Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells.

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in C. elegans zygotes that the feedback relies on CDC-42 phosphorylation at serine 71 by aPKC, which in turn results in aPKC dissociation from CDC-42. The dissociated aPKC then associates with PAR-3 clusters, which are transported anteriorly by actomyosin-based cortical flow. Moreover, the turnover of aPKC-mediated CDC-42 phosphorylation regulates the organisation of the actomyosin cortex that drives aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust polarisation of many cell types.

Going with the flow: insights from Caenorhabditis elegans zygote polarization.

Cell polarity is the asymmetric distribution of cellular components along a defined axis. Polarity relies on complex signalling networks between conserved patterning proteins, including the PAR (partitioning defective) proteins, which become segregated in response to upstream symmetry breaking cues. Although the mechanisms that drive the asymmetric localization of these proteins are dependent upon cell type and context, in many cases the regulation of actomyosin cytoskeleton dynamics is central to the transport, recruitment and/or stabilization of these polarity effectors into defined subcellular domains. The transport or advection of PAR proteins by an actomyosin flow was first observed in the Caenorhabditis elegans zygote more than a decade ago. Since then a multifaceted approach, using molecular methods, high-throughput screens, and biophysical and computational models, has revealed further aspects of this flow and how polarity regulators respond to and modulate it. Here, we review recent findings on the interplay between actomyosin flow and the PAR patterning networks in the polarization of the C. elegans zygote. We also discuss how these discoveries and developed methods are shaping our understanding of other flow-dependent polarizing systems. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.

Morphogenetic degeneracies in the actomyosin cortex.

One of the great challenges in biology is to understand the mechanisms by which morphogenetic processes arise from molecular activities. We investigated this problem in the context of actomyosin-based cortical flow in C. elegans zygotes, where large-scale flows emerge from the collective action of actomyosin filaments and actin binding proteins (ABPs). Large-scale flow dynamics can be captured by active gel theory by considering force balances and conservation laws in the actomyosin cortex. However, which molecular activities contribute to flow dynamics and large-scale physical properties such as viscosity and active torque is largely unknown. By performing a candidate RNAi screen of ABPs and actomyosin regulators we demonstrate that perturbing distinct molecular processes can lead to similar flow phenotypes. This is indicative for a 'morphogenetic degeneracy' where multiple molecular processes contribute to the same large-scale physical property. We speculate that morphogenetic degeneracies contribute to the robustness of bulk biological matter in development.

aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity.

The conserved polarity effector proteins PAR-3, PAR-6, CDC-42, and atypical protein kinase C (aPKC) form a core unit of the PAR protein network, which plays a central role in polarizing a broad range of animal cell types. To functionally polarize cells, these proteins must activate aPKC within a spatially defined membrane domain on one side of the cell in response to symmetry-breaking cues. Using the Caenorhabditis elegans zygote as a model, we find that the localization and activation of aPKC involve distinct, specialized aPKC-containing assemblies: a PAR-3-dependent assembly that responds to polarity cues and promotes efficient segregation of aPKC toward the anterior but holds aPKC in an inactive state, and a CDC-42-dependent assembly in which aPKC is active but poorly segregated. Cycling of aPKC between these distinct functional assemblies, which appears to depend on aPKC activity, effectively links cue-sensing and effector roles within the PAR network to ensure robust establishment of polarity.

Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy.

Although single-gene loss-of-function analyses can identify components of particular processes, important molecules are missed owing to the robustness of biological systems. Here we show that large-scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 Caenorhabditis elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologues. We experimentally confirmed functional roles predicted by the network and characterized through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.

Growth and differentiation of the retina and the optic tectum in the medaka fish requires olSfrp5.

Secreted Frizzled-Related Proteins (SFRPs) are extracellular modulators of Wnt and Bmp signaling. Previous studies in birds and fishes have shown that Sfrp1, a member of this family, is strongly expressed throughout the development of the eye contributing to the specification of the eye field, retina neurogenesis and providing guidance information to retina ganglion cell axons. Here, we report that in medaka fish (Oryzias latipes) the expression of olSfrp5, which is closely related to olSfrp1, largely overlaps with that of olSfrp1 in the eye, but is additionally expressed in the developing midbrain and gut primordium. Morpholino-based interference with olSfrp5 expression causes microphthalmia and reduction of the tectum size associated with an increase in apoptotic cell death in these structures. Furthermore, interference with the levels of olSfrp5 expression impairs the patterning of the ventral portion of the optic cup, leading in some cases to a fissure coloboma. These early defects are followed by an abnormal retinal and tectal neurogenesis. In particular, only reduced numbers of photoreceptor and RGC were generated in olSfrp5 morphants retinas. The results point to an important role of olSfrp5 in visual system formation and indicate that olSfrp1 and olSfrp5, despite their overlapping expression, have only partially redundant function during eye development.

