Biology subjects

Murphy, P. A.

Publications and source records attributed to Murphy, P. A..

4 recordsLinked to original sources

bioRxiv2021

Quantification of Myxococcus xanthus Aggregation and Rippling Behaviors: Deep-learning Transformation of Phase-contrast into Fluorescence Microscopy Images

Myxococcus xanthus bacteria are a model system for understanding pattern formation and collective cell behaviors. When starving, cells aggregate into fruiting bodies to form metabolically inert spores. During predation, cells self-organize into traveling cell-density waves termed ripples. Both phase-contrast and fluorescence microscopy are used to observe these patterns but each has its limitations. Phase-contrast images have higher contrast, but the resulting image intensities lose their correlation with cell density. The intensities of fluorescence microscopy images, on the other hand, are well correlated with cell density, enabling better segmentation of aggregates and better visualization of streaming patterns in between aggregates. However, fluorescence microscopy requires the engineering of cells to express fluorescent proteins and can be phototoxic to the cells. To combine the advantages of both imaging methodologies, we develop a generative adversarial network that converts phase-contrast into fluorescent images. By including an additional histogram-equalized output to the state-of-art pix2pixHD algorithm, our model generates accurate images of aggregates and streams, enabling the estimation of aggregate positions and sizes, but with small shifts of their boundaries. Further training on ripple patterns enables accurate estimation of the rippling wavelength. Our methods are thus applicable for many other phenotypic behaviors and pattern formation studies.

systems biology↗

bioRxiv2021

Splice Factor Polypyrimidine tract-binding protein 1 (Ptbp1) is Required for Immune Priming of the Endothelium in Atherogenic Disturbed Flow Conditions

NF{kappa}B mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through upregulation of adhesion molecules Icam1 and Vcam. The endothelium is "primed" for cytokine activation of NF{kappa}B by exposure to low and disturbed blood flow (LDF) in vivo and by LDF or static conditions in cultured cells. While priming leads to an exaggerated expression of Icam1 and Vcam following cytokine stimulation, the molecular underpinnings are not fully understood. We showed that alternative splicing of genes regulating NF{kappa}B signaling occurs during priming, but the functional implications of this are not known. We hypothesize that the regulation of splicing by RNA-binding splice factors is critical for priming. Here, we perform a CRISPR screen in cultured aortic endothelial cells to determine whether splice factors active in the response to LDF participate in endothelial cell priming. Using Icam1 and Vcam induction by TNF stimulation as a marker of priming, we identify polypyrimidine tract binding protein (Ptbp1) as a required splice factor. Ptbp1 expression is increased and its motifs are enriched nearby alternatively spliced exons in endothelial cells exposed to LDF in vivo in a platelet dependent manner, indicating its induction by early innate immune cell recruitment. At a mechanistic level, deletion of Ptbp1 inhibited NF{kappa}B nuclear translocation and transcriptional activation. These changes coincided with altered splicing of key components of the NF{kappa}B signaling pathway that were similarly altered in the LDF response. However, these splicing and transcriptional changes could be restored by expression of human PTBP1 cDNA in Ptbp1 deleted cells. In vivo, endothelial specific deletion of Ptbp1 reduced myeloid cell infiltration at regions of LDF in atherosclerotic mice. In human coronary arteries, PTBP1 expression correlates with expression of TNF pathway genes and amount of plaque. Together, our data suggest that Ptbp1, which is activated in the endothelium by innate immune cell recruitment in regions of LDF, is required for priming of the endothelium for subsequent NF{kappa}B activation and myeloid cell recruitment in vascular inflammation. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Plaque forms in low and disturbed flow regions of the vasculature, where endothelial cells are "primed" to respond to cytokines (e.g. TNF) with elevated levels of cell adhesion molecules via the NF{kappa}B signaling pathway. We show that the splice factor Ptbp1 (purple) mediates priming. Ptbp1 is induced in endothelial cells by platelet recruitment, promoting priming and subsequent myeloid cell infiltration into plaque. Mechanistically, Ptbp1 regulates splicing of genes involved in the NF{kappa}B signaling pathway and is required for efficient nuclear translocation of NF{kappa}B in endothelial cells. This provides new insight into the molecular mechanisms underlying an endothelial priming process that reinforces vascular inflammatory responses. C_FIG_DISPLAY

cell biology↗

bioRxiv2021

A Method for Rapid Flow-cytometric Isolation of Endothelial Nuclei and RNA from Archived Frozen Brain Tissue

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

cell biology↗

bioRxiv2021

Identification of Splice Regulators of Fibronectin-EIIIA and EIIIB by Direct Measurement of Exon Usage in a Flow-Cytometry Based CRISPR Screen

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

cell biology↗