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The PCH family protein, Cdc15p, recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe.

Carnahan RH, Gould KL - J. Cell Biol. (2003)

Bottom Line: Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants.Cdc15p also binds directly to Cdc12p.We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

ABSTRACT
Cytokinetic actin ring (CAR) formation in Schizosaccharomyces pombe requires two independent actin nucleation pathways, one dependent on the Arp2/3 complex and another involving the formin Cdc12p. Here we investigate the role of the S. pombe Cdc15 homology family protein, Cdc15p, in CAR assembly and find that it interacts with proteins from both of these nucleation pathways. Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants. Cdc15p also binds directly to Cdc12p. Cdc15p and Cdc12p not only display mutual dependence for CAR localization, but also exist together in a ring-nucleating structure before CAR formation. The disruption of these interactions in cdc15 cells is likely to be the reason for their complete lack of CARs. We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.

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Localization of Cdc15p–GFP. (A) Representative live cell images of cdc15-GFP sid4-GFP cells (KGY3362) from various cell cycle stages. Sid4p–GFP localization was used to monitor spindle pole bodies and to indicate cell cycle stage of cells. (B) Time-lapse images of an interphase cdc15-GFP cell (KGY3019) with two nonmotile spots indicated by arrowheads. The arrow indicates the formation of a smaller patch from a larger one. Time is indicated in seconds. (C) Time-lapse images of synchronized cdc15-GFP cdc25-22 cells (KGY3042) that had been arrested for 4 h at 36°C and released to 25°C for 10 min before the beginning of the time course at the early stages of ring formation. The arrowhead indicates a branched network of fibers encircling the medial region that is the initial Cdc15p ring structure. This network then coalesced to form a thick bright ring structure. Time is indicated in minutes. Bars, 5 μm.
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fig3: Localization of Cdc15p–GFP. (A) Representative live cell images of cdc15-GFP sid4-GFP cells (KGY3362) from various cell cycle stages. Sid4p–GFP localization was used to monitor spindle pole bodies and to indicate cell cycle stage of cells. (B) Time-lapse images of an interphase cdc15-GFP cell (KGY3019) with two nonmotile spots indicated by arrowheads. The arrow indicates the formation of a smaller patch from a larger one. Time is indicated in seconds. (C) Time-lapse images of synchronized cdc15-GFP cdc25-22 cells (KGY3042) that had been arrested for 4 h at 36°C and released to 25°C for 10 min before the beginning of the time course at the early stages of ring formation. The arrowhead indicates a branched network of fibers encircling the medial region that is the initial Cdc15p ring structure. This network then coalesced to form a thick bright ring structure. Time is indicated in minutes. Bars, 5 μm.

Mentions: Given that medial mitotic recruitment of Arp2/3 regulators is dependent on Cdc15p function, we wished to determine whether loss of these proteins affected Cdc15p localization. Consistent with the nonessential nature of wsp1, vrp1, or myo1 for cytokinesis, Cdc15p–GFP localization to the CAR appeared normal in the individual deletion strains (unpublished data). The live cell imaging analysis, however, revealed a previously unappreciated interphase localization of Cdc15p to numerous spots at cell tips in wild-type and wsp1, vrp1, or myo1 deletion cells (Fig. 3 A; see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200305012/DC1). We typically observed a small number (two to five per cell) of large bright nonmotile spots as well as numerous smaller fast-moving spots (Fig. 3 B; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200305012/DC1). Small motile spots could occasionally be observed emerging from the larger static spots (Fig. 3 B). Upon entry into mitosis, Cdc15p–GFP spots moved to the medial region of the cell where they were seemingly incorporated into the forming Cdc15p ring structure (Fig. 4 A; see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200305012/DC1). The Cdc15p ring initially appeared as a network of interconnected fibers encircling the medial region (Fig. 3 C; see Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200305012/DC1) that quickly coalesced into a thicker ring structure. The Cdc15p–GFP ring then constricted as cells divided, and subsequently, Cdc15p–GFP reformed as spots and relocalized to the old cell ends. Cdc15p–GFP spots were then detected at both cell ends when cells resumed bipolar growth. Because the localization and motility of Cdc15p–GFP spots were reminiscent of actin patches, we examined whether these structures were identical. We did not find a high degree of spatial overlap between Cdc15p and Myo1p, Wsp1p, Vrp1p, or the known actin patch protein Arc15p during interphase (unpublished data), suggesting that Cdc15p spots are distinct from actin patches.


