Reconstitution of the augmin complex provides insights into its architecture and function

ABSTRACT Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, through

the carboxyl terminus of its hDgt6 subunit to nucleate microtubules within spindles. However, augmin’s biochemical complexity has restricted analysis of its structural organization and

function. Here, we reconstitute human augmin and show that it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains _in vitro_

microtubule-binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to _Xenopus_ egg extracts promotes microtubule

aster formation, an activity enhanced by Ran–GTP. This activity requires microtubule binding, but not the characterized hDgt6 γ-tubulin-recruitment domain. Tetrameric sub-complexes induce

asters, but activity and microtubule bundling within asters are reduced compared with octameric complexes. Together, our findings shed light on augmin’s structural organization and

microtubule-binding properties, and define subunits required for its function in organizing microtubule-based structures. Access through your institution Buy or subscribe This is a preview

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ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS THE AUGMIN COMPLEX

ARCHITECTURE REVEALS STRUCTURAL INSIGHTS INTO MICROTUBULE BRANCHING Article Open access 26 September 2022 INTEGRATED MODEL OF THE VERTEBRATE AUGMIN COMPLEX Article Open access 13 April 2023

MOLECULAR ARCHITECTURE OF THE AUGMIN COMPLEX Article Open access 16 September 2022 REFERENCES * Walczak, C. E. & Heald, R. Mechanisms of mitotic spindle assembly and function. _Int. Rev.

Cytol._ 265, 111–158 (2008). Article  CAS  PubMed  Google Scholar  * Meunier, S. & Vernos, I. Microtubule assembly during mitosis---from distinct origins to distinct functions? _J. Cell

Sci._ 15, 2805–2814 (2012). Article  Google Scholar  * Teixidó-Travesa, N., Roig, J. & Lüders, J. The where, when and how of microtubule nucleation--one ring to rule them all. _J. Cell

Sci._ 125, 4445–4456 (2012). Article  PubMed  Google Scholar  * Lüders, J. & Stearns, T. Microtubule-organizing centres: a re-evaluation. _Nat. Rev. Mol. Cell Biol._ 8, 161–167 (2007).

Article  PubMed  Google Scholar  * Bettencourt-Dias, M. & Glover, D. M. Centrosome biogenesis and function: centrosomics brings new understanding. _Nat. Rev. Mol. Cell Biol._ 8, 451–463

(2007). Article  CAS  PubMed  Google Scholar  * Kollman, J. M., Merdes, A., Mourey, L. & Agard, D. A. Microtubule nucleation by γ-tubulin complexes. _Nat. Rev. Mol. Cell Biol._ 12,

709–721 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wadsworth, P. & Khodjakov, A. E pluribus unum: towards a universal mechanism for spindle assembly. _Trends Cell

Biol._ 14, 413–419 (2004). Article  CAS  PubMed  Google Scholar  * Walczak, C. E., Cai, S. & Khodjakov, A. Mechanisms of chromosome behaviour during mitosis. _Nat. Rev. Mol. Cell Biol._

11, 91–102 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Dumont, J. & Desai, A. Acentrosomal spindle assembly and chromosome segregation during oocyte meiosis. _Trends

Cell Biol._ 22, 241–249 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Karsenti, E. & Vernos, I. The mitotic spindle: a self-made machine. _Science_ 294, 543–547 (2001).

Article  CAS  PubMed  Google Scholar  * Kelly, A. E. & Funabiki, H. Correcting aberrant kinetochore microtubule attachments: an Aurora B-centric view. _Curr. Opin. Cell Biol._ 21, 51–58

(2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Goshima, G., Mayer, M., Zhang, N., Stuurman, N. & Vale, R. D. Augmin: a protein complex required for

centrosome-independent microtubule generation within the spindle. _J. Cell Biol._ 181, 421–429 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Uehara, R. et al. The augmin

complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells. _Proc. Natl Acad. Sci. USA_ 106, 6998–7003 (2009). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Lawo, S. et al. HAUS, the 8-subunit human Augmin complex, regulates centrosome and spindle integrity. _Curr. Biol._ 19, 816–826 (2009). Article  CAS

  PubMed  Google Scholar  * Colombié, N., Głuszek, A. A., Meireles, A. M. & Ohkura, H. Meiosis-specific stable binding of augmin to acentrosomal spindle poles promotes biased microtubule

assembly in oocytes. _PLoS Genet._ 9, e1003562 (2013). Article  PubMed  PubMed Central  Google Scholar  * Petry, S., Pugieux, C., Nédélec, F. J. & Vale, R. D. Augmin promotes meiotic

spindle formation and bipolarity in Xenopus egg extracts. _Proc. Natl Acad. Sci. USA_ 108, 14473–14478 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Petry, S., Groen, A.

