT and KIT Wsh groups.

MC degranulation
Blood samples were drawn into serum separation tubes and serum aliquots stored at −80°C until analysis. Histamine concentrations were assessed using EIA histamine kits according to the manufacturer's instructions (Beckman Coulter). Serum was acylated and added to histamine-coated plates with conjugate and incubated overnight at 4°C. Wells were washed and substrate added for 30 min at room temperature while shaking. Reactions were stopped and plates read at 405 nm. In vitro, BMMCs were incubated overnight at 37°C with 1 µg/mL IgE anti-DNP in 1 mL culture medium and stimulated for 1 h at 37°C with 10 ng/mL DNP-BSA antigen in 200 µL Hepes-Tyrode's buffer, pH 7.4. Culture supernatants from cells were collected and assayed for β-hexosaminidase.
After solubilization with 0.5% (vol/vol) Triton X-100 in Tyrode's buffer, enzymatic activities of β-hexosaminidase in supernatants and cell pellets were measured with pnitrophenyl N-acetyl-b-D-glucosaminide in 0.1 M sodium citrate (pH 4.5) for 60 min at 37°C. Reactions were stopped by addition of 0.2 M glycine (pH 10.7). Release of 4-p-nitrophenol product was detected by absorbance at 405 nm. Extent of degranulation was represented as 4-pnitrophenol absorbance in supernatants as a percentage of sum of absorbance in supernatants and in cell pellets solubilized in detergent.

Determination of MC survival
MCs were treated with or without IL-33 after withdrawing cytokines IL-3 and SCF, which are essential for MC survival. For MC apoptosis analysis, cells were stained with annexin V (eBioscience) at room temperature in the dark for 15 min, followed by the addition of propidium iodide. After staining, the percentage of apoptotic cells among 10,000 cells was analyzed using flow cytometry. The Q2 region represents late apoptotic cells, and the Q4 region represents early apoptotic cells. To evaluate MC survival and proliferation, a cell counting Kit-8 (CCK-8; Dojindo) was used according to the manufacturer's instructions.

Statistical analysis
Results were expressed as mean values ± SD. Differences among experimental groups were evaluated via Student's t-test or ANOVA when appropriate, and significance of differences between groups assessed via Newman-Keuls post hoc test. Analysis used statistical software package (GraphPad-Prism 7; GraphPad, La Jolla, CA, USA) and significance was defined as P < 0.05.