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First published online 24 June 2008
doi: 10.1242/jcs.026849

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In migrating cells, the Golgi complex and the position of the centrosome depend on geometrical constraints of the substratum

Cell Biology and Cell Biophysics Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany

View larger version (61K): [in this window] [in a new window]   Fig. 1. Cells on fibronectin-patterned lines are polarized. (A) Patterned, 6-µm thick fibronectin lines and Pll–g-PEG. (B) Bsc1 cells on a standard glass coverslip labelled for the actin network (green), the Golgi complex (red) and the nucleus (blue). (C) Bsc1 cells labelled as in B, grown on 6-µm fibronectin lines. (D) Ptk2 cells labelled as in B, grown on 6-µm fibronectin lines. (E) Centered projection of 100 Bsc1 cells grown on a standard coverslip demarcated to outline the mean cell size and shape, scale bar 10 µm (see supplementary material Fig. S1). (F) Centered and aligned (along the longest cell axis) projection of 100 Bsc1 cells grown on a standard coverslip (see supplementary material Fig. S1). (G) Centered and aligned (along the longest cell axis) projection of 100 Bsc1 cells grown on fibronectin line (see supplementary material Fig. S1). (H) Centered and aligned (along the longest cell axis) overlay of 50 Ptk2 cells grown on a standard coverslip (see supplementary material Fig. S1). (I) Centered and aligned (along the longest cell axis) overlay of 50 Ptk2 cells grown on fibronectin line (see supplementary material Fig. S1). (J) Threshold images of 38 Bsc1 cells grown on 6-µm fibronectin lines, fixed and analysed by immunofluorescence. Actin cell contour (black, phalloidin staining), nucleus (red, Hoechst staining) and Golgi (green, GM130 staining). Scale bars, 20 µm (A-D); 10 µm (E-I).

View larger version (61K): [in this window] [in a new window]   Fig. 2. The Golgi complex is displaced on patterned cells. (A) Distance between the border of the nucleus (0) and the Golgi complex (red box) in Bsc1 cells grown on a standard coverslip. (B) Distance between the border of the nucleus (0) and the Golgi complex (green box) in Bsc1 cells grown on 6 µm fibronectin lines. (C) Distance between the border of the nucleus (0) and the Golgi complex (red box) in Ptk2 cells grown on a standard coverslip. (D) Distance between the border of the nucleus (0) and the Golgi complex (green box) in Ptk2 cells grown on 6-µm fibronectin lines. (E) Bsc1 cells grown on 6-µm fibronectin lines labelled for the actin network (green), the Golgi complex (red) and the nucleus (blue). Scale bar, 20 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Migration leads to displacement of the Golgi complex. (A) Montage of the fast-migrating Bsc1 cell on a 6-µm fibronectin line (1 image/15 minutes; total time, 12.5 hours). (B) Montage of a slow-migrating Bsc1 cell on a 6-µm fibronectin line (1 image/15 minutes; total time, 12.25 hours). (C) Montage of a migrating Bsc1 cell, nucleus (H2B-HcRed), Golgi complex (GFP2-GalT) (1 image/30 minutes; total time, 10 hours). (D) Montage of a migrating Bsc1 cell centred on the nucleus (H2B-HcRed), Golgi complex (GalT-GFP2) (1 image/5 minutes; total time, 8.5 hours).

View larger version (36K): [in this window] [in a new window]   Fig. 4. The position of the Golgi is constant. (A) The position of the Golgi complex in Bsc1 cells grown on 6-µm fibronectin lines (B, back; I, intermediate; F, front). (B) The position of the Golgi complex in Bsc1 cells grown on 6-µm fibronectin lines after reorientation of the migration direction (B/B, initial back position and final back position; I/B, initial intermediate position and final back position; F/B, initial front position and final back position). (C) Sequence of the reorientation of the motility direction of a Bsc1 cell, Golgi (green), nucleus (red). Changes of direction are indicated by asterisks (*) when the cell reaches the coverslip border, and by a plus sign (+) when the cell encounters another cell. (D) Same as C omitting the transmission channel. (E) Kymograph of the complete sequence shown in C, centered on the nucleus (red). The Golgi complex is shown in green.

View larger version (80K): [in this window] [in a new window]   Fig. 5. The MTOC and the Golgi complex are moved to the new rear upon direction changes. (A-C) Staining of Bsc1 cells for (A) -tubulin (centrosomal protein), (B) GM130 (Golgi matrix protein), (C) nucleus (Hoechst staining). (D) Overlay of A-C. (E) Projection of 25 Bsc1 cells as described in supplementary material Fig. S1 for -tubulin (red), GM130 (green) and the nucleus (blue). (F) Sequence of the reorientation of a Bsc1 cell. Shown in alternation are transmission images and immunofluorescence images of the GalT-GFP signal (Golgi matrix protein, green) and the -tubulin cherry (Gtub-cherry) highlighting the centrosome position (red) taken at 0.5 frames/hour. Direction is indicated by arrows. Centrosome position is highlighted by asterisks.

View larger version (115K): [in this window] [in a new window]   Fig. 6. Golgi location in the zebrafish lateral line primordium (llp). (A) Scheme of the migrating llp on the Sdf1a strip. (B) Side view of a claudinB-GFP transgenic embryo at 42 hours post fertilization (hpf) showing an overview of the posterior lateral line. (C) Magnification of the inset in A showing that many cells in the group display cellular lamellipod-like extensions in the direction of migration. (D) Side view of the llp of a zebrafish embryo expressing gfp-gm130 (green) and lyntdTomato (red). (E) Detailed view of the inset in D. (F) Front cell of the primordium in an embryo expressing gfp-p115 (green) and lyntdTomato (red) showing the Golgi in the most posterior part of the cell. (G) Front cells of the primordium in a CldnB:GFP embryo with -tubulin cherry (Gtub-cherry) highlighting the centrosome position. (H,K-N) TEM micrographs of a parasagital section of a 40 hpf primordium. (H) Assembly of 12 images showing a side view across most of the primordium. Cells have been pseudo-coloured and insets are magnified below (k,l,m,n). (I) Longitudinal section of the primordium as represented by the arrows in H. The section goes through all the nuclei except in the front-most cell, which is slightly flatter. (J) Detailed view of the front-cell inset in I showing the Golgi complex abutting the plasma membrane in the back. (O) Inset from J magnified, showing a detailed view of the Golgi complex. (K-N) Magnification the insets indicated in H (labelled k, l, m, n) showing the close association of the Golgi complex with the centrosomes in different cells throughout the migrating tissue from the back of the primordium (K,M) to the front (L,N). Blue arrows, Golgi complex; red arrows, centrosome. Posterior is on the right and dorsal is to the top in all panels. The direction of migration is from the left to right as indicated by the arrow in B.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Cell migration models. (A) Top view of a migrating cell on a glass substrate. (B) Top view of a migrating cell on linear FN pattern. (C) Top view of a migrating neuron. (D) Top view of a migrating T lymphocyte. (E) Side view of a migrating cell on a glass substrate. (F) Side view of a migrating cell on linear FN pattern. (G) Side view of a migrating T lymphocyte.

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