- Title
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Bleb Expansion in Migrating Cells Depends on Supply of Membrane from Cell Surface Invaginations
- Authors
- Goudarzi, M., Tarbashevich, K., Mildner, K., Begemann, I., Garcia, J., Paksa, A., Reichman-Fried, M., Mahabaleshwar, H., Blaser, H., Hartwig, J., Zeuschner, D., Galic, M., Bagnat, M., Betz, T., Raz, E.
- Source
- Full text @ Dev. Cell
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Lack of directed membrane flow during bleb formation. Related to Figure 2. (A) An area of Farnesylated-EGFP labeled membrane adjacent to a forming bleb (arrowhead) was photobleached and the distribution of the fluorescence was assessed. Despite the expansion of the bleb, no directional material flow could be observed, 10 cells analyzed. (B) Photobleaching of Farnesylated-EGFP on the membrane of an inflating bleb (red arrowheads) reveals expansion of the dark area during bleb formation, 10 cells analyzed. (C) Photobleaching of a truncated non-internalizable, non-ligand binding form of Cxcr4b fused to EGFP. The photobleaching experiment reveals no membrane flow in the direction of the region where blebs are formed (white arrowheads), 5 cells were analyzed. (D) EGFP LifeAct–expressing PGC located within the tissue of a deyolked embryo (to allow lipid-dye labeling, see methods). The fluorescence level of the lipid dye FM4-64 observed in a single optical plane shows non-uniform signal intensity and a reduction in signal within the inflating bleb (arrowhead). (E) Normalized maximum and minimum intensity ratios of the lipid dye, FM4-64 over 3 Z-planes (maximum intensity projection) (n=16 cells). The graph shows median and interquartile range. P value was calculated using non-parametric Mann Whitney U test. Scale bars=5µm. |
The role of caveolae in blebbing. Related to Figure 4 (A) Two-color in-situ hybridization for caveolin1 (blue) and nanos3 (brown) mRNAs showing their co-expression within a PGC. (B) Global expression of Cav1a-GFP in PGCs and in somatic cells at 7hpf (upper panel) and 24hpf (lower panel), with the membrane of the PGCs labeled with mCherry-F (asterisks). Arrowheads point at membrane localization of the Cav1a-GFP to the plasma membrane of nearby somatic cells, while the PGC membrane is largely devoid of GFP signal. (C) Formation of blebs (arrowheads) at the front of a cav-1; cav-3 maternal-zygotic mutant migrating live PGC (upper panels, 8hpf) and proper arrival of the PGCs at the position where the gonad develops in 24hpf embryo of the same genotype (lower panel, 24hpf). The experiments were performed 3 times, with a total of 28 wild-type and 12 mz cav1; cav3 embryos assessed. Scale bar=5 µm. |
The role of vesicle trafficking in blebbing. Related to Figure 4 (A) Labeling of the Golgi apparatus in a migrating PGC using human beta 1, 4- galactosyltransferase target sequence fused to dsRed (dsRed-Gtransferase). The plasma membrane is labeled with EGFP-F or with mCherry-F (presented in green). Arrow shows the direction of migration and the position of the nucleus is outlined with the dashed-lines. Localization of different vesicle markers including VAMP8, Rab11a, Rab5c and Exo70 and their position relative to the forming bleb (asterisk). (B) 24 hpf embryos globally expressing control RNA or dominant negative (DN) forms of Exo70, Rab11a and Rab5c (upper two rows) and 24 hpf embryos expressing the same dominant negative forms preferentially in the PGCs by employing the nanos3 3’UTR (lower two rows). In all cases, the PGCs reach their clustering position. (C) PGCs expressing the dominant-negative of Exo70, Rab11a and Rab5c show a frequency of bleb formation similar to that of control cells. Scale bar=5 µm. Statistical analysis was conducted using the Kruskal-Wallis test, with P value= 0.1, ns. The graph presents the combined data from 3 independent experiments. The graphs show median and interquartile range. Ctrl stands for Control RNA. |
Cytoplamic flow during bleb formation. Related to Figure 1 Confocal image of a PGC labeled with farnesylated mCherry and cytoplasmic GFP. The asterisk in upper row marks the location of bleb inflation. The cytoplasmic GFP signal is presented in pseudocolors in the second row and third row presents an overlay of cytoplasm and membrane signals. Dashed rectangle marks the area of the forming bleb, magnified in the bottom (bleb) panel. Scale bar= 5µm. Similar observations were made in 13 cells and 38 blebs. The ratio between the cytoplasmic GFP signal intensity within the bleb divided by the prebleb cytoplasm signal intensity was not substantially different (median=0.98, interquartile range (0.91-1.03). |
Reprinted from Developmental Cell, 43(5), Goudarzi, M., Tarbashevich, K., Mildner, K., Begemann, I., Garcia, J., Paksa, A., Reichman-Fried, M., Mahabaleshwar, H., Blaser, H., Hartwig, J., Zeuschner, D., Galic, M., Bagnat, M., Betz, T., Raz, E., Bleb Expansion in Migrating Cells Depends on Supply of Membrane from Cell Surface Invaginations, 577-587.e5, Copyright (2017) with permission from Elsevier. Full text @ Dev. Cell