Bleb-associated motility of PGCs in the under-agarose migration assay

In the assay described above, BSA prevents nonspecific binding of the cells to the plastic surface of the Petri dish, while an agarose gel creates a second resistant surface.

Migration of PGCs in this assay suggests that they do not require specific cell-cell or extracellular matrix adhesion for motility. This is very much in line with the assumptions of the second model of bleb-associated motility that suggests the traction force for cell

Fig. 10. Isolated PGCs cultivated in extracellular matrix had high cellular dynamics, but did not initiate active migration. (A) Ventral explants were dissected from tailbud stage embryos injected with GFP_DELE mRNA to label PGCs. Explants were dissociated via enzymatic treatment. Isolated cells were either transferred on top of polymerized extracellular matrix (ECM), or premixed with ECM prior to polymerization. One sector of the culture dish was covered with 0,5% (m/v) agarose. Dorsal extract was added in a pocket made in the agarose gel to induce polarization of PGCs. Cells were cultivated in DFA, DMEM or 0.8x MBSH buffer conditions. (B) Representative time-lapse images of two PGCs (GFP-positive, green) cultivated on top of ECM. Images were recorded approximately 30 minutes after cell seeding. Cells formed bleb-like protrusions and had high cellular dynamics, but did not initiate migration. Merged images are generated from UV and normal transmitted light channels. Relative time after the first recorded image (hours : minutes : seconds) is indicated in the upper left corner of the images; scale bar – 50 µm; agarose gel with dorsal extract is towards upper side (C) Representative fluorescent time-lapse images of PGCs (GFP-positive, green) imbedded in the ECM. Despite high cellular dynamics, cells failed to initiate migration. Cells were cultivated up to 24 hours, but after several hours of cultivation cellular dynamics and formation of bleb-like protrusions were significantly decreased. Relative time after the first recorded image (minutes : seconds) is indicated in the upper left corner of the images; scale bar – 50 µm; agarose gel with dorsal extract is towards upper side .

migration to be independent of specific adhesion (Charras and Paluch, 2008; Fig. 5).

Moreover, when migrating PGCs get into contact with another cell, new interactions form that ‘anchor’ PGCs and prevent them from moving despite persisting cellular dynamics to resume or start migration (Fig. 13).

In contrast to the migration via lamellipodia, cells migrating via a bleb-associated mechanism do not exhibit constant actin polymerization at the leading edge (Charras and Paluch, 2008; Fackler and Grosse, 2008). To investigate actin filament distribution in migrating X. laevis PGCs, cells were isolated from tailbud stage embryos injected at 2-cell stage with chimeric mRNA LifeAct-GFP_DELE, consisting of actin sensor, LifeAct, fused to GFP ORF and Dead end LE. As described in section 3.3.2, in non-migrating PGCs actin cortex reassembles on the plasma membrane of the bleb, which is followed by the retraction of the protrusion. In PGCs that initiate active migration, re-polymerization of the actin filaments was not observed and retraction of the bleb-like protrusions did not occur. Instead, during in

Fig. 11. PGCs embedded in the agarose gel showed migratory behavior, but could not initiate migration.

(A) Ventral explants were dissected from tailbud stage embryos injected with GFP_DELE mRNA to label PGCs. Explants were dissociated via enzymatic treatment. Isolated PGCs (green) were transferred on top of a polymerized 0,1-0,5% (m/v) agarose gel and coated with another layer of the gel. One sector of the culture dish was covered with 2% (m/v) agarose. Dorsal extract was added in a pocket made in the 2%

agarose gel to induce polarization of PGCs. (B) Representative time-lapse images of a PGC (GFP-positive, green) recorded after approximately 20 minutes of cultivation. Cultivated PGCs had high cellular dynamics and exhibited migratory-like behavior, but could not initiate migration. After several hours of cultivation cells obtained stationary round morphology. Merged images are generated from UV and normal transmitted light channels. Relative time after the first recorded image (minutes : seconds) is indicated in the upper left corner of the images;scale bar: 100 µm; agarose gel with dorsal extract is towards upper side .

vitro PGC migration in the uder-agarose assay, actin filaments were enriched in the rear and the sides of the cell, but not in the leading edge (Fig 14). This confirms that X. laevis PGCs migrate via bleb-associated amoeboid movement and not via lamellipodia-based actin-dependent mechanism.

Previous results from our lab showed that phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is enriched in the bleb-like protrusions formed by isolated PGCs. Interference with endogenous PIP3 levels led to a decrease or absence of cell blebbing. It also resulted in the mislocalization during migration and loss of PGCs in vivo (Tarbashevich 2007; Tarbashevich

Fig. 12. X. laevis PGC can migrate in vitro in the under-agarose migration. (A) Ventral explants were dissected from tailbud stage embryos injected with GFP_DELE mRNA to label PGCs. Explants were dissociated via enzymatic treatment. Dissociated cells, including PGCs (green), were transferred between 0.5% (m/v) polymerized agarose gel and the bottom of a Petri dish. Prior to addition of agarose gel, in some experiments, the Petri dish was pre-coated with fibronectin or 5% (m/v) bovine serum albumin (BSA). (B) Time-lapse images of an under-agarose migration assay with a BSA-coated Petri dish. PGCs can be identified as GFP-positive (green) in contrast to the GFP-negative somatic cells. Red arrows indicate migrating PGC. Merged images are generated from UV and normal transmitted light channels. Relative time after the first recorded image (minutes : seconds) is indicated in the upper left corner of the images, scale bar: 20 µm.

Fig. 13. Surrounding cells can ‘anchor’ PGCs and prevent their migration in vitro in the under-agarose migration. (A) Ventral explants were dissected from tailbud stage embryos injected with GFP_DELE mRNA to label PGCs. Explants were dissociated via enzymatic treatment. Dissociated cells, including PGCs (green), were transferred between 0.5% (m/v) polymerized agarose gel and a 5% BSA-coated Petri dish in 0.8x MBSH buffer. (B) Time-lapse images of an under-agarose migration assay depicting an anchoring of migrating PGC (red arrow) by another cell (white arrow). Merged images are generated from UV and normal transmitted light channels. Relative time after the first recorded image (minutes : seconds) is indicated in the upper left corner of the images, scale bar: 20 µm.

et al., 2011). To visualize PIP3 distribution in migrating PGCs, cells were isolated from the embryos injected with chimeric mRNA GFP_GRPI_PH_DELE, consisting of pleckstrin homology (PH) domain of GRPI protein, used as a PIP3 sensor, fused to GFP ORF and Dead end LE. As a control, GFP_GRPI_PH_DELE-injected embryos were also co-injected with mRNA encoding membrane red fluorescent protein (mRFP) to visualize cell membrane. Distribution of mRFP served as a control to monitor the intracellular distribution of PIP3. Although PIP3 was enriched in the protrusions formed by non-migrating cells, no specific enrichment was observed during active migration (Fig. 15). This suggests that PIP3 enrichment in the bleb-like protrusions is required for polarization, but is not maintained during PGC migration.

3.2 PGCs isolated form neurula stage can migrate in the under-agarose