C., Ishihara, K., Mitchison, T. J. & Vale, R. D. Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2. _Cell_ 152, 768–777 (2013). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Kamasaki, T. et al. Augmin-dependent microtubule nucleation at microtubule walls in the spindle. _J. Cell Biol._ 202, 25–33 (2013). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Hayward, D., Metz, J., Pellacani, C. & Wakefield, J. G. Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven

mitotic spindle formation. _Dev. Cell_ 28, 81–93 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wu, G. et al. Hice1, a novel microtubule-associated protein required for

maintenance of spindle integrity and chromosomal stability in human cells. _Mol. Cell. Biol._ 28, 3652–3662 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Tan, S., Kern, R.

C. & Selleck, W. The pST44 polycistronic expression system for producing protein complexes in Escherichia coli. _Protein Expr. Purif._ 40, 385–95 (2005). Article  CAS  PubMed  Google

Scholar  * Subramanian, R. et al. Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein. _Cell_ 142, 433–443 (2010). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Trowitzsch, S., Bieniossek, C., Nie, Y., Garzoni, F. & Berger, I. New baculovirus expression tools for recombinant protein complex production.

_J. Struct. Biol._ 172, 45–54 (2010). Article  CAS  PubMed  Google Scholar  * Desai, A., Murray, A., Mitchison, T. J. & Walczak, C. E. The use of Xenopus egg extracts to study mitotic

spindle assembly and function _in vitro_. _Methods Cell Biol._ 61, 385–412 (1999). Article  CAS  PubMed  Google Scholar  * Suloway, C. et al. Automated molecular microscopy: the new Leginon

system. _J. Struct. Biol._ 151, 41–60 (2005). Article  CAS  PubMed  Google Scholar  * Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. _J. Struct. Biol._

157, 38–46 (2007). Article  CAS  PubMed  Google Scholar  * Hohn, M. et al. SPARX, a new environment for cryo-EM image processing. _J. Struct. Biol._ 157, 47–55 (2007). Article  CAS  PubMed 

Google Scholar  * Yang, Z., Fang, J., Chittuluru, J., Asturias, F. J. & Penczek, P. A. Iterative stable alignment and clustering of 2D transmission electron microscope images.

_Structure_ 20, 237–247 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Smith, M. B. et al. Interactive, computer-assisted tracking of speckle trajectories in fluorescence

microscopy: application to actin polymerization and membrane fusion. _Biophys. J._ 101, 1794–1804 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Folta-Stogniew, E. &

Williams, K. R. Determination of molecular masses of proteins in solution: implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory.

_J. Biomol. Tech._ 10, 51–63 (1999). CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank G. Goshima (Nagoya University, Japan) for the gift of UCHL5IP

antibody; and L. Pelletier (University of Toronto, Canada) for the antibodies against C14orf94 and Cep27. The SEC-LS/UV/RI instrumentation used for light-scattering analysis was supported by

a National Institutes of Health (NIH) award (1S10RR023748-01). TEM studies were conducted at the National Resource for Automated Molecular Microscopy, which is supported by the National

Institute of General Medical Sciences (9 P41 GM103310). K-C.H. was supported by the Kimberly Lawrence-Netter Cancer Research Discovery Fund at The Rockefeller University and is supported by

a Special Fellow Award from The Leukemia and Lymphoma Society. S.F. acknowledges postdoctoral support from an NIH National Research Service Award Fellowship (F32GM099380). R.A.M.

acknowledges support from the NIH (GM-052468). T.M.K. acknowledges support from the NIH (GM-65933). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Laboratory of Chemistry and Cell Biology,

Rockefeller University, New York, New York 10065, USA Kuo-Chiang Hsia, Alejandro Dottore, Qi Hao, Scott Forth, Yuta Shimamoto & Tarun M. Kapoor * Department of Integrated Structure and

Computational Biology, The Scripps Research Institute, La Jolla, California 92037, USA Elizabeth M. Wilson-Kubalek, Kuang-Lei Tsai & Ronald A. Milligan Authors * Kuo-Chiang Hsia View

author publications You can also search for this author inPubMed Google Scholar * Elizabeth M. Wilson-Kubalek View author publications You can also search for this author inPubMed Google

