Generation of human excitatory forebrain neurons by cooperative binding of proneural NGN2 and homeobox factor EMX1.

Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.

Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3.

Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5-3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5-3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non-phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5-3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.

The integrated stress response regulates BMP signalling through effects on translation.

BACKGROUND: Developmental pathways must be responsive to the environment. Phosphorylation of eIF2α enables a family of stress-sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Bone morphogenetic proteins (BMPs) regulate multiple developmental processes in organisms from insects to mammals. RESULTS: Here we show in Drosophila that GCN2 antagonises BMP signalling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2α phosphatase dPPP1R15 impairs developmental BMP signalling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signalling. Moreover, loss of d4E-BP, a target of crc, augments BMP signalling in vitro and rescues tissue development in vivo. CONCLUSION: These results identify a novel mechanism by which the ISR modulates BMP signalling during development.

The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.

α1-Antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming polymers that become trapped in inclusions of endoplasmic reticulum (ER). It is unclear whether these inclusions are connected to the main ER network in Z-α1-antitrypsin-expressing cells. Using live cell imaging, we found that despite inclusions containing an immobile matrix of polymeric α1-antitrypsin, small ER resident proteins can diffuse freely within them. Inclusions have many features to suggest they represent fragmented ER, and some are physically separated from the tubular ER network, yet we observed cargo to be transported between them in a cytosol-dependent fashion that is sensitive to N-ethylmaleimide and dependent on Sar1 and sec22B. We conclude that protein recycling occurs between ER inclusions despite their physical separation.-Dickens, J. A., Ordóñez, A., Chambers, J. E., Beckett, A. J., Patel, V., Malzer, E., Dominicus, C. S., Bradley, J., Peden, A. A., Prior, I. A., Lomas, D. A., Marciniak, S. J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.

New Concepts in Alpha-1 Antitrypsin Deficiency Disease Mechanisms.

Alpha-1 antitrypsin deficiency is predominantly caused by point mutations that alter the protein's folding. These mutations fall into two broad categories: those that destabilize the protein dramatically and lead to its post-translational degradation and those that affect protein structure more subtly to promote protein polymerization within the endoplasmic reticulum (ER). This distinction is important because it determines the cell's response to each mutant. The severely misfolded mutants trigger an unfolded protein response (UPR) that promotes improved protein folding but can kill the cell in the chronic setting. In contrast, mutations that permit polymer formation fail to activate the UPR but instead promote a nuclear factor-κB-mediated ER overload response. The ability of polymers to increase a cell's sensitivity to ER stress likely explains apparent inconsistencies in the alpha-1 antitrypsin-signaling literature that have linked polymers with the UPR. In this review we discuss the use of mutant serpins to dissect each signaling pathway.

Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation.

Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR.