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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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Actin filaments elongate rapidly. (A) An actin filament is tracked during successive images collected at ∼3-s intervals. It undergoes a breakage event at t = 90 s (arrow) and the newly created filament end (open arrowhead) remains stationary or shrinks slightly during the next 30 s. At t = 116 s, the filament end (open arrowhead) begins to grow and extends out of the field of view at t = 129 s. The average rate of growth for this filament, determined as shown in D, was 0.97 µm/s. A second filament, first appearing at t = 116 s, grows upward and toward the right at a rate of 1.7 µm/s. See Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). Bar, 2 µm. (B) Kymograph of the filament highlighted in A. The filament origin or base is at the right and the elongation extends toward the left. (C) Plot of pixel intensity as a function of position along the filament shown in A. The traces show two different time points: gray line, 100 s; black line, 132 s. Filament origin is plotted at the right and growth occurs toward the left. A black arrow marks the location where this filament crosses over another filament (see also Fig. 2 A; 132 s), resulting in a doubling of fluorescence intensity. The solid black line at 2000 represents the upper limit of pixel intensity for an individual actin filament, as determined with the analyses shown in Fig. S3. (D) Actin filament length as a function of time for several representative filaments is plotted and a line of best fit used to estimate rate of elongation. Closed squares, filament marked with open arrowheads in A; open circles, second filament growing upward and toward right in A; closed circles, example filament from a different time-lapse series.
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fig2: Actin filaments elongate rapidly. (A) An actin filament is tracked during successive images collected at ∼3-s intervals. It undergoes a breakage event at t = 90 s (arrow) and the newly created filament end (open arrowhead) remains stationary or shrinks slightly during the next 30 s. At t = 116 s, the filament end (open arrowhead) begins to grow and extends out of the field of view at t = 129 s. The average rate of growth for this filament, determined as shown in D, was 0.97 µm/s. A second filament, first appearing at t = 116 s, grows upward and toward the right at a rate of 1.7 µm/s. See Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). Bar, 2 µm. (B) Kymograph of the filament highlighted in A. The filament origin or base is at the right and the elongation extends toward the left. (C) Plot of pixel intensity as a function of position along the filament shown in A. The traces show two different time points: gray line, 100 s; black line, 132 s. Filament origin is plotted at the right and growth occurs toward the left. A black arrow marks the location where this filament crosses over another filament (see also Fig. 2 A; 132 s), resulting in a doubling of fluorescence intensity. The solid black line at 2000 represents the upper limit of pixel intensity for an individual actin filament, as determined with the analyses shown in Fig. S3. (D) Actin filament length as a function of time for several representative filaments is plotted and a line of best fit used to estimate rate of elongation. Closed squares, filament marked with open arrowheads in A; open circles, second filament growing upward and toward right in A; closed circles, example filament from a different time-lapse series.

Mentions: To correlate the nature of an actin-based structure with its average pixel intensity, we computer-generated multiple lines that scanned the entire surface of the observed field of view throughout a full time-lapse series (e.g., Fig. 1). After background subtraction, we were able to generate the full range of pixel intensities corresponding to different populations of cortical actin-based structures for growing (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1) and nongrowing cells (Fig. S3 B). The majority of pixels in the field of view had intensities below 1800 arbitrary units (a.u.). A direct comparison of these values with those generated by individual, hand-selection revealed that the faintest actin filaments had average pixel intensities of ∼1000 a.u., but the intensity varied substantially along their length (Fig. S3 C). As shown in a representative example of filament growth after severing, the newly elongated region has an average intensity similar to the mother filament, but varies by more than 500 a.u. along its length (Fig. 2 C). Importantly, where two actin filaments crossed, the pixel intensity nearly doubled (Fig. 2 C, arrow).


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)

Actin filaments elongate rapidly. (A) An actin filament is tracked during successive images collected at ∼3-s intervals. It undergoes a breakage event at t = 90 s (arrow) and the newly created filament end (open arrowhead) remains stationary or shrinks slightly during the next 30 s. At t = 116 s, the filament end (open arrowhead) begins to grow and extends out of the field of view at t = 129 s. The average rate of growth for this filament, determined as shown in D, was 0.97 µm/s. A second filament, first appearing at t = 116 s, grows upward and toward the right at a rate of 1.7 µm/s. See Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). Bar, 2 µm. (B) Kymograph of the filament highlighted in A. The filament origin or base is at the right and the elongation extends toward the left. (C) Plot of pixel intensity as a function of position along the filament shown in A. The traces show two different time points: gray line, 100 s; black line, 132 s. Filament origin is plotted at the right and growth occurs toward the left. A black arrow marks the location where this filament crosses over another filament (see also Fig. 2 A; 132 s), resulting in a doubling of fluorescence intensity. The solid black line at 2000 represents the upper limit of pixel intensity for an individual actin filament, as determined with the analyses shown in Fig. S3. (D) Actin filament length as a function of time for several representative filaments is plotted and a line of best fit used to estimate rate of elongation. Closed squares, filament marked with open arrowheads in A; open circles, second filament growing upward and toward right in A; closed circles, example filament from a different time-lapse series.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Actin filaments elongate rapidly. (A) An actin filament is tracked during successive images collected at ∼3-s intervals. It undergoes a breakage event at t = 90 s (arrow) and the newly created filament end (open arrowhead) remains stationary or shrinks slightly during the next 30 s. At t = 116 s, the filament end (open arrowhead) begins to grow and extends out of the field of view at t = 129 s. The average rate of growth for this filament, determined as shown in D, was 0.97 µm/s. A second filament, first appearing at t = 116 s, grows upward and toward the right at a rate of 1.7 µm/s. See Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1). Bar, 2 µm. (B) Kymograph of the filament highlighted in A. The filament origin or base is at the right and the elongation extends toward the left. (C) Plot of pixel intensity as a function of position along the filament shown in A. The traces show two different time points: gray line, 100 s; black line, 132 s. Filament origin is plotted at the right and growth occurs toward the left. A black arrow marks the location where this filament crosses over another filament (see also Fig. 2 A; 132 s), resulting in a doubling of fluorescence intensity. The solid black line at 2000 represents the upper limit of pixel intensity for an individual actin filament, as determined with the analyses shown in Fig. S3. (D) Actin filament length as a function of time for several representative filaments is plotted and a line of best fit used to estimate rate of elongation. Closed squares, filament marked with open arrowheads in A; open circles, second filament growing upward and toward right in A; closed circles, example filament from a different time-lapse series.
Mentions: To correlate the nature of an actin-based structure with its average pixel intensity, we computer-generated multiple lines that scanned the entire surface of the observed field of view throughout a full time-lapse series (e.g., Fig. 1). After background subtraction, we were able to generate the full range of pixel intensities corresponding to different populations of cortical actin-based structures for growing (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200806185/DC1) and nongrowing cells (Fig. S3 B). The majority of pixels in the field of view had intensities below 1800 arbitrary units (a.u.). A direct comparison of these values with those generated by individual, hand-selection revealed that the faintest actin filaments had average pixel intensities of ∼1000 a.u., but the intensity varied substantially along their length (Fig. S3 C). As shown in a representative example of filament growth after severing, the newly elongated region has an average intensity similar to the mother filament, but varies by more than 500 a.u. along its length (Fig. 2 C). Importantly, where two actin filaments crossed, the pixel intensity nearly doubled (Fig. 2 C, arrow).

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