Author response: CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation (2022)

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αβ-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules. Editor's evaluation This work investigates the mechanism by which CAMSAP2 nucleates microtubule asters in vitro, with implications for its role in the cell. The authors show the importance of CAMSAP condensates for aster formation and use electron microscopy to suggest that nucleation proceeds vs stabilization of protofilaments and sheets of tubulin. https://doi.org/10.7554/eLife.77365.sa0 Decision letter eLife's review process eLife digest Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in. Introduction Microtubules are dynamic tubular polymers that contribute to fundamental processes in cells, such as cell shape determination, chromosome segregation, cilium and flagellum formation, and molecular motor trafficking (Desai and Mitchison, 1997). α-tubulin and β-tubulin align head-to-tail to form protofilaments that associate laterally grow into the microtubules (Alushin et al., 2014; Nogales et al., 1999). The exposed β-tubulin end is called the plus-end, whereas the exposed α-tubulin end is called the minus-end. The microtubule plus-end is more dynamic than the minus-end; it dynamically alternates between growth and shrinkage. The dynamic events are regulated by many microtubule plus-end-tracking proteins (+TIPs) (Akhmanova and Steinmetz, 2015; Akhmanova and Steinmetz, 2008; Howard and Hyman, 2003). The minus-end is less dynamic and is responsible for determining the geometry of microtubule networks by stably anchoring at microtubule nucleation sites or the microtubule organizing centre (Akhmanova and Steinmetz, 2019; Dammermann et al., 2003). Despite the identification of many +TIPs, only a few minus-end-binding proteins (−TIPs) have been reported, including γ-tubulin and calmodulin-regulated spectrin-associated proteins (CAMSAPs). γ-Tubulin which forms a ring complex (γ-TuRC) is located at centrosomes to nucleate microtubules and stabilize the microtubule minus-ends (Akhmanova and Steinmetz, 2015). γ-TuRC serves as a nucleation template for centrosomal microtubule formation by aligning αβ-tubulins to form the tubular structure of the microtubule in a process called templated nucleation (Moritz et al., 1995; Roostalu and Surrey, 2017; Wieczorek et al., 2020; Zheng et al., 1995). CAMSAPs, the protein family composed of CAMSAP1-3 in vertebrates, Patronin in Drosophila melanogaster, and PaTRoNin (microtubule-binding protein) homolog (PTRN-1) in Caenorhabditis elegans, have been reported to be involved in non-centrosomal microtubule formation (Goodwin and Vale, 2010; Marcette et al., 2014; Meng et al., 2008; Richardson et al., 2014; Wu et al., 2016). Although several in vitro reconstitution experiments have shown differences among CAMSAPs, CAMSAPs generally bind to the growing microtubule minus-ends and strongly suppress the dynamicity of non-centrosomal microtubules in different cell types, including neurons and epithelial cells (Chuang et al., 2014; Hannak et al., 2002; Jiang et al., 2014; Martin et al., 2018; Nashchekin et al., 2016; Noordstra et al., 2016; Pongrakhananon et al., 2018; Sampaio et al., 2001; Tanaka et al., 2012; Toya et al., 2016; Wu et al., 2016; Yau et al., 2014). The minus-end side of the microtubule lattice, but not the very end, is recognized by the CKK domain, although some additional effects were detected by other regions of CAMSAPs (Atherton et al., 2019; Atherton et al., 2017). CAMSAPs have been reported to act in parallel with γ-TuRC-dependent microtubule nucleation, and the microtubule-severing protein katanin regulates the functions of the CAMSAPs (Jiang et al., 2018; Wang et al., 2015; Wu et al., 2016). CAMSAPs are rapidly recruited to and to decorate pre-existing nascent microtubule minus-ends to sustain non-centrosomal microtubules. On the other hand, some reports have demonstrated the possibility that CAMSAP-containing foci are involved in promoting microtubule