JIP3 regulates bi-directional organelle transport in neurons through its interaction with dynein and kinesin-1

The conserved MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. Whether and how JIP3's interaction with dynein and kinesin-1 contributes to this critical organelle clearance function is unclear. Using purified recombinant human proteins, we show that dynein light intermediate chain (DLIC) binds to the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in a hydrophobic pocket of the RH1 domain, previously shown to abrogate RILPL2 binding to myosin Va, abrogates the binding of JIP3/4 and RILP to DLIC without perturbing the interaction between the JIP3 RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3's leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bi-directional organelle transport.

Unveiling the Role Displayed by Penicillium digitatum PdMut3 Transcription Factor in Pathogen–Fruit Interaction

Zn2Cys6 transcription factors are unique to fungi and are involved in different regulatory functions. In this study, we have identified the Penicillium digitatumPdMut3 gene, which encodes a putative Zn (II) 2Cys6 DNA-binding protein. Elimination of PdMut3 in Pd1 strain caused increased virulence during citrus infection. The transcription of the PdMut3 gene showed a higher expression rate during fungal growth and less transcription during fruit infection. Furthermore, the deletion of the gene in the wild-type isolate of P. digitatum did not produce any modification of the sensitivity to different fungicides, indicating that the gene is not associated with resistance to fungicides. In contrast, PdMut3 null mutants showed a reduction in growth in minimal media, which was associated with severe alterations in conidiophore development and morphological alterations of the hyphae. Mutants showed greater sensitivity to compounds that interfere with the cell wall and an invasive growth block. Thus, PdMut3 might have an indirect role in fungi virulence through metabolism and peroxisomes development.

Concurrent Disruption of Genetic Interference and Increase of Genetic Recombination Frequency in Hybrid Rice Using CRISPR/Cas9

Manipulation of the distribution and frequency of meiotic recombination events to increase genetic diversity and disrupting genetic interference are long-standing goals in crop breeding. However, attenuation of genetic interference is usually accompanied by a reduction in recombination frequency and subsequent loss of plant fertility. In the present study, we generated null mutants of the ZEP1 gene, which encodes the central component of the meiotic synaptonemal complex (SC), in a hybrid rice using CRISPR/Cas9. The null mutants exhibited absolute male sterility but maintained nearly unaffected female fertility. By pollinating the zep1 null mutants with pollen from indica rice variety 93-11, we successfully conducted genetic analysis and found that genetic recombination frequency was greatly increased and genetic interference was completely eliminated in the absence of ZEP1. The findings provided direct evidence to support the controversial hypothesis that SC is involved in mediating interference. Additionally, the remained female fertility of the null mutants makes it possible to break linkage drag. Our study provides a potential approach to increase genetic diversity and fully eliminate genetic interference in rice breeding.

Leishmania parasite arginine deprivation response pathway influences the host macrophage lysosomal arginine sensing machinery