SFRP1 regulates the growth of retinal ganglion cell axons through the Fz2 receptor.

Axon growth is governed by the ability of growth cones to interpret attractive and repulsive guidance cues. Recent studies have shown that secreted signaling molecules known as morphogens can also act as axon guidance cues. Of the large family of Wnt signaling components, only Wnt4 and Wnt5 seem to participate directly in axon guidance. Here we show that secreted Frizzled-related protein 1 (SFRP1), a proposed Wnt signaling inhibitor, can directly modify and reorient the growth of chick and Xenopus laevis retinal ganglion cell axons. This activity does not require Wnt inhibition and is modulated by extracellular matrix molecules. Intracellularly, SFRP1 function requires G(alpha) protein activation, protein synthesis and degradation, and it is modulated by cyclic nucleotide levels. Because SFRP1 interacts with Frizzled-2 (Fz2) and interference with Fz2 expression abolishes growth cone responses to SFRP1, we propose a previously unknown function for this molecule: the ability to guide growth cone movement via the Fz2 receptor.

Morphogens as growth cone signalling molecules.

Morphogen signalling among cells is one of the most important mechanisms underlying the progressive patterning of embryos. Members of the hedgehog (Hh), wingless (Wnt), transforming growth factor-beta (TGFbeta), and fibroblast growth factor (Fgf) families of extracellular signalling molecules act as morphogens. Recent studies have demonstrated that members of these four families of proteins, secreted by well-characterised organiser centres in the central nervous system (CNS) as floor plate or midbrain-hindbrain boundary, are reused at later developmental stages to control axon growth. Here, we have summarised the evidence for this novel idea with a particular emphasis on those related to Shh and Wnt signalling-the object of some works in our laboratory.

Evaluation of SFRP1 as a candidate for human retinal dystrophies.

PURPOSE: Secreted Frizzled Related Proteins (SFRPs) are soluble molecules capable of modulating Wnt signalling. Different lines of evidence indicate that SFRP activity is related with the development and function of the retina photoreceptor cells as well as with their apoptotic degeneration associated with the onset of different cases of retinal dystrophy (RD). Because the genetic causes of many retinal dystrophies still need to be determined, we have asked whether mutations in the SFRP genes might be associated with retinal dystrophies. METHODS: Here we describe the genomic structure of SFRP1, SFRP2, and SFRP5 and a mutational screening of SFRP1 in 325 individuals affected by various non X-linked forms of inherited retinal disorders. RESULTS: Three polymorphic variants were identified. CONCLUSIONS: Our data, so far, exclude SFRP1 as a molecular cause of RD, since two out of three genetic variants of the gene were present in both RD patients and normal population.

SFRP1 modulates retina cell differentiation through a beta-catenin-independent mechanism.

Secreted frizzled related proteins (SFRPs) are soluble molecules capable of binding WNTS and preventing the activation of their canonical signalling cascade. Here we show that Sfrp1 contributes to chick retina differentiation with a mechanism that does not involve modifications in the transcriptional activity of beta-catenin. Thus, addition of SFRP1 to dissociated retinal cultures or retroviral mediated overexpression of the molecule consistently promoted retinal ganglion and cone photoreceptor cell generation, while decreasing the number of amacrine cells. Measure of the activity of the beta-catenin-responsive Tcf-binding site coupled to a luciferase reporter in transiently transfected retinal cells showed that Sfrp1 was unable to modify the basal beta-catenin transcriptional activity of the retina cells. Interestingly, a dominant-negative form of GSK3beta gave similar results to those of Sfrp1, and a phosphorylation-dependent inhibition of GSK3beta activity followed SFRP1 treatment of retina cells. Furthermore, retroviral mediated expression of a dominant-negative form of GSK3beta induced a retina phenotype similar to that observed after Sfrp1 overexpression, suggesting a possible involvement of this kinase in SFRP1 function.

DNA polymerase lambda, a novel DNA repair enzyme in human cells.

DNA polymerase lambda (pol lambda) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in pol lambda supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol lambda to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol lambda inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5'-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol lambda is a novel beta-like DNA polymerase. However, the high affinity of pol lambda for dNTPs (37-fold over pol beta) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol beta and pol lambda have nonredundant in vivo functions.