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Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes.

Fu Y, Wu G, Yang Z - J. Cell Biol. (2001)

Bottom Line: Rop GTPase-activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes.Finally, SAB oscillates and appears at the tip before growth.Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany and Plant Sciences, University of California, Riverside, California 92521, USA.

ABSTRACT
Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein-tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase-activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.

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GDI and RopGAP1 suppress transverse actin band formation induced by Rop1At or DN-rop1At overexpression. Coexpression of GFP-mTalin with one or more constructs was conducted, and GFP images were captured and analyzed as described in the legend to Fig. 3. (A) A control tube shows SAB at the tip. (B) A Rop1At-overexpressing tube shows a network of apical fine actin and subapical transverse actin band. (C) A tube overexpressing DN-rop1At shows clear apex and subapical transverse actin band. (D) No transverse actin band is observed in a tube cooverexpressing Rop1At with GDI, but extensive SABs are seen. The tube is less expanded than tubes overexpressing Rop1At alone. (E) Neither transverse actin band nor SAB is found in a tube cooverexpressing DN-rop1At and GDI. This tube is less expanded than the one overexpressing DN-rop1At alone. (F) No SABs are observed in tubes overexpressing RopGAP1. (G) A tube cooverexpressing Rop1At and RopGAP1. No transverse actin band is found, but a subapical actin collar (arrowhead) and weak apical SAB are recovered. (H) Frequency of transverse actin bands in tubes overexpressing DN-rop1At or Rop1At alone and in tubes cooverexpressing one of these genes with GDI. (I) Rop1At-induced transverse actin band is suppressed by RopGAP1 in a dose-dependent manner. 800 ng of LAT52::Rop1At plasmid DNA was mixed with indicated ratios of LAT52:RopGAP1 plasmid DNA and used for cobombardment with LAT52::GFP-mTalin. Arrowhead, SAB or actin network at the tip; arrow, actin collar or transverse actin band behind the apex; asterisk, F-actin–free apex; CK, tubes expressing GFP-mTalin alone. Bar, 10 μm.
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Figure 4: GDI and RopGAP1 suppress transverse actin band formation induced by Rop1At or DN-rop1At overexpression. Coexpression of GFP-mTalin with one or more constructs was conducted, and GFP images were captured and analyzed as described in the legend to Fig. 3. (A) A control tube shows SAB at the tip. (B) A Rop1At-overexpressing tube shows a network of apical fine actin and subapical transverse actin band. (C) A tube overexpressing DN-rop1At shows clear apex and subapical transverse actin band. (D) No transverse actin band is observed in a tube cooverexpressing Rop1At with GDI, but extensive SABs are seen. The tube is less expanded than tubes overexpressing Rop1At alone. (E) Neither transverse actin band nor SAB is found in a tube cooverexpressing DN-rop1At and GDI. This tube is less expanded than the one overexpressing DN-rop1At alone. (F) No SABs are observed in tubes overexpressing RopGAP1. (G) A tube cooverexpressing Rop1At and RopGAP1. No transverse actin band is found, but a subapical actin collar (arrowhead) and weak apical SAB are recovered. (H) Frequency of transverse actin bands in tubes overexpressing DN-rop1At or Rop1At alone and in tubes cooverexpressing one of these genes with GDI. (I) Rop1At-induced transverse actin band is suppressed by RopGAP1 in a dose-dependent manner. 800 ng of LAT52::Rop1At plasmid DNA was mixed with indicated ratios of LAT52:RopGAP1 plasmid DNA and used for cobombardment with LAT52::GFP-mTalin. Arrowhead, SAB or actin network at the tip; arrow, actin collar or transverse actin band behind the apex; asterisk, F-actin–free apex; CK, tubes expressing GFP-mTalin alone. Bar, 10 μm.

Mentions: To test the hypothesis that the transverse actin band is caused by ectopically localized Rop1At, we investigated the effect of guanine nucleotide dissociation inhibitor (GDI) overexpression on actin organization. In mammalian cells, GDIs act to recycle Rho GTPases from PM, and thus we suspect that Arabidopsis GDIs may have a similar role in pollen tubes. We found that overexpression of an Arabidopsis GDI in tobacco pollen tubes indeed suppressed transverse actin band formation (Fig. 4, B–E). As shown in Fig. 4 H, the transverse actin band was observed in 65% of tubes overexpressing Rop1At alone, but only in 20% of tubes cooverexpressing Rop1At and GDI. Similarly, 59% of tubes overexpressing DN-rop1At had a transverse actin band, whereas cooverexpression with GDI reduced the transverse actin band–containing tubes to 3.7% (Fig. 4 H). The GDI suppression of Rop1At-induced depolarized growth was tightly correlated with that of the transverse actin band (i.e., none of the tubes devoid of the transverse actin band had swollen tips or expanded tubes). Interestingly, a meshwork composed of SABs was observed in tubes cooverexpressing Rop1At and GDI, whereas tubes cooverexpressing DN-rop1At and GDI had an actin-free apex, as in tubes overexpressing DN-rop1At alone. Hence, these results not only provide evidence that ectopically localized Rop is responsible for the formation of abnormal actin bands behind the apex, but also suggest that Rop1At is involved in the regulation of SABs found in the extreme apex.


Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes.

