Combinations of deletion and missense variations of the dynein-2 DYNC2LI1 subunit found in skeletal ciliopathies cause ciliary defects

Cilia play crucial roles in sensing and transducing extracellular signals. Bidirectional protein trafficking within cilia is mediated by the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes, with the aid of kinesin-2 and dynein-2 motors. The dynein-2 complex drives retrograde trafficking of the IFT machinery after its transportation to the ciliary tip as an IFT cargo. Mutations in genes encoding the dynein-2-specific subunits (DYNC2H1, WDR60, WDR34, DYNC2LI1, and TCTEX1D2) are known to cause skeletal ciliopathies. We here demonstrate that several pathogenic variants of DYNC2LI1 are compromised regarding their ability to interact with DYNC2H1 and WDR60. When expressed in DYNC2LI1-knockout cells, deletion variants of DYNC2LI1 were unable to rescue the ciliary defects of these cells, whereas missense variants, as well as wild-type DYNC2LI1, restored the normal phenotype. DYNC2LI1-knockout cells coexpressing one pathogenic deletion variant together with wild-type DYNC2LI1 demonstrated a normal phenotype. In striking contrast, DYNC2LI1-knockout cells coexpressing the deletion variant in combination with a missense variant, which mimics the situation of cells of compound heterozygous ciliopathy individuals, demonstrated ciliary defects. Thus, DYNC2LI1 deletion variants found in individuals with skeletal ciliopathies cause ciliary defects when combined with a missense variant, which expressed on its own does not cause substantial defects.

Proteomimetics of Natural Regulators of JAK–STAT Pathway: Novel Therapeutic Perspectives

The JAK-STAT pathway is a crucial cellular signaling cascade, including an intricate network of Protein–protein interactions (PPIs) responsible for its regulation. It mediates the activities of several cytokines, interferons, and growth factors and transduces extracellular signals into transcriptional programs to regulate cell growth and differentiation. It is essential for the development and function of both innate and adaptive immunities, and its aberrant deregulation was highlighted in neuroinflammatory diseases and in crucial mechanisms for tumor cell recognition and tumor-induced immune escape. For its involvement in a multitude of biological processes, it can be considered a valuable target for the development of drugs even if a specific focus on possible side effects associated with its inhibition is required. Herein, we review the possibilities to target JAK–STAT by focusing on its natural inhibitors as the suppressor of cytokine signaling (SOCS) proteins. This protein family is a crucial checkpoint inhibitor in immune homeostasis and a valuable target in immunotherapeutic approaches to cancer and immune deficiency disorders.

Pros and Cons of Pharmacological Manipulation of cGMP-PDEs in the Prevention and Treatment of Breast Cancer

The cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers responsible for translating extracellular signals to intracellular biological responses in both normal and tumor cells. When these signals are aberrant or missing, cells may undergo neoplastic transformation or become resistant to chemotherapy. cGMP-hydrolyzing phosphodiesterases (PDEs) are attracting tremendous interest as drug targets for many diseases, including cancer, where they regulate cell growth, apoptosis and sensitization to radio- and chemotherapy. In breast cancer, PDE5 inhibition is associated with increased intracellular cGMP levels, which is responsible for the phosphorylation of PKG and other downstream molecules involved in cell proliferation or apoptosis. In this review, we provide an overview of the most relevant studies regarding the controversial role of PDE inhibitors as off-label adjuvants in cancer therapy.

Extracellular Signalling Modulates Scar/WAVE Complex Activity through Abi Phosphorylation

The lamellipodia and pseudopodia of migrating cells are produced and maintained by the Scar/WAVE complex. Thus, actin-based cell migration is largely controlled through regulation of Scar/WAVE. Here, we report that the Abi subunit—but not Scar—is phosphorylated in response to extracellular signalling in Dictyostelium cells. Like Scar, Abi is phosphorylated after the complex has been activated, implying that Abi phosphorylation modulates pseudopodia, rather than causing new ones to be made. Consistent with this, Scar complex mutants that cannot bind Rac are also not phosphorylated. Several environmental cues also affect Abi phosphorylation—cell-substrate adhesion promotes it and increased extracellular osmolarity diminishes it. Both unphosphorylatable and phosphomimetic Abi efficiently rescue the chemotaxis of Abi KO cells and pseudopodia formation, confirming that Abi phosphorylation is not required for activation or inactivation of the Scar/WAVE complex. However, pseudopodia and Scar patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Dictyostelium, in which Scar and Abi are both unphosphorylatable, can still form pseudopods, but migrate substantially faster. We conclude that extracellular signals and environmental responses modulate cell migration by tuning the behaviour of the Scar/WAVE complex after it has been activated.

Alternative Splicing of MAPKs in the Regulation of Signaling Specificity

The mitogen-activated protein kinase (MAPK) cascades transmit signals from extracellular stimuli to a variety of distinct cellular processes. The MAPKKs in each cascade specifically phosphorylate and activate their cognate MAPKs, indicating that this step funnels various signals into a seemingly linear pathway. Still, the effects of these cascades vary significantly, depending on the identity of the extracellular signals, which gives rise to proper outcomes. Therefore, it is clear that the specificity of the signals transmitted through the cascades is tightly regulated in order to secure the desired cell fate. Indeed, many regulatory components or processes that extend the specificity of the cascades have been identified. Here, we focus on a less discussed mechanism, that is, the role of distinct components in each tier of the cascade in extending the signaling specificity. We cover the role of distinct genes, and the alternatively spliced isoforms of MAPKKs and MAPKs, in the signaling specificity. The alternatively spliced MEK1b and ERK1c, which form an independent signaling route, are used as the main example. Unlike MEK1/2 and ERK1/2, this route’s functions are limited, including mainly the regulation of mitotic Golgi fragmentation. The unique roles of the alternatively spliced isoforms indicate that these components play an essential role in determining the proper cell fate in response to distinct stimulations.

