Microglia maintain structural integrity during fetal brain morphogenesis

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.</p

Decoding the Divergent Subcellular Location of Two Highly Similar Paralogous LEA Proteins

Many mitochondrial proteins are synthesized as precursors in the cytosol with an N-terminal mitochondrial targeting sequence (MTS) which is cleaved off upon import. Although much is known about import mechanisms and MTS structural features, the variability of MTS still hampers robust sub-cellular software predictions. Here, we took advantage of two paralogous late embryogenesis abundant proteins (LEA) from Arabidopsis with different subcellular locations to investigate structural determinants of mitochondrial import and gain insight into the evolution of the LEA genes. LEA38 and LEA2 are short proteins of the LEA_3 family, which are very similar along their whole sequence, but LEA38 is targeted to mitochondria while LEA2 is cytosolic. Differences in the N-terminal protein sequences were used to generate a series of mutated LEA2 which were expressed as GFP-fusion proteins in leaf protoplasts. By combining three types of mutation (substitution, charge inversion, and segment replacement), we were able to redirect the mutated LEA2 to mitochondria. Analysis of the effect of the mutations and determination of the LEA38 MTS cleavage site highlighted important structural features within and beyond the MTS. Overall, these results provide an explanation for the likely loss of mitochondrial location after duplication of the ancestral gene

Experimental determination of organelle targeting peptide cleavage sites using transient expression of GFP translational fusions

The majority of nuclear-encoded organellar proteins contain a cleavable presequence which is necessary for protein targeting and import into the correct cellular compartment. Knowledge about targeting-peptide cleavage sites is essential for the structural and functional characterization of the mature organellar proteins as well as for a deeper understanding of the import process. Because of the low consensus and high variability of presequences, bioinformatics of targeting-peptide cleavage fails to predict the length of the targeting peptide with high confidence. Therefore, we have developed a rapid and robust method to experimentally determine the cleavage site of the transit peptide for proteins imported into mitochondria or plastids. The protein precursor with GFP fused to its C-terminus is transiently expressed in cells (for animal proteins) or protoplasts (for plant proteins), allowing translocation into organelles, and removal of the transit peptide. After lysis, the matured protein is immunopurified using an anti-GFP antibody coupled to magnetic beads. The N-terminal amino sequence is then determined by Edman microsequencing or mass spectrometry. The method has been validated using proteins with known targeting peptide sequences and is suitable for animal and plant organelle-targeted proteins