Literature context: r 546 Invitrogen Cat#A-11010; RRID:AB_143156 Donkey anti-Rabbit IgG Alexa Fl
During meiosis, crossover recombination promotes the establishment of physical connections between homologous chromosomes, enabling their bipolar segregation. To ensure that persistent recombination intermediates are disengaged prior to the completion of meiosis, the Yen1(GEN1) resolvase is strictly activated at the onset of anaphase II. Whether controlled activation of Yen1 is important for meiotic crossing-over is unknown. Here, we show that CDK-mediated phosphorylation of Yen1 averts its pervasive recruitment to recombination intermediates during prophase I. Yen1 mutants that are refractory to phosphorylation resolve DNA joint molecules prematurely and form crossovers independently of MutLγ, the central crossover resolvase during meiosis. Despite bypassing the requirement for MutLγ in joint molecule processing and promoting crossover-specific resolution, unrestrained Yen1 impairs the spatial distribution of crossover events, genome-wide. Thus, active suppression of Yen1 function, and by inference also of Mus81-Mms4(EME1) and Slx1-Slx4(BTBD12) resolvases, avoids precocious resolution of recombination intermediates to enable meiotic crossover patterning.
Literature context: 546 Cell Signaling Cat#A-11010; RRID:AB_143156 Donkey Anti-Rabbit IgG (H+L) Po
Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states.
Literature context: cular Probes Cat#A11010; RRID:AB_143156 Alexa goat anti-mouse 546 Molec
Astrocytes are complex bushy cells that serve important functions through close contacts between their processes and synapses. However, the spatial interactions and dynamics of astrocyte processes relative to synapses have proven problematic to study in adult living brain tissue. Here, we report a genetically targeted neuron-astrocyte proximity assay (NAPA) to measure astrocyte-synapse spatial interactions within intact brain preparations and at synaptic distance scales. The method exploits resonance energy transfer between extracellularly displayed fluorescent proteins targeted to synapses and astrocyte processes. We validated the method in the striatal microcircuitry following in vivo expression. We determined the proximity of striatal astrocyte processes to distinct neuronal input pathways, to D1 and D2 medium spiny neuron synapses, and we evaluated how astrocyte-to-excitatory synapse proximity changed following cortical afferent stimulation, during ischemia and in a model of Huntington's disease. NAPA provides a simple approach to measure astrocyte-synapse spatial interactions in a variety of experimental scenarios. VIDEO ABSTRACT.
Literature context: t IgG Invitrogen Cat# A11010; RRID:AB_143156 Alexa Fluor 546 goat anti-mouse
Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration.
Literature context: Life Technologies Cat# A11010; RRID:AB_143156 Goat anti-Mouse IgG Alexa Fluor
Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.
Literature context: t#A11010; RRID:AB_143156 Alexa goat
Astrocytes are ubiquitous in the brain and are widely held to be largely identical. However, this view has not been fully tested, and the possibility that astrocytes are neural circuit specialized remains largely unexplored. Here, we used multiple integrated approaches, including RNA sequencing (RNA-seq), mass spectrometry, electrophysiology, immunohistochemistry, serial block-face-scanning electron microscopy, morphological reconstructions, pharmacogenetics, and diffusible dye, calcium, and glutamate imaging, to directly compare adult striatal and hippocampal astrocytes under identical conditions. We found significant differences in electrophysiological properties, Ca2+ signaling, morphology, and astrocyte-synapse proximity between striatal and hippocampal astrocytes. Unbiased evaluation of actively translated RNA and proteomic data confirmed significant astrocyte diversity between hippocampal and striatal circuits. We thus report core astrocyte properties, reveal evidence for specialized astrocytes within neural circuits, and provide new, integrated database resources and approaches to explore astrocyte diversity and function throughout the adult brain. VIDEO ABSTRACT.
