Biology subjects

Scott, M.

Publications and source records attributed to Scott, M..

25 records · Page 2Linked to original sources

bioRxiv2021

Growth-dependent heterogeneity in the DNA damage response in Escherichia coli

In natural environments bacteria are frequently exposed to sub-lethal levels of DNA damage which leads to the induction of a stress response (the SOS response in Escherichia coli). Natural environments also vary in nutrient availability, resulting in distinct physiological changes in bacteria which may have direct implications on their capacity to repair their chromosomes. Here, we evaluated the impact of varying the nutrient availability on the expression of the SOS response induced by chronic sub-lethal DNA damage in E. coli. The expression of the SOS regulon was found to be highly heterogeneous at the single-cell level in all growth conditions. Surprisingly, we observed a larger fraction of high SOS-induced cells in slow growth as compared with fast growth, despite a higher rate of SOS induction in fast growth. This counter-intuitive result can be explained by the dynamic balance between the rate of SOS induction and the division rates of cells exposed to DNA damage. Taken together, our data illustrates how cell division and physiology come together to produce growth-dependent heterogeneity in the DNA damage response.

cell biology↗

bioRxiv2020

A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi. For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi. Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen. IMPORTANCEGene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

microbiology↗

bioRxiv2020

Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation and migration. Erk is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response however, involves a Vegfr-dependent mechanism that initiates concomitant with the wound inflammatory response. This work thus reveals a highly dynamic sequence in regenerative angiogenesis that was not previously appreciated. Altogether, this study demonstrates the utility of a unique biosensor strain for analysing dynamic endothelial Erk-signalling events and validates a new resource for the study of vascular signalling in real-time.

developmental biology↗

bioRxiv2020

Comparative analysis of genome-wide DNA methylation identifies patterns that associate with conserved transcriptional programs in osteosarcoma.

Osteosarcoma is an aggressive tumor of the bone that primarily affects young adults and adolescents. Osteosarcoma is characterized by genomic chaos and heterogeneity. While inactivation of tumor suppressor p53 TP53 is nearly universal other high frequency mutations or structural variations have not been identified. Despite this genomic heterogeneity, key conserved transcriptional programs associated with survival have been identified across human, canine and induced murine osteosarcoma. The epigenomic landscape, including DNA methylation, plays a key role in establishing transcriptional programs in all cell types. The role of epigenetic dysregulation has been studied in a variety of cancers but has yet to be explored at scale in osteosarcoma. Here we examined genome-wide DNA methylation patterns in 24 human and 44 canine osteosarcoma samples identifying groups of highly correlated DNA methylation marks in human and canine osteosarcoma samples. We also link specific DNA methylation patterns to key transcriptional programs in both human and canine osteosarcoma. Building on previous work, we built a DNA methylation-based measure for the presence and abundance of various immune cell types in osteosarcoma. Finally, we determined that the underlying state of the tumor, and not changes in cell composition, were the main driver of differences in DNA methylation across the human and canine samples. SignificanceThis is the first large scale study of DNA methylation in osteosarcoma and lays the ground work for the exploration of DNA methylation programs that help establish conserved transcriptional programs in the context of different genomic landscapes.

cancer biology↗

bioRxiv2020

The role of startle fluctuation and non-response startle reflex in tracking amygdala dynamics

The startle reflex is considered a sensitive defensive reaction to potential threats that manifests as a unique eye blink-like pattern in the EMG. Eye blink EMG has a bell-shaped potential when startle probes are elicited, that strongly corresponds to amygdala activity. Considering how amygdala activity fluctuates over time in response to emotional and self-threatening stimuli, observing startle eyeblink size fluctuation over time could provide a cost-effective, convenient, and less resource intensive means for examining amygdala activity over time. Yet based on current standards in the literature, often startle evoked eye blink EMG values do not exhibit activity 3SDs from the mean eyeblink response, thus these trials are typically excluded from startle analyses. It stands to reason, however, that these trials may still index amygdala activity in a meaningful way. Through investigating the association between startle eyeblink amplitude, corresponding ERP amplitude, and underlying neural activity, the current study provides evidence that startle amplitudes exhibit a linear relationship with proxies for amygdala activity, e.g., N100 amplitudes and regions heavily interconnected with the amygdala. Specifically, the startle reflex correlates to large amount of brain regions in N100 time window in addition to the N100 amplitude. Thus, both valid and otherwise traditionally non-valid startle reflex responses appear to index amygdala activity and should be included accordingly. This approach could help salvage large amounts of meaningful data traditionally excluded from studies interested in amygdala responses to various stimuli over time.

neuroscience↗

bioRxiv2019

Development and testing of a novel Killer-Rescue self-limiting gene drive system in Drosophila melanogaster

We report the development and laboratory testing of a novel Killer-Rescue (K-R) self-limiting gene drive system in Drosophila melanogaster. This K-R system utilizes the well-characterized Gal4/UAS binary expression system and the Gal4 inhibitor, Gal80. Three killer (K) lines were tested; these used either an autoregulated UAS-Gal4 or UAS-Gal4 plus UAS-hid transgene. One universal rescue (R) line was used, UAS-Gal80, to inhibit Gal4 expression. The K lines are lethal and cause death in the absence of R. We show that Gal4 RNA levels are high in the absence of R. Death is possibly due to transcriptional squelching from high levels of Gal4. When R is present, Gal4 activation of Gal80 would lead to inhibition of Gal4 and prevent overexpression. With a single release ratio of 2:1 engineered K-R to wildtype, we find that K drives R through the population while the percent of wild type individuals decreases each generation. The choice of core promoter for a UAS-Gal4 construct strongly influences the K-R system. With the strong hsp70 core promoter, K was very effective but was quickly lost from the population. With the weaker DSCP core promoter, K persisted for longer allowing the frequency of individuals with at least one copy of R to increase to over 98%. This simple gene drive system could be readily adapted to other species such as mosquito disease vectors for driving anti-viral or anti-parasite genes.\n\nSignificanceHere we report the development and testing of a novel self-limiting gene drive system, Killer-Rescue, in Drosophila melanogaster. This system is composed of an auto-regulated Gal4 Killer (K) and a Gal4-activated Gal80 Rescue (R). Overexpression of Gal4 is lethal but in the presence of R, activation of Gal80 leads to much lower levels of Gal4 and rescue of lethality. We demonstrate that with a single 2:1 engineered to wildtype release, more than 98% of the population carry R after eight generations. We discuss how this Killer-Rescue system may be used for population replacement in a human health pest, Aedes aegypti, or for population suppression in an agricultural pest, Drosophila suzukii.

genetics↗

bioRxiv2018

Characterization of fluorescent proteins, promoters, and selectable markers for applications in the Lyme disease spirochete Borrelia burgdorferi

Lyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirochete Borrelia burgdorferi, is thus evermore pressing. Despite important advances in B. burgdorferi genetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multi-color imaging in B. burgdorferi. We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Lastly, we describe a set of constitutively active promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities in B. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, a B. burgdorferi lipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites of B. burgdorferi cells, highlighting the complex subcellular organization of this spirochete.\n\nIMPORTANCEGenetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacteriums transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and constitutively active promoters of different strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

microbiology↗