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Swift, J.

Publications and source records attributed to Swift, J..

At least 19 recordsLinked to original sources

Ageing impacts extracellular matrix turnover and remodelling in the kidney

At least 10% of the global population is impacted by chronic kidney disease (CKD) and ageing is a key risk factor. CKD is characterised by the build-up of extracellular matrix and a loss of functional nephrons. However, the mechanisms that maintain matrix homeostasis across the physiological lifespan remain elusive. Using {superscript 1}3C-lysine metabolic labelling, we quantified kidney matrix protein turnover in healthy mice at four timepoints (8, 22, 52, and 78 weeks). We found that basement membrane components, including collagen IV, laminin-521, nidogens and perlecan, were more long-lived over age, with collagen IV half-lives extending from weeks in young kidneys to years in aged kidneys, suggesting a reduced capacity for basement membrane renewal. The half-lives of fibrillar collagens I and III also increased over age up to forty-fold, which is consistent with minimal degradation. In contrast, collagen XV retained rapid turnover despite increased abundance, indicating a persistent role in tissue remodelling. Using peptide location fingerprinting to predict structural alterations and proteolytic processing we identified age-dependent meprin oligomerisation and altered nidogen-laminin interaction states. We predicted structural alterations within assembly domains of collagen VI and reduced accessibility of integrin-binding regions, suggesting altered microfibril organisation and cell-surface binding. Collagen XV had predicted structural changes across the NC1 domain encoding the matrikine restin, consistent with altered protease accessibility and matrikine release during ageing. These findings indicate that age-related kidney fibrosis is primarily caused by impaired matrix degradation, with protease accessibility and altered matrix interactions likely playing key roles in this remodeling process.

systems biology↗

HHO5: A key orchestrator of dose-dependent nitrogen signaling pathways in Arabidopsis

A major goal in agriculture is to engineer crops that can maintain yield with less nitrogen (N) fertilizer input. Major orchestrators of plant responses to N include members of the HRS1 HOMOLOG (HHO) family of transcription factors (TFs). However, HHO TFs have been difficult targets for functional studies in planta due to their redundancy. Here, we highlight a unique role for a phylogenetically diverged HHO TF, HHO5, whose expression is regulated in an N-dose dependent fashion and is specifically expressed in phloem. We found that an HHO5 single mutant displays significant misregulation of N-dose dependent genes and plant growth rates. HHO5 is also unique as it displays a dual activator/repressor activity on N-dose dependent gene regulation. HHO5 specifically acts as a direct gene repressor when binding DNA targets. In contrast, genes activated by HHO5 include indirect targets regulated by TFs downstream of HHO5 (TF2s). To validate the influence of HHO5 via its direct TF2s, we used validated TF2 data to build a gene regulatory network that links HHO5-TF2 targets to [~]70% of the N-dose genes regulated by HHO5 in planta. By these means, we define HHO5 as a novel dual activator/repressor of plant N-dose signaling. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/667803v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@11adaa9org.highwire.dtl.DTLVardef@9c221org.highwire.dtl.DTLVardef@a80eceorg.highwire.dtl.DTLVardef@163fcdf_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

Direct electrical stimulation of the human amygdala enhances recognition memory for objects but not scenes

Research from neuroscience studies using invasive neuroanatomy-inspired direct manipulations suggests the basolateral amygdala (BLA) mediates a generalized modulation of many different types of memory. In contrast, noninvasive, psychology-inspired indirect correlations suggest that specificity exists in how the BLA prioritizes experiences in memory. We used direct electrical stimulation of the BLA to investigate the specificity of the memory enhancement in the human brain. Patients undergoing intracranial monitoring via depth electrodes viewed object and scene images, half of which were followed by BLA stimulation. Stimulation enhanced long-term memory for object but not scene images. Furthermore, BLA stimulation elicited stronger evoked responses in the anterior vs. posterior medial temporal lobe (MTL), regions that preferentially process object and scene learning, respectively. These results suggest the BLA exerts an important influence over the specificity of what information is prioritized in memory, rather than a general enhancement of all memory, and provide insight into how BLA-MTL projections contribute to the dynamics of memory prioritization.

neuroscience↗

Stress drives plasticity in leaf maturation transcriptional dynamics

Leaf development is dynamic, adapting to environmental stress to optimize resource use. For example, in response to drought, Arabidopsis restricts leaf growth to enhance water use efficiency. While this plastic response is well described at the physiological level, the underlying transcriptional changes that facilitate adjustments in leaf development in response to stress remain poorly described. By constructing a [~]1 million single-nuclei transcriptome atlas, we demonstrate that drought stress limits leaf growth by advancing transcriptional responses related to maturation and aging. Notably, we find that these transcriptional changes scale with environmental stress, and help explain how shoot size can decline proportionally with stress intensity. Leveraging these insights, we increased leaf growth under stress by cell-type targeted upregulation of FERRIC REDUCTION OXIDASE 6 in the mesophyll.

plant biology↗

Golgins support extracellular matrix secretion by collectively maintaining the Golgi structure-function relationship

The secretion of extracellular matrix (ECM) proteins is vital to the maintenance of tissue health. One major control point of this process is the Golgi apparatus, whose dysfunction causes numerous connective tissue disorders. Golgi function is tightly linked to its structure, which is maintained by the cytoskeleton and Golgi organising proteins. We sought to investigate the role of two of these organising proteins, the golgins GMAP210 and Golgin-160, in ECM secretion. We found that loss of either protein had distinct impacts on Golgi organisation. GMAP210 loss caused cisternal fragmentation and dilation, alongside the accumulation of tubulovesicular structures. Meanwhile, Golgin-160 knockout lead to Golgi fragmentation and vesicle build-up. Nonetheless, loss of each protein had a similar impact on ECM secretion and glycosaminoglycan synthesis. We therefore propose that golgins are collectively required to create the correct physical-chemical space to support efficient ECM protein secretion and modification. This is the first time that Golgin-160 has been shown to be required for ECM secretion. SummaryIn this study, Thompson et al demonstrate that two cis-Golgi golgins, GMAP210 and Golgin-160, have distinct, non-redundant roles in maintaining Golgi organisation and that both are required to support the efficient secretion, assembly, and modification of extracellular matrix proteins.

cell biology↗

Human single-neuron activity is modulated by intracranial theta burst stimulation of the basolateral amygdala

Direct electrical stimulation of the human brain has been used for numerous clinical and scientific applications. At present, however, little is known about how intracranial stimulation affects activity at the microscale. In this study, we recorded intracranial EEG data from a cohort of patients with medically refractory epilepsy as they completed a visual recognition memory task. During the memory task, brief trains of intracranial theta burst stimulation (TBS) were delivered to the basolateral amygdala (BLA). Using simultaneous microelectrode recordings, we isolated neurons in the hippocampus, amygdala, orbitofrontal cortex, and anterior cingulate cortex and tested whether stimulation enhanced or suppressed firing rates. Additionally, we characterized the properties of modulated neurons, clustered presumed excitatory and inhibitory neurons by waveform morphology, and examined the extent to which modulation affected memory task performance. We observed a subset of neurons ([~]30%) whose firing rate was modulated by TBS, exhibiting highly heterogeneous responses with respect to onset latency, duration, and direction of effect. Notably, location and baseline activity predicted which neurons were most susceptible to modulation, although the impact of this neuronal modulation on memory remains unclear. These findings advance our limited understanding of how focal electrical fields influence neuronal firing at the single-cell level. HighlightsIndividual neurons in the human brain were responsive to theta burst stimulation Basolateral amygdala stimulation preferentially modulated neurons in the hippocampus, orbitofrontal cortex, and amygdala Excitatory and inhibitory neurons were modulated equally Neurons modulated by stimulation tended to have greater baseline firing rates Duration, onset, and valence of neuronal modulation were heterogeneous

neuroscience↗

Circadian and permanent pools of extracellular matrix co-exist in tendon tissue, but have distinct rates of turnover and differential responses to ageing

