Biology subjects

Breen, C.

Publications and source records attributed to Breen, C..

7 recordsLinked to original sources

bioRxiv2024

A spreading, multi-tissue wound signal initiates whole-body regeneration

ABSTRACTAnimal bodies are equipped to sense injuries and respond, and rapid activation of ERK signaling at wound sites has emerged as a recurring mechanism across metazoans. During whole-body regeneration, distant species including cnidarians, planarians and echinoderms activate ERK at wounds to drive early transcriptional changes. However, it is unclear to what extent these are distinct phenomena, utilizing a common effector pathway but with different upstream inputs and downstream functions, or are more deeply alike, sharing upstream inputs, activating cell type(s), or spatiotemporal dynamics. To facilitate thorough comparisons across animals, we examined wound-induced ERK during whole-body regeneration in the acoel Hofstenia miamia. ERK signaling is induced at wound sites within 10 minutes post-amputation and peaks in activity within one hour. ERK is required for head and tail regeneration and inhibiting ERK suppresses the expression of wound-induced genes, which are themselves required for regeneration. Quantification of spatial dynamics revealed that active ERK spreads rapidly away from the wound site during the first 30 minutes post-amputation and continues to expand in space through 3-6 hours, albeit at lower levels. Multiple cell types activate ERK including neoblasts (stem cells) and muscle, and ERK is spatially dynamic within both cell types, however, ERK activation in muscle does not require neoblasts. Finally, neuregulin-1, a putative ligand produced exclusively by muscle cells, is required for ERK activation at wounds. Altogether, our data identify a key signaling role of muscle cells in driving ERK activation in multiple cell types after injury, and describe a spatial spreading phenomenon with parallels to dynamic ERK patterns in other systems.

developmental biology↗

bioRxiv2024

Iron-sensitive RNA regulation by poly C binding proteins

Iron is essential for normal cellular function. Homeostatic responses to low iron availability have long been known to rely on posttranscriptional mechanisms. Poly C binding proteins (PCBPs) are essential RNA binding proteins that regulate alternative splicing, translation and RNA stability. They also serve as critical iron chaperones that manage intracellular iron flux. However, the impact of cellular iron levels on the PCBP-directed transcriptome has not been globally evaluated. We have found broad transcriptome changes in response to low iron availability consistent with numerous operant posttranscriptional mechanisms that sense iron. By comparing gene expression and alternative splicing directed by PCBPs to the iron-sensitive transcriptome, we found genes with iron-sensitive PCBP RNA regulation. Further, we demonstrate that cellular iron levels impact PCBP1 RNA binding. Our data show a large-scale RNA regulatory response to cellular iron levels and open the possibility for other iron-sensitive RNA binding proteins.

molecular biology↗

bioRxiv2024

Whole genome methylation sequencing in blood from persons with mild cognitive impairment and dementia due to Alzheimer's disease identifies cognitive status

INTRODUCTIONWhole genome methylation sequencing (WGMS) in blood identifies differential DNA methylation in persons with late-onset dementia due to Alzheimers disease (AD) but has not been tested in persons with mild cognitive impairment (MCI). METHODSWe used WGMS to compare DNA methylation levels at 25,244,219 CpG loci in 382 blood samples from 99 persons with MCI, 109 with AD, and 174 who are cognitively unimpaired (CU). RESULTSWGMS identified 9,756 differentially methylated positions (DMPs) in persons with MCI, including 1,743 differentially methylated genes encoding proteins in biological pathways related to synapse organization, dendrite development, and ion transport. 447 DMPs exhibit progressively increasing or decreasing DNA methylation levels between CU, MCI, and AD that correspond to cognitive status. DISCUSSIONWGMS identifies DMPs in known and newly detected genes in blood from persons with MCI and AD that support blood DNA methylation levels as candidate biomarkers of cognitive status.

