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First published online 9 September 2008
doi: 10.1242/jcs.033878

Journal of Cell Science 121, 3187-3195 (2008)
Published by The Company of Biologists 2008
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Dynein drives nuclear rotation during forward progression of motile fibroblasts

Jennifer R. Levy and Erika L. F. Holzbaur*

Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6085, USA


Figure 1
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  		Fig. 1. Dynein and dynactin localize to the leading edge of migrating cells and promote centrosome reorientation, leading-edge protrusion and nuclear migration. (A) Dynein and dynactin concentrate in cortical patches at the leading edge of migrating cells. NIH/3T3 cells were mock transfected, transfected with siRNAs targeting p150Glued or DHC, or transfected with a scrambled oligonucleotide control, then grown to confluency and the monolayer wounded. Six hours after wounding, cells were fixed and stained for {alpha}-tubulin and either p150Glued or DIC. Mock-transfected cells show patches of dynein and dynactin at the leading edge of migrating cells (arrowheads). Inset, overlay of cortical patch of p150Glued (red) and projecting MTs (green). Scale bar, 10 µm. (B) Western blot of protein lysates from cells that were mock transfected or transfected with siRNAs targeting p150Glued, DHC or β-catenin consistently show knockdown of target protein levels by 75-95%. Levels of DIC decrease in DHC knockdown cells, suggesting the dynein complex is being destabilized. By contrast, knockdown of β-catenin, which binds to DIC but is not part of the dynein motor complex (Ligon et al., 2001Go), does not destabilize DIC. (C) Centrosome reorientation is inhibited in dynein and dynactin knockdown cells. Orientation of centrosomes 6 hours after wounding. Centrosome orientation was determined by measuring the position of centrosomes, as determined by {gamma}-tubulin staining, in relation to Hoechst-stained nuclei. Centrosomes in the forward-facing 120 degree sector (green zone) were scored as reoriented. (D) Leading-edge extension is inhibited in dynein knockdown cells. Displacement of the leading edge over 45 minutes of wound healing was measured (n=15). (E) Tracks of nuclei centroids during migration of fibroblasts over 45 minutes. Paths are oriented so cells are migrating towards the top of the graph, each path starts at (0,0). Axis labels are in µm. (F) Rates of nuclear movement are decreased in dynein and dynactin knockdown cells. Nuclear velocity during migration was decreased in DHC and p150Glued siRNA cells, compared with mock-transfected cells (n=15). Error bars indicate s.e.m.; *P<0.05; **P<0.005.

 

Figure 2
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  		Fig. 2. Dynein-dependent nuclear rotations are enhanced after monolayer wounding. (A) A time series of a nucleus that rotates during cell migration. Red arrows indicate two nucleoli that stay in position relative to other nucleoli within the nucleus. This nucleus rotates counter-clockwise for the first 30 minutes, and then switches direction during the final 10 minutes of this timelapse. Scale bar, 10 µm. Time is minutes:seconds. (B) Tracks of nucleoli during migration of fibroblasts over 45 minutes. Large circular traces seen in mock-transfected cells demonstrate paths of nucleoli in nuclei that are rotating. These are rarely seen in DHC and p150Glued siRNA cells (n=15). Axis labels are in µm. (C) Particle tracking was used to track the duration of rotation of nuclei from cells at the wound edge during migration. A run was defined as rotation in a single direction without pausing (n=225 runs from 25 cells). (D) Angular velocity of nuclei in cells along the wound edge of wounded cultures is higher than that of nuclei in cells that have been grown to confluency, but not wounded (n=25). (E) Angular velocity of nuclei in mock-treated cells along the leading edge is greater than that of DHC and p150Glued siRNA cells, which rarely rotate and thus have a slower average angular velocity (n=15). Error bars indicate s.e.m.; *P<0.05.

 

Figure 3
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  		Fig. 3. Nuclear rotation includes ER closely associated with the nuclear membrane. (A) Cells transfected with dsRed2-ER were grown to confluency and wounded. The top panel shows a nucleus rotating clockwise during migration. White arrowhead indicates the position of a nucleolus during rotation. The bottom panel shows the morphology of the ER during the same timeframe. Some small fragments of the ER that are closely opposed to the nucleus rotate with it (black arrowhead, see also supplementary material Movie 3). Peripheral ER shows dynamic remodeling during the rotation. (B) Tracks of the paths of a single nucleolus and an ER particle within the cell shown in A during nuclear rotation over 45 minutes. Both follow the same circular path, indicating that they are both rotating in the same manner (see also supplementary material Movie 3). Time is minutes:seconds. Scale bar, 10 µm.

 

Figure 4
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  		Fig. 4. Nuclear rotations are not coupled to centrosome rotation. (A) Exogenous Centrin2 closely associates with endogenous {gamma} tubulin in puncta capable of MT nucleation, supporting its functional role as a centrosome marker. Cells transfected with dsRed1-Centrin2 (red) were subjected to nocodazole treatment. After a 5-minute wash, cells were fixed and stained for {gamma} tubulin (green) and {alpha} tubulin (blue). (B) Tracks of a single nucleolus (red) and a centrosome (blue) within the cell shown in C during nuclear rotation over 45 minutes. The cell is migrating towards the top of the graph, and the nucleus rotates while the centrosome remains between the nucleus and the leading edge. (C) Cells transfected with dsRed1-Centrin2 (red), a centrosomal marker, were grown to confluency and wounded. The arrowhead indicates a centrosome that stays positioned between the nucleus and the leading edge while the nucleus rotates clockwise (black arrow, also see supplementary material Movie 5). Time is minutes:seconds. Scale bars, 10 µm.

 

Figure 5
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  		Fig. 5. Nuclear rotations are independent of myosin II activity. (A) Tracks of nucleoli during migration of DMSO or blebbistatin-treated fibroblasts over 45 minutes (n=15). Axis labels are in µm. (B) Angular velocity of nuclei in the presence of myosin II inhibitor blebbistatin (n=25).

 

Figure 6
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  		Fig. 6. Nuclear rotations are independent of leading-edge and Golgi-localized dynein. (A) Cells treated with low doses of nocodazole (NZ) 5.5 hours after wounding maintain the MT network morphology (green), but lose patches of p150Glued (red) at the leading edge. (B) Dynactin patch accumulation is inhibited in cells treated with low doses of NZ. Patches are partially rescued 30 minutes after NZ washout. (C) Cells treated with BFA 5.5 hours after wounding maintain MT network morphology (green) but have fragmented Golgi (red, Golgi marker GM130). Scale bar, 10 µm. (D) Tracks of nucleoli treated with DMSO, BFA or NZ during migration of fibroblasts over 45 minutes. Axis labels are in µm. (E) Angular velocity nuclei of cells treated with DMSO (control), NZ or BFA (n=15). (F) Scheme of the two roles of dynein and dynactin in cell motility: (1) dynein and dynactin accumulate in cortical patches at the leading edge, where they interact with MTs and mediate centrosome and MT orientation during motility; and (2) dynein and dynactin interact with the nuclear envelope and transport the nucleus along MTs on the sides of the nucleus. Error bars indicate s.e.m.; **P<0.005.

 

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© The Company of Biologists Ltd 2008