Biology subjects

Spiller, D.

Publications and source records attributed to Spiller, D..

6 recordsLinked to original sources

bioRxiv2024

Interplay between material properties and cellular effects drives distinct pattern of interaction of graphene oxide with cancer and non-cancer cells

Understanding how graphene oxide (GO) interacts with cells is crucial for its safe and efficient biomedical applications. Despite extensive research, a systematic investigation using a panel of cell lines, thoroughly characterized label-free nanomaterials, and complementary analytical techniques is lacking. Here, we examined the uptake of thin GO sheets with distinct lateral dimensions in 13 cell lines: 8 cancer (HeLa, A549, PC3, DU-145, LNCaP, SW-480, SH-SY5Y, U87-MG) and 5 non-cancer (BEAS-2B, NIH/3T3, PNT-2, HaCaT, 293T), using confocal microscopy, transmission electron microscopy, and flow cytometry. Our results reveal a striking difference in GO uptake: non-cancer cells internalized GO efficiently, while in cancer cells, GO predominantly interacted with the plasma membrane, showing minimal to no internalization. Comparison to other nanomaterials (polystyrene beads and graphene flakes) confirmed that cancer cells internalize materials similarly to non-cancer cells, indicating GO-specific interactions. We identified that GO thickness plays a pivotal role in this differential uptake. Additionally, GO disrupted the actin cytoskeleton, impairing migration in cancer but not in non-cancer cells. We propose that thin GO sheets act as a cue upon interaction with the plasma membrane of cancer cell lines, subsequently inducing actin filaments disruption leading to impaired endocytosis, migration activity, and reduced capacity of cancer cells towards GO uptake.

cell biology↗

bioRxiv2024

Collagen fibril formation at the plasma membrane occurs independently from collagen secretion

Collagen fibrils are the primary supporting scaffold of vertebrate tissues but how they are assembled is unclear. Here, using CRISPR-tagging of type I collagen and SILAC labelling, we elucidate the cellular mechanism for the spatiotemporal assembly of collagen fibrils, in cultured fibroblasts. Our findings reveal multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiate the processes of collagen secretion and fibril assembly and identify the crucial involvement of endocytosis in regulating fibril formation. By employing Col1a1 knockout fibroblasts we demonstrate the incorporation of exogenous collagen into nucleation sites at the plasma membrane through these recycling mechanisms. Our study sheds light on the assembly process and its regulation in health and disease. Mass spectrometry data are available via ProteomeXchange with identifier PXD036794.

cell biology↗

bioRxiv2021

Quantitative characterisation of low abundant yeast mitochondrial proteins reveals compensation for haplo-insufficiency in different environments

Quantification of low abundant membrane-binding proteins such as transcriptional factors and chaperones has been proved difficult even with the most sophisticated analytical technologies. Here we exploit and optimise the non-invasive Fluorescence Correlation Spectroscopy (FCS) for quantitation of low abundance proteins and as proof of principle we choose two interacting proteins involved in fission of mitochondria in yeast, Fis1p and Mdv1p. In Saccharomyces cerevisiae the recruitment of Fis1p and Mdv1p to mitochondria is essential for the scission of the organelles and the retention of functional mitochondrial structures in the cell. We used FCS in single, GFP-labelled live yeast cells to quantify the protein abundance in homozygote and heterozygote cells, and to investigate the impact of the environments on protein copy number, bound/unbound protein state and mobility kinetics. Both proteins were observed to localise predominantly at mitochondrial structures with the Mdv1p bound state increasing significantly in a strictly respiratory environment. Moreover, a compensatory mechanism which controls Fis1p abundance upon deletion of one allele was observed in Fis1p but not in Mdv1p, suggesting differential regulation of Fis1p and Mdv1p protein expression.

biochemistry↗

bioRxiv2021

Complexities in the role of acetylation dynamics in modifying inducible gene activation parameters

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. However, several studies have shown that histone acetylation dynamics rather than hyperacetylation per se are important determinants in gene activation, particularly at inducible genes. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous DUSP1 activation kinetics and that acetylation levels influence allele activation frequencies. Our data therefore point to a complex interplay between acetylation dynamics and target gene induction, which cannot simply be explained by a unified response to acetylation activity. Instead, acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner.

molecular biology↗

bioRxiv2019

Dynamic interactions and intracellular fate of label-free GO within mammalian cells: role of lateral sheet size

Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 m). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.

cell biology↗

bioRxiv2019

Identification of a novel therapeutic agent for Inflammatory Bowel Disease guided by systems medicine

ObjectiveInflammatory bowel diseases cause significant morbidity and mortality. Aberrant NF-{kappa}B signalling is strongly associated with these conditions, and several established drugs influence the NF-{kappa}B signalling network to exert their effect. This study aimed to identify drugs which alter NF-{kappa}B signalling and may be repositioned for use in inflammatory bowel disease. DesignThe SysmedIBD consortium established a novel drug-repurposing pipeline based on a combination of in-silico drug discovery and biological assays targeted at demonstrating an impact on NF-kappaB signalling, and a murine model of IBD. ResultsThe drug discovery algorithm identified several drugs already established in IBD, including corticosteroids. The highest-ranked drug was the macrolide antibiotic Clarithromycin, which has previously been reported to have anti-inflammatory effects in aseptic conditions. Clarithromycins effects were validated in several experiments: it influenced NF-{kappa}B mediated transcription in murine peritoneal macrophages and intestinal enteroids; it suppressed NF-{kappa}B protein shuttling in murine reporter enteroids; it suppressed NF-{kappa}B (p65) DNA binding in the small intestine of mice exposed to LPS, and it reduced the severity of dextran sulphate sodium-induced colitis in C57BL/6 mice. Clarithromycin also suppressed NF-{kappa}B (p65) nuclear translocation in human intestinal enteroids. ConclusionsThese findings demonstrate that in-silico drug repositioning algorithms can viably be allied to laboratory validation assays in the context of inflammatory bowel disease; and that further clinical assessment of clarithromycin in the management of inflammatory bowel disease is required.

systems biology↗