The PCH family protein, Cdc15p, recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe.

Carnahan RH, Gould KL - J. Cell Biol. (2003)

Localization of Cdc15p–GFP. (A) Representative live cell images of cdc15-GFP sid4-GFP cells (KGY3362) from various cell cycle stages. Sid4p–GFP localization was used to monitor spindle pole bodies and to indicate cell cycle stage of cells. (B) Time-lapse images of an interphase cdc15-GFP cell (KGY3019) with two nonmotile spots indicated by arrowheads. The arrow indicates the formation of a smaller patch from a larger one. Time is indicated in seconds. (C) Time-lapse images of synchronized cdc15-GFP cdc25-22 cells (KGY3042) that had been arrested for 4 h at 36°C and released to 25°C for 10 min before the beginning of the time course at the early stages of ring formation. The arrowhead indicates a branched network of fibers encircling the medial region that is the initial Cdc15p ring structure. This network then coalesced to form a thick bright ring structure. Time is indicated in minutes. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172828&req=5

fig3: Localization of Cdc15p–GFP. (A) Representative live cell images of cdc15-GFP sid4-GFP cells (KGY3362) from various cell cycle stages. Sid4p–GFP localization was used to monitor spindle pole bodies and to indicate cell cycle stage of cells. (B) Time-lapse images of an interphase cdc15-GFP cell (KGY3019) with two nonmotile spots indicated by arrowheads. The arrow indicates the formation of a smaller patch from a larger one. Time is indicated in seconds. (C) Time-lapse images of synchronized cdc15-GFP cdc25-22 cells (KGY3042) that had been arrested for 4 h at 36°C and released to 25°C for 10 min before the beginning of the time course at the early stages of ring formation. The arrowhead indicates a branched network of fibers encircling the medial region that is the initial Cdc15p ring structure. This network then coalesced to form a thick bright ring structure. Time is indicated in minutes. Bars, 5 μm.
Mentions: Given that medial mitotic recruitment of Arp2/3 regulators is dependent on Cdc15p function, we wished to determine whether loss of these proteins affected Cdc15p localization. Consistent with the nonessential nature of wsp1, vrp1, or myo1 for cytokinesis, Cdc15p–GFP localization to the CAR appeared normal in the individual deletion strains (unpublished data). The live cell imaging analysis, however, revealed a previously unappreciated interphase localization of Cdc15p to numerous spots at cell tips in wild-type and wsp1, vrp1, or myo1 deletion cells (Fig. 3 A; see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200305012/DC1). We typically observed a small number (two to five per cell) of large bright nonmotile spots as well as numerous smaller fast-moving spots (Fig. 3 B; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200305012/DC1). Small motile spots could occasionally be observed emerging from the larger static spots (Fig. 3 B). Upon entry into mitosis, Cdc15p–GFP spots moved to the medial region of the cell where they were seemingly incorporated into the forming Cdc15p ring structure (Fig. 4 A; see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200305012/DC1). The Cdc15p ring initially appeared as a network of interconnected fibers encircling the medial region (Fig. 3 C; see Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200305012/DC1) that quickly coalesced into a thicker ring structure. The Cdc15p–GFP ring then constricted as cells divided, and subsequently, Cdc15p–GFP reformed as spots and relocalized to the old cell ends. Cdc15p–GFP spots were then detected at both cell ends when cells resumed bipolar growth. Because the localization and motility of Cdc15p–GFP spots were reminiscent of actin patches, we examined whether these structures were identical. We did not find a high degree of spatial overlap between Cdc15p and Myo1p, Wsp1p, Vrp1p, or the known actin patch protein Arc15p during interphase (unpublished data), suggesting that Cdc15p spots are distinct from actin patches.

Bottom Line: Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants.Cdc15p also binds directly to Cdc12p.We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

ABSTRACT
Cytokinetic actin ring (CAR) formation in Schizosaccharomyces pombe requires two independent actin nucleation pathways, one dependent on the Arp2/3 complex and another involving the formin Cdc12p. Here we investigate the role of the S. pombe Cdc15 homology family protein, Cdc15p, in CAR assembly and find that it interacts with proteins from both of these nucleation pathways. Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants. Cdc15p also binds directly to Cdc12p. Cdc15p and Cdc12p not only display mutual dependence for CAR localization, but also exist together in a ring-nucleating structure before CAR formation. The disruption of these interactions in cdc15 cells is likely to be the reason for their complete lack of CARs. We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.

Show MeSH
Related in: MedlinePlus