Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing.

In Drosophila, female development is governed by a single RNA-binding protein, Sex-lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Sxl expression in female flies is maintained by an auto-regulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now, it remained unclear how males prevent accidental triggering of the Sxl expression cascade and protect themselves against runaway protein production. Here, we identify the protein Sister-of-Sex-lethal (Ssx) as an inhibitor of Sxl auto-regulatory splicing. Sxl and Ssx have a comparable RNA-binding specificity and compete for binding to RNA regulatory elements present in the Sxl transcript. In cultured Drosophila cells, Sxl-induced changes to alternative splicing can be reverted by the expression of Ssx. Moreover, in adult male flies ablation of the ssx gene results in a low level of productive Sxl mRNA splicing and Sxl protein production in isolated, clonal cell populations. In sum, this demonstrates that Ssx safeguards male animals against Sxl protein production to reinforce a stable, male-specific gene expression pattern.

Redundant regulation of localization and protein stability of DmPar3.

Apical-basal polarity is an important characteristic of epithelia and Drosophila neural stem cells. The conserved Par complex, which consists of the atypical protein kinase C and the scaffold proteins Baz and Par6, is a key player in the establishment of apical-basal cell polarity. Membrane recruitment of Baz has been reported to be accomplished by several mechanisms, which might function in redundancy, to ensure the correct localization of the complex. However, none of the described interactions was sufficient to displace the protein from the apical junctions. Here, we dissected the role of the oligomerization domain and the lipid-binding motif of Baz in vivo in the Drosophila embryo. We found that these domains function in redundancy to ensure the apical junctional localization of Baz: inactivation of only one domain is not sufficient to disrupt the function of Baz during apical-basal polarization of epithelial cells and neural stem cells. In contrast, mutation of both domains results in a strongly impaired protein stability and a phenotype characterized by embryonic lethality and an impaired apical-basal polarity in the embryonic epithelium and neural stem cells, resembling a baz-loss of function allele. Strikingly, the binding of Baz to the transmembrane proteins E-Cadherin, Echinoid, and Starry Night was not affected in this mutant protein. Our findings reveal a redundant function of the oligomerization and the lipid-binding domain, which is required for protein stability, correct subcellular localization, and apical-basal cell polarization.

Evolutionary conservation of Kv3.1 in the barn owl Tyto alba.

For prey capture in the dark, the barn owl Tyto alba has evolved into an auditory specialist with an exquisite capability of sound localization. Adaptations include asymmetrical ears, enlarged auditory processing centers, the utilization of minute interaural time differences, and phase locking along the entire hearing range up to 10 kHz. Adaptations on the molecular level have not yet been investigated. Here, we tested the hypothesis that divergence in the amino acid sequence of the voltage-gated K(+) channel Kv3.1 contributes to the accuracy and high firing rates of auditory neurons in the barn owl. We therefore cloned both splice variants of Kcnc1, the gene encoding Kv3.1. Both splice variants, Kcnc1a and Kcnc1b, encode amino acids identical to those of the chicken, an auditory generalist. Expression analyses confirmed neural-restricted expression of the channel. In summary, our data reveal strong evolutionary conservation of Kcnc1 in the barn owl and point to other genes involved in auditory specializations of this animal. The data also demonstrate the feasibility to address neuroethological questions in organisms with no reference genome by molecular approaches. This will open new avenues for neuroethologists working in these organisms.

Controlling the master-upstream regulation of the tumor suppressor LKB1.

The tumor suppressor LKB1 is an essential serine/threonine kinase, which regulates various cellular processes such as cell metabolism, cell proliferation, cell polarity, and cell migration. Germline mutations in the STK11 gene (encoding LKB1) are the cause of the Peutz-Jeghers syndrome, which is characterized by benign polyps in the intestine and a higher risk for the patients to develop intestinal and extraintestinal tumors. Moreover, mutations and misregulation of LKB1 have been reported to occur in most types of tumors and are among the most common aberrations in lung cancer. LKB1 activates several downstream kinases of the AMPK family by direct phosphorylation in the T-loop. In particular the activation of AMPK upon energetic stress has been intensively analyzed in various diseases, including cancer to induce a metabolic switch from anabolism towards catabolism to regulate energy homeostasis and cell survival. In contrast, the regulation of LKB1 itself has long been only poorly understood. Only in the last years, several proteins and posttranslational modifications of LKB1 have been analyzed to control its localization, activity and recognition of substrates. Here, we summarize the current knowledge about the upstream regulation of LKB1, which is important for the understanding of the pathogenesis of many types of tumors.

Imaging in Biologically-Relevant Environments with AFM Using Stiff qPlus Sensors.

High-resolution imaging of soft biological samples with atomic force microscopy (AFM) is challenging because they must be imaged with small forces to prevent deformation. Typically, AFM of those samples is performed with soft silicon cantilevers (k ≈ 0.1-10 N/m) and optical detection in a liquid environment. We set up a new microscope that uses a stiff qPlus sensor (k ≥ 1 kN/m). Several complex biologically-relevant solutions are non-transparent, and even change their optical properties over time, such as the cell culture medium we used. While this would be problematic for AFM setups with optical detection, it is no problem for our qPlus setup which uses electrical detection. The high stiffness of the qPlus sensor allows us to use small amplitudes in frequency-modulation mode and obtain high Q factors even in liquid. The samples are immersed in solution in a liquid cell and long tips are used, with only the tip apex submerged. We discuss the noise terms and compare the minimal detectable signal to that of soft cantilevers. Atomic resolution of muscovite mica was achieved in various liquids: H2O, Tris buffer and a cell culture medium. We show images of lipid membranes in which the individual head groups are resolved.

Structural basis for the interaction between the cell polarity proteins Par3 and Par6.

Polarity is a fundamental property of most cell types. The Par protein complex is a major driving force in generating asymmetrically localized protein networks and consists of atypical protein kinase C (aPKC), Par3, and Par6. Dysfunction of this complex causes developmental abnormalities and diseases such as cancer. We identified a PDZ domain-binding motif in Par6 that was essential for its interaction with Par3 in vitro and for Par3-mediated membrane localization of Par6 in cultured cells. In fly embryos, we observed that the PDZ domain-binding motif was functionally redundant with the PDZ domain in targeting Par6 to the cortex of epithelial cells. Our structural analyses by x-ray crystallography and NMR spectroscopy showed that both the PDZ1 and PDZ3 domains but not the PDZ2 domain in Par3 engaged in a canonical interaction with the PDZ domain-binding motif in Par6. Par3 thus has the potential to recruit two Par6 proteins simultaneously, which may facilitate the assembly of polarity protein networks through multivalent PDZ domain interactions.

Membrane-binding and activation of LKB1 by phosphatidic acid is essential for development and tumour suppression.

The serine/threonine kinase LKB1 regulates various cellular processes such as cell proliferation, energy homeostasis and cell polarity and is frequently downregulated in various tumours. Many downstream pathways controlled by LKB1 have been described but little is known about the upstream regulatory mechanisms. Here we show that targeting of the kinase to the membrane by a direct binding of LKB1 to phosphatidic acid is essential to fully activate its kinase activity. Consequently, LKB1 mutants that are deficient for membrane binding fail to activate the downstream target AMPK to control mTOR signalling. Furthermore, the in vivo function of LKB1 during development of Drosophila depends on its capacity to associate with membranes. Strikingly, we find LKB1 to be downregulated in malignant melanoma, which exhibit aberrant activation of Akt and overexpress phosphatidic acid generating Phospholipase D. These results provide evidence for a fundamental mechanism of LKB1 activation and its implication in vivo and during carcinogenesis.