Fu Y, Wu G, Yang Z - J. Cell Biol. (2001)

GDI and RopGAP1 suppress transverse actin band formation induced by Rop1At or DN-rop1At overexpression. Coexpression of GFP-mTalin with one or more constructs was conducted, and GFP images were captured and analyzed as described in the legend to Fig. 3. (A) A control tube shows SAB at the tip. (B) A Rop1At-overexpressing tube shows a network of apical fine actin and subapical transverse actin band. (C) A tube overexpressing DN-rop1At shows clear apex and subapical transverse actin band. (D) No transverse actin band is observed in a tube cooverexpressing Rop1At with GDI, but extensive SABs are seen. The tube is less expanded than tubes overexpressing Rop1At alone. (E) Neither transverse actin band nor SAB is found in a tube cooverexpressing DN-rop1At and GDI. This tube is less expanded than the one overexpressing DN-rop1At alone. (F) No SABs are observed in tubes overexpressing RopGAP1. (G) A tube cooverexpressing Rop1At and RopGAP1. No transverse actin band is found, but a subapical actin collar (arrowhead) and weak apical SAB are recovered. (H) Frequency of transverse actin bands in tubes overexpressing DN-rop1At or Rop1At alone and in tubes cooverexpressing one of these genes with GDI. (I) Rop1At-induced transverse actin band is suppressed by RopGAP1 in a dose-dependent manner. 800 ng of LAT52::Rop1At plasmid DNA was mixed with indicated ratios of LAT52:RopGAP1 plasmid DNA and used for cobombardment with LAT52::GFP-mTalin. Arrowhead, SAB or actin network at the tip; arrow, actin collar or transverse actin band behind the apex; asterisk, F-actin–free apex; CK, tubes expressing GFP-mTalin alone. Bar, 10 μm.
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Figure 4: GDI and RopGAP1 suppress transverse actin band formation induced by Rop1At or DN-rop1At overexpression. Coexpression of GFP-mTalin with one or more constructs was conducted, and GFP images were captured and analyzed as described in the legend to Fig. 3. (A) A control tube shows SAB at the tip. (B) A Rop1At-overexpressing tube shows a network of apical fine actin and subapical transverse actin band. (C) A tube overexpressing DN-rop1At shows clear apex and subapical transverse actin band. (D) No transverse actin band is observed in a tube cooverexpressing Rop1At with GDI, but extensive SABs are seen. The tube is less expanded than tubes overexpressing Rop1At alone. (E) Neither transverse actin band nor SAB is found in a tube cooverexpressing DN-rop1At and GDI. This tube is less expanded than the one overexpressing DN-rop1At alone. (F) No SABs are observed in tubes overexpressing RopGAP1. (G) A tube cooverexpressing Rop1At and RopGAP1. No transverse actin band is found, but a subapical actin collar (arrowhead) and weak apical SAB are recovered. (H) Frequency of transverse actin bands in tubes overexpressing DN-rop1At or Rop1At alone and in tubes cooverexpressing one of these genes with GDI. (I) Rop1At-induced transverse actin band is suppressed by RopGAP1 in a dose-dependent manner. 800 ng of LAT52::Rop1At plasmid DNA was mixed with indicated ratios of LAT52:RopGAP1 plasmid DNA and used for cobombardment with LAT52::GFP-mTalin. Arrowhead, SAB or actin network at the tip; arrow, actin collar or transverse actin band behind the apex; asterisk, F-actin–free apex; CK, tubes expressing GFP-mTalin alone. Bar, 10 μm.
Mentions: To test the hypothesis that the transverse actin band is caused by ectopically localized Rop1At, we investigated the effect of guanine nucleotide dissociation inhibitor (GDI) overexpression on actin organization. In mammalian cells, GDIs act to recycle Rho GTPases from PM, and thus we suspect that Arabidopsis GDIs may have a similar role in pollen tubes. We found that overexpression of an Arabidopsis GDI in tobacco pollen tubes indeed suppressed transverse actin band formation (Fig. 4, B–E). As shown in Fig. 4 H, the transverse actin band was observed in 65% of tubes overexpressing Rop1At alone, but only in 20% of tubes cooverexpressing Rop1At and GDI. Similarly, 59% of tubes overexpressing DN-rop1At had a transverse actin band, whereas cooverexpression with GDI reduced the transverse actin band–containing tubes to 3.7% (Fig. 4 H). The GDI suppression of Rop1At-induced depolarized growth was tightly correlated with that of the transverse actin band (i.e., none of the tubes devoid of the transverse actin band had swollen tips or expanded tubes). Interestingly, a meshwork composed of SABs was observed in tubes cooverexpressing Rop1At and GDI, whereas tubes cooverexpressing DN-rop1At and GDI had an actin-free apex, as in tubes overexpressing DN-rop1At alone. Hence, these results not only provide evidence that ectopically localized Rop is responsible for the formation of abnormal actin bands behind the apex, but also suggest that Rop1At is involved in the regulation of SABs found in the extreme apex.

Bottom Line: Rop GTPase-activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes.Finally, SAB oscillates and appears at the tip before growth.Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany and Plant Sciences, University of California, Riverside, California 92521, USA.

ABSTRACT
Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein-tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase-activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.

Show MeSH
Related in: MedlinePlus