Disentangling bias between Gq, GRK2, and arrestin3 recruitment to the M3 muscarinic acetylcholine receptor

G protein-coupled receptors (GPCRs) transmit extracellular signals to the inside by activation of intracellular effector proteins. Different agonists can promote differential receptor-induced signaling responses – termed bias – potentially by eliciting different levels of recruitment of effector proteins. As activation and recruitment of effector proteins might influence each other, thorough analysis of bias is difficult. Here, we compared the efficacy of seven agonists to induce G protein, G protein-coupled receptor kinase 2 (GRK2), as well as arrestin3 binding to the muscarinic acetylcholine receptor M3 by utilizing FRET-based assays. In order to avoid interference between these interactions, we studied GRK2 binding in the presence of inhibitors of Gi and Gq proteins and analyzed arrestin3 binding to prestimulated M3 receptors to avoid differences in receptor phosphorylation influencing arrestin recruitment. We measured substantial differences in the agonist efficacies to induce M3R-arrestin3 versus M3R-GRK2 interaction. However, the rank order of the agonists for G protein- and GRK2-M3R interaction was the same, suggesting that G protein and GRK2 binding to M3R requires similar receptor conformations, whereas requirements for arrestin3 binding to M3R are distinct.

Lipid droplets as endogenous intracellular microlenses

Using a single biological element as a photonic component with well-defined features has become a new intriguing paradigm in biophotonics. Here we show that endogenous lipid droplets in the mature adipose cells can behave as fully biocompatible microlenses to strengthen the ability of microscopic imaging as well as detecting intra- and extracellular signals. By the assistance of biolenses made of the lipid droplets, enhanced fluorescence imaging of cytoskeleton, lysosomes, and adenoviruses has been achieved. At the same time, we demonstrated that the required excitation power can be reduced by up to 73%. The lipidic microlenses are finely manipulated by optical tweezers in order to address targets and perform their real-time imaging inside the cells. An efficient detecting of fluorescence signal of cancer cells in extracellular fluid was accomplished due to the focusing effect of incident light by the lipid droplets. The lipid droplets acting as endogenous intracellular microlenses open the intriguing route for a multifunctional biocompatible optics tool for biosensing, endoscopic imaging, and single-cell diagnosis.

How do spike collisions affect spike sorting performance?

Recently, a new generation of devices have been developed to record neural activity simultaneously from hundreds of electrodes with a very high spatial density, both for in vitro and in vivo applications. While these advances enable to record from many more cells, they also dramatically increase the amount overlapping "synchronous" spikes (colliding in space and/or in time), challenging the already complicated process of spike sorting (i.e. extracting isolated single-neuron activity from extracellular signals). In this work, we used synthetic ground-truth recordings to quantitatively benchmark the performance of state-of-the-art spike sorters focusing specifically on spike collisions. Our results show that while modern template-matching based algorithms are more accurate than density-based approaches, all methods, to some extent, failed to detect synchronous spike events of neurons with similar extracellular signals. Interestingly, the performance of the sorters is not largely affected by the the spiking activity in the recordings, with respect to average firing rates and spike-train correlation levels.

An extracellular matrix damage sensor signals through membrane-associated kinase DRL-1 to mediate cytoprotective responses in Caenorhabditis elegans

We and others previously identified circumferential bands of collagen named annular furrows as key components of a damage sensor in the cuticle of Caenorhabditis elegans that regulates cytoprotective genes. Mutation or loss of non-collagen secreted proteins OSM-7, OSM-8, and OSM-11 activate the same cytoprotective responses without obvious changes to the cuticle indicating that other extracellular proteins are involved. Here, we used RNAi screening to identify protein kinase DRL-1 as a key modulator of cytoprotective gene expression and stress resistance in furrow and extracellular OSM protein mutants. DRL-1 functions downstream from furrow disruption and is expressed in cells that induce cytoprotective genes. DRL-1 is not required for expression of cytoprotective genes under basal or oxidative stress conditions consistent with specificity to extracellular signals. DRL-1 was previously shown to regulate longevity via a ‘Dietary Restriction-Like’ state, but it functions downstream from furrow disruption by a distinct mechanism. The kinase domain of DRL-1 is related to mammalian MEKK3, and MEKK3 is recruited to a plasma membrane osmosensor complex by a scaffold protein. In C. elegans, DRL-1 contains an atypical hydrophobic C-terminus with predicted transmembrane domains and is constitutively expressed at or near the plasma membrane where it could function to receive extracellular damage signals for cells that mount cytoprotective responses.

The mechanosensitive channel Piezo1 cooperates with Semaphorin to control neural crest migration

Cells are permanently exposed to a multitude of different kind of signals; however how cells respond to simultaneous extracellular signals within a complex in vivo environment is poorly understood. Here, we studied the role of the mechanosensitive ion channel Piezo1 on the migration of the neural crest (NC), a multipotent embryonic cell population. We identify that Piezo1 is required for the migration of Xenopus cephalic NC. We show that loss of Piezo1 promotes focal adhesion turnover and cytoskeletal dynamics by controlling Rac1 activity, leading to increased speed of migration. Moreover, overactivation of Rac1, due to Piezo1 inhibition, counteracts cell migration inhibitory signals by Semaphorins 3A and 3F, generating aberrant neural crest invasion in vivo. Thus, we find that, for directional migration in vivo, neural crest cells require a tight regulation of Rac1, by Semaphorins and Piezo1. We reveal here that a balance between a myriad of signals through Rac1 dictates cell migration in vivo, a mechanism that is likely to be conserved in other cell migration processes.