Scholar * Alejandro Dottore View author publications You can also search for this author inPubMed Google Scholar * Qi Hao View author publications You can also search for this author

inPubMed Google Scholar * Kuang-Lei Tsai View author publications You can also search for this author inPubMed Google Scholar * Scott Forth View author publications You can also search for

this author inPubMed Google Scholar * Yuta Shimamoto View author publications You can also search for this author inPubMed Google Scholar * Ronald A. Milligan View author publications You

can also search for this author inPubMed Google Scholar * Tarun M. Kapoor View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K-C.H. carried

out experiments in Figs 1–4 and assembled all figures. Q.H. helped purify and characterize proteins. Acquisition and interpretation of the electron microscopy and image analysis data shown

in Fig. 5 were carried out by E.M.W-K., K-L.T. and R.A.M. Single-molecule assays in Fig. 3 were carried out by A.D. K-C.H. and Y.S. carried out experiments involving _Xenopus_ egg extracts

(Figs 6–8). S.F. quantitatively analysed aster morphology (Fig. 8). All authors helped write the manuscript. T.M.K. directed the project and helped design experiments and prepare the 

manuscript. CORRESPONDING AUTHOR Correspondence to Tarun M. Kapoor. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. INTEGRATED SUPPLEMENTARY

INFORMATION SUPPLEMENTARY FIGURE 1 Biochemical characterization of augmin sub-complexes. (A–C) Oligomeric states of Hice1⋅hDgt6Δ(433–955) (A), tetramer-I (Hice1⋅hDgt6 (1–432)⋅UCHL5IP⋅Cep27)

(B), and tetramer-II (Hice1⋅hDgt6 (1–432)⋅His-C14orf94⋅Ccdc5) (C) were analysed by size exclusion chromatography coupled with light scattering. Recombinant sub-complexes elute earlier than

expected for a globular protein of equivalent molecular mass. Elution volumes of molecular weight standards used in the size exclusion chromatography are indicated. The average molecular

mass for sub-complexes analysed by light scattering are indicated (lines across the respective elution peaks). (D) Observed results from light scattering experiments (Obs) are presented

beside molecular weights calculated using standards (Cal). (E) Purified hexamer (Hice1⋅hDgt6 (1–432)⋅UCHL5IP⋅Cep27⋅His-C14orf94⋅Ccdc5) was analysed by SDS-PAGE, followed by staining with

Coomassie blue. (F) Western blot analysis of the hexameric complex with the indicated antibodies. (G) C14orf94 (a.a. 1–188) ⋅full length Ccdc5 hetero-dimer was examined by size exclusion

chromatography (Superdex 200 16/60). Peak fractions (between 60 and 80 ml; volumes indicted) were analysed by SDS-PAGE (staining with Coomassie blue). Void volume (Vo) is indicated. The star

indicates a GST-containing peak. Absorbance (a.u.) is 280 nm. SUPPLEMENTARY FIGURE 2 Analysis of subunits in the augmin holo-complex. (A) Recombinant holo-complex

(Hice1⋅GFP-hDgt6⋅UCHL5IP⋅GFP-Cep27⋅C14orf94⋅Ccdc5⋅His-hDgt3⋅hDgt5) was analysed by SDS-PAGE, followed by staining with Coomassie blue. (b-d) Western blots were carried out using the

indicated antibodies. GFP-tagged Cep27 within purified tetramer-I was also detected using anti-GFP antibody (B). The star indicates a GFP-containing degradation product of GFP-Cep27.

SUPPLEMENTARY FIGURE 3 Single molecule analysis of augmin complexes. (A–F) Fluorescence intensity average and distribution analysis of single particles for each complex adhered to a glass

surface: (A) Hice1⋅hDgt6Δ(433–955) (intensity average 5,400 ± 1,800, _N_ = 593 particles), (B) tetramer-I (GFP-Cep27) (intensity average 4,800 ± 1,800, _N_ = 600 particles), (C) tetramer-I

(GFP-hDgt6 (1–432)) (intensity average 5,400 ± 2,500, _N_ = 631 particles), (D) octamer[hDgt6D(433–955)] (intensity average 4,500 ± 2,000, _N_ = 626 particles), (E) holo-complex (8,300 ±

7,000, _N_ = 662 particles). (F) Monomeric GFP (intensity average 4,800 ± 1,700, _N_ = 577 particles) was used as reference. Intensity averages are reported as mean ± s.d. Three or more

independent experiments were analysed. (G–K) The mean square displacements for Hice1⋅hDgt6Δ(433–955) (G), tetramer-I (GFP-Cep27) (H), tetramer-I (GFP-hDgt6 (1–432)) (I), octamer