Heavy carbon isotopes in the tendons of people who grew up in the age of nuclear bomb testing have shown that the extracellular matrix (ECM), assembled during development, stays with us for life. However, recent work suggests that type-I collagen in ECM-rich mouse tendon exists in two pools: a permanent matrix, and a more soluble, circadian-regulated matrix. Despite this, the underlying regulation of such distinct pools is not understood. Here, we demonstrate using stable isotope labelling coupled with mass spectrometry proteomics that circadian and permanent matrix pools have significantly different half-lives. Furthermore, the properties of the matrix pools are altered during development and ageing. Tail tendon tissue was harvested from mice fed on a heavy-lysine diet; protein was then extracted for analysis using a sequential two-step protocol. The first, soluble fraction ( F1) was found to contain intracellular proteins, and a range of core and associated extracellular matrix proteins, including a pool of type-I collagen shown to be circadian-regulated. The remaining fraction ( F2) contained primarily collagens, including type-I collagen which did not show rhythmicity. In adult mice, matrix proteins extracted in the F1 pool had significantly shorter half-lives than F2, including type-I collagen which had half-lives of 4 {+/-} 2 days in F1, compared to 700 {+/-} 100 days in F2. Circadian-regulated matrix proteins were found to have significantly faster turnover than non-circadian in adult mice, but this distinction was lost in older animals. This work identifies protein turnover as the underlying mechanism for the circadian/permanent model of tendon matrix, and suggests a loss of circadian regulation as a characteristic of ECM ageing.

molecular biology↗

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↗

Single nuclei sequencing reveals C4 photosynthesis is based on rewiring of ancestral cell identity networks

In multicellular systems changes to the patterning of gene expression drive modifications in cell function and trait evolution. One striking example is found in more than sixty plant lineages where compartmentation of photosynthesis between cell types allowed the evolution of the efficient C4 pathway from the ancestral C3 state. The molecular events enabling this transition are unclear. We used single nuclei sequencing to generate atlases for C3 rice and C4 sorghum during photomorphogenesis. Our analysis revealed that initiation of photosynthesis gene expression is conditioned by cell identity. In both species a conserved cistrome defines each cell type, and photosynthesis genes switching expression from mesophyll in rice to bundle sheath in sorghum acquire hallmarks of bundle sheath identity. The sorghum bundle sheath has also acquired gene networks associated with C3 guard cells. We conclude C4 photosynthesis is based on rewiring in cis that exapts cell identity networks of C3 plants.

plant biology↗

Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel(R) SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel(R)Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel(R) Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels(R) with laminin. Laminin was found to require negatively charged PeptiGel(R) Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

cell biology↗

Drought Recovery Induced Immunity Confers Pathogen Resistance

All organisms experience stress as an inevitable part of life, from single-celled microorganisms to complex multicellular beings. The ability to recover from stress is a fundamental trait that determines the overall resilience of an organism, yet stress recovery is understudied. To begin unraveling the stress recovery process we studies recovery from drought stress in Arabidopsis thaliana. We performed a fine-scale time series of bulk RNA sequencing starting 15 minutes after rehydration following moderate drought. We reveal that drought recovery is a rapid process involving the activation of thousands of recovery-specific genes. To capture these rapid recovery responses in different leaf cell types, we performed single-nucleus transcriptome analysis at the onset of post-drought recovery, identifying a cell type-specific transcriptional state developing within 15 minutes of rehydration independently across cell types. Furthermore, we reveal a recovery-induced activation of the immune system that occurs independent of infection, which enhances pathogen resistance in vivo in A. thaliana, wild tomato (Solanum pennellii) and domesticated tomato (Solanum lycopersicum cv. M82). Since rehydration promotes microbial proliferation and thereby increases the risk of infection1-2, drought recovery-induced immunity may be crucial for plant survival in natural environments. These findings indicate that drought recovery coincides with a preventive defense response, unraveling the complex regulatory mechanisms that facilitate stress recovery in different plant cell types.