nucleation independently of γ-TuRC (Atherton et al., 2019; Jiang et al., 2018; Nashchekin et al., 2016). Thus, the mechanism by which CAMSAP2 proteins contribute to non-centrosomal microtubule organization has remained controversial. Recent studies suggest that microtubules in cells often nucleate independently of the γ-TuRC template (Hannak et al., 2002; O’Toole et al., 2012; Sampaio et al., 2001; Tsuchiya and Goshima, 2021; Yau et al., 2014). Therefore, we examined the roles of CAMSAPs in microtubule nucleation and microtubule network formation in vitro. In this study, we discovered the fundamental role of CAMSAP2 as a strong nucleator of microtubule formation from soluble αβ-tubulins. Our studies demonstrated that CAMSAP2 co-condenses with tubulins to stimulate spontaneous nucleation of microtubules without the γ-TuRC template. Many microtubule nucleation intermediates were observed inside the CAMSAP2-tubulin condensate during the early stages of microtubule polymerization. The CAMSAP2-tubulin condensate grows into the nucleation centre and mediates aster-like microtubule network formation in vitro, demonstrating the importance of CAMSAP2 in non-centrosomal, γ-tubulin-independent microtubule nucleation processes. Results Profiles of CAMSAP2 constructs used in in vitro assays Initially, we examined the biochemical properties of the recombinant CAMSAP2 constructs used in this study by SDS-PAGE and size exclusion chromatography (Figure 1A). As shown in Figure 1B and C, full-length CAMSAP2 constructs with (GFP-CAMSAP2-FL) and without GFP-tag (CAMSAP2-FL) were purified to show a single peak in size exclusion chromatography. GFP-CAMSAP2-FL was eluted slightly earlier than the non-tagged construct due to the size of the GFP-tag, indicating that both constructs should be in the same oligomeric state. The molecular mass of CAMSAP2-FL was further examined by multi-angle light scattering (MALS) as 122 ± 1.1 kDa (Figure 1—figure supplement 1). Considering that the estimated molecular weight of CAMSAP2-FL is 167 kDa, it likely exists as a monomer in the solution. Figure 1 with 1 supplement see all Download asset Open asset Functional study of recombinant calmodulin-regulated spectrin-associated protein 2 (CAMSAP2). (A) Schematic representation of the full-length CAMSAP2 constructs used in this study. CH, calponin-homology domain; MBD, microtubule-binding domain; CC, coiled-coil domain; CKK, C-terminal domain common to CAMSAP1 and two other mammalian proteins, KIAA1078 and KIAA1543. (B) (C) Size exclusion chromatography and SDS-PAGE of the peak fraction of (B) full-length CAMSAP2 and (C) GFP-CAMSAP2. (D) Total internal reflection fluorescence images of polarity-marked microtubules (magenta) decorated with purified GFP-CAMSAP2 (green). The minus-end segment of the microtubule is brighter than the plus-end segment. Figure 1—source data 1 Figure 1 SDS-PAGE gel of the full-length calmodulin-regulated spectrin-associated protein 2. https://cdn.elifesciences.org/articles/77365/elife-77365-fig1-data1-v1.jpg Download elife-77365-fig1-data1-v1.jpg We then analysed the microtubule binding pattern of GFP-CAMSAP2-FL by total internal reflection fluorescence (TIRF) microscopy. The results showed that GFP-CAMSAP2-FL bound to the microtubule lattice preferentially via the minus-ends, consistent with previous reports (Atherton et al., 2017; Jiang et al., 2014; Figure 1D). These observations collectively indicate that the CAMSAP2 proteins used in this study retained their original ability to bind to the microtubule minus-ends. CAMSAP2 reduces the microtubule nucleation barrier Microtubule nucleation from αβ-tubulin heterodimers (hereafter called tubulin in the Results section) can occur spontaneously in vitro (Kuchnir and Leibler, 1995; Voter and Erickson, 1984). De novo microtubule nucleation in vitro requires a critical tubulin concentration greater than 20 µM (CcMT nucleation) (Wieczorek et al., 2015). This concentration is considerably higher than that required for microtubule elongation from seeds, which is typically