Candida albicans cell wall mannosidases, Dfg5 and Dcw1, regulate chitin synthesis

In Candida albicans chitin synthesis is important for cell wall integrity and may also have a role in emergence of drug-resistance. Our past studies showed that cell wall mannosidases, Dfg5 and Dcw1, regulate HOG MAPK signaling. In this study, we investigated how Dfg5 and Dcw1 regulate chitin synthesis by affecting HOG, PKC and Calcium-Calcineurin signaling pathways. DFG5 and DCW1 heterologous mutants (ES1 & ES195) and a conditional mutant (ES195+methionine/cysteine) were utilized. WT SC5314 served as negative control and Hog1 knock-out mutant as positive control. Fluorescence microscopy of calcofluor white (CFW) stained mutant and control strains was performed to observe chitin accumulation. Quantitative PCR analysis was performed to measure the relative expression of chitin synthases CHS1, CHS2, CHS3 and CHS8. Incubation with chitinase was done to determine cell separation using light microscopy and scanning electron microscopy (SEM) analysis. Fluorescence microscopy showed significantly increased chitin accumulation in the mutants as compared to wild type. Chitin accumulation was observed mainly at the budding sites indicating a cause for defective cell separation phenotype. Incubation with chitinase led to cell separation in the mutants. CHS2, CHS3 and CHS8 expression was observed to be significantly upregulated in the conditional mutant and HOG1 mutant as compared to the wild type. This upregulation was also observed when the cell wall integrity PKC pathway was activated. However, activation of the Calcium-calcineurin pathway downregulated chitin synthase expression in the mutants. Our data indicates that Dfg5 and Dcw1 regulate expression of chitin synthases via HOG MAPK, PKC and Calcium-calcineurin signaling pathways.

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin, which extends up to 1 kb from early, efficient replication origins. The CMG holohelicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S-phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin which extends up to one kilobase from early, efficient replication origins. The CMG holo-helicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex, but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.

The cellular basis of protease activated receptor type 2 (PAR2) evoked mechanical and affective pain

ABSTRACTProtease-activated receptor type-2 (PAR2) has long been implicated in inflammatory and visceral pain, but the cellular basis of PAR2-evoked pain has not been delineated. While many studies have attributed PAR2-evoked pain to sensory neuron expression, RNA-sequencing experiments are ambiguous on detection of F2rl1 mRNA. Moreover, many pharmacological tools for PAR2 have been shown to be non-specific as they also act on the Mas-related (Mrg) family of g-protein coupled receptors (GPCRs) that are highly enriched in sensory neurons. We sought to bring clarity to the cellular basis of PAR2 pain. We developed a PAR2 conditional mutant mouse by loxp targeting of exon 2 of the F2rl1 gene and specifically deleted PAR2 in all sensory neurons using the PirtCre mouse line. Our behavioral findings show that PAR2 agonist-evoked mechanical hyperalgesia and facial grimacing, but not thermal hyperalgesia, is completely dependent on PAR2 expression in sensory neurons that project to the hindpaw in male and female mice. F2rl1 mRNA is expressed in a discrete population (~4%) of sensory neurons that also express the Nppb and IL31ra genes. This cell population has previously been implicated in itch, but our work shows that PAR2 activation in these cells causes clear pain-related behaviors from the skin. Our findings clarify the mechanism through which proteases, like tryptase and elastase, cause pain via PAR2 activation in a small subset of nociceptors.

Ephrin-B2 Paces Neuronal Production in the Developing Neocortex

ABSTRACTBackgroundDuring mammalian cerebral cortex development, different types of projection neurons are produced in a precise temporal order and in stereotypical numbers. The mechanisms regulating timely generation of neocortex projection neurons and ensuring production in sufficient numbers of each neuronal identity is only partially understood.ResultsHere, we show that ephrin-B2, a member of the Eph:ephrin cell-to-cell communication pathway, sets the neurogenic tempo in the neocortex. Indeed, conditional mutant embryos for ephrin-B2 exhibit a transient delay in neurogenesis and acute stimulation of Eph signaling by in utero injection of synthetic ephrin-B2 led to a transient increase in neuronal production. Using genetic approaches we show that ephrin-B2 acts on neural progenitors to control their differentiation in a juxtacrine manner. Unexpectedly, we observed that perinatal neuron numbers recovered following both loss or gain of ephrin-B2, highlighting the ability of neural progenitors to adapt their behavior to the state of the system in order to produce stereotypical numbers of neurons.ConclusionsAltogether, our data uncover a role for ephrin-B2 in embryonic neurogenesis and emphasizes the plasticity of neuronal production in the neocortex.

