Pals1a and aPKCλ are not essential for Schwann cell migration, division or myelination in zebrafish.

BACKGROUND: Schwann cells (SCs) are specialized glial cells of the peripheral nervous system that produce myelin and promote fast action potential propagation. In order to myelinate, SCs engage in a series of events that include migration and division along axons, followed by extensive cytoskeletal rearrangements that ensure axonal ensheathment and myelination. SCs are polarized and extend their processes along an abaxonal-adaxonal axis. Here, we investigate the role of the apical polarity proteins, Pals1a, and aPKCλ, in SC behavior during zebrafish development. RESULTS: We analyzed zebrafish nok and has mutants deficient for pals1a and aPKCλ function respectively. Using live imaging, transmission electron microscopy and whole mount immunostaining, we show that SCs can migrate and divide appropriately, exhibit normal radial sorting, express myelin markers and ensheath axons on time in has and nok mutants. CONCLUSIONS: Pals1a and aPKCλ are not essential for SC migration, division or myelination in zebrafish.

Timely Schwann cell division drives peripheral myelination in vivo via the laminin/cAMP pathway.

Schwann cells (SCs) migrate along peripheral axons and divide intensively to generate the right number of cells prior to axonal ensheathment; however, little is known regarding the temporal and molecular control of their division and its impact on myelination. We report that Sil, a spindle pole protein associated with autosomal recessive primary microcephaly, is required for temporal mitotic exit of SCs. In sil-deficient cassiopeia (csp-/-) mutants, SCs fail to radially sort and myelinate peripheral axons. Elevation of cAMP, but not Rac1 activity, in csp-/- restores myelin ensheathment. Most importantly, we show a significant decrease in laminin expression within csp-/- posterior lateral line nerve and that forcing Laminin 2 expression in csp-/- fully restores the ability of SCs to myelinate. Thus, we demonstrate an essential role for timely SC division in mediating laminin expression to orchestrate radial sorting and peripheral myelination in vivo.

Rgs4 is a regulator of mTOR activity required for motoneuron axon outgrowth and neuronal development in zebrafish.

The Regulator of G protein signaling 4 (Rgs4) is a member of the RGS proteins superfamily that modulates the activity of G-protein coupled receptors. It is mainly expressed in the nervous system and is linked to several neuronal signaling pathways; however, its role in neural development in vivo remains inconclusive. Here, we generated and characterized a rgs4 loss of function model (MZrgs4) in zebrafish. MZrgs4 embryos showed motility defects and presented reduced head and eye sizes, reflecting defective motoneurons axon outgrowth and a significant decrease in the number of neurons in the central and peripheral nervous system. Forcing the expression of Rgs4 specifically within motoneurons rescued their early defective outgrowth in MZrgs4 embryos, indicating an autonomous role for Rgs4 in motoneurons. We also analyzed the role of Akt, Erk and mechanistic target of rapamycin (mTOR) signaling cascades and showed a requirement for these pathways in motoneurons axon outgrowth and neuronal development. Drawing on pharmacological and rescue experiments in MZrgs4, we provide evidence that Rgs4 facilitates signaling mediated by Akt, Erk and mTOR in order to drive axon outgrowth in motoneurons and regulate neuronal numbers.

Rho GTPases Signaling in Zebrafish Development and Disease.

Cells encounter countless external cues and the specificity of their responses is translated through a myriad of tightly regulated intracellular signals. For this, Rho GTPases play a central role and transduce signals that contribute to fundamental cell dynamic and survival events. Here, we review our knowledge on how zebrafish helped us understand the role of some of these proteins in a multitude of in vivo cellular behaviors. Zebrafish studies offer a unique opportunity to explore the role and more specifically the spatial and temporal dynamic of Rho GTPases activities within a complex environment at a level of details unachievable in any other vertebrate organism.

Elmo1 function, linked to Rac1 activity, regulates peripheral neuronal numbers and myelination in zebrafish.

