Compartment-specific Transcription Factors Research Articles

Systematic Identification of Cell-Cell Communication Networks in the Developing Brain.

SummarySince the generation of cell-type specific knockout models, the importance of inter-cellular communication between neural, vascular, and microglial cells during neural development has been increasingly appreciated. However, the extent of communication between these major cell populations remains to be systematically mapped. Here, we describe EMBRACE (embryonic brain cell extraction using FACS), a method to simultaneously isolate neural, mural, endothelial, and microglial cells to more than 94% purity in ∼4 h. Utilizing EMBRACE we isolate, transcriptionally analyze, and build a cell-cell communication map of the developing mouse brain. We identify 1,710 unique ligand-receptor interactions between neural, endothelial, mural, and microglial cells in silico and experimentally confirm the APOE-LDLR, APOE-LRP1, VTN-KDR, and LAMA4-ITGB1 interactions in the E14.5 brain. We provide our data via the searchable “Brain interactome explorer”, available at https://mpi-ie.shinyapps.io/braininteractomeexplorer/. Together, this study provides a comprehensive map that reveals the richness of communication within the developing brain.

Hippo Signaling Circuit and Divergent Tissue Growth in Mammalian Eye.

Vertebrate organ development is accompanied by demarcation of tissue compartments, which grow coordinately with their neighbors. Hence, perturbing the coordinative growth of neighboring tissue compartments frequently results in organ malformation. The growth of tissue compartments is regulated by multiple intercellular and intracellular signaling pathways, including the Hippo signaling pathway that limits the growth of various organs. In the optic neuroepithelial continuum, which is partitioned into the retina, retinal pigment epithelium (RPE) and ciliary margin (CM) during eye development, the Hippo signaling activity operates differentially, as it does in many tissues. In this review, we summarize recent studies that have explored the relationship between the Hippo signaling pathway and growth of optic neuroepithelial compartments. We will focus particularly on the roles of a tumor suppressor, neurofibromin 2 (NF2), whose expression is not only dependent on compartment-specific transcription factors, but is also subject to regulation by a Hippo-Yap feedback signaling circuit.

Metal-dependent SpoIIE oligomerization stabilizes FtsZ during asymmetric division in Bacillus subtilis.

SpoIIE is a bifunctional protein involved in asymmetric septum formation and in activation of the forespore compartment-specific transcription factor σF through dephosphorylation of SpoIIAA-P. The phosphatase activity of SpoIIE requires Mn2+ as a metal cofactor. Here, we show that the presence of a metal cofactor also influences SpoIIE oligomerization and asymmetric septum formation. Absence of Mn2+ from sporulation medium results in a delay of the formation of polar FtsZ-rings, similar to a spoIIE null mutant. We purified the entire cytoplasmic part of the SpoIIE protein, and show that the protein copurifies with bound metals. Metal binding both stimulates SpoIIE oligomerization, and results in the formation of larger oligomeric structures. The presence of SpoIIE oligomers reduces FtsZ GTP hydrolysis activity and stabilizes FtsZ polymers in a light scattering assay. Combined, these results indicate that metal binding is not just required for SpoIIE phosphatase activity but also is important for SpoIIE's role in asymmetric septum formation.

Functional and Phylogenetic Characterization of Proteins Detected in Various Nematode Intestinal Compartments*[S

The parasitic nematode intestine is responsible for nutrient digestion and absorption, and many other processes essential for reproduction and survival, making it a valuable target for anthelmintic drug treatment. However, nematodes display extreme biological diversity (including occupying distinct trophic habitats), resulting in limited knowledge of intestinal cell/protein functions of fundamental or adaptive significance. We developed a perfusion model for isolating intestinal proteins in Ascaris suum (a parasite of humans and swine), allowing for the identification of over 1000 intestinal A. suum proteins (using mass spectrometry), which were assigned to several different intestinal cell compartments (intestinal tissue, the integral and peripheral intestinal membranes, and the intestinal lumen). A multi-omics analysis approach identified a large diversity of biological functions across intestinal compartments, based on both functional enrichment analysis (identifying terms related to detoxification, proteolysis, and host-parasite interactions) and regulatory binding sequence analysis to identify putatively active compartment-specific transcription factors (identifying many related to intestinal sex differentiation or lifespan regulation). Orthologs of A. suum proteins in 15 other nematodes species, five host species, and two outgroups were identified and analyzed. Different cellular compartments demonstrated markedly different levels of protein conservation; e.g. integral intestinal membrane proteins were the most conserved among nematodes (up to 96% conservation), whereas intestinal lumen proteins were the most diverse (only 6% conservation across all nematodes, and 71% with no host orthologs). Finally, this integrated multi-omics analysis identified conserved nematode-specific intestinal proteins likely performing essential functions (including V-type ATPases and ABC transporters), which may serve as promising anthelmintic drug or vaccine targets in future research. Collectively, the findings provide valuable new insights on conserved and adaptive features of nematode intestinal cells, membranes and the intestinal lumen, and potential targets for parasite treatment and control.

Compartment-specific transcription factors orchestrate angiogenesis gradients in the embryonic brain

Prevailing notions of cerebral vascularization imply that blood vessels sprout passively into the brain parenchyma from pial vascular plexuses to meet metabolic needs of growing neuronal populations. Endothelial cells, building blocks of blood vessels, are thought to be homogeneous in the brain with respect to their origins, gene expression patterns and developmental mechanisms. These current notions that cerebral angiogenesis is regulated by local environmental signals contrast with current models of cell-autonomous regulation of neuronal development. Here we demonstrate that telencephalic angiogenesis in mice progresses in an orderly, ventral-to-dorsal gradient regulated by compartment-specific homeobox transcription factors. Our data offer new perspectives on intrinsic regulation of angiogenesis in the embryonic telencephalon, call for a revision of the current models of telencephalic angiogenesis and support novel roles for endothelial cells in brain development.

Signalling network with a bistable hysteretic switch controls developmental activation of the σF transcription factor in Bacillus subtilis

The sporulation process of the bacterium Bacillus subtilis unfolds by means of separate but co-ordinated programmes of gene expression within two unequal cell compartments, the mother cell and the smaller forespore. sigmaF is the first compartment-specific transcription factor activated during this process, and it is controlled at the post-translational level by a partner-switching mechanism that restricts sigmaF activity to the forespore. The crux of this mechanism lies in the ability of the anti-sigma factor SpoIIAB (AB) to form alternative complexes either with sigmaF, holding it in an inactive form, or with the anti-anti-sigma factor SpoIIAA (AA) and a nucleotide, either ATP or ADP. In the complex with AB and ATP, AA is phosphorylated on a serine residue and released, making AB available to capture sigmaF in an inactive complex. Subsequent activation of sigmaF requires the intervention of the SpoIIE serine phosphatase to dephosphorylate AA, which can then attack the AB-sigmaF complex to induce the release of sigmaF. By incorporating biochemical, biophysical and genetic data from the literature we have constructed an integrative mathematical model of this partner-switching network. The model predicts that the self-enhancing formation of a long-lived complex of AA, AB and ADP transforms the network into an essentially irreversible hysteretic switch, thereby explaining the sharp, robust and irreversible activation of sigmaF in the forespore compartment. The model also clarifies the contributions of the partly redundant mechanisms that ensure correct spatial and temporal activation of sigmaF, reproduces the behaviour of various mutants and makes strong, testable predictions.

The cell differentiation protein SpoIIE contains a regulatory site that controls its phosphatase activity in response to asymmetric septation.

Starvation induces Bacillus subtilis to initiate a -simple, two-cell developmental process that begins with an asymmetric cell division. Activation of the first compartment-specific transcription factor, sigmaF, is coupled to this morphological event. SpoIIE, a bifunctional protein, is essential for the compartment-specific activation of sigmaF and also has a morphogenic activity required for asymmetric cell division. SpoIIE consists of three domains: a hydrophobic N-terminal domain, which targets the protein to the membrane; a central domain, involved in oligomerization of SpoIIE and interaction with the cell division protein FtsZ; and a C-terminal domain comprising a PP2C protein phosphatase. Here, we report the isolation of mutations at the very beginning of the central domain of spoIIE, which are capable of activating sigmaF inde-pendently of septum formation. Purified mutant proteins showed the same phosphatase activity as the wild-type protein in vitro. The mutant proteins were fully functional in respect of their localization to sites of asymmetric septation and support of asymmetric division. The data provide strong evidence that the phosphatase domain of SpoIIE is tightly regulated in a way that makes it respond to the formation of the asymmetric septum.

Direct interaction between the cell division protein FtsZ and the cell differentiation protein SpoIIE.

SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate in Bacillus subtilis. First, SpoIIE is needed for the normal formation of the asymmetrically positioned septum that forms early in sporulation and separates the mother cell from the prespore compartment. Secondly, SpoIIE is essential for the activation of the first compartment-specific transcription factor sigma(F) in the prespore. After initiation of sporulation, SpoIIE localizes to the potential asymmetric cell division sites near one or both cell poles. Localization of SpoIIE was shown to be dependent on the essential cell division protein FtsZ. To understand how SpoIIE is targeted to the asymmetric septum we have now analysed its interaction with FtsZ in vitro. Using the yeast two-hybrid system and purified FtsZ, and full-length and truncated SpoIIE proteins, we demonstrate that the two proteins interact directly and that domain II and possibly domain I of SpoIIE are required for the interaction. Moreover, we show that SpoIIE interacts with itself and suggest that this self-interaction plays a role in assembly of SpoIIE into the division machinery.

