Microglia are CNS resident macrophages, and they play important roles in neural development and function. Recent studies have suggested that murine microglia arise from a single source, the yolk sac (YS), yet these studies lack spatial resolution to define the bona fide source(s) for microglia. Here, using light-induced high temporal-spatial resolution fate mapping, we challenge this single-source view by showing that microglia in zebrafish arise from multiple sources. The embryonic/larval microglia originate from the rostral blood island (RBI) region, the equivalent of mouse YS for myelopoiesis, whereas the adult microglia arise from the ventral wall of dorsal aorta (VDA) region, a tissue also producing definitive hematopoiesis in mouse. We further show that the VDA-region-derived microglia are Runx1 dependent, but cMyb independent, and developmentally regulated differently from the RBI region-derived microglia. Our study establishes a new paradigm for investigating the development and function of distinct microglia populations.
Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD–RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.
As the primary microtubule-organizing centers, centrosomes require ␥-tubulin for microtubule nucleation and organization. Located in close vicinity to centrosomes, the Golgi complex is another microtubule-organizing organelle in interphase cells. CDK5RAP2 is a ␥-tubulin complex-binding protein and functions in ␥-tubulin attachment to centrosomes. In this study, we find that CDK5RAP2 localizes to the Golgi complex in an ATPand centrosome-dependent manner and associates with Golgi membranes independently of microtubules. CDK5RAP2 contains a centrosome-targeting domain with its core region highly homologous to the Motif 2 (CM2) of centrosomin, a functionally related protein in Drosophila. This sequence, referred to as the CM2-like motif, is also conserved in related proteins in chicken and zebrafish. Therefore, CDK5RAP2 may undertake a conserved mechanism for centrosomal localization. Using a mutational approach, we demonstrate that the CM2-like motif plays a crucial role in the centrosomal and Golgi localization of CDK5RAP2. Furthermore, the CM2-like motif is essential for the association of the centrosome-targeting domain to pericentrin and AKAP450. The binding with pericentrin is required for the centrosomal and Golgi localization of CDK5RAP2, whereas the binding with AKAP450 is required for the Golgi localization. Although the CM2-like motif possesses the activity of Ca 2؉ -independent calmodulin binding, binding of calmodulin to this sequence is dispensable for centrosomal and Golgi association. Altogether, CDK5RAP2 may represent a novel mechanism for centrosomal and Golgi localization.
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