Pal D et al. (2020), Rac and Arp2/3-Nucleated Actin Networks Antagon...

ECB-ART-49448

Front Cell Dev Biol 2020 Oct 20;8:591141. doi: 10.3389/fcell.2020.591141.

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Rac and Arp2/3-Nucleated Actin Networks Antagonize Rho During Mitotic and Meiotic Cleavages.

Pal D , Ellis A , Sepúlveda-Ramírez SP , Salgado T , Terrazas I , Reyes G , De La Rosa R , Henson JH , Shuster CB .

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In motile cells, the activities of the different Rho family GTPases are spatially segregated within the cell, and during cytokinesis there is evidence that this may also be the case. But while Rho's role as the central organizer for contractile ring assembly is well established, the role of Rac and the branched actin networks it promotes is less well understood. To characterize the contributions of these proteins during cytokinesis, we manipulated Rac and Arp2/3 activity during mitosis and meiosis in sea urchin embryos and sea star oocytes. While neither Rac nor Arp2/3 were essential for early embryonic divisions, loss of either Rac or Arp2/3 activity resulted in polar body defects. Expression of activated Rac resulted in cytokinesis failure as early as the first division, and in oocytes, activated Rac suppressed both the Rho wave that traverses the oocyte prior to polar body extrusion as well as polar body formation itself. However, the inhibitory effect of Rac on cytokinesis, polar body formation and the Rho wave could be suppressed by effector-binding mutations or direct inhibition of Arp2/3. Together, these results suggest that Rac- and Arp2/3 mediated actin networks may directly antagonize Rho signaling, thus providing a potential mechanism to explain why Arp2/3-nucleated branched actin networks must be suppressed at the cell equator for successful cytokinesis.

