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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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Dynamic redistribution of ARP3 and PIN1 during axial division of BY-2 cells. Dual visualization of actin microfilaments by GFP–FABD2 and RFP–ARP3 (A, D, G, J, M), and the distribution of ARP3 (B, E, H, K, N) and PIN1 (C, F, I, L, O) is shown for the symmetric unicellular (A–C), the asymmetric unicellular (D–F), the bicellular (G–I), the tricellular (J–L), and the quadricellular (M–O) state. Bars=20 μm. The arrows point to the increased density of the ARP3 signal in the distal halves of terminal cells (H, K, N). The asterisks label the ‘younger’ terminal cell of tricellular files (K, L). The white lines (O) point at the sites of beginning disintegration of the file of beginning disintegration of the file in smaller subsets (bicellular files).
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fig6: Dynamic redistribution of ARP3 and PIN1 during axial division of BY-2 cells. Dual visualization of actin microfilaments by GFP–FABD2 and RFP–ARP3 (A, D, G, J, M), and the distribution of ARP3 (B, E, H, K, N) and PIN1 (C, F, I, L, O) is shown for the symmetric unicellular (A–C), the asymmetric unicellular (D–F), the bicellular (G–I), the tricellular (J–L), and the quadricellular (M–O) state. Bars=20 μm. The arrows point to the increased density of the ARP3 signal in the distal halves of terminal cells (H, K, N). The asterisks label the ‘younger’ terminal cell of tricellular files (K, L). The white lines (O) point at the sites of beginning disintegration of the file of beginning disintegration of the file in smaller subsets (bicellular files).

Mentions: To test for possible correlations between ARP3 (as a marker for actin nucleation) and PIN1 (as a marker for cell polarity) during divisional patterning (Fig. 6), the localization of both markers was followed through the formation of the axial, pluricellular files in BY-2 cells. ARP3 was again visualized by an RFP fusion transiently expressed in a GFP–FABD2 background. PIN1 (from A. thaliana) was visualized as a fusion with GFP under control of the homologous PIN1 promoter.


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

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

Dynamic redistribution of ARP3 and PIN1 during axial division of BY-2 cells. Dual visualization of actin microfilaments by GFP–FABD2 and RFP–ARP3 (A, D, G, J, M), and the distribution of ARP3 (B, E, H, K, N) and PIN1 (C, F, I, L, O) is shown for the symmetric unicellular (A–C), the asymmetric unicellular (D–F), the bicellular (G–I), the tricellular (J–L), and the quadricellular (M–O) state. Bars=20 μm. The arrows point to the increased density of the ARP3 signal in the distal halves of terminal cells (H, K, N). The asterisks label the ‘younger’ terminal cell of tricellular files (K, L). The white lines (O) point at the sites of beginning disintegration of the file of beginning disintegration of the file in smaller subsets (bicellular files).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Dynamic redistribution of ARP3 and PIN1 during axial division of BY-2 cells. Dual visualization of actin microfilaments by GFP–FABD2 and RFP–ARP3 (A, D, G, J, M), and the distribution of ARP3 (B, E, H, K, N) and PIN1 (C, F, I, L, O) is shown for the symmetric unicellular (A–C), the asymmetric unicellular (D–F), the bicellular (G–I), the tricellular (J–L), and the quadricellular (M–O) state. Bars=20 μm. The arrows point to the increased density of the ARP3 signal in the distal halves of terminal cells (H, K, N). The asterisks label the ‘younger’ terminal cell of tricellular files (K, L). The white lines (O) point at the sites of beginning disintegration of the file of beginning disintegration of the file in smaller subsets (bicellular files).
Mentions: To test for possible correlations between ARP3 (as a marker for actin nucleation) and PIN1 (as a marker for cell polarity) during divisional patterning (Fig. 6), the localization of both markers was followed through the formation of the axial, pluricellular files in BY-2 cells. ARP3 was again visualized by an RFP fusion transiently expressed in a GFP–FABD2 background. PIN1 (from A. thaliana) was visualized as a fusion with GFP under control of the homologous PIN1 promoter.

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