Summary Astrocytes exist throughout the nervous system and are proposed to affect neural circuits and behavior. However, studying astrocytes has proven difficult because of the lack of tools permitting astrocyte selective genetic manipulations. Here, we report the generation of Aldh1l1-Cre/ERT2 transgenic mice to selectively target astrocytes in vivo. We characterised Aldh1l1-Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections and crosses to RiboTag, Ai95 and new Cre-dependent membrane tethered Lck-GCaMP6f knock-in mice that we also generated. Two-to-three weeks after tamoxifen induction, Aldh1l1-Cre/ERT2 selectively targeted essentially all adult (P80) brain astrocytes with no detectable neuronal contamination, resulting in expression of cytosolic and Lck-GCaMP6f and permitting subcellular astrocyte calcium imaging during startle responses in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and determination of the adult cortical astrocyte transcriptome. Thus, we provide well characterised, easy-to-use resources with which to selectively study astrocytes in situ and in vivo in multiple experimental scenarios.

Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo.Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin-or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.The poxviruses form a group of large DNA viruses that includes variola virus, the causative agent of smallpox disease. Though smallpox itself has been eradicated, the fear of biological warfare and the recent occurrence of accidental monkeypox virus transmission between species (22) and of eczema vaccinatum (33) have alerted us to the potential danger of new emerging diseases. In addition, the potential applications of poxviruses as improved vaccines (34) and as oncolytic agents for cancer therapy (57) have raised new interest in poxvirus biology.Vaccinia virus, the well-studied prototype of the Orthopoxvirus genus in the family Poxviridae, has a wide range of infectivity in many cell lines and animals (20). It produces several forms of infectious particles, of which the vaccinia intracellular mature virus (IMV) is the most abundant in cells (see reference 14 and references therein). An IMV is enclosed by a single envelope and contains more than 70 viral proteins (11,45,65).The molecular mechanism of vaccinia IMV entry remains largely unknown. IMV binds to ubiquitous cellular attachment factors, such as glycosaminoglycans (12, 27) and the extracellular matrix protein laminin (10). It is not known whether IMV recognizes additional cellular coreceptors to trigger the postbinding fusion step, although virus entry through fusion with the plasma membrane (3, 9, 19, 37) or intracellular compartments (16, 58) has been reported. Interestingly, IMV has been shown to trigger cellular signaling during virus entry (2, 37, 44), but the molecular pathway of virus uptake has not been characterized.In this study, we characterized the mechanism by whi...

Activation of glial cells following axon injury is mediated by a positive feedback loop downstream of the glial phagocytic receptor Draper, allowing the strength of the response to match the severity of injury.

Draper/Ced-1/MEGF-10 is an engulfment receptor that promotes clearance of cellular debris in C. elegans, Drosophila and mammals. Draper signals through an evolutionarily conserved Src family kinase cascade to drive cytoskeletal rearrangements and target engulfment through Rac1. Glia also alter gene expression patterns in response to axonal injury but pathways mediating these responses are poorly defined. We show Draper is cell autonomously required for glial activation of transcriptional reporters after axonal injury. We identify TNF receptor associated factor 4 (TRAF4) as a novel Draper binding partner that is required for reporter activation and phagocytosis of axonal debris. TRAF4 and misshapen (MSN) act downstream of Draper to activate c-Jun N-terminal kinase (JNK) signalling in glia, resulting in changes in transcriptional reporters that are dependent on Drosophila AP-1 (dAP-1) and STAT92E. Our data argue injury signals received by Draper at the membrane are important regulators of downstream transcriptional responses in reactive glia.