Nakamoto, R. | bioRxiv Science

bioRxiv2025

Spatial patterning of transcriptional and regulatory programs in the primate subcortex

Mammalian brain cell identity is shaped by intrinsic factors and external context. We present a spatially resolved transcriptomic and gene regulatory atlas of cell types across all subcortical regions in a primate, the common marmoset. Dense sampling and cross-species integration revealed spatially precise neuronal assemblies, including in complex midbrain and diencephalic structures. Chromatin accessibility and transcriptional identity are spatially tuned within and across subcortical structures; spatial gradients within hippocampal subfields are orchestrated by graded transcription factors acting through graded enhancers. The primate-expanded thalamic GABAergic population shares transcriptional and regulatory syntax with conserved midbrain populations, reflecting an evolutionary adaptation compared with rodents. Similar regional expression across cell types can arise by distinct regulatory architectures, as for telencephalic astrocytes and neurons. Conversely, distant cell types can share regulatory programs despite divergent identities: striatal GABAergic medium spiny neurons and telencephalic glutamatergic neurons share a postsynaptic regulatory program despite divergent lineage, region, and neurotransmitter identity.

neuroscience↗

bioRxiv2019

Disulfide chaperone knock-outs enable in-vivo double spin-labeling of an outer-membrane transporter

Recent advances in the application of EPR spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer-membrane proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli outer-membrane vitamin B12 transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated outer membranes or reconstituted systems, and the ability to label and perform EPR in cells is expected to be applicable to a range of other bacterial outer-membrane proteins.\n\nStatement of SignificanceEPR spectroscopy is an important method to characterize the structure and dynamics of membrane proteins, and recent efforts demonstrate that pulse EPR can be used to examine the extracellular surface of outer membrane proteins in live bacteria. In the present work, we show that pairs of cysteine residues in the Escherichia coli vitamin B12 transporter, BtuB, cannot be spin-labeled in wild-type strains, but can be labeled with the use of certain null mutants in the periplasmic disulfide bond formation, Dsb, system. These mutants also facilitate efficient spin-labeling of cysteines located on the periplasmic surface of BtuB. Distance measurements using pulse EPR provide evidence that the behavior of BtuB is different in the bacterial cell than it is in purified systems.

biophysics↗