Peripheral nervous system development involves a tight coordination of neuronal birth and death and a substantial remodelling of the myelinating glia cytoskeleton to achieve myelin wrapping of its projecting axons. However, how these processes are coordinated through time is still not understood. We have identified engulfment and cell motility 1, Elmo1, as a novel component that regulates (i) neuronal numbers within the Posterior Lateral Line ganglion and (ii) radial sorting of axons by Schwann cells (SC) and myelination in the PLL system in zebrafish. Our results show that neuronal and myelination defects observed in elmo1 mutant are rescued through small GTPase Rac1 activation. Inhibiting macrophage development leads to a decrease in neuronal numbers, while peripheral myelination is intact. However, elmo1 mutants do not show defective macrophage activity, suggesting a role for Elmo1 in PLLg neuronal development and SC myelination independent of macrophages. Forcing early Elmo1 and Rac1 expression specifically within SCs rescues elmo1-/- myelination defects, highlighting an autonomous role for Elmo1 and Rac1 in radial sorting of axons by SCs and myelination. This uncovers a previously unknown function of Elmo1 that regulates fundamental aspects of PNS development.

Reduced recruitment of 53BP1 during interstrand crosslink repair is associated with genetically inherited attenuation of mitomycin C sensitivity in a family with Fanconi anemia.

The Fanconi anemia (FA) pathway is implicated in the repair of DNA interstrand crosslinks (ICL). In this process, it has been shown that FA factors regulate the choice for DNA double strand break repair towards homologous recombination (HR). As this mechanism is impaired in FA deficient cells exposed to crosslinking agents, an inappropriate usage of non-homologous end joining (NHEJ) leads to the accumulation of toxic chromosomal abnormalities. We studied a family with two FANCG patients and found a genetically inherited attenuation of mitomycin C sensitivity resulting in-vitro in an attenuated phenotype for one patient or in increased resistance for two healthy relatives. A heterozygous mutation in ATM was identified in these 3 subjects but was not directly linked to the observed phenotype. However, the attenuation of ICL sensitivity was associated with a reduced recruitment of 53BP1 during the course of ICL repair, and increased HR levels. These results further demonstrate the importance of favoring HR over NHEJ for the survival of cells challenged with ICLs.

HLA-G inhibition of NK-cell cytolytic function is uncoupled from tumor cell lipid raft reorganization.

HLA-G is a non-classical HLA class I molecule with tolerogenic properties and restricted tissue distribution. The expression of HLA-G can be induced by tumors thus providing an efficient way to escape the anti-tumoral immune response. Although lipid rafts regulate diverse immunological mechanisms their relationship with HLA-G remains controversial. Our results show that HLA-G-mediated inhibition of both the interaction between NK and tumor cells, and of intracellular calcium flux in NK cells conjugated to their target cells were independent of lipid raft integrity. In addition, cytotoxicity assays indicated that HLA-G continued to efficiently inhibit NK-cell cytolytic function in several different tumor cells independently of lipid raft integrity. Confocal microscopy with 3D reconstruction combined with biochemical analysis showed that HLA-G was mainly localized outside the lipid rafts of tumor cells after cross-linking with specific antibody and remained excluded from lipid rafts during interaction with the ILT2 inhibitory receptor of NK cells. This study indicates that the inhibitory function of HLA-G is uncoupled from lipid raft organization, further distinguishing HLA-G from classical HLA molecules and providing novel information in the understanding of tumor immune escape mechanism mediated through HLA-G.

Inhibition of human gamma delta [corrected] T-cell antitumoral activity through HLA-G: implications for immunotherapy of cancer.

Vγ9Vδ2 T cells play a crucial role in the antitumoral immune response through cytokine production and cytotoxicity. Although the expression of the immunomodulatory molecule HLA-G has been found in diverse tumors, its impact on Vγ9Vδ2 T-cell functions remains unknown. Here we showed that soluble HLA-G inhibits Vγ9Vδ2 T-cell proliferation without inducing apoptosis. Moreover, soluble HLA-G inhibited the Vγ9Vδ2 T-cell production of IFN-γ induced by phosphoantigen stimulation. The reduction in Vγ9Vδ2 T-cell IFN-γ production was also induced by membrane-bound or soluble HLA-G expressed by tumor cell lines. Finally, primary tumor cells inhibited Vγ9Vδ2 T-cell proliferation and IFN-γ production through HLA-G. In this context, HLA-G impaired Vγ9Vδ2 T-cell cytotoxicity by interacting with ILT2 inhibitory receptor. These data demonstrate that HLA-G inhibits the anti-tumoral functions of Vγ9Vδ2 T cells and imply that treatments targeting HLA-G could optimize Vγ9Vδ2 T-cell-mediated immunotherapy of cancer.