Literature context: it Alexa Fluor-546 (Invitrogen; RRID:AB_143156) was applied in a 1 : 500 dilut
Members of the protein kinase D (PKD) family of serine/threonine kinases are known to exert diverse roles in neuronal stress responses. Here, we show the transient activation and nuclear translocation of endogenous PKD upon oxidative stress induced by H2 O2 treatment in primary neuronal cultures. Using pharmacological inhibition, we show that PKD activity protects neurons from oxidative stress-induced cell death. Although members of the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF kappaB) pathway were phosphorylated upon H2 O2 treatment, it was found that the neuronal response to oxidative stress is not executed through the nuclear translocation and activity of RelA. On the other hand, we demonstrate for the first time in neuronal cells, the association of green fluorescent protein-tagged kinase inactive PKD1 with mitochondrial membranes in vivo and the presence of PKD activity in the close vicinity of mitochondria in vitro. Our findings thus support the notion that the neuroprotective role of PKD is exerted independently from NF kappaB signaling and suggest a potential mitochondrial function for PKD in cultured neurons.
Literature context: . A11010; RRID:AB_143156) for 2 day
In Drosophila melanogaster olfactory sensory neurons (OSNs) establish synapses with projection neurons (PNs) and local interneurons within antennal lobe (AL) glomeruli. Substantial knowledge regarding this circuitry has been obtained by functional studies, whereas ultrastructural evidence of synaptic contacts is scarce. To fill this gap, we studied serial sections of three glomeruli using electron microscopy. Ectopic expression of a membrane-bound peroxidase allowed us to map synaptic sites along PN dendrites. Our data prove for the first time that each of the three major types of AL neurons is both pre- and postsynaptic to the other two types, as previously indicated by functional studies. PN dendrites carry a large proportion of output synapses, with approximately one output per every three input synapses. Detailed reconstructions of PN dendrites showed that these synapses are distributed unevenly, with input and output sites partially segregated along a proximal-distal gradient and the thinnest branches carrying solely input synapses. Moreover, our data indicate synapse clustering, as we found evidence of dendritic tiling of PN dendrites. PN output synapses exhibited T-shaped presynaptic densities, mostly arranged as tetrads. In contrast, output synapses from putative OSNs showed elongated presynaptic densities in which the T-bar platform was supported by several pedestals and contacted as many as 20 postsynaptic profiles. We also discovered synaptic contacts between the putative OSNs. The average synaptic density in the glomerular neuropil was about two synapses/µm(3) . These results are discussed with regard to current models of olfactory glomerular microcircuits across species.
Literature context: ylated goat anti-rabbit IgG, or Alexa Fluor 546-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR)
Local neurons in the vertebrate retina are instrumental in transforming visual inputs to extract contrast, motion, and color information and in shaping bipolar-to-ganglion cell transmission to the brain. In Drosophila, UV vision is represented by R7 inner photoreceptor neurons that project to the medulla M6 stratum, with relatively little known of this downstream substrate. Here, using R7 terminals as references, we generated a 3D volume model of the M6 stratum, which revealed a retinotopic map for UV representations. Using this volume model as a common 3D framework, we compiled and analyzed the spatial distributions of more than 200 single M6-specific local neurons (M6-LNs). Based on the segregation of putative dendrites and axons, these local neurons were classified into two families, directional and nondirectional. Neurotransmitter immunostaining suggested a signal routing model in which some visual information is relayed by directional M6-LNs from the anterior to the posterior M6 and all visual information is inhibited by a diverse population of nondirectional M6-LNs covering the entire M6 stratum. Our findings suggest that the Drosophila medulla M6 stratum contains diverse LNs that form repeating functional modules similar to those found in the vertebrate inner plexiform layer.
Literature context: #A11010, RRID:AB_143156), Alexa 48
Paranodal axoglial junctions are critical for maintaining the segregation of axonal domains along myelinated axons; however, the proteins required to organize and maintain this structure are not fully understood. Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC) are proteins enriched at paranodes that are expressed by neurons and oligodendrocytes. To identify the specific function of DCC expressed by oligodendrocytes in vivo, we selectively eliminated DCC from mature myelinating oligodendrocytes using an inducible cre regulated by the proteolipid protein promoter. We demonstrate that DCC deletion results in progressive disruption of the organization of axonal domains, myelin ultrastructure, and myelin protein composition. Conditional DCC knock-out mice develop balance and coordination deficits and exhibit decreased conduction velocity. We conclude that DCC expression by oligodendrocytes is required for the maintenance and stability of myelin in vivo, which is essential for proper signal conduction in the CNS.