genomics↗

bioRxiv2024

Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake; i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis. Author SummaryBlood-stage trypanosome parasites have a specialised invagination on the cell surface named the flagellar pocket (FP), where invariant essential nutrient receptors are located. The pocket houses diverse proteins, including a transferrin receptor (TfR), which facilitates uptake of host transferrin-bound iron for survival. Several FP proteins, including TfR, are linked to complex sugar molecules (carbohydrates), the functions of which are not well understood. Complex carbohydrates are made by enzymes called glycosyltransferases (GTs) and previously we partially inhibited complex carbohydrate synthesis by deletion of either TbGT8 or TbGT10. However, mutant parasites lacking either one of these enzymes survived, suggesting functional redundancy. Here, we created a parasite mutant that lacks both TbGT8 and TbG10 to understand the combined effect of losing both enzymes. The mutant parasites showed a decreased ability to uptake tomato lectin, a protein that specifically binds to these sugar conjugates in the FP, indicating a reduction in carbohydrate complexity. Despite reduced complexity in the sugar structures attached to TfR, its critical function in transferrin/iron uptake remained effective. Furthermore, the mutants remained viable in culture and in animal models, challenging previous assumptions about the necessity and function of these carbohydrate conjugates. Our findings imply a greater flexibility and redundancy in the carbohydrate complex roles than previously appreciated.

molecular biology↗

bioRxiv2023

Mechanical positive feedback and biochemical negative feedback combine to generate complex contractile oscillations in cytokinesis

Contractile force generation by the cortical actomyosin cytoskeleton is essential for a multitude of biological processes. The actomyosin cortex behaves as an active material that drives local and large-scale shape changes via cytoskeletal remodeling in response to biochemical cues and feedback loops. Cytokinesis is the essential cell division event during which a cortical actomyosin ring generates contractile force to change cell shape and separate two daughter cells. Our recent work with active gel theory predicts that actomyosin systems under the control of a biochemical oscillator and experiencing mechanical strain will exhibit complex spatiotemporal behavior, but cytokinetic contractility was thought to be kinetically simple. To test whether active materials in vivo exhibit spatiotemporally complex kinetics, we used 4-dimensional imaging with unprecedented temporal resolution and discovered sections of the cytokinetic cortex undergo periodic phases of acceleration and deceleration. Quantification of ingression speed oscillations revealed wide ranges of oscillation period and amplitude. In the cytokinetic ring, activity of the master regulator RhoA pulsed with a timescale of approximately 20 seconds, shorter than that reported for any other biological context. Contractility oscillated with 20-second periodicity and with much longer periods. A combination of in vivo and in silico approaches to modify mechanical feedback revealed that the period of contractile oscillation is prolonged as a function of the intensity of mechanical feedback. Effective local ring ingression is characterized by slower speed oscillations, likely due to increased local stresses and therefore mechanical feedback. Fast ingression also occurs where material turnover is high, in vivo and in silico. We propose that downstream of initiation by pulsed RhoA activity, mechanical positive feedback, including but not limited to material advection, extends the timescale of contractility beyond that of biochemical input and therefore makes it robust to fluctuations in activation. Circumferential propagation of contractility likely allows sustained contractility despite cytoskeletal remodeling necessary to recover from compaction. Our work demonstrates that while biochemical feedback loops afford systems responsiveness and robustness, mechanical feedback must also be considered to describe and understand the behaviors of active materials in vivo.

cell biology↗

bioRxiv2023

Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange

Members of the SLC26 family constitute a conserved class of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the ten functional paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in maintaining pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter, Sawicka, & Dutzler, 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a coupled exchanger of chloride with bicarbonate and presumably also oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism.

biophysics↗

bioRxiv2022

Long-ranged formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution. Yet, how morphogen gradients form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. We utilise fluorescence correlation spectroscopy (FCS) and perturbations to Bcd, to dissect Bcd dynamics at multiple spatial and temporal locations. In both the cytoplasm and nucleus, we find two dynamic modes for Bicoid diffusion dynamics, consisting of fast and slow populations of Bcd. Surprisingly, there are spatial differences in Bcd diffusivity along the AP-axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in the Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. We subsequently investigated which binding elements of Bcd are playing a role in its dynamics. In the nucleus, we demonstrate that the slower mode of Bcd transport is due to Bcd DNA binding. Addition of the Bcd homeodomain to eGFP::NLS can qualitatively replicate the observed Bcd concentration profile, suggesting this domain is the primary region regulating Bcd dynamics. This study provides a detailed analysis of morphogen dynamics at different spatial and temporal locations, revealing multiple modes of transport. These results explain how a long-ranged gradient can form while retaining a steep profile through much of its range.

developmental biology↗