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	FIGURE 1. Actin dynamics in the dividing sea urchin embryo. Frames from time-lapse movies of L. variegatus embryos injected with either recombinant Lifeact-GFP (A–E, final intracellular concentration of 0.21 μM) or Lifeact-GFP and C3 Transferase (F–J, final intracellular concentration 0.27 μM). Bar, 30 μm. Time points denote minutes post-nuclear envelope breakdown (NEB). (E,J) Corresponding kymographs of sections taken through the long axis of the spindle as denoted by the rectangles in panels (A,F). Prior to nuclear envelope breakdown a bright rim of perinuclear actin is visible (A,F). Upon mitotic entry, microvilli and their rootlets shorten, and there is a gradual increase in deep cytoplasmic actin initiating at the cortex and accumulating inward up until anaphase onset (C,E,H,J). (K) Kymograph of cortical microvillar dynamics in the polar regions of control- and C3-injected embryos, taken from regions denoted by the small rectangles in Panels (B,G). (L) Quantification of cytoplasmic actin levels beginning 1 min post-NEB up until the metaphase-anaphase transition (Mean ± SEM, n = 6 cells per condition). While there was not a qualitative effect of Rho inactivation on cytoplasmic actin, there was a significant increase in cytoplasmic Lifeact fluorescence in C3-injected embryos. (M) Quantitation of the increases in cortical thickness and microvillar length that accompanies the metaphase-anaphase transition in control and C3-injected embryos (n = 6 cells per condition, whiskers denote minimum and maximum values), where a value of 1.0 represents length or thickness 1 min prior to the metaphase-anaphase transition. While there was no significant difference in the growth of microvilli, Rho inactivation blocked the thickening of actin cortex. ****p < 0.0001.
	FIGURE 2. Activated Rac suppresses actin dynamics and cytokinesis. (A) Cleavage rates for L. variegatus embryos injected with WT, Q61L or Q61L/F37A Rac mRNA (final intracellular concentration 2.5 μg/ml) and recombinant Lifeact-GFP (final intracellular concentration 0.2 μM) immediately after fertilization and cultured at 20°C until un-injected controls completed cytokinesis. Mean ± SEM for three experimental replicates, with >21 cells per experiment. *p < 0.001. (B) Furrow ingression profiles for injected cells, beginning at the initiation of furrowing (mean ± SEM, n = 5 per condition). (C) Frames from time-lapse movies of L. variegatus embryos injected with mRNA encoding WT or mutant Rac. Time points denote minutes post-NEB. Bar, 30 μm. Rectangles in panels (a,g,l) denote the region used to generate the kymographs in panels (e,k,p). Small rectangles denote regions used to generate the kymographs in panel (D). Bar, 30 μm. (D) Kymograph highlighting microvillar growth and cortical actin dynamics during mitotic exit in WT or mutant Rac-expressing cells. (E) Quantification of cytoplasmic actin levels beginning 1 min post-NEB up until the metaphase-anaphase transition (Mean ± SEM, n = 6 cells per condition). Activated Rac (Q61L, red) suppressed the increase in cytoplasmic actin in comparison to WT (gray), whereas activated Rac containing the effector-binding mutation F37A (Q61L/F37A, magenta) had no effect on cytoplasmic actin levels. (F) Quantitation of the increases in cortical thickness and microvillar length that accompanies the metaphase-anaphase transition in embryos expressing WT and mutant Rac (n = 6 cells per condition, whiskers denote minimum and maximum values), where a value of 1.0 represents length or thickness 1 min prior to the metaphase-anaphase transition. Activated Rac blocked both cortical thickening and microvillar elongation, but these actin dynamics were rescued in embryos expressing the double mutant that is incapable of promoting WAVE-mediated Arp2/3 activation. p < 0.005, ****p < 0.0001.
	FIGURE 3. Activated Rac suppresses cytokinesis through Arp2/3. (A,B) S. purpuratus embryos probed for Arp3 (top row in grayscale, red in the merged image), tubulin (cyan), and DNA (White). Bar, 30 μm. As cytokinesis progresses, Arp2/3 is increasingly excluded from the furrow region. ***p < 0.0001. (C) Arp2/3 delays but does not block cytokinesis. Embryos were treated with 0.1% DMSO or 100 μM CK-666 prior to nuclear envelope breakdown, and furrow diameter was measured by Nomarski/DIC starting at the initiation of furrowing (mean ± SEM, n = 13 for DMSO, n = 12 for CK-666). (D) Comparison of the rates of furrowing (time to 50% ingression), p < 0.05. (E) Quantification of cytoplasmic actin levels in either DMSO- or CK-666-treated