doi: 10.1096/fj.14-250019. Epub 2014 Mar 20.
Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases
Affiliations
- PMID:24652946
- PMCID: PMC4062827
- DOI: 10.1096/fj.14-250019
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Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases
Amélie Robert et al. FASEB J.2014 Jul.
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Abstract
Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.
Keywords: cytoskeleton; motor proteins; particle tracking.
© FASEB.
Figures
Movement of vimentin ULFs in SW13 cells.A,B) GFP-tagged Y117L mutant vimentin was expressed in SW13 vim+ cells containing endogenous vimentin (A) or in vimentin-deficient SW13 vim− (B).C) Temporal color-code representation of GFP-ULF movement in a cell shown inB. Images were captured every second for 1 min, then color coded and merged for a qualitative representation of ULF trajectories. Time color-code bar is at left. Rainbows correspond to motile ULFs, and white dots correspond to stationary ULFs. Enlargement of the boxed area (at right) shows an example of a linear trajectory (rainbow picture) and the corresponding image sequence. See also Supplemental Movie S1. Scale bar = 10 μm.D) Trajectories of ULFs were analyzed using Diatrack software in ≥10 cells/experiment. Graph shows the mean frequency distribution of trajectory length for trajectories that are ≥3 μm long.
ULFs interact with microtubules and F-actin.A) GFP-ULF-expressing cells were transfected with TagRFP-EB3 that decorates microtubules. Microtubule network images were captured at the beginning and end of the time-lapse sequence of GFP-ULF. Cells were treated with vinblastine (5 nM) to minimize microtubule dynamics during the imaging period, and the image sequence of GFP-ULF was superimposed with the static microtubule image. Left panel: first frame of the image sequence, showing alignment of GFP-ULF with microtubules. Right panel: enlargement of the boxed area shows movement of ULFs along a microtubule (white arrows). See also Supplemental Movie S2.B) GFP-ULF interaction with actin. GFP-ULF-expressing cells were fixed after the following treatment: control (untreated), nocodazole (10 μM for 1 h), and Lat B (5 μM for 1 h). F-actin was stained using rhodamine-phalloidin. Enlargements show colocalization of ULF with actin cables in untreated and nocodazole-treated cells, or with residual actin aggregates into Lat B-treated cells. Scale bars = 10 μm.
F-actin suppresses microtubule-dependent transport of ULFs. GFP-ULF expressing cells were incubated for 30 min at 4°C to depolymerize microtubules, then transferred to 37°C for 1 h in medium containing: nocodazole (10 μM to prevent microtubule polymerization), Lat B (5 μM to depolymerize F-actin), or nocodazole and Lat B together (to prevent microtubule and F-actin polymerization). Control cells were incubated at 30 min at 4°C and then warmed at 37°C 60 min before imaging.A) Temporal color-code representation of ULF motility (see Materials and Methods and Fig. 1). Time color-code bar is shown in the control panel. Note that Lat B treatment increased ULF transport, as revealed by the number of rainbow tracks corresponding to motile ULFs. See also Supplemental Movie S3. Scale bar = 10 μm.B,C) Quantitative analysis of motility. Graphs show the frequency distribution of ULF trajectory length (B) and the percentage of trajectories longer than 6 μm (C).
Microtubule dynamics is not required for ULF transport. GFP-ULF-expressing cells were transfected with TagRFP-EB3. Left panel; temporal color coding from the 60-frame projection of EB3 (1 frame/s) revealed the EB3 comet evolution at the tip of growing microtubules as an indicator of microtubule dynamics. Right panel; 10 nM vinblastine for 5 min is sufficient to stop microtubule dynamics, since EB3 comets at the microtubule tips are absent. Time color-code bar is shown in the control panel. Scale bar = 10 μm. Insets: enlargement of the first frame corresponding to the boxed area. Arrows point to the EB3 comets, which are present in the control cells.B) Vinblastine (10 nM) was added for 5 to 30 min to GFP-ULF cells treated or not with 5 μM Lat B. Graph shows the percentage of ULF tracks longer than 6 μm. Inhibition of microtubule dynamics does not substantially change the movement.
