We have identified the yeast CRT1 gene as an effector of the DNA damage and replication checkpoint pathway. CRT1 encodes a DNA-binding protein that recruits the general repressors Ssn6 and Tup1 to the promoters of damage-inducible genes. Derepression of the Crt1 regulon suppresses the lethality of mec1 and rad53 null alleles and is essential for cell viability during replicative stress. In response to DNA damage and replication blocks, Crt1 becomes hyperphosphorylated and no longer binds DNA, resulting in transcriptional induction. CRT1 is autoregulated and is itself induced by DNA damage, indicating the existence of a negative feedback pathway that facilitates return to the repressed state after elimination of damage. The inhibition of an autoregulatory repressor in response to DNA damage is a strategy conserved throughout prokaryotic and eukaryotic evolution.

We cloned the C. elegans gene ced-1, which is required for the engulfment of cells undergoing programmed cell death. ced-1 encodes a transmembrane protein similar to human SREC (Scavenger Receptor from Endothelial Cells). We showed that ced-1 is expressed in and functions in engulfing cells. The CED-1 protein localizes to cell membranes and clusters around neighboring cell corpses. CED-1 failed to cluster around cell corpses in mutants defective in the engulfment gene ced-7. Motifs in the intracellular domain of CED-1 known to interact with PTB and SH2 domains were necessary for engulfment but not for clustering. Our results indicate that CED-1 is a cell surface phagocytic receptor that recognizes cell corpses. We suggest that the ABC transporter CED-7 promotes cell corpse recognition by CED-1, possibly by exposing a phospholipid ligand on the surfaces of cell corpses.

Inhibition of DNA synthesis prevents mitotic entry through the action of the S-phase checkpoint. We have isolated S-phase arrest-defective {sad) mutants that show lethality in the presence of the DNA synthesis inhibitor hydroxyurea (HU). Several of these mutants show phenotypes consistent with inappropriate mitotic entry in the presence of unreplicated DNA, indicating a defect in the S-phase checkpoint, sadl mutants are additionally defective for the G^ and Gj DNA damage checkpoints, and for DNA damage-induced transcription of RNR2 and RNR3. The transcriptional response to DNA damage requires activation of the Dunl protein kinase. Activation of Dunl in response to replication blocks or DNA damage is blocked in sadl mutants. The HU sensitivity of sadl mutants is suppressed by mutations in CKSl, a subunit of the p34^"*^^* kinase, further establishing a link between cell cycle progression and lethality, sadl mutants are allelic to rad53, a radiation-sensitive mutant. SADl encodes an essential protein kinase. The observation that SADl controls three distinct checkpoints suggests a common mechanism for cell cycle arrest at these points. Together, these observations implicate protein phosphorylation in the cellular response to DNA damage and replication blocks. A successful eukaryotic cell division requires that cer tain cellular processes occur in a defined order and are coupled so that the initiation of one event is dependent on the completion of another. In most cell types, entry into mitosis is dependent on the completion of DNA synthesis. Cells blocked for DNA replication arrest in S phase and delay mitotic initiation. Cell cycle arrest in response to S-phase inhibition is attributable to the pres ence of a feedback control or checkpoint mechanism (for reviews on cell cycle checkpoints, see Hartwell and

Apoptotic cells in animals are engulfed by phagocytic cells and subsequently degraded inside phagosomes. To study the mechanisms controlling the degradation of apoptotic cells, we developed time-lapse imaging protocols in developing Caenorhabditis elegans embryos and established the temporal order of multiple events during engulfment and phagosome maturation. These include sequential enrichment on phagocytic membranes of phagocytic receptor cell death abnormal 1 (CED-1), large GTPase dynamin (DYN-1), phosphatidylinositol 3-phosphate (PI(3)P), and the small GTPase RAB-7, as well as the incorporation of endosomes and lysosomes to phagosomes. Two parallel genetic pathways are known to control the engulfment of apoptotic cells in C. elegans. We found that null mutations in each pathway not only delay or block engulfment, but also delay the degradation of engulfed apoptotic cells. One of the pathways, composed of CED-1, the adaptor protein CED-6, and DYN-1, controls the rate of enrichment of PI(3)P and RAB-7 on phagosomal surfaces and the formation of phagolysosomes. We further identified an essential role of RAB-7 in promoting the recruitment and fusion of lysosomes to phagosomes. We propose that RAB-7 functions as a downstream effector of the CED-1 pathway to mediate phagolysosome formation. Our work suggests that phagocytic receptors, which were thought to act specifically in initiating engulfment, also control phagosome maturation through the sequential activation of multiple effectors such as dynamin, PI(3)P, and Rab GTPases.

Dynamins are large GTPases that act in multiple vesicular trafficking events. We identified 14 loss-of-function alleles of the C. elegans dynamin gene, dyn-1, that are defective in the removal of apoptotic cells. dyn-1 functions in engulfing cells to control the internalization and degradation of apoptotic cells. dyn-1 acts in the genetic pathway composed of ced-7 (ABC transporter), ced-1 (phagocytic receptor), and ced-6 (CED-1's adaptor). DYN-1 transiently accumulates to the surface of pseudopods in a manner dependent on ced-1, ced-6, and ced-7, but not on ced-5, ced-10, or ced-12. Abnormal vesicle structures accumulate in engulfing cells upon dyn-1 inactivation. dyn-1 and ced-1 mutations block the recruitment of intracellular vesicles to pseudopods and phagosomes. We propose that DYN-1 mediates the signaling of the CED-1 pathway by organizing an intracellular vesicle pool and promoting vesicle delivery to phagocytic cups and phagosomes to support pseudopod extension and apoptotic cell degradation.

Inhibition of DNA synthesis induces transcription of DNA damage-inducible genes and prevents mitotic entry through the action of the S phase checkpoint. We have isolated a mutant, dun2, defective for both of these responses. DUN2 is identical to POL2, encoding DNA polymerase epsilon (pol epsilon). Unlike sad1 mutants defective for multiple cell cycle checkpoints, pol2 mutants are defective only for the S phase checkpoint and the activation of DUN1 kinase necessary for the transcriptional response to damage. Interallelic complementation and mutation analysis indicate that pol epsilon contains two separable essential domains, an N-terminal polymerase domain and a C-terminal checkpoint domain unique to epsilon polymerases. We propose that DNA pol epsilon acts as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks.

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Background: Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism. Methods: Electron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms. Results: Our results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain-and loss-offunction experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p. Conclusion: Hypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.