[hDgt6Δ(433–955)] (J), holo-complex (K) are shown. Diffusion coefficients (_D_) are calculated as half the slope of the fit line. Three or more independent experiments were analysed. (L)

Image of GMPCPP-stabilized microtubules (X-rhodamine- and biotin-labelled) (top), GFP-tagged complexes (middle, maximum intensity projections from 300 images in the time-lapse sequence) and

corresponding kymographs (below) are shown for GFP-tagged tetramer-I (GFP-hDgt6 (1–432)) that also has a GFP on hDgt6, as is the case for the dimer. Scale bars, horizontal, 2 μm; vertical, 2

s. (M) The region highlighted (box) in each kymograph is also shown in greater detail as a montage. Scale bar, 2 μm. (N) Binding of individual molecules to a microtubule was tracked to

compute the CDF of the dwell time for the complex. Mean dwell time 〈_t_〉 and relative amplitude (in parentheses) were obtained by fitting to bi-exponential functions (grey curve). Data from

three or more independent experiments were analysed. SUPPLEMENTARY FIGURE 4 Electron microscopy analyses of augmin sub-complexes. (A,B) Negative stain electron micrographs of taxol

stabilized microtubules in the presence of (A) Hice1⋅hDgt6Δ(433–955) and (B) GFP-tagged tetramer-I. Scale bar, 40 nm. Microtubule without tetramer-I binding is indicated by arrow. (C)

SDS-PAGE analysis (stained with Coomassie blue) of purified tetramer-I with MBP-tagged Hice1 C-terminus. (D) Negative stain electron micrograph of the MBP-tagged tetramer-I (Hice1-MBP⋅hDgt6

(1–432)⋅UCHL5IP⋅Cep27) particles. Three representative class averages of individual particles (_N_ = 82, 52 and 49) are shown. The position of the MBP-tag is indicated (arrow). Scale bar, 10

nm (E,F) Negative stain electron micrograph of tetramer-I (Hice1⋅hDgt6 (1–432)⋅UCHL5IP⋅Cep27) (E) and tetramer-II (Hice1⋅hDgt6 (1–432)⋅C14orf94⋅Ccdc5) (F) particles. A field of either

tetramer-I or -II particles adsorbed onto a glow-discharged carbon grid was stained with 2% (w/v) uranyl acetate and processed for imaging. Scale bar, 100 nm. SUPPLEMENTARY FIGURE 5 Analysis

of metaphase spindle localization of augmin complexes. (A) Western blot analysis of crude _Xenopus_ egg extracts (10-fold dilution in CSF-XB buffer, 7 ml) with anti-xCcdc5 antibody (lane

6). Equal volumes (7 ml) of serial dilutions of recombinant xCcdc5 (a.a. 1–204) were analysed by SDS-PAGE to determine endogenous xCccdc5 concentration (lane 1–5). Concentrations of

recombinant xCcdc5 protein are indicated. (B) Localization of endogenous augmin in the metaphase spindles assembled in _Xenopus_ egg extracts, fixed, spun down on coverslips and stained with

anti-xCcdc5 and anti-α-tubulin antibodies. xCcdc5 antibody staining, left panels; and α-tubulin antibody staining (middle panels); and overlays (right panels; microtubules, red; xCcdc5,

green; and DNA, blue), are shown. Scale bar, 10 μm. (C) Additional fluorescence images of metaphase spindles in the presence GFP-tagged octamer[hDgt6Δ(433–955). GFP (left panels);

X-rhodamine (middle panels); and overlays (right panels; microtubules, red; GFP, green; and DNA, blue), are shown. Scale bar, 10 μm. (D,F) Fluorescence images of metaphase spindles in the

presence of GFP-tagged Hice1⋅hDgt6Δ(433–955) (D) and GFP protein (F) (left panels, 15 nM); X-rhodamine (middle panels); and overlays (right panels; microtubules, red; GFP, green; and DNA,

blue), are shown for spindles in D,F. Scale bar, 10 μm. (E,G) Linescans for GFP fluorescence (green) and X-rhodamine (red) along the long axis of the spindle are shown. (H) The intensity of

the GFP image in the presence of GFP-tagged octamer[hDgt6Δ(433–955), Hice1Δ(1–140)] was adjusted to visualize GFP signal at the spindle poles. (I) Localization analysis of augmin

holo-complex in metaphase spindles using _Xenopus_ egg extracts. Recombinant GFP-tagged holo-complex (6 nM) (left panels); tubulin (X-rhodamine-labelled, middle panels); and overlays (right

panels; tubulin, red; GFP, green; and DNA, blue), are shown. Scale bar, 10 μm. SUPPLEMENTARY FIGURE 6 Microtubule aster formation in the presence of augmin complexes. (A,B) Aster assembly in

the presence of Ran(Q69L) and GFP-tagged octamer[hDgt6Δ(433–955)] (A) or holo-complex (B) at 6 nM. (C) Immunodepletion of xCcdc5 from _Xenopus_egg extracts using anti-xCcdc5 antibody.

