Blebbing-associated motility as a basis for PGC migration in the endoderm

In Xenopus, PGCs initiate active migration at developmental stage 24, when they disperse from the cluster that they had formed in the endoderm (Nishiumi et al., 2005).

Similar to Drosophila and zebrafish, active migration of Xenopus PGCs within the endoderm is coupled to changes of their locomotive activity. Prior to migration (stage 18 and 24), isolated PGCs show little protrusion formation and exhibit mainly a spherical round morphology, similar to somatic endodermal cells. Upon dispersal at stage 28, isolated PGCs acquire an elongated shape, correlating with the onset of migratory activity. At stage 33/34, PGCs exhibit a high level of cellular dynamics that is characterized by the formation of numerous bleb-like protrusions and migratory activity. In these stages, PGCs alternate between locomotive and pausing phases (Fig. 3). At stage 41, isolated PGCs exhibit a reduced tendency to form bleb-like protrusions and locomotive activity (Terayama et al., 2013).

Migration of zebrafish and Xenopus PGCs within the endoderm occurs via a blebbing-associated mechanism (Tarbashevich et al., 2011; Terayama et al., 2013). Blebs are pressure-driven plasma membrane protrusions formed by the cells. In majority of the animal cells plasma membrane is tightly bound to the underlying cortex formed from actin, myosin, cortex-membrane linker ERM (ezrin, radixin and moesin) proteins and some other associated proteins. Myosin motors constantly keep the cortex under tension, applying a pressure on the cytoplasm. If disruption occurs either in the cortex or at the interface between cortex and plasma membrane, the internal pressure of the cell generated by acto-myosin contraction drives the cytoplasm to flow into this space. As a result, a spherical, cellular bleb-like protrusion is formed (Fig. 4a). It is not clear what causes these events. Most bleb-likely, in nonpolarized cells bleb initiation occurs randomly throughout the plasma membrane, but it also can be caused by some external triggers (Charras and Paluch, 2008; Fackler and Grosse, 2008). In nonpolarized cells the expansion of the bleb lasts 5-30 seconds (Fig. 4b). In these cells, the acto-myosin cortex reassembles on the plasma membrane (Fig. 4c) and the bleb retracts to the initial position (Fig. 4d). In migratory polarized cells, formation of the blebs occurs preferentially at the leading edge. In these cells, cortex re-polymerization on the surface of the bleb is followed by a new disruption of cortex-plasma membrane interactions

Fig. 3. PGC motility and morphology during different developmental stages of X.laevis embryos. At stage 24 PGCs initiate active migration from the cluster they formed in the endoderm. At this stage isolated PGCs have mostly round morphology and form bleb-like protrusions. During tailbud stage (St. 24-44), they migrate anteriorly and dorsally around the gut. Isolated PGCs at these stages alternate between migratory elongated shape and round shape with bleb-like protrusions. During the migration via dorsal mesentery (St. 45), isolated PGCs form filopodia-like protrusions. Embryos are drawn after Nieuwkoop and Faber, 1994. Vertical sections perpendicular to the anterior-posterior with the positions of PGCs (pink dots) in the embryo are indicated. The morphology of isolated PGCs is indicated according to Terayama et al., 2013;

Heasman and Wylie, 1978.

before the bleb is retracted. This results in the formation of a new bleb at the leading edge before the retraction of a former bleb occurs. The position of the bleb at the leading edge can also be stabilized by newly formed interactions with extracellular substrates. In both of these cases, the plasma membrane of the bleb is not retracted to its initial position, and the cell translocates to a new position with the help of acto-myosin cortex contraction in the rear (Charras and Paluch, 2008).

Lipid bilayer composition is also important for the rigidity of the cortex-membrane interaction. It has been shown, that phosphatidylinositol (4,5)-bisphosphate (PIP2) increases the level of cortex-membrane adhesion, while the sequestering of PIP2 results in the decrease of the adhesion energy (Raucher et al., 2000). PIP2 has many structural and anchoring functions and serve as a precursor for the second messengers inositol (1,4,5)-trisphosphate (IP3), calcium, and diacylglycerol (DAG) that may act in parallel with PIP2 in regulating cytoskeletal structure. The presence of PIP2 facilitates actin polymerization in

Fig. 4. The life cycle of a bleb. (a) The initiation of the bleb formation can be the result of a local detachment of the cortex from the membrane (left) or from a local rupture of the cortex (right). (b) The expansion of the bleb is supported by the flow of the cytoplasm into the bleb, caused by the intracellular hydrostatic pressure through the remaining cortex (left) or through the cortex hole (right). The bleb base can be increased by detaching the cortex from the plasma membrane. (c) At the end of the bleb expansion stage, the cortex starts its reassembly under the plasma membrane of the bleb. (d) Retraction of the bleb, dependent on the Rho-ROCK-myosin II machinery, restores the initial shape of the cell (taken from Charras and Paluch, 2008).

multiple ways (Sechi and Wehland, 2000; Yin and Janmey, 2003), while generation of IP3 and DAG, following phospholipase C (PLC)-mediated hydrolysis of PIP2, lead to solubilization of the actin network (Meberg, 2000; McGough et al., 2003). In addition, PIP2 induces conformational changes in vinculin, talin and ERM family proteins to promote anchoring of the actin cytoskeleton to the plasma membrane (Sechi and Wehland, 2000). PLC-mediated hydrolysis of PIP2 and the downstream activation of Ca2+/CaM (calmodulin) and protein kinase C (PKC) also influence actin-myosin based contractility. Ca2+/CaM activates myosin regulatory light chain kinase (MLCK), leading to phosphorylation of the myosin regulatory light chain (MLC) (Iwasaki et al., 2001). Similarly, PKC has been shown to phosphorylate and activate MLC (Naka et al., 1988; Varlamova et al., 2001). Moreover, PIP2 is a substrate for phosphatidylinositol-3-OH kinase (PI3K) to generate phosphatidylinositol (3,4,5)-trisphosphate (PIP3), an effector of different downstream targets of PI3K signaling cascades (Katso et al., 2001).

Therefore, PIP2 concentration could be directly involved in the regulation of membrane-cortex interactions reducing or increasing local interactions between the cytoskeleton and the plasma membrane. Alternatively, PIP2 can also indirectly regulate adhesion by modulating signaling cascades that alter the cortical cytoskeleton structure.

Actin polymerization and myosin activity are required for the locomotion and protrusion formation of Xenopus PGCs. In isolated non-migrating PGCs, actin filaments are localized to the periphery of the cell, forming a cortex underlying the plasma membrane, while in the migratory PGCs actin filaments can be found in the rear, but not at the leading edge of the cell (Terayama et al., 2012). Interestingly, in contrast to the migration of Xenopus PGCs in vitro, formation of actin brushes at the leading edge is required for PGC migration in the zebrafish in vivo (Kardash et al., 2010; discussed in chapter 1.3.2). Similar to the zebrafish, however, inhibition of actin polymerization and myosin activity by chemical inhibitors in Xenopus PGCs results in a loss of protrusion formation and cell locomotion (Terayama et al., 2013). In the same study, similar results were obtained by inhibition of RhoA/ROCK signaling, which regulates bleb formation by phosphorylating myosin light chain (MLC), as described by Fackler and Grosse, 2008.