Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch.

The serine-threonine kinase Akt plays a fundamental role in cell survival, metabolism, proliferation, and migration. To keep these essential processes under control, Akt activity and stability must be tightly regulated; otherwise, life-threatening conditions might prevail. Although it is well understood that phosphorylation regulates Akt activity, much remains to be known about how its stability is maintained. Here, we characterize BAG5, a chaperone regulator, as a novel Akt-interactor and substrate that attenuates Akt stability together with Hsp70. BAG5 switches monoubiquitination to polyubiquitination of Akt and increases its degradation caused by Hsp90 inhibition and Hsp70 overexpression. Akt interacts with BAG5 at the linker region that joins the first and second BAG domains and phosphorylates the first BAG domain. The Akt-BAG5 complex is formed in serum-starved conditions and dissociates in response to HGF, coincident with BAG5 phosphorylation. BAG5 knockdown attenuated Akt degradation and facilitated its activation, whereas the opposite effect was caused by BAG5 overexpression. Altogether, our results indicate that Akt stability and signaling are dynamically regulated by BAG5, depending on growth factor availability.

Zebrafish Primordial Germ Cell Migration.

Similar to many other organisms, zebrafish primordial germ cells (PGCs) are specified at a location distinct from that of gonadal somatic cells. Guided by chemotactic cues, PGCs migrate through embryonic tissues toward the region where the gonad develops. In this process, PGCs employ a bleb-driven amoeboid migration mode, characterized by low adhesion and high actomyosin contractility, a strategy used by other migrating cells, such as leukocytes and certain types of cancer cells. The mechanisms underlying the motility and the directed migration of PGCs should be robust to ensure arrival at the target, thereby contributing to the fertility of the organism. These features make PGCs an excellent model for studying guided single-cell migration in vivo. In this review, we present recent findings regarding the establishment and maintenance of cell polarity that are essential for motility and discuss the mechanisms by which cell polarization and directed migration are controlled by chemical and physical cues.

A mathematical model for bleb regulation in zebrafish primordial germ cells.

Blebs are cell protrusions generated by local membrane-cortex detachments followed by expansion of the plasma membrane. Blebs are formed by some migrating cells, e.g. primordial germ cells of the zebrafish. While blebs occur randomly at each part of the membrane in unpolarized cells, a polarization process guarantees the occurrence of blebs at a preferential site and thereby facilitates migration toward a specified direction. Little is known about the factors involved in the controlled and directed bleb generation, yet recent studies revealed the influence of an intracellular flow and the stabilizing role of the membrane-cortex linker molecule Ezrin. Based on this information, we develop and analyse a coupled bulk-surface model describing a potential cellular mechanism by which a bleb could be induced at a controlled site. The model rests upon intracellular Darcy flow and a diffusion-advection-reaction system, describing the temporal evolution from a homogeneous to a strongly anisotropic Ezrin distribution. We prove the well-posedness of the mathematical model and show that simulations qualitatively correspond to experimental observations, suggesting that indeed the interaction of an intracellular flow with membrane proteins can be the cause of the Ezrin redistribution accompanying bleb formation.

Chemokine-biased robust self-organizing polarization of migrating cells in vivo.

To study the mechanisms controlling front-rear polarity in migrating cells, we used zebrafish primordial germ cells (PGCs) as an in vivo model. We find that polarity of bleb-driven migrating cells can be initiated at the cell front, as manifested by actin accumulation at the future leading edge and myosin-dependent retrograde actin flow toward the other side of the cell. In such cases, the definition of the cell front, from which bleb-inhibiting proteins such as Ezrin are depleted, precedes the establishment of the cell rear, where those proteins accumulate. Conversely, following cell division, the accumulation of Ezrin at the cleavage plane is the first sign for cell polarity and this aspect of the cell becomes the cell back. Together, the antagonistic interactions between the cell front and back lead to a robust polarization of the cell. Furthermore, we show that chemokine signaling can bias the establishment of the front-rear axis of the cell, thereby guiding the migrating cells toward sites of higher levels of the attractant. We compare these results to a theoretical model according to which a critical value of actin treadmilling flow can initiate a positive feedback loop that leads to the generation of the front-rear axis and to stable cell polarization. Together, our in vivo findings and the mathematical model, provide an explanation for the observed nonoriented migration of primordial germ cells in the absence of the guidance cue, as well as for the directed migration toward the region where the gonad develops.

Gßγ recruits and activates P-Rex1 via two independent binding interfaces.

Gßγ marks the inner side of the plasma membrane where chemotactic GPCRs activate Rac to lead the assembly of actin filaments that push the cell to move forward. Upon dissociation from heterotrimeric Gi, Gßγ recruits and activates P-Rex1, a Rac guanine nucleotide exchange factor (RacGEF). This cytosolic chemotactic effector is kept inactive by intramolecular interactions. The mechanism by which Gßγ stimulates P-Rex1 has been debated. We hypothesized that Gßγ activates P-Rex1 by a two-step mechanism based on independent interaction interfaces to recruit and unroll this RacGEF. Using pulldown assays, we found that Gßγ binds P-Rex1-DH/PH as well as PDZ-PDZ domains. These domains and the DEP-DEP tandem interact among them and dissociate upon binding with Gßγ, arguing for a stimulatory allosteric effect. In addition, P-Rex1 catalytic activity is inhibited by its C-terminal domain. To discern P-Rex1 recruitment from activation, we studied Q-Rhox, a synthetic RhoGEF having the PDZ-RhoGEF catalytic DH/PH module, insensitive to Gßγ, swapped into P-Rex1. Gßγ recruited Q-Rhox to the plasma membrane, indicating that Gßγ/PDZ-PDZ interaction interface plays a role on P-Rex1 recruitment. In conclusion, we reconcile previous findings and propose a mechanistic model of P-Rex1 activation; accordingly, Gßγ recruits P-Rex1 via the Gßγ/PDZ-PDZ interface followed by a second contact involving the Gßγ/DH/PH interface to unleash P-Rex1 RacGEF activity at the plasma membrane.