approximately 1–2 µM and is called the critical concentration for microtubule polymerization (CcMT polymerization). To test the nucleation ability of the purified pig tubulins used in this study, we performed a spin-down spontaneous nucleation assay through microtubule co-pelleting experiments in vitro (Wieczorek et al., 2015). We slightly modified the protocol to detect the functional CAMSAP2-FL; it can bind to microtubules at 37°C and become re-solubilized on ice along with the microtubule depolymerization. Nucleation was induced by 60 min of incubation at 37°C, accompanied by high-speed centrifugation. The pellets with or without CAMSAP2-FL were re-suspended and incubated on ice for 30 min for depolymerization. The samples were centrifuged again to eliminate non-specific aggregation, and the supernatant was applied for SDS-PAGE analysis (Figure 2A). We detected de novo nucleation of microtubules (CcMT nucleation) just above the tubulin concentration of 20 µM, which is consistent with a previous report and our result by pelleting assay without modification (Wieczorek et al., 2015; Figure 2A and C, and Figure 2—figure supplement 1). Figure 2 with 1 supplement see all Download asset Open asset Calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) stimulates microtubule nucleation. (A) SDS-PAGE gels from a spin-down spontaneous nucleation assay of tubulin showing the total tubulin after 60 min of polymerization at 37°C (left gel). Polymerized tubulin was pelleted by centrifugation and then depolymerized on ice and centrifuged to remove debris, and the supernatant was subjected to SDS-PAGE (right gel). (B) SDS-PAGE gels from a spin-down spontaneous nucleation assay of tubulin with 1 µM CAMSAP2-FL showing the total tubulin-CAMSAP2 (left gel) and the polymerized/depolymerized tubulin (right gel). (C) Plots of the depolymerized tubulin concentrations determined by pelleting assay against the total tubulin concentrations determined by the reaction on SDS-PAGE gel from three independent assays (mean ± SD). The depolymerized tubulin from the tubulin assay is turquoise green, and that combined with CAMSAP2-FL is orange. The concentrations of tubulin greater than 0.1 µM are fitted with a trend line that has an x-intercept of 23.8 ± 0.96 µM (CcMT nucleation). (D) SDS-PAGE gels from a spin-down spontaneous nucleation assay of 10 µM tubulin with 10–1000 nM CAMSAP2-FL in the PEM (100 mM PIPES pH 6.8, 1 mM MgCl2, 1 mM EGTA, and 1 mM GTP) or PEM + 100 mM KCl. (E) Plots of the depolymerized pelleting assay in the different concentration of the CAMSAP2-FL with different buffer with SD from three independent assays (mean ± SD). Figure 2—source data 1 SDS-PAGE gel from a spin-down spontaneous nucleation assay of tubulin showing the total tubulin after 60 min of polymerization at 37°C. https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data1-v1.jpg Download elife-77365-fig2-data1-v1.jpg Figure 2—source data 2 SDS-PAGE gel of depolymerized tubulin after spin-down pelleting assay. https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data2-v1.jpg Download elife-77365-fig2-data2-v1.jpg Figure 2—source data 3 SDS-PAGE gel from a spin-down spontaneous nucleation assay of tubulin with 1 µM calmodulin-regulated spectrin-associated protein 2-FL showing the total tubulin after 60 min of polymerization at 37°C. https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data3-v1.jpg Download elife-77365-fig2-data3-v1.jpg Figure 2—source data 4 SDS-PAGE gel of depolymerized tubulin with 1 µM calmodulin-regulated spectrin-associated protein 2-FL after spin-down pelleting assay. https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data4-v1.jpg Download elife-77365-fig2-data4-v1.jpg Figure 2—source data 5 Quantification of the depolymerized tubulin concentrations determined by pelleting assay. https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data5-v1.docx Download elife-77365-fig2-data5-v1.docx Figure 2—source data 6 SDS-PAGE gel from a spin-down spontaneous nucleation assay of 10 µM tubulin with 