embryos beginning 1 min post-NEB up until the metaphase-anaphase transition (Mean ± SEM, n = 6 cells per condition). (F) Quantitation of the increases in cortical thickness and microvillar length that accompanies the metaphase-anaphase transition in DMSO or CK-666-treated embryos expressing (n = 6 cells per condition, whiskers denote minimum and maximum values), where a value of 1.0 represents length or thickness 1 min prior to the metaphase-anaphase transition. No significant differences were detected for either actin population. (G) Arp2/3 inhibition rescues cytokinesis in embryos expressing activated Rac. Q61L Rac data replicated from Figure 2A. Mean ± SEM for three experimental replicates, >24 cells per experiment. *p < 0.001. (H) Cytoplasmic actin levels in embryos expressing Q61L Rac in the absence or presence of CK-666, beginning 1 min post-NEB up until the metaphase-anaphase transition (Mean ± SEM, n = 6 cells per condition). (I) Quantitation of the increases in cortical thickness and microvillar length that accompanies the metaphase-anaphase transition in embryos expressing Q61L Rac in the absence or presence of CK-666 (n = 6 cells per condition, whiskers denote minimum and maximum values), where a value of 1.0 represents length or thickness 1 min prior to the metaphase-anaphase transition. p = 0.005.
	FIGURE 4. Rac and Arp2/3 are required for polar body extrusion in sea star oocytes. (A) First polar body extrusion in P. miniata oocytes co-expressing Lifeact (white) and GFP-tubulin (red), rGBD-GFP (white) and mCherry-EMTB (red), and ArpC-GFP (white) and mCherry-EMTB (red). Time points indicate minutes post-hormone activation. Arrow denotes protruding polar body. Bar, 50 μm. (B) P. miniata oocytes co-expressing Lifeact (white) and GFP-tubulin (red) were activated with maturation hormone and 40 min following germinal vesicle breakdown, were treated with 0.1% DMSO or 100 μM CK-666. The spindle in the CK-666 sample is positioned in the Z axis of the image. The spindle failed to rotate or form a polar body protrusion. Bar, 50 μm (C) Quantification of polar body formation in DMSO or CK-666 samples for three experimental replicates, 100 oocytes scored per condition per experiment (Mean ± SEM, **p < 0.0001). (D) P. miniata oocytes co-expressing Lifeact (white), GFP-tubulin (red) and either wild-type (WT) or dominant-negative (T17N) Rac. In contrast to WT Rac-injected oocytes, T17N Rac-expressing oocytes failed in polar body formation after spindles failed to dock at the cortex. Bar, 50 μm. (E) Quantification of polar body formation in WT or T17N Rac-expressing oocytes for three experimental replicates, 100 oocytes scored per condition per experiment (Mean ± SEM, *p < 0.001).
	FIGURE 5. Rac and Arp2/3 directly antagonizes Rho activation during meiosis I. (A) Quantification of polar body formation in P. miniata oocytes injected WT, Q61L or Q61L/F37A Rac (final intracellular concentration 1.8 μg/ml), 200 oocytes scored per condition per experiment (Mean ± SEM, *p < 0.001; **p < 0.0001). (B) Cortical Rho activity during the surface contraction wave (SCW) in oocytes expressing WT Rac (gray), Q61L Rac (red), Q61L/F37A Rac (magenta) or Q61L Rac oocytes treated with 100 μM CK-666 (blue). Rhotekin-GFP fluorescence was measured for the entire cortex, where time 0 denotes the initiation of the SCW at the vegetal pole. Mean ± SEM, 7 oocytes per condition. (C) SCW and polar body extrusion in P. miniata oocytes co-expressing rGBD-GFP (white), mCherry-EMTB (red) and Rac variants described in panel (B). Time point indicate minutes post-initiation of the SCW. Bar, 50 μm. (D) Examples of Rho activity associated with meiotic spindles after cytokinesis failure in Q61L Rac-expressing cells. The first panel is the same cell shown in panel (B). Bar, 50 μm.
	FIGURE 6. Model for Rac-Arp2/3 signaling during mitotic and meiotic divisions. (A) In the presence of WT Rac, the centralspindlin complex recruits Ect2 to promote equatorial Rho activity, which in turn stimulates formin-nucleated actin, myosin II activation and contractile ring assembly. Simultaneously, centralspindlin blocks Rac and Arp2/3 activity, downregulating branched actin networks at the cell equator. (B) During meiosis, Rho is activated at the vegetal pole, inducing a surface contractile wave that converges on the animal pole where the centralspindlin complex organizes the ring that completes cytokinesis and polar body extrusion. Rac (activated by an unidentified exchange factor) promotes Arp2/3-mediated branched actin that is required for proper positioning of the spindle and polar body protrusion.

