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. 2011 Jun;23(6):2302-13.
doi: 10.1105/tpc.111.087940. Epub 2011 Jun 21.

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis

Arun Sampathkumar  1 , Jelmer J Lindeboom, Seth Debolt, Ryan Gutierrez, David W Ehrhardt, Tijs Ketelaar, Staffan Persson
Affiliations

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis

Arun Sampathkumar et al. Plant Cell. 2011 Jun.

Abstract

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.

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Figures

Figure 1.
Figure 1.
Jasplakinolide Affects MT Orientation through Stabilization of the Actin Cytoskeleton. (A) and (B) Organization of the actin cytoskeleton in mock-treated (A) and jasplakinolide-treated (B) hypocotyl cells of GFP:FABD-expressing 5-d-old etiolated seedlings. (C) and (D) MT organization in control (C) and jasplakinolide-treated (5 μM for 3 h [D]) hypocotyl cells of YFP:TUA5-expressing 5-d-old etiolated seedlings. (E) Histogram documenting distribution of MT angles with regard to the growth axis. (F) vg and vs were measured for the leading ends of single MTs (± SE). Histograms are presented showing shrinkage (red), growth (green), and pause (blue). (G) Actin fragments reside in transient coincidence with cortical MTs after jasplakinolide treatment (5 μM for 6 h). White arrowheads indicate actin fragments aligned with MTs. Scale bars = 5 μm.
Figure 2.
Figure 2.
Coalignment between Cortical AFs and MTs in Arabidopsis Hypocotyl Cells. (A) to (C) Dual-labeled GFP:FABD (A) and mCherry:TUA5 (B), and merge of A and B (C), observed in hypocotyl cells of 3-d-old etiolated seedlings. Carets enclose regions of co-occurrence of the two channels. (D) Selected frames from a time series of a GFP:FABD and mCherry:TUA5 dual-labeled line. Red arrowheads indicate regions where AFs appear static after MT alignment. White arrowhead indicates that AFs emerge from a lower focal plane, and yellow carets enclose the region of MT and AF coalignment. The far right panel shows a kymograph corresponding to the dashed white line, with time on the vertical axis. (E) AF bending and straightening events facilitate alignment between AFs and MTs. Yellow arrowheads indicate the region of the AF that exhibits bending, and yellow carets enclose the region of the coaligned MT and AF. The far right panel shows a kymograph corresponding to the white dashed line, with time on the vertical axis. (F) Kymograph covering the entire cell length showing multiple static AF events (green enclosure) that coincided with MTs (red enclosure). Only AFs that were static for more than 30 s were considered. (G) A fluorescence intensity plot of a transect of the kymograph shown in (F [white dashed line]). a.u., arbitrary units. Scale bars = 5 μm.
Figure 3.
Figure 3.
Light-Induced Rearrangements of the Cytoskeleton. (A) Histogram showing actin–MT interaction over time (n = 4 cells). (B) Changes in average MT angle over time during the process of imaging (n = 3 cells). Error bars represent SE. (C) Selected frames from a time series of a GFP:FABD and mCherry:TUA5 dual-labeled line at the hypocotyl cell cortex in etiolated 3-d-old seedlings after light exposure. (D) Changes in relative fluorescence intensity of AFs over time (n = 3 cells). Error bars represent SE. a.u., arbitrary units. Scale bars = 5 μm.
Figure 4.
Figure 4.
Recovery of the Actin Cytoskeleton following latB Treatment and Washout Requires MTs. (A) Newly formed AFs coincide with MTs 3 h after latB washout. (B) Fluorescence intensity plot of the GFP:FABD and mCherry:TUA5 signals across a transect of an elongating hypocotyl cell (cyan line shown in [A]). a.u., arbitrary units. (C) Selected frames from a time series of a GFP:FABD and mCherry:TUA5 dual-labeled line at the hypocotyl cell cortex in etiolated 3-d-old seedlings 5 h after latB washout. White arrowheads indicate the movement of small AF fragments along MTs. Scale bar = 5 μm. (D) Selected frames from a time series of a GFP:FABD and mCherry:TUA5 dual-labeled line at the cell cortex 5 h after latB washout. Yellow carets enclose small actin structures that move along the MT plus end (yellow vertical line). Bottom panel, fluorescence intensity plot of the GFP:FABD and mCherry:TUA5 signals along the cyan line in top panel. (E) Kymograph of the cyan line in image (D). Scale bars = 1 μm.
Figure 5.
Figure 5.
Mutual Dependence of the Actin and MT Cytoskeleton Assayed by Recovery after Depolymerization. (A) and (B) One micromolar latB treatment of GFP:FABD and mCherry:TUA5 dual labeled etiolated 3-d-old seedlings for 16 h followed by washout of latB and recovery of the actin cytoskeleton for 6 h (A) and 20 h (B). (C) and (D) One micromolar latB and 20 μM oryzalin treatment for 16 h followed by washout of latB and recovery of the actin cytoskeleton for 5 h (C) and 20 h (D). (E) and (F) Oryzalin treatment of GFP:FABD and mCherry:TUA5 dual labeled lines for 16 h followed by washout of oryzalin and recovery of MTs for 5 h (E) and 20 h (F). (G) and (H) LatB and oryzalin treatment for 16 h followed by washout of oryzalin and recovery of MTs for 5 h (G) and 20 h (H). Scale bars = 5 μm.
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