Contribution of partner switching and SpoIIAA cycling to regulation of sigmaF activity in sporulating Bacillus subtilis.

sigmaF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-sigma factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or sigmaF depends on the nature of the adenine nucleotide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, sigmaF-SpoIIAB-ADP, and sigmaF-SpoIIAB-ATP. The results suggested that SpoIIAB's choice of binding partner is unlikely to depend on the ATP/ADP ratio in the cell. The intracellular concentrations of sigmaF, SpoIIAB, SpoIIAA, and SpoIIAA-phosphate (SpoIIAA-P) were measured by quantitative immunoblotting between 0 and 3 h after the beginning of sporulation (t0 to t3). sigmaF and SpoIIAB were barely detectable at t0, but their concentrations increased in parallel to reach maxima at about t1.5. SpoIIAA-P increased steadily to a maximum at t3, but nonphosphorylated SpoIIAA was detectable only from t1.5, reached a maximum at t2.5, and then declined. Kinetic studies of the phosphorylation of SpoIIAA catalyzed by SpoIIAB suggested that the reaction was limited by a very slow release of one of the products (SpoIIAA-P or ADP) from SpoIIAB, with a turnover of about once per 20 min. This remarkable kinetic property provides an unexpected mechanism for the regulation of sigmaF. We propose that when SpoIIE (which dephosphorylates SpoIIAA-P) is active at the same time as SpoIIAB, SpoIIAA cycles repeatedly between the phosphorylated and nonphosphorylated forms. This cycling sequesters SpoIIAB in a long-lived complex and prevents it from inhibiting sigmaF.

Role of interactions between SpoIIAA and SpoIIAB in regulating cell-specific transcription factor σF of Bacillus subtilis

Genetic experiments have suggested that sigma F, the first compartment-specific transcription factor in sporulating B. subtilis, is regulated by an anti-sigma factor SpoIIAB and an anti-anti-sigma factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of sigma F-directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA-phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA-phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-sigma F activity, whereas SpoIIAAS58A, which cannot be phosphorylated, makes complexes with SpoIIAB in the presence of ADP or ATP and has constitutive anti-anti-sigma F activity both in vivo and in vitro. It seems likely that the alternative reactions of SpoIIAA and SpoIIAB, involving ADP or ATP, regulate the anti-anti-sigma capacity of SpoIIAA and hence the activity of sigma F.

σF, the first compartment-specific transcription factor of B. subtilis, is regulated by an anti-σ factor that is also a protein kinase

The establishment of compartment-specific transcription in sporulating cells of B. subtilis is governed at the level of the activity of transcription factor σ F. Genetic experiments have suggested that SpoIIAA and SpoIIAB, the other products of the σ F operon, are involved in regulating σ F activity. This activity is inhibited in the predivisional cell but specifically released from inhibition in the prespore about 1.5 hr after sporulation is induced. We now show that purified SpoIIAB Inhibits transcription directed by σ F in vitro. We note that the amino acid sequence of SpoIIAB shows some similarity to a group of bacterial histidine protein kinases, and we find that SpoIIAB is indeed a protein kinase that phosphorylates SpoIIAA on a serine residue. We suggest that this phosphorylation is responsible for the compartment-specific release of σ F activity, perhaps through the formation of a tight complex between SpoIIAB and phosphorylated SpoIIAA.

Deletion of spoIIAB blocks endospore formation in Bacillus subtilis at an early stage

During an early stage of endospore formation in Bacillus subtilis, the cell divides asymmetrically into two compartments that follow different developmental paths. The differential expression of genes in these two compartments is controlled in part by the production of compartment-specific transcription factors, sigma G and sigma K. It is not known how sigma G accumulation is restricted to one of the two compartments, the forespore. However, the observations that sigma F directs transcription of the structural gene for sigma G and that sigma F activity can be modified by the product of a gene, spoIIAB, has led us to investigate the role of spoIIAB during sporulation. We have isolated mutants that carry deletion alleles of spoIIAB. Electron microscopic examination of these mutants revealed that these mutations blocked endospore formation at an early stage before septation and caused extensive cell lysis. The spoIIAB deletion alleles caused hyperexpression of genes that are normally expressed exclusively in the forespore compartments of sporulating wild-type cells, whereas these alleles reduced expression of other genes, including spoIIE, which is expressed before septation in wild-type cells. These observations confirm that spoIIAB is essential for sporulation and are consistent with models in which the product of spoIIAB plays a role in regulating the timing and/or compartment specificity of sigma F- and sigma G-directed transcription.