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. 2019 Jul 1;30(14):1645-1654.
doi: 10.1091/mbc.E19-02-0126. Epub 2019 May 15.

Spindle-F-actin interactions in mitotic spindles in an intact vertebrate epithelium

Angela M Kita  1   2 , Zachary T Swider  1   2 , Ivan Erofeev  3 , Mary C Halloran  4   5 , Andrew B Goryachev  3 , William M Bement  1   2   4
Affiliations

Spindle-F-actin interactions in mitotic spindles in an intact vertebrate epithelium

Angela M Kita et al. Mol Biol Cell. .

Abstract

Mitotic spindles are well known to be assembled from and dependent on microtubules. In contrast, whether actin filaments (F-actin) are required for or are even present in mitotic spindles has long been controversial. Here we have developed improved methods for simultaneously preserving F-actin and microtubules in fixed samples and exploited them to demonstrate that F-actin is indeed associated with mitotic spindles in intact Xenopus laevis embryonic epithelia. We also find that there is an "F-actin cycle," in which the distribution and organization of spindle F-actin changes over the course of the cell cycle. Live imaging using a probe for F-actin reveals that at least two pools of F-actin are associated with mitotic spindles: a relatively stable internal network of cables that moves in concert with and appears to be linked to spindles, and F-actin "fingers" that rapidly extend from the cell cortex toward the spindle and make transient contact with the spindle poles. We conclude that there is a robust endoplasmic F-actin network in normal vertebrate epithelial cells and that this network is also a component of mitotic spindles. More broadly, we conclude that there is far more internal F-actin in epithelial cells than is commonly believed.