References [+] :

Argiros, Centralspindlin and chromosomal passenger complex behavior during normal and Rappaport furrow specification in echinoderm embryos. 2012, Pubmed, Echinobase

	FIGURE 1. Actin dynamics in the dividing sea urchin embryo. Frames from time-lapse movies of L. variegatus embryos injected with either recombinant Lifeact-GFP (A–E, final intracellular concentration of 0.21 μM) or Lifeact-GFP and C3 Transferase (F–J, final intracellular concentration 0.27 μM). Bar, 30 μm. Time points denote minutes post-nuclear envelope breakdown (NEB). (E,J) Corresponding kymographs of sections taken through the long axis of the spindle as denoted by the rectangles in panels (A,F). Prior to nuclear envelope breakdown a bright rim of perinuclear actin is visible (A,F). Upon mitotic entry, microvilli and their rootlets shorten, and there is a gradual increase in deep cytoplasmic actin initiating at the cortex and accumulating inward up until anaphase onset (C,E,H,J). (K) Kymograph of cortical microvillar dynamics in the polar regions of control- and C3-injected embryos, taken from regions denoted by the small rectangles in Panels (B,G). (L) Quantification of cytoplasmic actin levels beginning 1 min post-NEB up until the metaphase-anaphase transition (Mean ± SEM, n = 6 cells per condition). While there was not a qualitative effect of Rho inactivation on cytoplasmic actin, there was a significant increase in cytoplasmic Lifeact fluorescence in C3-injected embryos. (M) Quantitation of the increases in cortical thickness and microvillar length that accompanies the metaphase-anaphase transition in control and C3-injected embryos (n = 6 cells per condition, whiskers denote minimum and maximum values), where a value of 1.0 represents length or thickness 1 min prior to the metaphase-anaphase transition. While there was no significant difference in the growth of microvilli, Rho inactivation blocked the thickening of actin cortex. ****p < 0.0001.
	FIGURE 2. Activated Rac suppresses actin dynamics and cytokinesis. (A) Cleavage rates for L. variegatus embryos injected with WT, Q61L or Q61L/F37A Rac mRNA (final intracellular concentration 2.5 μg/ml) and recombinant Lifeact-GFP (final intracellular concentration 0.2 μM) immediately after fertilization and cultured at 20°C until un-injected controls completed cytokinesis. Mean ± SEM for three experimental replicates, with >21 cells per experiment. *p < 0.001. (B) Furrow ingression profiles for injected cells, beginning at the initiation of furrowing (mean ± SEM, n = 5 per condition). (C) Frames from time-lapse movies of L. variegatus embryos injected with mRNA encoding WT or mutant Rac. Time points denote minutes post-NEB. Bar, 30 μm. Rectangles in panels (a,g,l) denote the region used to generate the kymographs in panels (e,k,p). Small rectangles denote regions used to generate the kymographs in panel (D). Bar, 30 μm. (D) Kymograph highlighting microvillar growth and cortical actin dynamics during mitotic exit in WT or mutant Rac-expressing cells. (E) Quantification of cytoplasmic actin levels beginning 1 min post-NEB up until the metaphase-anaphase transition (Mean ± SEM, n = 6 cells per condition). Activated Rac (Q61L, red) suppressed the increase in cytoplasmic actin in comparison to WT (gray), whereas activated Rac containing the effector-binding mutation F37A (Q61L/F37A, magenta) had no effect on cytoplasmic actin levels. (F) Quantitation of the increases in cortical thickness and microvillar length that accompanies the metaphase-anaphase transition in embryos expressing WT and mutant Rac (n = 6 cells per condition, whiskers denote minimum and maximum values), where a value of 1.0 represents length or thickness 1 min prior to the metaphase-anaphase transition. Activated Rac blocked both cortical thickening and microvillar elongation, but these actin dynamics were rescued in embryos expressing the double mutant that is incapable of promoting WAVE-mediated Arp2/3 activation. p < 0.005, ****p < 0.0001.
