Unraveling the complexity of Drosophila immune cells: a focus on blood cell heterogeneity, plasticity, and dynamics.

Until recently, Drosophila immune cells, named hemocytes, were only characterized and clustered into subpopulations based on morphology, function, and a limited number of marker genes. Thus, the cells were subdivided into plasmatocytes, crystal cells, and lamellocytes, and the population of Drosophila plasmatocytes has been thought to be a relatively homogenous sub-group. However, new single-cell RNA sequencing approaches revealed a complex heterogeneity and plasticity within this cell population. Particularly, the transcriptional profile of hemocytes changes in response to the significant environmental changes during the transition from the larval to the pupal stage of development. Additionally, the here identified complex heterogeneity of Drosophila immune cells includes cells derived from embryonic and lymph gland precursors with a highly migratory and immune responsive Posterior Signaling Center (PSC) niche-like blood cell type that persists into the adult fly. Those niche-like progenitor cells could be potential precursor cells for known hemocyte subpopulations like the lamellocytes. However, so far, lamellocytes have only been reported to differentiate from progenitor hemocytes in response to infestation by parasitoid wasps. Hemocytes rely on the ability to rapidly adapt to different immune challenges and migrate to locations where they are needed. This is only possible because of the highly regulated actin cytoskeleton. Dynamic remodeling of this dense network – especially inside the lamellipodium - is highly regulated and a crucial step for the necessary cell shape changes to allow locomotion and efficient immune defense. A key regulator, which builds lamellipodial protrusions and thereby drives cell migration, is the Arp2/3 complex, which in turn is activated by the hetero-pentameric WAVE regulatory complex. The role of phosphorylation in regulating WAVE, an indispensable part of the complex, has been addressed in various in vitro studies. However, the in vivo relevance of WAVE phosphorylation on actin dynamics is still poorly understood and further investigated in this study. CK1α is a constitutively active and ubiquitously expressed serine/threonine kinase, which is involved in regulating many cellular processes ranging from cell division, and signaling to circadian rhythm and has now emerged as an essential regulator of WAVE. MARCM induced ck1α missense mutant hemocytes phenocopy WAVE depletion resulting in the disruption of the actin network that causes reduced lamellipodia formation and impaired migratory behavior. Rescue experiments using a phosphorylation-deficient mutation in the CK1α target sequence within the VCA domain of WAVE outline the dependency on CK1α phosphorylation for WAVE stability. Remarkably, loss of phosphorylation leads to proteasomal degradation of WAVE. This suggests that WAVE has a basal level of phosphorylation by CK1α, which protects it from degradation and thus promotes its function in vivo

Optogenetic tools for manipulation of cyclic nucleotides functionally coupled to cyclic nucleotide‐gated channels

Background and Purpose The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers regulating numerous biological processes. Malfunctional cNMP signalling is linked to diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in Caenorhabditis elegans is restricted to soluble adenylyl cyclases, the membrane‐bound Blastocladiella emersonii CyclOp and hyperpolarizing rhodopsins; yet missing are membrane‐bound photoactivatable adenylyl cyclases and hyperpolarizers based on K+ currents. Experimental Approach For the characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic nucleotide‐gated channels in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to depolarize or hyperpolarize cells, we performed behavioural analyses, measured cNMP content in vitro, and compared in vivo expression levels. Key Results We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine‐control of cGMP levels. We established photoactivatable membrane‐bound adenylyl cyclases, based on mutated versions (“A‐2x”) of Blastocladiella and Catenaria (“Be,” “Ca”) CyclOp, as N‐terminal YFP fusions, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two‐component optogenetics, we introduced the cAMP‐gated K+‐channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A‐2x), or YFP‐BeCyclOp(A‐2x). As an alternative, we implemented the B. emersonii cGMP‐gated K+‐channel BeCNG1 together with BeCyclOp. Conclusion and Implications We established a comprehensive suite of optogenetic tools for cNMP manipulation, applicable in many cell types, including sensory neurons, and for potent hyperpolarization.Deutsche Forschungsgemeinschaft (DFG) http://dx.doi.org/10.13039/501100001659Peer Reviewe

New aspects of p66Shc in ischemia reperfusion injury and cardiovascular diseases

Although reactive oxygen species (ROS) act as crucial factors in the onset and progression of a wide array of diseases, they are also involved in numerous signal pathways related to cell metabolism, growth and survival. ROS are produced at various cellular sites and a general consensus exists that mitochondria generate the largest amount, especially in cardiomyocytes. However, the identification of the most relevant sites within mitochondria, the interaction among the various sources, and the events responsible for the increase in ROS formation under pathological conditions remain issues that are highly debated, but far from convincing conclusions. Here, we review information linking the adaptor protein p66Shc with cardiac injury induced by ischemia and reperfusion (I/R), including the contribution of risk factors, such as metabolic syndrome and aging. In response to several stimuli, p66Shc migrates into mitochondria where it catalyzes electron transfer from cytochrome c to oxygen resulting in hydrogen peroxide formation. Deletion of p66Shc has been shown to reduce I/R injury as well as vascular abnormalities related to diabetes and aging. On the other hand, p66Shc-induced ROS formation is involved in insulin signaling and might contribute to self-endogenous defenses against mild I/R injury. Besides physiological and pathological aspects, for the first time available information is reviewed on compounds or conditions modulating p66Shc expression and activity