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Tobacco Arp3 is localized to actin-nucleating sites in vivo.

Maisch J, Fiserová J, Fischer L, Nick P - J. Exp. Bot. (2009)

Bottom Line: These dots could be shown to decorate actin filaments by expressing RFP-ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)-FABD (fimbrin actin-binding domain 2).The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP-ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file.These findings are interpreted in terms of position-dependent differences of actin organization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Botany 1, University of Karlsruhe, Kaiserstrasse 2, D-76128 Karlsruhe, Germany. jan.maisch@bio.uni-karlsruhe.de

ABSTRACT
The polarity of actin is a central determinant of intracellular transport in plant cells. To visualize actin polarity in living plant cells, the tobacco homologue of the actin-related protein 3 (ARP3) was cloned and a fusion with the red fluorescent protein (RFP) was generated. Upon transient expression of these fusions in the tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2), punctate structures were observed near the nuclear envelope and in the cortical plasma. These dots could be shown to decorate actin filaments by expressing RFP-ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)-FABD (fimbrin actin-binding domain 2). When actin filaments were disrupted by latrunculin B or by prolonged cold treatment, and subsequently allowed to recover, the actin filaments reformed from the RFP-ARP3 structures, that therefore represented actin nucleation sites. The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP-ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file. These findings are interpreted in terms of position-dependent differences of actin organization.

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Transiently expressed RFP–ARP3 decorates actin filaments in tobacco BY-2 cells that stably express GFP–FABD2. (A–D) Co-localization of RFP–ARP3 and GFP–FABD2 in a focal section of the cortical region. (E–H) Co-localization of RFP–ARP3 and GFP–FABD2 in a focal section of the perinuclear region with cytopasmic strands. (I–L) Localization of RFP–ARP3 during the recovery of actin filaments after latrunculin B treatment (500 nM, 10 h). Details boxed in A, E, and I are shown in B, F, and J for the GFP–FABD2 fluorescence, in C, G, and K for the RFP–ARP3 fluorescence, and in D, H, and L for dual fluorescence, respectively. The arrow in E highlights, as an example, a site of actin filament branching. Bars=20 μm.
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fig3: Transiently expressed RFP–ARP3 decorates actin filaments in tobacco BY-2 cells that stably express GFP–FABD2. (A–D) Co-localization of RFP–ARP3 and GFP–FABD2 in a focal section of the cortical region. (E–H) Co-localization of RFP–ARP3 and GFP–FABD2 in a focal section of the perinuclear region with cytopasmic strands. (I–L) Localization of RFP–ARP3 during the recovery of actin filaments after latrunculin B treatment (500 nM, 10 h). Details boxed in A, E, and I are shown in B, F, and J for the GFP–FABD2 fluorescence, in C, G, and K for the RFP–ARP3 fluorescence, and in D, H, and L for dual fluorescence, respectively. The arrow in E highlights, as an example, a site of actin filament branching. Bars=20 μm.

Mentions: The localization of RFP-labelled ARP3 proteins was analysed upon particle bombardment with this construct into BY-2 GFP-FABD2 cells using projections of serial optical sections. The ARP3 signals appeared as distinct dots and clearly decorated all arrays of the actin cytoskeleton, namely cortical microfilaments, the perinuclear network, and the transvacuolar strands (Fig. 3). The dots were also frequently found at points of actin filament branching (for instance Fig. 3E, white arrow).


Tobacco Arp3 is localized to actin-nucleating sites in vivo.

Maisch J, Fiserová J, Fischer L, Nick P - J. Exp. Bot. (2009)

Transiently expressed RFP–ARP3 decorates actin filaments in tobacco BY-2 cells that stably express GFP–FABD2. (A–D) Co-localization of RFP–ARP3 and GFP–FABD2 in a focal section of the cortical region. (E–H) Co-localization of RFP–ARP3 and GFP–FABD2 in a focal section of the perinuclear region with cytopasmic strands. (I–L) Localization of RFP–ARP3 during the recovery of actin filaments after latrunculin B treatment (500 nM, 10 h). Details boxed in A, E, and I are shown in B, F, and J for the GFP–FABD2 fluorescence, in C, G, and K for the RFP–ARP3 fluorescence, and in D, H, and L for dual fluorescence, respectively. The arrow in E highlights, as an example, a site of actin filament branching. Bars=20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2651461&req=5

fig3: Transiently expressed RFP–ARP3 decorates actin filaments in tobacco BY-2 cells that stably express GFP–FABD2. (A–D) Co-localization of RFP–ARP3 and GFP–FABD2 in a focal section of the cortical region. (E–H) Co-localization of RFP–ARP3 and GFP–FABD2 in a focal section of the perinuclear region with cytopasmic strands. (I–L) Localization of RFP–ARP3 during the recovery of actin filaments after latrunculin B treatment (500 nM, 10 h). Details boxed in A, E, and I are shown in B, F, and J for the GFP–FABD2 fluorescence, in C, G, and K for the RFP–ARP3 fluorescence, and in D, H, and L for dual fluorescence, respectively. The arrow in E highlights, as an example, a site of actin filament branching. Bars=20 μm.
Mentions: The localization of RFP-labelled ARP3 proteins was analysed upon particle bombardment with this construct into BY-2 GFP-FABD2 cells using projections of serial optical sections. The ARP3 signals appeared as distinct dots and clearly decorated all arrays of the actin cytoskeleton, namely cortical microfilaments, the perinuclear network, and the transvacuolar strands (Fig. 3). The dots were also frequently found at points of actin filament branching (for instance Fig. 3E, white arrow).

Bottom Line: These dots could be shown to decorate actin filaments by expressing RFP-ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)-FABD (fimbrin actin-binding domain 2).The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP-ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file.These findings are interpreted in terms of position-dependent differences of actin organization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Botany 1, University of Karlsruhe, Kaiserstrasse 2, D-76128 Karlsruhe, Germany. jan.maisch@bio.uni-karlsruhe.de

ABSTRACT
The polarity of actin is a central determinant of intracellular transport in plant cells. To visualize actin polarity in living plant cells, the tobacco homologue of the actin-related protein 3 (ARP3) was cloned and a fusion with the red fluorescent protein (RFP) was generated. Upon transient expression of these fusions in the tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2), punctate structures were observed near the nuclear envelope and in the cortical plasma. These dots could be shown to decorate actin filaments by expressing RFP-ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)-FABD (fimbrin actin-binding domain 2). When actin filaments were disrupted by latrunculin B or by prolonged cold treatment, and subsequently allowed to recover, the actin filaments reformed from the RFP-ARP3 structures, that therefore represented actin nucleation sites. The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP-ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file. These findings are interpreted in terms of position-dependent differences of actin organization.

Show MeSH
Related in: MedlinePlus