PLoS Genetics最新文献

Mitosis and meiosis have two mechanisms for regulating the accuracy of chromosome segregation: error correction and the spindle assembly checkpoint (SAC). We have investigated the function of several checkpoint proteins in meiosis I of Drosophila oocytes. Increased localization of several SAC proteins was found upon depolymerization of microtubules by colchicine. However, unattached kinetochores or errors in biorientation of homologous chromosomes do not induce increased SAC protein localization. Furthermore, the metaphase I arrest does not depend on SAC genes, suggesting the APC is inhibited even if the SAC is not functional. Two SAC proteins, ROD of the ROD-ZW10-Zwilch (RZZ) complex and MPS1, are also required for the biorientation of homologous chromosomes during meiosis I, suggesting an error correction function. Both proteins aid in preventing or correcting erroneous attachments and depend on SPC105R for localization to the kinetochore. We have defined a region of SPC105R, amino acids 123-473, that is required for ROD localization and biorientation of homologous chromosomes at meiosis I. Surprisingly, ROD removal from kinetochores and movement towards spindle poles, termed "streaming," is independent of the dynein adaptor Spindly and is not linked to the stabilization of end-on attachments. Instead, meiotic RZZ streaming appears to depend on cell cycle stage and may be regulated independently of kinetochore attachment or biorientation status. We also show that Spindly is required for biorientation at meiosis I, and surprisingly, the direction of RZZ streaming.

Some animals can regenerate large missing regions of their nervous system, requiring mechanisms to restore the pattern, numbers, and wiring of diverse neuron classes. Because injuries are unpredictable, regeneration must be accomplished from an unlimited number of starting points. Coordinated regeneration of neuron-glia architecture is thus a major challenge and remains poorly understood. In planarians, neurons and glia are regenerated from distinct progenitors. We found that planarians first regenerate neurons expressing a Delta-encoding gene, delta-2, at key positions in the central and peripheral nervous systems. Planarian glia are specified later from dispersed Notch-1-expressing mesoderm-like phagocytic progenitors. Inhibition of delta-2 or notch-1 severely reduced glia in planarians, but did not affect the specification of other phagocytic cell types. Loss of several delta-2-expressing neuron classes prevented differentiation of the glia associated with them, whereas transplantation of delta-2-expressing photoreceptor neurons was sufficient for glia formation at an ectopic location. Our results suggest a model in which patterned delta-2-expressing neurons instruct phagocytic progenitors to locally differentiate into glia, presenting a mechanism for coordinated regeneration of numbers and pattern of cell types.

Bacterial cytokinesis begins with polymerization of the tubulin homologue FtsZ into a ring-like structure at midcell, the Z-ring, which recruits the late cell division proteins that synthesize the division septum. Assembly of FtsZ is carefully regulated and supported by a dozen conserved cell division proteins. Generally, these proteins are not essential, but removing more than one is in many cases lethal. Therefore, it is still not fully clear how the different protein components contribute to cell division, and whether there is a minimal set of proteins that can execute cell division. In this study, we tried to find the minimal set of proteins that is required to establish an active Z-ring in the model bacterium Bacillus subtilis. By making use of known suppressor mutations we were able to find a gene deletion route that eventually enabled us the remove eight conserved cell division proteins: ZapA, MinC, MinJ, UgtP, ClpX, Noc, EzrA and FtsA. Only FtsZ and its membrane anchor SepF appeared to be required for Z-ring formation. Interestingly, SepF is also the FtsZ anchor in archaea, and both proteins date back to the Last Universal Common Ancestor (LUCA). Viability of the multiple deletion mutant was not greatly affected, although the frequency of cell division was considerably reduced. Whole genome sequencing suggested that the construction of this minimal divisome strain was also possible due to the accumulation of suppressor mutations. After extensive phenotypic testing of these mutations, we found an unexpected cell division regulation function for the branched chain amino acid transporter BraB, which may be related to a change in fatty acid composition. The implications of these findings for the role of SepF, and the construction of a minimal cell division machinery are discussed.

A bidirectional nucleus-mitochondria communication is essential for homeostasis and stress. By acting as critical molecules, the nuclear-encoded lncRNAs (nulncRNAs) have been implicated in the nucleus-to-mitochondria anterograde regulation. However, role of mitochondrial-derived lncRNAs (mtlncRNAs) in the mitochondria-to-nucleus retrograde regulation remains elusive. Here, we identify functional implication of the mtlncRNAs MDL1AS, lncND5 and lncCyt b in retrograde regulation. Mediated by HuR and PNPT1 proteins, the mtlncRNAs undergo a mitochondria-to-nucleus traveling and then regulate a network of nuclear genes. Moreover, as an example of the functional consequence, we showed that the nuclear-translocated lncCyt b cooperates with the splicing factor hnRNPA2B1 to influence several aspects of cell metabolism including glycolysis, possibly through their regulatory effect on the post-transcriptional processing of related nuclear genes. This study advances our knowledge in mitochondrial biology and provides new insights into the role of mtlncRNAs in mitochondria-nucleus communications.