xCcdc5 bands in crude (A), IgG-depleted (B) and xCcdc5-depleted (C) egg extracts were analysed by western blots. Equal volumes of egg extract samples with the same dilution were analysed by

SDS-PAGE. Supernatant fractions (S) and bead bound fractions (P) are indicated. IgG and xCcdc5 bands are indicated. Three independent experiments were performed and depletion was found to be

∼60%. (D) A representative image of microtubule aster formed in the presence of Ran(Q69L) and GFP-tagged tetramer-II at 15 nM. Tubulin (X-rhodamine-labelled), left panel; and GFP

fluorescence, right panel, are shown. Scale bar, 10 μm. (E) Analysis of microtubule (rhodamine signal) and augmin (GFP signal) levels in asters. Ratios of average fluorescence at 1 μm versus

5 mm radius for tetramer-II (15 nM) induced asters are shown. (_N_ = 19 asters; s.d. was determined from data pooled from 3 independent experiments.) (F–H) Representative images after polar

transformation of asters induced by RanQ69L alone (F) and in the presence of Ran(Q69L) and holo-complex (15 nM) (G) and octamer[hDgt6Δ(433–955)] (15 nM) (H). (I–M) Representative images of

microtubule asters induced by Ran(Q69L) alone (I) and in the presence of Ran(Q69L) and GFP-tagged holo-complex (15 nM) (J), octamer[hDgt6Δ(433–955)] (15 nM) (K), octamer[hDgt6Δ(433–955)] (60

nM) (L) or tetramer-II (60 nM) (M). Three half-circle (180 degree) regions (at 4 and 8, and 12 μm radii) used for determination of coefficient of variation are indicated in first image for

each panel (dashed white lines). SUPPLEMENTARY FIGURE 7 Analysis of aster number and microtubule intensity in asters promoted by addition of recombinant augmin complexes to egg extracts.

Individual fluorescence images of X-rhodamine-labelled microtubules were taken automatically using a motorized XY stage and then 400 captured images were stitched into one composite image

using NIS-Element software. A representative single image from each composite image is shown in the bottom inset. Augmin complexes and Ran(Q69L) were added to extracts, incubated for 10 min

(or as noted), fixed and processed. (A) Representative composite images of microtubule asters in the presence of buffer control, GFP-tagged octamer[hDgt6Δ(433–955)] and holo-complex. (B)

Representative composite images of microtubule asters in the presence of GFP-tagged octamer[hDgt6Δ(433–955), Hice1Δ(1–140)] and octamer[hDgt6Δ(433–955)] with Ran(Q69L). (C) Representative

composite images of microtubule asters in the presence of indicated GFP-tagged octamer[hDgt6Δ(433–955)] concentrations. (D) Representative composite images of microtubule asters in the

presence of GFP-tagged octamer[hDgt6Δ(433–955)] without Ran(Q69L). Scale bars, 1 mm (composite image); 100 mm (single image). (E) Total microtubule intensity in asters induced in the

presence of Ran(Q69L) and GFP-tagged tetramer-II or octamer[hDgt6Δ(433–955)] at 60 nM. (F) Total microtubule intensity in asters induced by of Ran(Q69L) alone and in the presence of

Ran(Q69L) and GFP-tagged holo-complex or octamer[hDgt6Δ(433–955)] at 15 nM. s.d. was determined from data pooled from at least 3 independent experiments. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Information (PDF 1245 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Hsia, KC., Wilson-Kubalek, E., Dottore,

A. _et al._ Reconstitution of the augmin complex provides insights into its architecture and function. _Nat Cell Biol_ 16, 852–863 (2014). https://doi.org/10.1038/ncb3030 Download citation

* Received: 15 August 2013 * Accepted: 16 July 2014 * Published: 31 August 2014 * Issue Date: September 2014 * DOI: https://doi.org/10.1038/ncb3030 SHARE THIS ARTICLE Anyone you share the

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