Loss of Fbxw7 in Sertoli cells impairs testis development and causes infertility in mice†

F-box and WD-40 domain protein 7 (Fbxw7) is a component of the Skp1-Cdc53/Cullin-F-box-protein complex (SCF/β-TrCP), which is an E3 ubiquitin ligase that mediates protein degradation. This complex has recently been shown to negatively regulate spermatogonial stem cell self-renewal; however, its roles in Sertoli cell (SC) proliferation, differentiation, and function remain to be established. In this study, we generated conditional mutant mice with SC-specific deletion of Fbxw7 via the Cre-loxP system. Fbxw7 deficiency in SCs impaired testis development, which is characterized by age-dependent tubular atrophy, excessive germ cell loss, and spermatogenic arrest, and the mutant males were infertile at 7 months old. Fbxw7 ablation also compromised cytoskeletal organization and cell polarity of SCs, as well as integrity of the blood-testis barrier. In addition, the transcript levels of cell markers for germ cells, Leydig cells, and SCs were significantly decreased in Fbxw7 mutant mice. Importantly, protein levels of GATA-4, a transcription factor that plays a crucial role in SC maturation and testis development, were progressively decreased in control SCs after postnatal day 14, whereas levels were aberrantly elevated in Fbxw7-deleted SCs. Interestingly, the Gata-4 messenger RNA levels remained stable following Fbxw7 deletion. Fbxw7 silencing in SCs also induced progressive Leydig cell inefficiency and testosterone insufficiency. Collectively, these results demonstrate that Fbxw7 expression is required for SC maturation and function, potentially through degradation of GATA-4, to support pubertal testis development and spermatogenesis.

Metabolomics profiling reveals new aspects of dolichol biosynthesis in Plasmodium falciparum

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Polyprenols have been hardly detectable in eukaryotic cells under normal conditions with the exception of plants and sporulating yeast. Our metabolomics studies revealed that cis-polyisoprenoids are more prevalent and diverse in the parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was also observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite’s development. In addition, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite. Isotopic labeling and metabolomic analyses of a conditional mutant of PfCPT or PfPPRD suggest that polyprenols may be able to substitute dolichols in their biological functions when dolichol synthesis is impaired in Plasmodium.

CEP290 myosin-tail homology domain is essential for protein confinement between inner and outer segments in photoreceptors

ABSTRACTMutations in CEP290 cause various ciliopathies involving retinal degeneration. CEP290 proteins localize to the ciliary transition zone and are thought to act as a gatekeeper that controls ciliary protein trafficking. However, precise roles of CEP290 in photoreceptors and pathomechanisms of retinal degeneration in CEP290-associated ciliopathies are not sufficiently understood. Using Cep290 conditional mutant mice, in which the C-terminal myosin-tail homology domain is disrupted after the connecting cilium is assembled, we show that CEP290, more specifically the myosin-tail homology domain of CEP290, is essential for protein confinement between the inner and the outer segments. Inner segment plasma membrane proteins including STX3, SNAP25, and IMPG2 rapidly accumulate in the outer segment upon disruption of the myosin-tail homology domain. In contrast, localization of endomembrane proteins is not altered. Trafficking and confinement of most outer segment-resident proteins appear to be unaffected or only minimally affected in this mouse model. One notable exception is RHO, which exhibits severe mislocalization to inner segments from the initial stage of degeneration. Similar mislocalization phenotypes were observed in rd16 mice. These results suggest that failure of protein confinement at the connecting cilium and consequent accumulation of inner segment membrane proteins in the outer segment combined with insufficient RHO delivery is part of the disease mechanisms that cause retinal degeneration in CEP290-associated ciliopathies. Our study provides insights into the pathomechanisms of retinal degenerations associated with compromised ciliary gates.