Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

During cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a β-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the β- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies β-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a β-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.


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(1). For the quantification of the numbers of multi-nucleate cells, each data point collected presents the percentage of multi-nucleate cells in a population of 30 cells within an optic field of the ×60/1.4 NA oil-immersion objective lens. At least 6 random fields were observed in each independent experiment (a total of 200 cells) and at least 20 random fields from three independent experiments were analyzed for each group (a total of 600 cells). Under experimental conditions in this study, an optical field of the ×60/1.4 NA oil-immersion objective lens includes 30-40 HeLa cells in average, with at least 30 cells possessing intact nucleus that are not at the edge of an observable field.
(2)The quantification of the numbers of cells with DIAPH1 or IQGAP1 at the cortex was performed using identical methods described above. Quantifications in (1) and (2) involve observation of a "yes" or a "no" phenotype (eg. mono-nucleate vs. binucleate, proteins localizing to the cortex vs. not localizing to the cortex) thus hundreds of cells in each independent experiment can be quantified within reasonable time.
(3) The number of blebs around the cortex, or length of long axis of the cell was determined in individual cell and >30 cells (in anaphase B) in each independent experiment were analyzed, with a total of 100 cells (in anaphase B) from three independent experiments were analyzed for each condition.
(4) The number of PLA signals around the cortex, the ratios of PLA signals between the two poles, the ratio of protein fluorescence intensity or blebs between two poles were determined in individual cell and >15 cells in each independent experiments were analyzed, with a total of 50 cells from three independent experiments were analyzed for each group. Quantifications in (3) and (4) involve numerical measurements of the subcellular structure (blebs), micron-scale mitotic geometry (length of long axis), individual fluorescent foci (PLA signals) or protein fluorescence intensity distribution in each individual cell thus tens of cells in each independent experiment were quantified within reasonable time.
(5) To quantify the rate of furrow ingression, a total of 5 cells in each live-cell imaging group were analyzed. To quantify the extent of cell elongation, a total of 10 cells in each live-cell imaging group were analyzed.
(6) In AFM live-cell analysis, at least 9 mitotic cells were measured in each independent experiment and 3 independent experiments were performed, with a total of at least 27 cells examined in each condition.
(7) In BLI affinity assays, each binding sequence between a ligand and an analyte was repeated in at least three independent experiments.
(8) For in vitro protein binding assays, at least three independent binding assays were performed for each condition with similar results.
In all in vitro binding assays and all in vivo fixed cell analysis, no data were excluded. In live-cell imaging analysis monitoring furrow ingression or cell elongation dynamics, cells that underwent continuous metaphase-arrest within observation period (120min), or apoptosis during early anaphase, that were induced by photon-toxicity were excluded from subsequent analyses.
In all in vitro binding assays, or in vivo assays including all microscopic analysis, at least three independent experiments were perform in each condition. In BLI affinity assays each binding sequence was repeated in at least three independent experiment. In all live-cell imaging analysis, at least 5 cells over three independent experiments were analyzed. All replications were successful.
In the binucleate phenotype analysis and DIAPH1/IQGAP1 cortical localization analysis, the selection of an observable optical field in each independent experiment was random. In these experiments, the numbers of optical fields (at least 6 fields) among each independent experiment of each group were randomized, adding up to a total of at least 20 fields in each group. In all fixed-cell analysis, live-cell imaging analysis, or AFM analysis the number of cells observed/analyzed in each independent experiment are not predetermined.
All experiments were done with a control group set up in order to examine the effects of a certain variable (different from that of control) in that experiment thus blinding is not applicable to this study.