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. 2019 May 31:15:274-281.
doi: 10.1016/j.isci.2019.04.022. Epub 2019 Apr 28.

Centrosomal Actin Assembly Is Required for Proper Mitotic Spindle Formation and Chromosome Congression

Matthias Plessner  1 , Julian Knerr  2 , Robert Grosse  3
Affiliations

Centrosomal Actin Assembly Is Required for Proper Mitotic Spindle Formation and Chromosome Congression

Matthias Plessner et al. iScience. .

Abstract

Cytoskeletal cross talk between actin filaments and microtubules is a common mechanism governing the assembly of cellular structures, i.e., during filopodia formation or cilia organization. However, potential actin-microtubule interactions during mammalian cell divisions are less well understood. At mitotic entry, centrosomes propagate the formation of the mitotic spindle, thereby aligning individual chromosomes to the metaphase plate, a process coined chromosome congression. Here, we identify actin filament assembly spatially defined at centrosomes contemporaneously with spindle microtubules forming during prometaphase. We show that pharmacological Arp2/3 complex inhibition as well as overexpression of the Arp2/3 complex inhibitory protein Arpin decreased spindle actin. As a consequence, mitotic spindle formation is impaired, which resulted in disorganized chromosome congression and ultimately mitotic defects in non-transformed cells. Thus centrosomal Arp2/3 complex activity plays a role in the maintenance of genomic integrity during mitosis.

Keywords: Biological Sciences; Cell Biology; Functional Aspects of Cell Biology; Organizational Aspects of Cell Biology.

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Figures

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Graphical abstract
Figure 1
Figure 1
Formation of Perinuclear and Spindle Actin during Prometaphase (A) Confocal scans of glutaraldehyde-fixed NIH 3T3 cells in indicated z direction, stained for F-actin (phalloidin, green, and black and white) and DNA (DAPI, blue). Note the aster-like F-actin structures below and on top of the nucleus (at 6.0 and 9.0 μm) as well as perinuclear F-actin (7.5 μm). Scale bar, 5 μm. (B) Incidence of spindle actin during prometaphase in different mammalian cell lines. Data are shown as mean of two independent experiments together with individual data points; n > 13 cells in prometaphase per data point. (C) Cropped, orthogonal projections of phalloidin staining (black and white) as indicated in (A) (7.5 μm) by dashed, violet lines. Scale bar, 5 μm. Arrowheads indicate F-actin structures protruding into the nuclear compartment.
Figure 2
Figure 2
Spindle Actin Is Branched and Precedes Kinetochore MTs (A) Confocal live-cell imaging of RPE-1 cells, stably expressing shuttling actin-chromobody-TagGFP2 (actin, green), stained with SiR-tubulin (MTs, magenta). Spindle actin is assembled at centrosomes and microtubules during formation of the mitotic spindle (arrowheads). Scale bar, 5 μm; time stamp, min:s. (B) Quantification of spindle actin duration in NIH 3T3 and RPE-1 cells. Data are shown as mean + SD of two independent experiments together with individual data points; n > 5 events per cell line. (C) Confocal live-cell imaging of an individual centrosome (asterisk, 00:00) in NIH 3T3 cells during prometaphase, stably expressing shuttling actin-chromobody (actin, green) and mCherry-β-tubulin (MTs, red). Individual actin filaments aligning with forming spindle MTs are indicated by arrowheads. Scale bar, 1.5 μm; time stamp, min:s. (D) Quantification of spindle actin branch angle in NIH 3T3 cells, as indicated in (C) (00:01) by dashed, violet lines. Data are shown as a scatter box plot; n = 11 actin filament branches. (E) Confocal live-cell imaging of NIH 3T3 cells stably expressing shuttling actin-chromobody (actin, green) and mCherry-β-tubulin (MTs, red) during prometaphase. Kymographs corresponding to the violet arrows are shown on the right and illustrate actin assembly before MT polymerization on individual tracks at the equatorial plane. Scale bar, 5 μm; time stamp, min:s.
Figure 3
Figure 3
The Arp2/3 Complex Nucleates Spindle Actin, and Spindle Actin Is Required for Mitotic Spindle Formation (A) Centrosomal Arp2/3 complex localization in inter- and prometaphase. NIH 3T3 cells were stained with anti-Arp2 (green) and Centrin 1 (red). Arrowheads indicate centrosomes, and the individual centrosome in interphase is shown as a magnification. For prometaphase, a maximum intensity projection was calculated to show both centrosomes in a single image. Scale bar, 10 μm or 1 μm (magnification). (B) Chromosome congression in NIH 3T3 cells stably expressing mCherry-β-tubulin (MTs, red). Cells were stained with SiR-DNA (magenta) and treated with indicated inhibitors. See also Videos S6, S7, and S8. Scale bar, 5 μm; time stamp, min:s. (C) Quantification of integrated sAC-TagGFP2 fluorescence intensities at centrosomes with indicated treatments during prometaphase. Data are normalized to DMSO and presented as box plots; n = 6 (CK-689, CK-666, SMIFH2) or 7 (DMSO, CK-869) events from two independent experiments. (D) Live-cell imaging of NIH 3T3 cells labeled as in (B) treated with CK-666 during mitotic progression. Note the lack of centrosome-mediated MT assembly, whereas chromatin-mediated MT nucleation is not affected (white arrowheads). Scale bar, 5 μm; time stamp, min:s.
Figure 4
Figure 4
Spindle Actin Inhibition Results in Mitotic Defects (A) Image examples of synchronized RPE-1 cells, stably expressing the chromatin marker H2B-mCherry (red) and treated with indicated compounds at 00:00. Mitotic events are indicated by arrowheads. Scale bar, 25 μm; time stamp, h:min. (B) Quantification of mitotic defects as shown in (A). Data are presented as mean + SD; n > 15 mitotic events, pooled from three independent experiments. (C) RPE-1 cells, stably expressing the chromatin marker H2B-mCherry (red) were transfected with GFP or GFP-Arpin and synchronized at the G2/M border. Image examples show mitotic progression after release of the mitotic block. Mitotic events are indicated by arrowheads. Scale bar, 25 μm; time stamp, h:min. (D) Quantification of mitotic defects as shown in (C). Data are presented as mean + SD; n > 7 mitotic events. Immunoblot indicates expression and molecular weight of Arpin.
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