10–1000 nM calmodulin-regulated spectrin-associated protein 2-FL in the PEM. https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data6-v1.jpg Download elife-77365-fig2-data6-v1.jpg Figure 2—source data 7 SDS-PAGE gel from a spin-down spontaneous nucleation assay of 10 µM tubulin with 10–1000 nM calmodulin-regulated spectrin-associated protein 2-FL in the PEM + 100 mM KCl. https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data7-v1.jpg Download elife-77365-fig2-data7-v1.jpg Figure 2—source data 8 Quantification of the depolymerized tubulin concentrations determined by pelleting assay. https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data8-v1.docx Download elife-77365-fig2-data8-v1.docx We then evaluated the effect of CAMSAP2 on microtubule polymerization by a co-pelleting assay. We found that tubulin can be pelleted from 1.0 to 2.0 µM in the presence of 1.0 µM CAMSAP2-FL, indicating that tubulins could polymerize into microtubules or tubulin oligomers at a close concentration to the CcMT polymerization with CAMSAP2-FL (Figure 2B and C). CAMSAP2 drastically reduced the nucleation barrier to a level close to the theoretical limit of microtubule polymerization. In this assay, both tubulin and CAMSAP2-FL in the pellet were recovered from the supernatant after incubation on ice, suggesting that CAMSAP2-FL co-precipitated with tubulins and this precipitation is reversible. We further tested microtubule polymerization at the different concentrations of the CAMSAP2-FL from 10 nM to 1.0 µM in the tubulin polymerization buffer (PEM) or in the PEM supplemented with 100 mM KCl, which mimics a physiological condition. Polymerization was detected from 250 nM CAMSAP2-FL in PEM, and 500 nM CAMSAP2-FL in PEM +100 mM KCl, albeit the polymerization efficiency considerably decreased by increasing the salt concentration (Figure 2D and E). These results suggest that CAMSAP2 significantly enhances the nucleation and polymerization of tubulin. The enhancement effect still exists in the physiological condition, although it became milder than the tubulin polymerization buffer. CAMSAP2 recruits tubulins through liquid-liquid phase separation Recent studies of microtubule-associated proteins, TPX2 and Tau, showed that these proteins undergo liquid-liquid phase separation (LLPS) to interact with microtubules through an internal long disordered region (King and Petry, 2020; Tan et al., 2019). CAMSAP2 also possesses a long disordered region at the middle portion, as predicted by program PONDR, indicating its potential for phase separation (Figure 3A; Romero et al., 2001). We thus investigated the droplet formation of CAMSAP2 in the polymerization PEM buffer, finding that both CAMSAP2-FL and GFP-CAMSAP2-FL formed droplets (Figure 3B). We next evaluated the concentration of CAMSAP2 or salt on the droplet formation. By examining a series of different GFP-CAMSAP2-FL and salt concentrations, we found that 10 nM GFP-CAMSAP2-FL, which approximates a physiological concentration, could form the droplet under the salt concentration of less than 400 mM, which is similar to TPX2 (King and Petry, 2020; Figure 3C and D). Figure 3 with 1 supplement see all Download asset Open asset Calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) forms co-condensate with tubulin in vitro. (A) Intrinsic disorder prediction of CAMSAP2 by PONDR. CH, calponin-homology domain; MBD, microtubule-binding domain; CC, coiled-coil domain; CKK, C-terminal domain common to CAMSAP1 and two other mammalian proteins, KIAA1078 and KIAA1543. (B) Fluorescent image of GFP-CAMSAP2-FL condensates (top) and DIC image of CAMSAP2-FL condensates (bottom). (C, D) Phase diagram of 1 µM GFP-CAMSAP2-FL with indicated salt concentrations (C) and different concentrations of GFP-CAMSAP2-FL with 100 mM KCl (D). (E) Fusion of the GFP-CAMSAP2-FL condensate (also see Video 1). (F) Fluorescence recovery after photobleaching of GFP-CAMSAP2-FL condensates, acquired via confocal microscopy and (G) quantification. Time 00:00 (minutes:seconds) corresponds immediately