Adaptation to existence outside the womb is a key event in the life of a mammal. The absence of macrophages in rats with a homozygous mutation in the colony-stimulating factor 1 receptor (Csf1r) gene (Csf1rko) severely compromises pre-weaning somatic growth and maturation of organ function. Transfer of wild-type bone marrow cells (BMT) at weaning rescues tissue macrophage populations permitting normal development and long-term survival. To dissect the phenotype and function of macrophages in postnatal development, we generated transcriptomic profiles of all major organs of wild-type and Csf1rko rats at weaning and in selected organs following rescue by BMT. The transcriptomic profiles revealed subtle effects of macrophage deficiency on development of all major organs. Network analysis revealed a common signature of CSF1R-dependent resident tissue macrophages that includes the components of complement C1Q (C1qa/b/c genes). Circulating C1Q was almost undetectable in Csf1rko rats and rapidly restored to normal levels following BMT. Tissue-specific macrophage signatures were also identified, notably including sinus macrophage populations in the lymph nodes. Their loss in Csf1rko rats was confirmed by immunohistochemical localisation of CD209B (SIGNR1). By 6-12 weeks, Csf1rko rats succumb to emphysema-like pathology associated with the selective loss of interstitial macrophages and granulocytosis. This pathology was reversed by BMT. Along with physiological rescue, BMT precisely regenerated the abundance and expression profiles of resident macrophages. The exception was the brain, where BM-derived microglia-like cells had a distinct expression profile compared to resident microglia. In addition, the transferred BM failed to restore blood monocyte or CSF1R-positive bone marrow progenitors. These studies provide a model for the pathology and treatment of CSF1R mutations in humans and the innate immune deficiency associated with prematurity.

The ability to manipulate gene activity and control transgene expression is essential to study gene function. While several genetic tools for modifying genes or controlling expression separately are available for Caenorhabditis elegans, there are no genetic approaches to generate mutations that simultaneously disrupt gene function and provide genetic access to the cells expressing the disrupted gene. To achieve this, we developed a versatile gene trap strategy based on cGAL, a GAL4-UAS bipartite expression system for C. elegans. We designed a cGAL gene trap cassette and used CRISPR/Cas9 to insert it into the target gene, creating a bicistronic operon that simultaneously expresses a truncated endogenous protein and the cGAL driver in the cells expressing the target gene. We demonstrate that our cGAL gene trap strategy robustly generated loss-of-function alleles. Combining the cGAL gene trap lines with different UAS effector strains allowed us to rescue the loss-of-function phenotype, observe the gene expression pattern, and manipulate cell activity spatiotemporally. We show that, by recombinase-mediated cassette exchange (RMCE) via microinjection or genetic crossing, the cGAL gene trap lines can be further engineered in vivo to easily swap cGAL with other bipartite expression systems' drivers, including QF/QF2, Tet-On/Tet-Off, and LexA, to generate new gene trap lines with different drivers at the same genomic locus. These drivers can be combined with their corresponding effectors for orthogonal transgenic control. Thus, our cGAL-based gene trap is versatile and represents a powerful genetic tool for gene function analysis in C. elegans, which will ultimately provide new insights into how genes in the genome control the biology of an organism.

Mutations of the Cullin-3 (Cul3) E3 ubiquitin ligase are associated with autism and schizophrenia, neurological disorders characterized by sleep disturbances and altered synaptic function. Cul3 engages dozens of adaptor proteins to recruit hundreds of substrates for ubiquitination, but the adaptors that impact sleep and synapses remain ill-defined. Here we implicate Insomniac (Inc), a conserved protein required for normal sleep and synaptic homeostasis in Drosophila, as a Cul3 adaptor. Inc binds Cul3 in vivo, and mutations within the N-terminal BTB domain of Inc that weaken Inc-Cul3 associations impair Inc activity, suggesting that Inc function requires binding to the Cul3 complex. Deletion of the conserved C-terminus of Inc does not alter Cul3 binding but abolishes Inc activity in the context of sleep and synaptic homeostasis, indicating that the Inc C-terminus has the properties of a substrate recruitment domain. Mutation of a conserved, disease-associated arginine in the Inc C-terminus also abolishes Inc function, suggesting that this residue is vital for recruiting Inc targets. Inc levels are negatively regulated by Cul3 in neurons, consistent with Inc degradation by autocatalytic ubiquitination, a hallmark of Cullin adaptors. These findings link Inc and Cul3 in vivo and support the notion that Inc-Cul3 complexes are essential for normal sleep and synaptic function. Furthermore, these results indicate that dysregulation of conserved substrates of Inc-Cul3 complexes may contribute to altered sleep and synaptic function in autism and schizophrenia associated with Cul3 mutations.