Membrane redistributions through multi-intercellular exchanges and serial trogocytosis.

Trogocytosis is a rapid transfer between cells of membranes and associated proteins. Trogocytic exchanges have been investigated between different cell types, mainly in two-cell systems, involving one donor and one acceptor cell type. Here, we studied trogocytosis in a more complex system, involving not only several immune cell subsets but also multiple tumor cells. We show that CD4(+) T cells, CD8(+) T cells and monocytes can acquire membrane patches and the intact proteins they contain from different tumor cells by multiple simultaneous trogocytoses. The trogocytic capabilities of CD4(+) and CD8(+) T cells were found to be similar, but inferior to that of autologous monocytes. Activated peripheral-blood mononuclear cells (PBMCs) may also exchange membranes between themselves in an all-autologous system. For this reason, monocytes are capable of acquiring membranes from multiple tumor cell sources, and transfer them again to autologous T cells, along with some of their own membranes (serial trogocytosis). Our data illustrate the extent of membrane exchanges between autologous activated immune effector cells and their environment, and how the cellular content of the local environment, including "bystander" cells, may impact the functions of immune effector cells.

ILT2/HLA-G interaction impairs NK-cell functions through the inhibition of the late but not the early events of the NK-cell activating synapse.

Natural killer (NK) cells play a crucial role in the antitumoral responses through cytolytic function and cytokine production. Expression of HLA-G at the surface of tumoral cells confers a protection against NK-cell cytolysis through its interaction with the ILT2 inhibitory receptor. Even though the role of this interaction on the inhibition of NK-cell cytotoxicity is well established, its effect on the molecular events occurring at the NK/target-cell synapse is not well characterized. We found that the interaction of the inhibitory receptor ILT2 with HLA-G inhibited the polarization of NK-cell lytic granules and the microtubule organizing center (MTOC) as well as the accumulation of filamentous actin (F-actin) at the area of contact. However, it did not affect the recruitment of the activatory receptor CD2 at the NK/target-cell interface. Even though CD2 was accumulated to the NK-cell synapse, the interaction of ILT2 with HLA-G efficiently inhibited intracellular calcium mobilization and IFN-gamma polarized production of NK cells. These results indicate that while the ILT2/HLA-G interaction leads to the inhibition of NK-cell functions, it displays differential effects on cytoskeleton reorganization and CD2 localization at the NK-cell synapse.-Favier, B., LeMaoult, J., Lesport, E., Carosella, E. D. ILT2/HLA-G interaction impairs NK-cell functions through the inhibition of the late but not the early events of the NK-cell-activating synapse.

Human melanoma cell secreting human leukocyte antigen-G5 inhibit natural killer cell cytotoxicity by impairing lytic granules polarization toward target cell.

Human leukocyte antigen-G (HLA-G) is a nonclassical tolerogenic molecule that can be expressed either as membrane bound (HLA-G1) or secreted (HLA-G5) isoforms. Upregulation of HLA-G1 or HLA-G5 expression by tumor cells constitutes an efficient way to escape from antitumoral immune responses. The inhibitory role of HLA-G1 on NK cell cytotoxicity is well characterized; however, that of the HLA-G5 isoform secreted by tumor is poorly understood. Our results indicate that the HLA-G5 isoform secreted by M8 melanoma cells is able to protect them from natural killer leukemia cell line (NKL) cytotoxicity. Analysis of NKL/M8-HLA-G5 conjugates by confocal microscopy demonstrates that the inhibition of NKL cytotoxic activity resulted from an impairment of NKL actin reorganization and perforin granules polarization toward M8-HLA-G5 target cell. This study also indicates that HLA-G5 soluble isoform remains evenly distributed in the cytoplasm of M8-HLA-G5 conjugated to NKL cells, suggesting that HLA-G5 does not require to polarize toward effector cell to induce efficient inhibition. These results highlight the inhibitory mechanisms mediated through HLA-G5 leading to tumor escape from NK cell cytotoxicity.