ATP depletion inhibits ULF transport. ATP in cells was depleted by incubation with 20 mM sodium azide (NaN3) in Ca2+, Mg2+-containing PBS. Lat B (5 μM) was added to stimulate microtubule-dependent transport. Controls include NaN3 washout and incubation with NaN3 in the presence of 50 mM glucose to allow ATP production by glycolysis.A) Temporal color-code representation of ULF motility before and after NaN3 treatment.B) Graph shows the percentage of ULF tracks longer than 6 μm, which is completely inhibited by NaN3 treatment.C) GFP-ULF binding to microtubules in Lat B- and NaN3-treated cells. GFP-ULF-expressing cells were fixed and stained by an antibody against tubulin. Scale bar = 10 μm.
Inhibition of dynein with ciliobrevin blocks ULF transport. GFP-ULF cells were incubated with serum-free medium supplemented with 5 μM Lat B for 15 min; then 50 μM ciliobrevin was added for 1 h.A) Temporal color coding of GFP-ULF position shows that ciliobrevin treatment inhibits ULF. Scale bar = 10 μm.B) Graph shows the percentage of ULF tracks longer than 6 μm, which are completely inhibited by the ciliobrevin treatment. For the ciliobrevin washout condition, ciliobrevin-treated cells were washed twice with PBS and incubated for 1 h in completed medium in the presence of Lat B to reverse ciliobrevin action.
ROCK is a negative regulator of ULF transport along microtubules.A) F-actin staining of GFP-ULF cells fixed 1 h after treatment with 10 μM Y-27632. Note that the Y-27632 treatment inhibited actin bundling into stress fibers but did not affect ULF interaction with F-actin.B) Lat B-treated GFP-ULF cells were treated with 10 μM Y-27632 for 1 h before and during live-cell imaging of GFP-ULF motility. Temporal color coding of ULF position revealed the presence of very long trajectories in Y-27632-treated cells as compared to control. Scale bars = 10 μm.C) Graph of the frequency distribution of ULF trajectory length shows that Y-27632 treatment increased the frequency of motile ULFs and the length of trajectories.
PAK2 is a positive regulator of ULF transport along microtubules.A,B) GFP-ULF cells were incubated for 1 h with 5 μM Lat B in the absence or presence of 10 μM IPA-3 before imaging of GFP-ULF motility.A) Temporal color coding of GFP-ULF position shows that IPA-3 treatment inhibits ULF transport along microtubules. Scale bar = 10 μm.B) Graph of the frequency distribution of ULF trajectory length shows that IPA-3 treatment decreased the frequency of motile ULF and the length of trajectories.C) GFP-ULF cells were transfected twice with scrambled (all star), PAK1, PAK2, or PAK1 and PAK2 siRNAs. Cell lysates were prepared 96 h after the first transfection. Western blot analysis reveal ≥80 and 90% knockdown efficiency for PAK1 and PAK2, respectively. Level of expression of GFP-ULF revealed by the vimentin antibody was used as a loading control.D) Imaging of GFP-ULF motility was performed 96 h after transfection with the indicated siRNAs. Graph shows the frequency distribution of ULF trajectory length > 6 μm. Note that PAK2 siRNA treatment decreased the frequency of motile ULFs and the length of trajectories, while PAK1 siRNA alone had no effect.E) Lysosomes were labeled using Lysotracker Red (Molecular Probes) in control or 10 μM IPA-3-treated GFP-ULF-expressing cells. Cell imaging was performed in the presence of 5 μM Lat B to reproduce the same conditions where PAK inhibition inhibited GFP-ULF transport. Graph of the frequency distribution of lysosome trajectory length shows that IPA-3 treatment had no effect on lysosome transport.
References
- Minin A. A., Moldaver M. V. (2008) Intermediate vimentin filaments and their role in intracellular organelle distribution. Biochem. Biokhim. 73, 1453–1466 - PubMed
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