after photobleaching. The graph shows the fluorescence recovery process of one of the four quantified droplets in Figure 3—figure supplement 1. (H) (I) GFP-CAMSAP2-FL and tubulin formed co-condensate in the physiological buffer (PEM with 100 mM KCl) and microtubule polymerization buffer (PEM). Figure 3—source data 1 Quantification of the fluorescence recovery after photobleaching of GFP-calmodulin-regulated spectrin-associated protein 2-FL condensates, acquired via confocal microscopy. https://cdn.elifesciences.org/articles/77365/elife-77365-fig3-data1-v1.docx Download elife-77365-fig3-data1-v1.docx To evaluate the nature of the CAMSAP2 condensate, we observed their liquid fluidity. We increased the concentration of GFP-CAMSAP2-FL to 4 µM to observe condensates efficiently. Once two CAMSAP2 condensates came close to each other, they fused and became a single large condensate (Figure 3E, Video 1). We also observed the fluorescence recovery after photobleaching (FRAP) in a CAMSAP2 condensate and found a slow recovery of the fluorescence (Figure 3F and G, Figure 3—figure supplement 1, and Video 2). These results suggest that the CAMSAP2 condensates possess high viscosity. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Fusion of the calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) condensate. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Fluorescence recovery after photobleaching of GFP-calmodulin-regulated spectrin-associated protein 2-FL condensates, acquired via confocal microscopy. The video was recorded immediately after photobleaching. We then tested whether CAMSAP2 can recruit tubulins into its condensate. In the physiological condition (PEM with 100 mM KCl), tubulin alone did not form any droplet but formed co-condensate only when mixed with GFP-CAMSAP2-FL (Figure 3H). These findings suggest that CAMSAP2 forms condensates by phase separation mechanism, into which tubulins are recruited. CAMSAP2 and tubulin co-condense to form aster-like structure To investigate how CAMSAP2 regulates the nucleation and polymerization of microtubules in connection with phase separation, we co-condensed tubulin and GFP-CAMSAP2-FL and observed them by fluorescence microscopy. Ten micromolar tetramethylrhodamine (TMR)-labelled tubulin was co-incubated with 10–1000 nM full-length GFP-CAMSAP2-FL at 37°C for 10 min in the physiological condition (PEM with 100 mM KCl) (Figure 4A). These molecules co-condensed together, and importantly microtubules extended radially from the condensates (Figure 4B), as observed for the TPX2 aster-like structure (Roostalu et al., 2015; Schatz et al., 2003). The appearance of this structure was reminiscent of the centrosomal aster; thus, we named it ‘Cam2-aster’. Cam2-asters became detectable above 250 nM of GFP-CAMSAP2-FL, and the number of asters increased as the CAMSAP2 concentration raised from 250 to 1000 nM, consistent with our pelleting assay (Figure 3B and D). Figure 4 with 1 supplement see all Download asset Open asset Tubulin is incorporated into calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) condensates to form aster-like structure. (A) The procedure used to obtain the data in panels (B) and (C). GFP-CAMSAP2-FL, 10 μM tubulin, and 0.5 μM tetramethylrhodamine (TMR)-tubulin were mixed in BRB80 supplemented with 100 mM KCl and incubated for 10 min at 37°C. The solution was directly transferred onto a coverslip and observed by fluorescence microscopy. (B) Representative image of asters. GFP-CAMSAP2-FL (0.5 μM), tubulin (10 μM), and TMR-tubulin (0.5 μM) were co-incubated. (C) Quantification of the numbers of asters in solutions containing 10 μM tubulin, 0.5 μM TMR-tubulin, and 10, 25, 50, 100, 250, 500, and 1000 nM GFP-CAMSAP2-FL. The results of three independent assays are shown with dots. (D) Schematic showing reconstitution of CAMSAP2-containing foci. CAMSAP2 condensates were formed as described in Figure 3 and fixed on the coverslip by an anti-GFP antibody. (E) Soluble tubulins were incorporated into CAMSAP2 condensates within 1 min. (F) Schematic