	FIGURE 3. Activated Rac suppresses cytokinesis through Arp2/3. (A,B) S. purpuratus embryos probed for Arp3 (top row in grayscale, red in the merged image), tubulin (cyan), and DNA (White). Bar, 30 μm. As cytokinesis progresses, Arp2/3 is increasingly excluded from the furrow region. ***p < 0.0001. (C) Arp2/3 delays but does not block cytokinesis. Embryos were treated with 0.1% DMSO or 100 μM CK-666 prior to nuclear envelope breakdown, and furrow diameter was measured by Nomarski/DIC starting at the initiation of furrowing (mean ± SEM, n = 13 for DMSO, n = 12 for CK-666). (D) Comparison of the rates of furrowing (time to 50% ingression), p < 0.05. (E) Quantification of cytoplasmic actin levels in either DMSO- or CK-666-treated embryos beginning 1 min post-NEB up until the metaphase-anaphase transition (Mean ± SEM, n = 6 cells per condition). (F) Quantitation of the increases in cortical thickness and microvillar length that accompanies the metaphase-anaphase transition in DMSO or CK-666-treated embryos expressing (n = 6 cells per condition, whiskers denote minimum and maximum values), where a value of 1.0 represents length or thickness 1 min prior to the metaphase-anaphase transition. No significant differences were detected for either actin population. (G) Arp2/3 inhibition rescues cytokinesis in embryos expressing activated Rac. Q61L Rac data replicated from Figure 2A. Mean ± SEM for three experimental replicates, >24 cells per experiment. *p < 0.001. (H) Cytoplasmic actin levels in embryos expressing Q61L Rac in the absence or presence of CK-666, beginning 1 min post-NEB up until the metaphase-anaphase transition (Mean ± SEM, n = 6 cells per condition). (I) Quantitation of the increases in cortical thickness and microvillar length that accompanies the metaphase-anaphase transition in embryos expressing Q61L Rac in the absence or presence of CK-666 (n = 6 cells per condition, whiskers denote minimum and maximum values), where a value of 1.0 represents length or thickness 1 min prior to the metaphase-anaphase transition. p = 0.005.
	FIGURE 4. Rac and Arp2/3 are required for polar body extrusion in sea star oocytes. (A) First polar body extrusion in P. miniata oocytes co-expressing Lifeact (white) and GFP-tubulin (red), rGBD-GFP (white) and mCherry-EMTB (red), and ArpC-GFP (white) and mCherry-EMTB (red). Time points indicate minutes post-hormone activation. Arrow denotes protruding polar body. Bar, 50 μm. (B) P. miniata oocytes co-expressing Lifeact (white) and GFP-tubulin (red) were activated with maturation hormone and 40 min following germinal vesicle breakdown, were treated with 0.1% DMSO or 100 μM CK-666. The spindle in the CK-666 sample is positioned in the Z axis of the image. The spindle failed to rotate or form a polar body protrusion. Bar, 50 μm (C) Quantification of polar body formation in DMSO or CK-666 samples for three experimental replicates, 100 oocytes scored per condition per experiment (Mean ± SEM, **p < 0.0001). (D) P. miniata oocytes co-expressing Lifeact (white), GFP-tubulin (red) and either wild-type (WT) or dominant-negative (T17N) Rac. In contrast to WT Rac-injected oocytes, T17N Rac-expressing oocytes failed in polar body formation after spindles failed to dock at the cortex. Bar, 50 μm. (E) Quantification of polar body formation in WT or T17N Rac-expressing oocytes for three experimental replicates, 100 oocytes scored per condition per experiment (Mean ± SEM, *p < 0.001).
	FIGURE 5. Rac and Arp2/3 directly antagonizes Rho activation during meiosis I. (A) Quantification of polar body formation in P. miniata oocytes injected WT, Q61L or Q61L/F37A Rac (final intracellular concentration 1.8 μg/ml), 200 oocytes scored per condition per experiment (Mean ± SEM, *p < 0.001; **p < 0.0001). (B) Cortical Rho activity during the surface contraction wave (SCW) in oocytes expressing WT Rac (gray), Q61L Rac (red), Q61L/F37A Rac (magenta) or Q61L Rac oocytes treated with 100 μM CK-666 (blue). Rhotekin-GFP fluorescence was measured for the entire cortex, where time 0 denotes the initiation of the SCW at the vegetal pole. Mean ± SEM, 7 oocytes per condition. (C) SCW and polar body extrusion in P. miniata oocytes co-expressing rGBD-GFP (white), mCherry-EMTB (red) and Rac variants described in panel (B). Time point indicate minutes post-initiation of the SCW. Bar, 50 μm. (D) Examples of Rho activity associated with meiotic spindles after cytokinesis failure in Q61L Rac-expressing cells. The first panel is the same cell shown in panel (B). Bar, 50 μm.
	FIGURE 6. Model for Rac-Arp2/3 signaling during mitotic and meiotic divisions. (A) In the presence of WT Rac, the centralspindlin complex recruits Ect2 to promote equatorial Rho activity, which in turn stimulates formin-nucleated actin, myosin II activation and contractile ring assembly. Simultaneously, centralspindlin blocks Rac and Arp2/3 activity, downregulating branched actin networks at the cell equator. (B) During meiosis, Rho is activated at the vegetal pole, inducing a surface contractile wave that converges on the animal pole where the centralspindlin complex organizes the ring that completes cytokinesis and polar body extrusion. Rac (activated by an unidentified exchange factor) promotes Arp2/3-mediated branched actin that is required for proper positioning of the spindle and polar body protrusion.