Astrocytes are complex glial cells with numerous fine cellular processes that infiltrate the neuropil and interact with synapses. The mechanisms that control the establishment of astrocyte morphology ...are unknown, and it is unclear whether impairing astrocytic infiltration of the neuropil alters synaptic connectivity. Here we show that astrocyte morphogenesis in the mouse cortex depends on direct contact with neuronal processes and occurs in parallel with the growth and activity of synaptic circuits. The neuroligin family cell adhesion proteins NL1, NL2, and NL3, which are expressed by cortical astrocytes, control astrocyte morphogenesis through interactions with neuronal neurexins. Furthermore, in the absence of astrocytic NL2, the formation and function of cortical excitatory synapses are diminished, whereas inhibitory synaptic function is enhanced. Our findings highlight a previously undescribed mechanism of action for neuroligins and link astrocyte morphogenesis to synaptogenesis. Because neuroligin mutations have been implicated in various neurological disorders, these findings also point towards an astrocyte-based mechanism of neural pathology.
The emergence of antibiotic resistance in bacteria has led to the investigation of alternative treatments, such as phage therapy. In this study, we examined the interactions between the ...nucleus-forming jumbo phage ФKZ and antibiotic treatment against Pseudomonas aeruginosa. Using the fluorescence microscopy technique of bacterial cytological profiling, we identified mechanism-of-action-specific interactions between antibiotics that target different biosynthetic pathways and ФKZ infection. We found that certain classes of antibiotics strongly inhibited phage replication, while others had no effect or only mildly affected progression through the lytic cycle. Antibiotics that caused an increase in host cell length, such as the cell wall active antibiotic ceftazidime, prevented proper centering of the ФKZ nucleus via the PhuZ spindle at midcell, leading us to hypothesize that the kinetic parameters of the PhuZ spindle evolved to match the average length of the host cell. To test this, we developed a computational model explaining how the dynamic properties of the PhuZ spindle contribute to phage nucleus centering and why some antibiotics affect nucleus positioning while others do not. These findings provide an understanding of the molecular mechanisms underlying the interactions between antibiotics and jumbo phage replication.
Organoid technologies have become a powerful emerging tool to model liver diseases, for drug screening, and for personalized treatments. These applications are, however, limited in their capacity to ...generate functional hepatocytes in a reproducible and efficient manner. Here, we generated and characterized the hepatic organoid (eHEPO) culture system using human induced pluripotent stem cell (iPSC)-derived EpCAM-positive endodermal cells as an intermediate. eHEPOs can be produced within 2 weeks and expanded long term (>16 months) without any loss of differentiation capacity to mature hepatocytes. Starting from patient-specific iPSCs, we modeled citrullinemia type 1, a urea cycle disorder caused by mutations in the argininosuccinate synthetase (ASS1) enzyme. The disease-related ammonia accumulation phenotype in eHEPOs could be reversed by the overexpression of the wild-type ASS1 gene, which also indicated that this model is amenable to genetic manipulation. Thus, eHEPOs are excellent unlimited cell sources to generate functional hepatic organoids in a fast and efficient manner.
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•iPSC-derived EpCAM+ endodermal cells generate functional hepatic organoids (eHEPOs)•eHEPOs can be stably maintained in long-term culture•An organoid-based model for a urea cycle disorder (citrullinemia) is generated•Genetic manipulation of disease-specific eHEPOs rescues disease phenotype
Erdal and colleagues generated a hepatic organoid (eHEPO) culture system using human iPSC-derived EpCAM-positive endodermal cells. eHEPOs can be produced within 2 weeks and expanded long term (>16 months) without any loss of differentiation capacity to mature hepatocytes. Starting from patient-specific iPSCs, a urea cycle disorder, citrullinemia, was modeled and disease phenotype was rescued via overexpression of wild-type ASS1 gene.
The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcare's most potent antibiotics are becoming obsolete. Approximately two-thirds of the world's ...antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 104 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.
The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and ...somatic-specific transcriptional programs remain unknown.
We employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes.
Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.
The emergence of antibiotic resistance in bacteria has led to the investigation of alternative treatments, such as phage therapy. In this study, we examined the interactions between the ...nucleus-forming jumbo phage PHIKZ and antibiotic treatment against Pseudomonas aeruginosa. Using the fluorescence microscopy technique of bacterial cytological profiling, we identified mechanism-of-action-specific interactions between antibiotics that target different biosynthetic pathways and PHIKZ infection. We found that certain classes of antibiotics strongly inhibited phage replication, while others had no effect or only mildly affected progression through the lytic cycle. Antibiotics that caused an increase in host cell length, such as the cell wall active antibiotic ceftazidime, prevented proper centering of the PHIKZ nucleus via the PhuZ spindle at midcell, leading us to hypothesize that the kinetic parameters of the PhuZ spindle evolved to match the average length of the host cell. To test this, we developed a computational model explaining how the dynamic properties of the PhuZ spindle contribute to phage nucleus centering and why some antibiotics affect nucleus positioning while others do not. These findings provide an understanding of the molecular mechanisms underlying the interactions between antibiotics and jumbo phage replication.
In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against host immune ...factors. By segregating the genome from the host cytoplasm, however, the 'phage nucleus' introduces the need to specifically translocate messenger RNA and proteins through the nuclear shell and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear-shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggest that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging and may also participate in messenger RNA and/or protein translocation.
To protect themselves from the constant threat of bacteriophage (phage) infection, bacteria have evolved diverse immune systems including restriction‐modification, CRISPR‐Cas, and many others. Here, ...we describe the discovery of a two‐protein transcriptional regulator module associated with hundreds of CBASS immune systems and demonstrate that this module drives the expression of its associated CBASS system in response to DNA damage. We show that the helix‐turn‐helix transcriptional repressor CapH binds the promoter region of its associated CBASS system to repress transcription until it is cleaved by the metallopeptidase CapP. CapP is activated in vitro by single‐stranded DNA, and in cells by DNA‐damaging drugs. Together, CapH and CapP drive increased expression of their associated CBASS system in response to DNA damage. We identify CapH‐ and CapP‐related proteins associated with diverse known and putative bacterial immune systems including DISARM and Pycsar antiphage operons. Overall, our data highlight a mechanism by which bacterial immune systems can sense and respond to a universal signal of cell stress, potentially enabling multiple immune systems to mount a coordinated defensive response against an invading pathogen.
Synopsis
Bacterial anti‐phage defense mechanisms include the CBASS immune systems. Here, a pair of proteins is found to control CBASS gene expression in response to DNA damage, with bioinformatics showing that similar proteins regulate a broad set of bacterial defense systems.
Hundreds of bacterial CBASS operons are located adjacent to a pair of genes termed capH and capP.
CapH is a DNA‐binding helix‐turn‐helix family transcriptional repressor.
CapP is a zinc metallopeptidase that specifically cleaves CapH in the presence of single‐stranded DNA.
CapH and CapP upregulate their associated CBASS system in response to DNA damage.
In addition to CBASS, CapP and CapH homologs are associated with Pycsar, DISARM, and other bacterial defense systems.
The CapH/CapP transcriptional regulator module exemplifies how bacterial anti‐phage immunity may be harnessed for other stress responses.
Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems
. In response, one family of large bacteriophages uses a ...nucleus-like compartment to protect its replicating genomes by excluding host defence factors
. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.
Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic ...adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.
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•Astrocyte-secreted hevin is a pre- and postsynaptic organizer•Hevin induces thalamocortical synapse formation by bridging NRX1α and NL1•Hevin is required for recruitment of NL1 and NMDAR to excitatory synapses in vivo•Astrocyte-secreted hevin is necessary for ocular dominance plasticity
Astrocytes promote synapse formation and plasticity by secreting a protein that bridges non-interacting isoforms of neurexin and neuroligin, suggesting an additional layer of complexity to the isoform code that dictates synaptic adhesion.
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