Extensive interaction between the host and pathogen metabolic networks decidedly shapes the outcome of infection. Infection with Leishmania donovani, an intracellular protozoan parasite, leads to a competition for arginine between the host and the parasite. L. donovani transports arginine via a high-affinity transporter LdAAP3, encoded by the two genes LdAAP3.1 and LdAAP3.2. Earlier reports show that upon arginine starvation, cultured Leishmania parasites promptly activate an Arginine Deprivation Response (ADR) pathway, resulting in the stoichiometric up-regulation of LdAAP3.2 mRNA, protein and activity. Lysosomes, on the other hand, are known to employ a specific sensor and an arginine-activated amino acid transporter, solute carrier family 38 member 9 (SLC38A9) that monitors intra-lysosome arginine sufficiency and subsequently up-regulates cellular mTORkinase activity. The present study investigates the interaction between Leishmania and macrophage-lysosome arginine sensing machinery. We show that infection with L. donovani activates SLC38A9 arginine sensing in the human monocyte like-macrophage cell line (THP-1) when grown under physiological concentrations of arginine (0.1 mM). However, supplementing the macrophage growth medium with excess arginine (1.5 mM) followed by infection led to the down-regulation of SLC38A9. Similarly, THP-1 cells infected with LdAAP3.2 null mutants grown in 0.1 mM arginine resulted in reduced expression of SLC38A9 and mTOR. These results indicate that inside the host macrophage, Leishmania overcome low arginine levels by up-regulating the transport of arginine via LdAAP3 and SLC38A9 signalling. Furthermore, while LdAAP3.2 null mutants were impaired in their ability to develop inside THP-1 macrophages, their infectivity and intracellular growth were restored in SLC38A9 silenced macrophages. This study provides the first identification of regulatory role of SLC38A9 in the expression and role of LdAAP3.Author SummaryLeishmania donovani, the causative agent of kala-azar, exhibits a digenetic life cycle. Following infection of the mammalian host, promastigotes differentiate into intracellular amastigotes within the phagolysosome of macrophages. Arginine is a central point of competition between the host and the pathogen. L. donovani senses lack of arginine in the surrounding micro-environment and activates a unique ADR pathway, thus upregulating the expression of the arginine transporter (LdAAP3). The arginine-activated amino acid transporter SLC38A9 localizes to the lysosome surface of mammalian cells and acts as a sensor that transmits information about arginine levels in the lysosome lumen to the mechanistic target of rapamycin (mTOR) kinase. In the present study, we identified the functional interaction of host SLC38A9 and parasite LdAAP3 in macrophages infected with L. donovani. We report that host SLC38A9 upregulation is critical for enhancing and maintaining high LdAAP3 levels in intracellular L. donovani. Our results decode crucial information regarding the molecular mechanism involved in the arginine sensing response in L. donovani-infected host cells. These findings increase our understanding of the interaction of signalling intermediates during Leishmania infection which may lead to the discovery of novel therapeutic interventions.

Exo1-protected DNA nicks direct crossover formation in meiosis

In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is critical for the viability of haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday Junction (dHJ) intermediates. This dHJ resolution step involves the actions Rad2/XPG family nuclease Exo1 and the Mlh1- Mlh3 mismatch repair endonuclease. At present little is known about how these factors act in meiosis at the molecular level. Here we show that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 required for interactions with DNA, such as bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase specifically reduced the crossover levels of exo1 DNA binding mutants to levels approaching the exo1 null. In addition, our work identified a role for Exo1 in crossover interference that appears independent of its resection activity. Together, these studies provide experimental evidence for Exo1 protected nicks being critical for the formation of meiotic crossovers and their distribution.

Synergistic roles of homeodomain proteins UNC-62 homothorax and MLS-2 HMX/NKX in the specification of olfactory neurons in Caenorhabditis elegans

General identity of the Caenorhabditis elegans AWC olfactory neuron pair is specified by the OTX/OTD transcription factor CEH-36 and the HMG-box transcription factor SOX-2, followed by asymmetrical differentiation of the pair into two distinct subtypes, default AWCOFF and induced AWCON, through a stochastic signaling event. The HMX/NKX transcription factor MLS-2 regulates the expression of ceh-36 to specify general AWC identity. However, general AWC identity is lost in only one of the two AWC cells in the majority of mls-2 null mutants displaying defective general AWC identity, suggesting that additional transcription factors have a partially overlapping role with MLS-2 in the specification of general AWC identity. Here we identify a role of unc-62, encoding a homothorax/Meis/TALE homeodomain protein, in the specification of general AWC identity. As in mls-2 null mutants, unc-62 null mutants showed an incomplete penetrance in loss of general AWC identity. However, unc-62; mls-2 double mutants display a nearly complete penetrance of identity loss in both AWC cells. Thus, unc-62 and mls-2 have a partially overlapping function in the specification of general AWC identity. Furthermore, our genetic results suggest that mls-2 and unc-62 act cell autonomously in promoting the AWCON subtype. Together, our findings reveal the sequential roles of the unc-62 and mls-2 pair in AWC development, specification of general AWC identity in early embryogenesis and asymmetric differentiation of AWC subtypes in late embryogenesis.