Protein ubiquitination is usually coupled with proteasomal degradation and is crucial in regulating protein quality. The E3 ubiquitin-protein ligase SCF (Skp1-Cullin-F-box) complex directly recognizes the target substrate via interaction between the F-box protein and the substrate. F-box protein is the determinant of substrate specificity. The limited number of identified ubiquitin ligase-substrate pairs is a major bottleneck in the ubiquitination field. Penicillium oxalicum contains many transcription factors, such as BrlA, CreA, XlnR, and Ace1, conserved in filamentous fungi that regulate the fungal development and transcription of (hemi)cellulase genes. Transcription factor Ace1 (also known as SltA) positively correlated with fungal growth and conidiation and negatively correlated with the expression of (hemi)cellulase genes. A ubiquitin ligase-substrate pair, SCFFbx23-Ace1, is identified in P. oxalicum. Most of PoFbx23 is present in free form within the nucleus. A small portion of PoFbx23 associates with Skp1 to form PoFbx23-Skp1 heterodimer or assembles with the three invariable core components (Skp1, Cul1, and Rbx1) of SCF to form the SCFFbx23 complex. Under glucose signal, PoFbx23 absence (Δfbx23) results in decreased transcription levels of the brlA gene which encodes the master regulator for asexual development and six spore pigmentation genes (abrB→abrA→aygB→arpA→arpB→albA) which encode the proteins in the dihydroxynaphthalene-melanin pathway, along with impaired conidiation. Under cellulose signal, transcription levels of (hemi)cellulase genes in the Δfbx23 mutant are significantly upregulated. When PoFbx23 is present, PoAce1 exists as a full-length version and several low-molecular-weight degraded versions. PoAce1 has polyubiquitin modification. Deleting the Pofbx23 gene does not affect Poace1 gene transcription but results in the remarkable accumulation of all versions of the PoAce1 protein. Accumulated PoAce1 protein is a dysfunctional form that no longer binds promoters of the target gene, including the cellulase genes cbh1 and eg1, the hemicellulase gene xyn11A, and the pigmentation-related gene abrB. PoFbx23 acts as a transcriptional coactivator, recognizing and activating PoAce1, allowing the latter to regulate the transcription of target genes with different effects (activating or repressing) under different signals.

Proteins with nuclear localization sequences (NLSs) are directed into the cell nucleus through interactions between the NLS and importin proteins. NLSs are generally short motifs rich in basic amino acids; however, identifying NLSs can be challenging due to the lack of a universally conserved sequence. In this study, we characterized the sequence specificity of an essential and conserved NLS in Mcm3, a subunit of the replicative DNA helicase. Through mutagenesis and AlphaFold 3 (AF3) modeling, we demonstrate that the precise positioning of basic residues within the NLS is critical for nuclear transport of Mcm3 through optimal interactions with importin. Disrupting these interactions impairs the nuclear import of Mcm3, resulting in defective chromatin loading of the MCM complex and poor cell growth. Our results provide a structure-guided framework for predicting and analyzing monopartite NLSs, which, despite lacking a single consensus sequence, retain key characteristics shared between the NLSs of Mcm3 and the SV40 large T antigen.

Development of multicellular organisms requires well-orchestrated interplay between cell-intrinsic transcription factors and cell-cell signaling. One set of highly conserved transcription factors that plays diverse roles in development is the SoxC group. C. elegans contains a sole SoxC protein, SEM-2. SEM-2 is essential for embryonic development, and for specifying the sex myoblast (SM) fate in the postembryonic mesoderm, the M lineage. We have identified a novel partial loss-of-function sem-2 allele that has a proline to serine change in the C-terminal tail of the highly conserved DNA-binding domain. Detailed analyses of mutant animals harboring this point mutation uncovered new functions of SEM-2 in the M lineage. First, SEM-2 functions antagonistically with LET-381, the sole C. elegans FoxF/C forkhead transcription factor, to regulate dorsoventral patterning of the M lineage. Second, in addition to specifying the SM fate, SEM-2 is essential for the proliferation and diversification of the SM lineage. Finally, SEM-2 appears to directly regulate the expression of hlh-8, which encodes a basic helix-loop-helix Twist transcription factor and plays critical roles in proper patterning of the M lineage. Our data, along with previous studies, suggest an evolutionarily conserved relationship between SoxC and Twist proteins. Furthermore, our work identified new interactions in the gene regulatory network (GRN) underlying C. elegans postembryonic development and adds to the general understanding of the structure-function relationship of SoxC proteins.