Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.

Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.The SOS response in Escherichia coli provides the coordination between DNA damage sensing and the cellular responses to DNA damage (reviewed in reference 22). The primary SOS signal, single-stranded DNA (ssDNA), activates RecA in a ternary complex with ATP as a transcriptional regulator (44) and as a DNA repair protein (reviewed in reference 41). The transcriptional induction of the SOS regulon leads to increased expression of certain DNA repair genes (including RecA itself) and also elicits transient cell cycle arrest by the expression of sfiA, a cell division inhibitor (22). The activation of RecA as a repair protein leads to immediate repair of the primary damage that initiated the SOS signal. Although different in mechanism, the DNA damage checkpoints could provide a similar coordination between DNA damage sensing and repair in eukaryotes. First conceptualized as an active cell cycle control system in response to DNA damage in Saccharomyces cerevisiae (29,89), DNA damage checkpoints were later shown to control also DNA damage-induced gene expression in this organism (3). DNA damage checkpoints and DNA repair serve a common purpose to secure survival and genomic stability after DNA damage. Indirect effects of the DNA damage checkpoints on DNA repair have been discussed before (reviewed in references 18, 85, and 87), but a direct coupling of the DNA damage sensing capabilities of the checkpoint system with DNA damage repair pathways has not been identified yet.The DNA damage checkpoints in eukaryotes relay a signal in response to DNA damage to transiently delay the entry into the S or M phases, to slow down the ongoing DNA replication, or to arrest in meiotic prophase (reviewed in references 29, 62, and 87). They also elicit DNA damage-induced transcription of many genes, including some coding for DNA repair proteins (87, 93). Moreover, a related DNA replication block checkpoint ensures the dependency of M phase on a...

Rad51p is a eukaryotic homolog of RecA, the central homologous pairing and strand exchange protein in Escherichia coli. Rad54p belongs to the Swi2p/Snf2p family of DNA‐stimulated ATPases. Both proteins are also important members of the RAD52 group which controls recombinational DNA damage repair of double‐strand breaks and other DNA lesions in Saccharomyces cerevisiae. Here we demonstrate by genetic, molecular and biochemical criteria that Rad51 and Rad54 proteins interact. Strikingly, overexpression of Rad54p can functionally suppress the UV and methyl methanesulfonate sensitivity caused by a deletion of the RAD51 gene. However, no suppression was observed for the defects of rad51 cells in the repair of γ‐ray‐induced DNA damage, mating type switching or spontaneous hetero‐allelic recombination. This suppression is genetically dependent on the presence of two other members of the recombinational repair group, RAD55 and RAD57. Our data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair. As both proteins are conserved throughout evolution from yeasts to humans, a similar protein–protein interaction may be expected in other organisms.

The transforming protozoan Theileria recruits Plk1, a host kinase that regulates mitosis, to its surface and engages spindle microtubules to secure its division and inheritance into both daughter cells.

SummaryEukaryotic cells can employ autophagy to defend themselves against invading pathogens. Upon infection by Plasmodium berghei sporozoites, the host hepatocyte targets the invader by labelling the parasitophorous vacuole membrane (PVM) with the autophagy marker protein LC3. Until now, it has not been clear whether LC3 recruitment to the PVM is mediated by fusion of autophagosomes or by direct incorporation. To distinguish between these possibilities, we knocked out genes that are essential for autophagosome formation and for direct LC3 incorporation into membranes. The CRISPR/Cas9 system was employed to generate host cell lines deficient for either FIP200, a member of the initiation complex for autophagosome formation, or ATG5, responsible for LC3 lipidation and incorporation of LC3 into membranes. Infection of these knockout cell lines with P. berghei sporozoites revealed that LC3 recruitment to the PVM indeed depends on functional ATG5 and the elongation machinery, but not on FIP200 and the initiation complex, suggesting a direct incorporation of LC3 into the PVM. Importantly, in P. berghei- infected ATG5−/− host cells, lysosomes still accumulated at the PVM, indicating that the recruitment of lysosomes follows an LC3-independent pathway. | INTRODUCTIONMalaria, with more than 200 million estimated cases and more than 400 thousand deaths per year, remains one of the most devastating diseases worldwide (World Health Organization, 2015). Once Plasmodium sporozoites are injected by an infected female Anopheles mosquito during blood feeding, they migrate to the liver and infect hepatocytes by the formation of a parasitophorous vacuole (PV).Although the PV membrane (PVM) originates from invagination of the host cell plasma membrane, it is remodelled by the parasite upon infection (Spielmann, Montagna, Hecht, & Matuschewski, 2012).Inside the vacuole, the parasite starts its exoerythrocytic development by transforming into a multinuclear schizont that undergoes massive replication. Finally, several thousand erythrocyte-infective merozoites are formed and safely released into the blood within merosomes (Sturm et al., 2006). Within the hepatocyte, the Plasmodium parasite shows one of the fastest replication rates among eukaryotes, necessitating that massive amounts of nutrients are obtained from the host cell (Bano, Romano, Jayabalasingham, & Coppens, 2007).At the same time, the parasite needs to escape defence mechanisms of the host cell. homeostasis, cells can use selective autophagy to remove damaged or deleterious elements from the cytoplasm. Selective autophagy can also serve as a cellular antimicrobial defence against intracellular pathogens, a process called xenophagy (Deretic & Levine, 2009;Knodler & Celli, 2011;Levine, Mizushima, & Virgin, 2011;Mostowy, 2013). Starvation-induced and selective autophagy are both characterised by the formation of double membrane autophagosomes that sequester the autophagic cargo (Levine et al., 2011;Mizushima & Komatsu, 2011). In mammalian cells, starvation initiates autophag...