Figure 2.
MTs coordinate protrusion-retraction dynamics. (a) Cells migrating under agarose along a soluble CCL19 gradient were fixed and stained for α-tubulin and the nucleus (DAPI). Boxed regions indicate trailing edge (purple) or pioneering (gray) MTs toward the leading edge. Right panel: Line profile of α-tubulin distribution along the anterior-posterior polarization axis, derived from the purple line in the left panel of n = 10 cells. Scale bar, 10 µm. (b) Angular distribution of automatically detected MT growth events according to EB3 signals along the anterior-posterior polarization axis. (c) MT dynamics during directed migration. EMTB-mCherry expressing DC migrating under a pad of agarose. Gray box indicates the protrusive cell front, whereas the purple boxed area denotes the contractile trailing edge. Growth (white arrowheads) and shrinkage (purple arrowheads) frequencies of individual MT filaments (according to EMTB labeling) were assessed in protrusive (front, white box) versus contractile (back, purple box) areas of the same migratory cell. Red dotted lines indicate cell edges. Growth events and catastrophes ≥1 µm were tracked for n = 10 filaments each in the respective region of n = 8 cells. Mean ± SD. Annotation above columns indicates results of unpaired Student’s t test; ****, P ≤ 0.0001. See also Video 2. Scale bar, 10 µm (left image) and 5 µm (right panels). (d) Acetylated (acetyl.)-tubulin staining in DCs fixed while migrating under agarose. Blue line indicates position of the nucleus; red line, cell outline. Insets show the area around the MTOC. Levels of acetylation were assessed by measuring the mean fluorescence intensity (MFI) of acetylated tubulin along individual α-tubulin filaments, directed toward the front (gray) or back (purple) of n = 87 filaments from N = 3 experiments. Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of unpaired Student’s t test; ****, P ≤ 0.0001. Scale bar, 10 µm. (e) Time-lapse sequence of control or PST-1 treated cells stained with TAMRA, which were locally photoactivated (red boxes) during migration under agarose. Right panel indicates the frequency of local retractions upon photoactivation of control or PST-1–treated DCs during migration (n = 26 cells per condition ± SD from N = 3 experiments). Annotation above columns indicates results of unpaired Student’s t test; *, P ≤ 0.05, ****, P ≤ 0.0001. Scale bar, 10 µm. See also Video 3. Retr., retracting.

MTs coordinate protrusion-retraction dynamics. (a) Cells migrating under agarose along a soluble CCL19 gradient were fixed and stained for α-tubulin and the nucleus (DAPI). Boxed regions indicate trailing edge (purple) or pioneering (gray) MTs toward the leading edge. Right panel: Line profile of α-tubulin distribution along the anterior-posterior polarization axis, derived from the purple line in the left panel of n = 10 cells. Scale bar, 10 µm. (b) Angular distribution of automatically detected MT growth events according to EB3 signals along the anterior-posterior polarization axis. (c) MT dynamics during directed migration. EMTB-mCherry expressing DC migrating under a pad of agarose. Gray box indicates the protrusive cell front, whereas the purple boxed area denotes the contractile trailing edge. Growth (white arrowheads) and shrinkage (purple arrowheads) frequencies of individual MT filaments (according to EMTB labeling) were assessed in protrusive (front, white box) versus contractile (back, purple box) areas of the same migratory cell. Red dotted lines indicate cell edges. Growth events and catastrophes ≥1 µm were tracked for n = 10 filaments each in the respective region of n = 8 cells. Mean ± SD. Annotation above columns indicates results of unpaired Student’s t test; ****, P ≤ 0.0001. See also Video 2. Scale bar, 10 µm (left image) and 5 µm (right panels). (d) Acetylated (acetyl.)-tubulin staining in DCs fixed while migrating under agarose. Blue line indicates position of the nucleus; red line, cell outline. Insets show the area around the MTOC. Levels of acetylation were assessed by measuring the mean fluorescence intensity (MFI) of acetylated tubulin along individual α-tubulin filaments, directed toward the front (gray) or back (purple) of n = 87 filaments from N = 3 experiments. Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of unpaired Student’s t test; ****, P ≤ 0.0001. Scale bar, 10 µm. (e) Time-lapse sequence of control or PST-1 treated cells stained with TAMRA, which were locally photoactivated (red boxes) during migration under agarose. Right panel indicates the frequency of local retractions upon photoactivation of control or PST-1–treated DCs during migration (n = 26 cells per condition ± SD from N = 3 experiments). Annotation above columns indicates results of unpaired Student’s t test; *, P ≤ 0.05, ****, P ≤ 0.0001. Scale bar, 10 µm. See also Video 3. Retr., retracting.

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