Mangiameli, S. M. | Search bioRxiv

bioRxiv2024

Subcellular Level Spatial Transcriptomics with PHOTON

The subcellular localization of RNA is closely linked to its function. Many RNA species are partitioned into organelles and other subcellular compartments for storage, processing, translation, or degradation. Thus, capturing the subcellular spatial distribution of RNA would directly contribute to the understanding of RNA functions and regulation. Here, we present PHOTON (Photoselection of Transcriptome over Nanoscale), a method which combines high resolution imaging with high throughput sequencing to achieve spatial transcriptome profiling at subcellular resolution. We demonstrate PHOTON as a versatile tool to accurately capture the transcriptome of target cell types in situ at the tissue level such as granulosa cells in the ovary, as well as RNA content within subcellular compartments such as the nucleolus and the stress granule. Using PHOTON, we also reveal the functional role of m6A modification on mRNA partitioning into stress granules. These results collectively demonstrate that PHOTON is a flexible and generalizable platform for understanding subcellular molecular dynamics through the transcriptomic lens.

genomics↗

bioRxiv2022

Photoselective sequencing: microscopically-guided genomic measurements with subcellular resolution

In biological systems, spatial organization is interconnected with genome function and regulation. However, methods that couple high-throughput genomic and epigenomic profiling with spatial information are lacking. Here, we developed Photoselective Sequencing, a spatially-informed DNA sequencing method to assay collections of cells or subcellular regions that share a unifying morphological trait. In Photoselective Sequencing, we prepare a blocked fragment library within a fixed biological specimen. Guided by fluorescence imaging, we remove the block in specific regions of interest using targeted illumination with near-UV light, ultimately allowing high-throughput sequencing of the selected fragments. To validate Photoselective Sequencing, we profile chromatin openness in fluorescently-labeled cell types within the mouse brain and demonstrate strong agreement with published single-cell ATAC-seq data. Using Photoselective Sequencing, we characterize the accessibility profiles of oligodendrocyte-lineage cells within the cortex and corpus-callosum regions of the brain. We develop a new computational strategy for decomposing bulk accessibility profiles by individual cell types, and report a relative enrichment of oligodendrocyte-progenitor-like cells in the cortex. Finally, we leverage Photoselective Sequencing for unbiased profiling of DNA at the nuclear periphery, a key chromatin organizing region. We compare and contrast the Photoselective Sequencing profile with lamin ChIP-seq data, and identify features beyond lamin interaction that are correlated with positioning at the nuclear periphery. These results collectively demonstrate that Photoselective Sequencing is a flexible and generalizable platform for exploring the interplay of spatial structures with genomic and epigenomic properties.

genomics↗

bioRxiv2016

The replisomes remain spatially proximal throughout the cell cycle in bacteria

The positioning of the DNA replication machinery (replisome) has been the subject of several studies. Two conflicting models for replisome localization have been proposed: In the Factory Model, sister replisomes remain spatially colocalized as the replicating DNA is translocated through a stationary replication factory. In the Track Model, sister replisomes translocate independently along a stationary DNA track and the replisomes are spatially separated for the majority of the cell cycle. Here, we used time-lapse imaging to observe and quantify the position of fluorescently labeled processivity-clamp (DnaN) complexes throughout the cell cycle in two highly-divergent bacterial model organisms: Bacillus subtilis and Escherichia coli. Because DnaN is a core component of the replication machinery, its localization patterns should be an appropriate proxy for replisome positioning in general. We present automated statistical analysis of DnaN positioning in large populations, which is essential due to the high degree of cell-to-cell variation. We find that both bacteria show remarkably similar DnaN positioning, where any potential separation of the two replication forks remains below the diffraction limit throughout the majority of the replication cycle. Additionally, the localization pattern of several other core replisome components is consistent with that of DnaN. These data altogether indicate that the two replication forks remain spatially colocalized and mostly function in close proximity throughout the replication cycle.The conservation of the observed localization patterns in these highly divergent species suggests that the subcellular positioning of the replisome is a functionally critical feature of DNA replication.\n\nAuthor SummaryCell proliferation depends on efficient replication of the genome. Bacteria typically have a single origin of replication on a circular chromosome. After replication initiation, two replisomes assemble at the origin and each copy one of the two arms of the chromosome until they reach the terminus. There have been conflicting reports about the subcellular positioning and putative co-localization of the two replication forks during this process. It has remained controversial whether the two replisomes remain relatively close to each other with the DNA being pulled through, or separate as they translocate along the DNA like a track. Existing studies have relied heavily on snapshot images and these experiments cannot unambiguously distinguish between these two models: i.e. two resolvable forks versus two pairs of co-localized forks. The ability of replication to re-initiate before cell division in bacterial cells further complicates the interpretation of these types of imaging studies. In this paper, we use a combination of snapshot imaging, time-lapse imaging, and quantitative analysis to measure the fraction of time forks are co-localized during each cell cycle. We find that the forks are co-localized for the majority ( 80%) of the replication cycle in two highly-divergent model organisms: B. subtilis and E. coli. Our observations are consistent with proximal localization of the two forks, but also some transient separations of sister forks during replication. The conserved behavior of sub-cellular positioning of the replisomes in these two highly divergent species implies a potential functional relevance of this feature.

cell biology↗