Bmmp influences wing shape by regulating anterior-posterior and proximal-distal axis development

Insect wings are subject to strong selective pressure, resulting in the evolution of remarkably diverse wing shapes that largely determine flight capacity. However, the genetic basis and regulatory mechanisms underlying wing shape development are not well understood. The silkworm Bombyx mori micropterous ( mp ) mutant exhibits shortened wing length and enlarged vein spacings , albeit without changes in total wing area. Thus, the mp mutant comprises a valuable genetic resource for studying wing shape development. In this study, we used molecular mapping to identify the gene responsible for the mp phenotype and designated it Bmmp . Phenotype-causing mutations were identified as indels and single nucleotide polymorphisms in non-coding regions. These mutations resulted in decreased Bmmp mRNA levels and changes in transcript isoform composition. Bmmp null mutants were generated by CRISPR/Cas9 and exhibited significantly smaller wings. By examining the expression of genes critical to wing development in wildtype and Bmmp null mutants, we found that Bm mp exerts its function by coordinately modulating anterior-posterior and proximal-distal axis development. We also studied a Drosophila mp mutant and found that Bmmp is functionally conserved in Drosophila . The Drosophila mp mutant strain exhibits curly wings of reduced size and a complete loss of flight capacity. Our results increase our understanding of the mechanisms underpinning insect wing development and reveal potential targets for pest control.

WDR31 is a novel ciliopathy protein displaying functional redundancy with GTPase-activating proteins ELMOD and RP2 in recruiting BBSome to cilium

The term “ciliopathy” refers to a group of over 35 rare disorders characterized by defective cilia and many overlapping clinical features, such as hydrocephalus, cerebellar vermis hypoplasia, polydactyly, and retinopathy. Even though many genes have been implicated in ciliopathies, the genetic pathogenesis in certain cases remains still undisclosed. Here, we identified a homozygous truncating variant in WDR31 in a patient with a typical ciliopathy phenotype encompassing congenital hydrocephalus, polydactyly, and renal agenesis. WDR31 is an evolutionarily conserved protein that localizes to the cilium and cilia-related compartment. Analysis from zebrafish supports the role of WDR31 in regulating the cilia morphology. The CRISPR/Cas9 knock-in (p.Arg261del) C. elegans model of the patient variant (p.Arg268*) reproduced several cilia-related defects observed in wdr-31 null mutants. Mechanistic analysis from C. elegans revealed that WDR-31 functions redundantly with ELDM-1 (ELMOD protein) and RPI-2 (RP2) to regulate the IFT trafficking through controlling the cilia entry of the BBSome. This work revealed WDR31 as a new ciliopathy protein that regulates IFT and BBSome trafficking.

Bacterial type 1A topoisomerases maintain the stability of the genome by preventing and dealing with R-loop-and nucleotide excision repair-dependent topological stress.

E. coli type 1A topoisomerases (topos), topo I (topA) and topo III (topB) have both relaxation and decatenation activities. B. subtilis and E. coli topA topB null cells can survive owing to DNA amplifications allowing overproduction of topo IV, the main cellular decatenase that can also relax supercoiling. We show that overproducing human topo IB, a relaxase but not a decatenase, can substitute for topo IV in allowing E. coli topA null but not topA topB null cells to survive. Deleting topB exacerbates phenotypes of topA null mutants including hypernegative supercoiling, R-loop formation, and RNase HI-sensitive replication, phenotypes that are not corrected by topo IV overproduction. These phenotypes lead to Ter DNA amplification causing a chromosome segregation defect that is corrected by topo IV overproduction. Furthermore, topA topB null mutants not overproducing topo IV acquire uvrB or uvrC mutations, revealing a nucleotide excision repair (NER)-dependent problem with replication fork progression. Thus, type IA topos maintain the stability of the genome by providing essential relaxase and decatenase activities to prevent and solve topological stress related to R-loops and NER. Moreover, excess R-loop formation is well tolerated in cells that have enough topoisomerase activity to support the subsequent replication-related topological stress.