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Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array.

Staiger CJ, Sheahan MB, Khurana P, Wang X, McCurdy DW, Blanchoin L - J. Cell Biol. (2009)

Bottom Line: Remodeling of the cortical actin array also features filament buckling and straightening events.These observations indicate a mechanism inconsistent with treadmilling.Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hansen Life Sciences Research Building, Purdue University, West Lafayette, IN 47907, USA.

ABSTRACT
Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskeleton in growing and nongrowing epidermal cells. One population of filaments in the cortical array, which most likely represent single actin filaments, is randomly oriented and highly dynamic. These filaments grow at rates of 1.7 microm/s, but are generally short-lived. Instead of depolymerization at their ends, actin filaments are disassembled by severing activity. Remodeling of the cortical actin array also features filament buckling and straightening events. These observations indicate a mechanism inconsistent with treadmilling. Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly.

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Latrunculin treatment reduces the filament elongation rate in a dose-dependent manner. (A) Representative cell treated with 1 µM LatB has numerous, short filaments or fragments as well as several large cables. Actin filaments or fragments grow rather slowly, if at all. Time points indicate elapsed time from start of video sequence. Bar, 2 µm. See Video 9 for full time-lapse series (available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). (B) Kymograph of the single filament marked in A. This short filament undergoes slow elongation and rapid shrinkage events. (C) Plot of average rate (±SD) of filament elongation in control and LatB-treated cells. Treatments with 100 nM and 1 µM LatB for 1–10 min significantly reduce the rate of filament elongation (P < 0.0001).
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fig5: Latrunculin treatment reduces the filament elongation rate in a dose-dependent manner. (A) Representative cell treated with 1 µM LatB has numerous, short filaments or fragments as well as several large cables. Actin filaments or fragments grow rather slowly, if at all. Time points indicate elapsed time from start of video sequence. Bar, 2 µm. See Video 9 for full time-lapse series (available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). (B) Kymograph of the single filament marked in A. This short filament undergoes slow elongation and rapid shrinkage events. (C) Plot of average rate (±SD) of filament elongation in control and LatB-treated cells. Treatments with 100 nM and 1 µM LatB for 1–10 min significantly reduce the rate of filament elongation (P < 0.0001).

Mentions: Latrunculin B (LatB) binds tightly to plant monomeric actin (Kd = 74 nM) and reduces polymeric actin levels in live cells (Gibbon et al., 1999). Here, we used LatB to test whether filament growth rates were dependent on the available monomer concentration. Growing epidermal cells were treated with either 100-nm or 1-µM LatB and imaged by VAEM within 2–10 min. After several minutes, 1 µM LatB reduced the dynamics and overall length of actin filaments (Fig. 5 A; Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). Individual actin filaments became short fragments with reduced stochastic dynamics (Fig. 5 B). Filament growth and severing still occurred; however, the elongation rate was just 0.15 ± 0.2 µm/s (n = 47; Fig. 5 C). Lower concentrations of LatB (100 nM) still had significant effects on stochastic dynamics (Table II). Elongation rates in the presence of 100 nM LatB were 0.6 ± 0.2 µm/s (n = 51), nearly threefold less than in control cells (Table II; Fig. 5 C). Surprisingly, severing frequency was slightly elevated in the presence of 100 nM LatB (Table II). The combination of lower growth rates and constant severing activity resulted in a net reduction of average filament length. In control cells, the maximum length of growing filaments was 14.8 ± 6.4 µm (n = 78), whereas this value was reduced to 4.4 ± 1.6 µm (n = 57) and 2.4 ± 1.5 µm (n = 49) in cells treated with 100-nM or 1-µM LatB, respectively.


Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array.

Staiger CJ, Sheahan MB, Khurana P, Wang X, McCurdy DW, Blanchoin L - J. Cell Biol. (2009)

Latrunculin treatment reduces the filament elongation rate in a dose-dependent manner. (A) Representative cell treated with 1 µM LatB has numerous, short filaments or fragments as well as several large cables. Actin filaments or fragments grow rather slowly, if at all. Time points indicate elapsed time from start of video sequence. Bar, 2 µm. See Video 9 for full time-lapse series (available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). (B) Kymograph of the single filament marked in A. This short filament undergoes slow elongation and rapid shrinkage events. (C) Plot of average rate (±SD) of filament elongation in control and LatB-treated cells. Treatments with 100 nM and 1 µM LatB for 1–10 min significantly reduce the rate of filament elongation (P < 0.0001).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654301&req=5

fig5: Latrunculin treatment reduces the filament elongation rate in a dose-dependent manner. (A) Representative cell treated with 1 µM LatB has numerous, short filaments or fragments as well as several large cables. Actin filaments or fragments grow rather slowly, if at all. Time points indicate elapsed time from start of video sequence. Bar, 2 µm. See Video 9 for full time-lapse series (available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). (B) Kymograph of the single filament marked in A. This short filament undergoes slow elongation and rapid shrinkage events. (C) Plot of average rate (±SD) of filament elongation in control and LatB-treated cells. Treatments with 100 nM and 1 µM LatB for 1–10 min significantly reduce the rate of filament elongation (P < 0.0001).
Mentions: Latrunculin B (LatB) binds tightly to plant monomeric actin (Kd = 74 nM) and reduces polymeric actin levels in live cells (Gibbon et al., 1999). Here, we used LatB to test whether filament growth rates were dependent on the available monomer concentration. Growing epidermal cells were treated with either 100-nm or 1-µM LatB and imaged by VAEM within 2–10 min. After several minutes, 1 µM LatB reduced the dynamics and overall length of actin filaments (Fig. 5 A; Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). Individual actin filaments became short fragments with reduced stochastic dynamics (Fig. 5 B). Filament growth and severing still occurred; however, the elongation rate was just 0.15 ± 0.2 µm/s (n = 47; Fig. 5 C). Lower concentrations of LatB (100 nM) still had significant effects on stochastic dynamics (Table II). Elongation rates in the presence of 100 nM LatB were 0.6 ± 0.2 µm/s (n = 51), nearly threefold less than in control cells (Table II; Fig. 5 C). Surprisingly, severing frequency was slightly elevated in the presence of 100 nM LatB (Table II). The combination of lower growth rates and constant severing activity resulted in a net reduction of average filament length. In control cells, the maximum length of growing filaments was 14.8 ± 6.4 µm (n = 78), whereas this value was reduced to 4.4 ± 1.6 µm (n = 57) and 2.4 ± 1.5 µm (n = 49) in cells treated with 100-nM or 1-µM LatB, respectively.

Bottom Line: Remodeling of the cortical actin array also features filament buckling and straightening events.These observations indicate a mechanism inconsistent with treadmilling.Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hansen Life Sciences Research Building, Purdue University, West Lafayette, IN 47907, USA.

ABSTRACT
Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskeleton in growing and nongrowing epidermal cells. One population of filaments in the cortical array, which most likely represent single actin filaments, is randomly oriented and highly dynamic. These filaments grow at rates of 1.7 microm/s, but are generally short-lived. Instead of depolymerization at their ends, actin filaments are disassembled by severing activity. Remodeling of the cortical actin array also features filament buckling and straightening events. These observations indicate a mechanism inconsistent with treadmilling. Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly.

Show MeSH
Related in: MedlinePlus