showing CAMSAP2-containing foci with soluble tubulin and GFP-CAMSAP2-FL. (G) CAMSAP2 in solution induced aster formation from CAMSAP2 condensates in a dose-dependent manner. Representative images for 0 and 50 nM GFP-CAMSAP2-FL are (H) Quantification of microtubule formation from CAMSAP2 The of CAMSAP2 condensates with microtubules among total CAMSAP2 condensates are shows the results of three independent (I) images of aster formation. Tubulin (10 μM), TMR-tubulin (0.5 μM), and GFP-CAMSAP2-FL were incubated with CAMSAP2 condensates fixed on microtubules from CAMSAP2 condensates were observed The indicate 5 3 and The CAMSAP2 concentration required for aster formation in vitro was higher than the physiological concentration of CAMSAP2 of et al., 2014). reports have demonstrated that CAMSAPs are not only but also at structures such as and through the with some proteins et al., and these CAMSAPs that are to structures microtubule We also observed the condensates of CAMSAP2 in cells and during the polymerization of microtubules (Figure supplement 1). We thus that the stabilization of CAMSAP2 the efficiency of microtubule polymerization. To test this we fixed GFP-CAMSAP2-FL on the coverslip with the condition in which GFP-CAMSAP2-FL forms condensates (Figure was fixed on the coverslip through the GFP-CAMSAP2 condensates were then applied (Figure by the of tubulin, and by microscopy. Tubulin was incorporated into the CAMSAP2 foci within 1 although polymerization of microtubules was observed (Figure We that microtubule polymerization from CAMSAP2 foci CAMSAP2 in and we supplemented 10–1000 nM of GFP-CAMSAP2-FL (Figure As a microtubule formation was induced from the CAMSAP2 foci. GFP-CAMSAP2-FL at a concentration of 50 nM in solution was for microtubule polymerization from the although higher concentrations of GFP-CAMSAP2-FL further increased the microtubule polymerization efficiency (Figure and Thus, when CAMSAP2 is fixed as observed in its concentration required for microtubule elongation could be we acquired images of microtubules using GFP-CAMSAP2-FL and tubulin by microscopy. We found that dynamic polymerization and of microtubules at CAMSAP2-containing foci with the of GFP-CAMSAP2-FL (Figure 3 and These findings suggest that the fixed foci of CAMSAP2 act as a for microtubule with CAMSAP2 to the formation of microtubule asters. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg of aster formation. Tubulin (10 μM), TMR-tubulin (0.5 μM), and GFP-calmodulin-regulated spectrin-associated protein 2 were incubated with CAMSAP2 condensates fixed on The size of the is The is Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg of aster formation from 1. microtubules from calmodulin-regulated spectrin-associated protein 2 condensates were The size of the is The is of Cam2-asters by electron microscopy To of formation, we electron We observed condensates of CAMSAP2-FL by electron microscopy (Figure with light microscopy the CAMSAP2 condensates were we mixed tubulin and CAMSAP2-FL and observed them by in various 10 µM tubulin was incubated with 1 µM CAMSAP2-FL at 37°C for 30 microtubule from each condensate (Figure as found in the experiments (Figure Microtubule from the condensates further a microtubule (Figure CAMSAP2 homolog which is reported to stimulate microtubule nucleation and growth in vitro (Roostalu et al., also the ability to the microtubule aster (Figure supplement the aster formation be a common among Figure 5 with 3 see all Download asset Open asset Nucleation and aster formation of calmodulin-regulated spectrin-associated protein 2 (CAMSAP2). Representative electron microscopy images are shown from at three independent (A) of 1 µM CAMSAP2-FL incubated at for 30 min. (B) of 10 µM tubulin with 1 µM of CAMSAP2-FL after incubation at 37°C for 10 min. microtubule structures were of tubulin with CAMSAP2-FL incubated at various were also in Figure supplement 2. (C) of 30 µM tubulin with 3 µM CAMSAP2-FL after incubation at 37°C for 10 min at different Cam2-asters are indicated by the The