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Figures

FIGURE 1:
FIGURE 1:
Comparison of fixation protocols for preservation of endoplasmic F-actin. (A) Sample stained with phalloidin after overnight washing. Cortical F-actin is abundant and internal F-actin is sparse and disorganized. (A′) Orthogonal view of the epithelium in A. (B) PDA-fixed sample. Cortical and endoplasmic F-actin are abundant. F-actin cables extend from the nucleus (empty arrowhead), run parallel to the nucleus (solid arrowheads), and are organized in spindle-like structures (arrows). (B′) Orthogonal view of epithelium shown in B; arrows point to the same structure seen in B. (C–H) PDA-fixed cells. (C) Mitotic cell with F-actin cable extending from a spindle-like structure toward cortex. (D) Interphase cell with F-actin cables emanating from one side of the nucleus. (E) Interphase cell with F-actin cables emanating from the nucleus. (F) Mitotic cell with F-actin cables organized in spindle shape. (G) Mitotic cell with F-actin cables organized in spindle shape. (H) Presumptive telophase cell with extensive endoplasmic F-actin cables. (I) Interphase, metaphase, and telophase zebrafish blastomeres fixed with the PDA protocol showing abundant endoplasmic F-actin; arrows and arrowheads mark apparently identical structures to those seen in B. (J) Metaphase and telophase RPE cells fixed with the PDA protocol showing abundant endoplasmic F-actin; arrows mark apparent spindle poles. Scale bars = 10 µm.
FIGURE 2:
FIGURE 2:
Mitotic spindle-associated F-actin in fixed samples. (A) PDA-fixed X. laevis epithelial cells labeled for DNA (mCherry-H2B, blue), microtubules (MTs; anti–α-tubulin, magenta), and F-actin (phalloidin, green). Clusters of F-actin punctae (solid arrowheads) are evident throughout the cell cycle as are F-actin cables (empty arrowheads). (B) Metaphase cells prepared as in A; F-actin punctae concentrate around spindle poles; bright F-actin cables run from the cortex toward the spindle poles (arrows). (C) PDA-fixed cells without anti-tubulin staining. In late G2 cables and punctae are abundant; in metaphase a long, bright cable (arrows) runs from the cortex toward the expected site of the spindle pole; in anaphase cables run between separating sister chromosomes. (D) Paraformaldehyde fixed, methanol extracted samples stained for microtubules (anti–α-tubulin, magenta) and actin (anti–γ-actin, green) have both spindle-associated F-actin cables and punctae. (E) Cold-acetone–fixed cells double labeled for DNA (mCherry-H2B, magenta), and F-actin (phalloidin, green) have spindle-associated F-actin cables and punctae. Scale bars = 10 µm.
FIGURE 3:
FIGURE 3:
F-actin is coupled to the mitotic spindle. (A) Top, still image from a movie of live X. laevis epithelial cell expressing GFP-UtrCH (UtrCH, green) and mCh-α-tubulin (α-tub, magenta). Bottom, UtrCH alone. (A′) Kymograph drawn from the white rectangle in A showing parallel oscillations of UtrCH and α-tubulin (see Supplemental Movie 1). (B) Still images from the sample prepared as in A; optical flow field for each probe overlaid on each image (white arrows). Horizontal scale bars = 10 µm; vertical scale bar = 2 min. (C) Scatter plot showing correlation coefficients for optical flow of GFP-UtrCH and mCh-α-tubulin. N = 17 cells from 12 different embryos; bars represent mean ± SD.
FIGURE 4:
FIGURE 4:
Spindle pole–targeted F-actin fingers. (A) Top, still image from a movie of live X. laevis epithelial cell expressing GFP-UtrCH (UtrCH, green) and mCh-α-tubulin (α-tub, magenta). Bottom, UtrCH alone. Bright F-actin “finger” (arrowhead) extends from the cortex toward the spindle pole. (A′) Vertical montage of 23 frames from the white rectangle in A showing the extension and retraction dynamics of a single F-actin finger (see Supplemental Movie 2). (B) Montage of the cell prepared as in A showing the dynamics of an F-actin finger (see Supplemental Movie 2). (C) Montage of the cell prepared as in A showing several F-actin fingers (see Supplemental Movie 2). Scale bars = 10 µm. Time in min:s. (D) Schematic representation of angles measured to determine the spindle pole targeting of F-actin fingers. (D′) Scatterplot showing measured angles; n = 62 fingers from 40 different cells in 16 embryos; ***, P < 0.0001; bars represent mean ± SD.
FIGURE 5:
FIGURE 5:
A formin inhibitor perturbs spindle F-actin, spindle length, and mitotic duration. (A) Images of PDA-fixed X. laevis epithelial cells labeled for DNA (mCherry-H2B, blue), microtubules (MTs; anti–α-tubulin, magenta), and F-actin (phalloidin, green) after treatment with SMIFH2 or DMSO. Scale bars = 10 µm. (B) SMIFH2-induced loss of spindle F-actin. Each dot represents a single cell (n = 11 cells from two DMSO-treated embryos and 14 cells from three SMIFH2-treated embryos), *, P = 0.045; bars represent mean ± SD. (C) SMIFH2-induced spindle shortening. N = 38 cells from nine DMSO-treated embryos and 31 cells from eight SMIFH2-treated embryos; ***, P < 0.0001; bars represent mean ± SD. (D) Images from a movie of X. laevis epithelial cells coexpressing GFP-α-tubulin (abbreviated MTs, green) and mCh-H2B (DNA, magenta) and treated either with SMIFH2 or DMSO. Empty arrowheads mark the location of the separating anaphase chromosomes. Scale bars = 10 µm. Time is in min:s. (E) SMIFH2-induced lengthening of mitosis as measured from nuclear envelope breakdown to anaphase (n = 10 cells from six DMSO-treated embryos and six cells from four SMIFH2-treated embryos); ***, P < 0.0001; bars represent mean ± SD.
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References

    1. Abràmoff MD, Niessen WJ, Viergever MA. (2000). Objective quantification of the motion of soft tissues: an application to orbital soft tissue motion. IEEE Trans Med Imag , 986–995. - PubMed
    1. Aubin JE, Weber K, Osborn M. (1979). Analysis of actin and microfilament-associated proteins in the mitotic spindle and cleavage furrow of PtK2 cells by immunofluorescence microscopy. A critical note. Exp Cell Res , 93–109. - PubMed
    1. Azoury J, Lee KW, Georget V, Rassinier P, Leader B, Verlhac MH. (2008). Spindle positioning in mouse oocytes relies on a dynamic meshwork of actin filaments. Curr Biol , 1514–1519. - PubMed
    1. Barak LS, Nothnagel EA, DeMarco EF, Webb WW. (1981). Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-­diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement? Proc Natl Acad Sci USA , 3034–3038. - PMC - PubMed
    1. Burdyniuk M, Callegari A, Mori M, Nédélec F, Lénárt P. (2018). F-actin nucleated on chromosomes coordinates their capture by microtubules in oocyte meiosis. J Cell Biol , 2661–2674. - PMC - PubMed

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