Counterregulation of cAMP-directed kinase activities controls ciliogenesis

The primary cilium emanates from the cell surface of growth-arrested cells and plays a central role in vertebrate development and tissue homeostasis. The mechanisms that control ciliogenesis have been extensively explored. However, the intersection between GPCR signaling and the ubiquitin pathway in the control of cilium stability is unknown. Here, we observe that cAMP elevation promotes cilia resorption. At centriolar satellites, we identify a multimeric complex nucleated by PCM1 that includes two kinases, NEK10 and PKA, and the E3 ubiquitin ligase CHIP. We show that NEK10 is essential for ciliogenesis in mammals and for the development of medaka fish. PKA phosphorylation primes NEK10 for CHIP-mediated ubiquitination and proteolysis resulting in cilia resorption. Dearangement of this control mechanism occurs in proliferative and genetic disorders. These findings unveil a pericentriolar kinase signalosome that efficiently links the cAMP cascade with the ubiquitin-proteasome system, controlling essential aspects of ciliogenesis

High-Resolution Electron Microscopy of Semiconductor Heterostructures and Nanostructures

This chapter briefly describes the fundamentals of high-resolution electron microscopy techniques. In particular, the Peak Pairs approach for strain mapping with atomic column resolution, and a quantitative procedure to extract atomic column compositional information from Z-contrast high-resolution images are presented. It also reviews the structural, compositional, and strain results obtained by conventional and advanced transmission electron microscopy methods on a number of III–V semiconductor nanostructures and heterostructures

The Impact of miRNAs in Health and Disease of Retinal Pigment Epithelium

MicroRNAs (miRNAs), a class of non-coding RNAs, are essential key players in the control of biological processes in both physiological and pathological conditions. miRNAs play important roles in fine tuning the expression of many genes, which often have roles in common molecular networks. miRNA dysregulation thus renders cells vulnerable to aberrant fluctuations in genes, resulting in degenerative diseases. The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells that resides between the light-sensitive photoreceptors (PR) and the choriocapillaris. The demanding physiological functions of RPE cells require precise gene regulation for the maintenance of retinal homeostasis under stress conditions and the preservation of vision. Thus far, our understanding of how miRNAs function in the homeostasis and maintenance of the RPE has been poorly addressed, and advancing our knowledge is central to harnessing their potential as therapeutic agents to counteract visual impairment. This review focuses on the emerging roles of miRNAs in the function and health of the RPE and on the future exploration of miRNA-based therapeutic approaches to counteract blinding diseases

Electric-Field-Induced Deformation Dynamics of a Single Nematic Disclination

Disclinations in nematic liquid crystals usually adopt a straight shape in order to minimize their elastic energy. Once created in the course of a nonequilibrium process such as a temperature quench from the isotropic to the nematic phase, the topologically stable disclinations of half-integer strength either annihilate each other in pairs of opposite strength or form topologically unstable disclinations of integer strength. In this article, we demonstrate that the annihilation process can be inhibited and the defects can be deformed by an applied electric field. We study the disclination lines in the deep uniaxial nematic phase, located at the boundary between two different types of walls, the so-called π wall (a planar soliton stabilized by the surface anchoring) and the Brochard-Léger (BL) wall stabilized by the applied electric field. By changing the electric voltage, one can control the energy of director deformations associated with the two walls and thus control the deformation and dynamics of the disclination line. At small voltages, the disclinations are straight lines connecting the opposite plates of the cell, located at the two ends of the π walls. The π walls tend to shrink. When the voltage increases above EF, the Fréedericksz threshold, the BL walls appear and connect pairs of disclinations along a path complementary to the π wall. At E>2EF, the BL walls store sufficient energy to prevent shrinking of the π walls. Reconstruction of the three-dimensional director configuration using a fluorescent confocal polarizing microscopy demonstrates that the disclinations are strongly bent in the region between the π and the BL walls. The distortions and the related dynamics are associated with the transformation of the BL wall into two surface disclination lines; we characterize it experimentally as a function of the applied electric field, the cell thickness, and the sample temperature. A simple model captures the essential details of the experimental data.</p

Loss of Ciliary Gene Bbs8 Results in Physiological Defects in the Retinal Pigment Epithelium

Primary cilia are sensory organelles vital for developmental and physiological processes. Their dysfunction causes a range of phenotypes including retinopathies. Although primary cilia have been described in the retinal pigment epithelium (RPE), little is known about their contribution to biological processes within this tissue. Ciliary proteins are increasingly being identified in non-ciliary locations and might carry out additional functions, disruption of which possibly contributes to pathology. The RPE is essential for maintaining photoreceptor cells and visual function. We demonstrate that upon loss of Bbs8, predominantly thought to be a ciliary gene, the RPE shows changes in gene and protein expression initially involved in signaling pathways and developmental processes, and at a later time point RPE homeostasis and function. Differentially regulated molecules affecting the cytoskeleton and cellular adhesion, led to defective cellular polarization and morphology associated with a possible epithelial-to-mesenchymal transition (EMT)-like phenotype. Our data highlights the benefit of combinatorial “omics” approaches with in vivo data for investigating the function of ciliopathy proteins. It also emphasizes the importance of ciliary proteins in the RPE and their contribution to visual disorders, which must be considered when designing treatment strategies for retinal degeneration

MLL4-associated condensates counterbalance Polycomb-mediated nuclear mechanical stress in Kabuki syndrome

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid–liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus

The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation

The primary cilium is a microtubule-based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X-linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin-proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G-protein-coupled receptor (GPCR)-cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2-UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non-phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders

Optimization of silver nanoparticles production by laser ablation in water using a 150-ps laser

Silver nanoparticles were synthesized by laser ablation in liquid (water) using a 150-ps Nd:YAG laser. Due to their extraordinary characteristics, especially when obtained by this method providing high purity and high stability of colloids, silver NPs are nowadays highly important in various applications. The objective of this study was to optimize the process parameters in order to achieve the highest possible yield while retaining small particle size. Yield/mass concentration of the obtained particles was measured depending on different parameters: time of irradiation, pulse energy, position regarding the focus, and number of irradiation locations. The conditions providing relatively high yield, small particle size, highest production rate, and highest efficiency are 7 mJ, 15-min irradiation time (9000 pulses), and target position similar to 4 mm in front of the lens focus. The results are compared with the results obtained by the longer nanosecond as well as the ultrashort pulsed lasers. A possible physical explanation is given