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Spindle-F-actin interactions in mitotic spindles in an intact vertebrate epithelium.
Kita AM
,
Swider ZT
,
Erofeev I
,
Halloran MC
,
Goryachev AB
,
Bement WM
.
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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.
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:. 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:. 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:. 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:. 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.
Supplementary Figure 1
A. Scatterplot showing the occurrence of fingers in Xenops laevis epithelial cells in interphase (0±0 fingers) vs metaphase (1.5±2.2 fingers). B-F. Histograms showing the distribution of B. finger lengths, C. extension speeds, D. retraction speeds, E. percent distance spanned to the spindle pole, and F. total duration. Values on histograms represent mean±SD. G. Metaphase and telophase RPE cells fixed with the PDA protocol showing different degrees of F-actin occupying the mitotic spindle (presumptive spindle poles are marked with arrows). H. Interphase and metaphase RPE cells fixed with the PDA protocol. The arrowhead marks clear cytoplasmic punctae not obviously seen in mitotic cells. H’. Orthogonal views of the regions boxed in H showing cytoplasmic punctae in the interphase cell (1; arrowheads) and a robust endoplasmic network of actin in the metaphase cell (2). Horizontal scale bars = 10µm, vertical scale bars = 5µm.
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