plant biology↗

Mimicking genuine drought responses using a high throughput plate assay

Soil-free assays that induce water stress are routinely used to investigate drought responses in the plant Arabidopsis thaliana. Due to their ease of use, the research community often relies on polyethylene glycol (PEG), mannitol and salt (NaCl) treatments to reduce the water potential of agar media, and thus induce drought conditions in the laboratory. However, while these types of stress can create phenotypes that resemble those of water deficit experienced by soil-grown plants, it remains unclear how these treatments compare at the transcriptional level. Here, we demonstrate that these different methods of lowering water potential elicit both shared and distinct transcriptional responses in Arabidopsis shoot and root tissue. When we compared these transcriptional responses to those found in Arabidopsis roots subject to vermiculite drying, we discovered many genes induced by vermiculite drying were repressed by low water potential treatments on agar plates (and vice versa). Additionally, we also tested another method for lowering water potential of agar media. By increasing the nutrient content and tensile strength of agar, we show the hard agar (HA) treatment can be leveraged as a high-throughput assay to investigate natural variation in Arabidopsis growth responses to low water potential.

plant biology↗

Loss of cytoskeletal proteostasis links dysregulation of cell size and mechanotransduction in mesenchymal stem cell senescence

Tissues are maintained by homeostatic feedback mechanisms where cells respond to, but also modify, the chemical and mechanical properties of the surrounding extracellular matrix. Mesenchymal stem cells (MSCs) resident in the marrow niche experience a diverse mechanical environment, but ageing can affect the composition and quality of bone and marrow tissues. Here we quantified the effect of replication-induced senescence on MSC morphology and their ability to correctly respond to different substrate stiffnesses. The matrix proteome was found to be sensitive to substrate stiffness, but pharmacological inhibition of cellular contractility perturbed this response, decreasing levels of tenascin-C, fibulins and fibronectin. Similar decreases in these mechanosensitive proteins were observed in senescent cells, suggested a decoupling of mechanotransduction pathways. Intracellular proteomic and transcriptomic analyses confirmed a decrease in components of the cytoskeletal chaperone complex CCT/TRiC in senescent MSCs. Finally, pharmacological inhibition of CCT/TRiC was able to partially recapitulate senescence-associated morphological changes in non-senescent MSCs. These results demonstrate a senescence-mediated perturbation to cytoskeletal homeostasis, pathways of mechanotransduction and the secretion of matrix proteins required for tissue maintenance.

cell biology↗

Novel Laser Capture Microdissection-Proteomic Analysis Identifies Spatially Distinct Extracellular Matrix Signatures in the Core and Infiltrating Edge of Human Glioblastoma.

BackgroundGlioblastoma is the most common and aggressive primary brain tumour in adults. Hallmarks of glioblastoma include its intra-tumoural heterogeneity and extensive invasion of the surrounding brain. Glioblastoma is known to remodel the extracellular matrix (ECM) of the brain, resulting in altered mechanical properties and the establishment of a tumour-promoting microenvironment. How changes in the expression and spatial distribution of ECM constituents within glioblastoma contribute to invasion and disease progression is still unclear. MethodsHere we report on a protocol for laser-capture microdissection coupled with mass spectrometry (LCM-proteomics) that allowed a spatially resolved and unbiased analysis of the regional ECM proteome (matrisome) in formalin-fixed and paraffin-embedded (FFPE) samples of human glioblastoma. We investigated five molecularly characterised hemispheric adult glioblastomas where the brain/tumour interface and tumour epicentre were represented in the surgical specimens and snap-frozen tissue was available. LCM-proteomic analysis was validated with immunohistochemistry. ResultsLCM-proteomics identified 53 matrisome proteins in FFPE tissue, demonstrating comparable performance with conventional analysis of snap-frozen tissue. The analysis revealed distinct matrisome components in the brain/tumour interface versus the tumour epicentre. Guided by data from LCM-proteomic analysis, immunostaining for tenascin-R confirmed greater staining in the brain/tumour interface, whilst expression of fibronectin was higher in the tumour epicentre. ConclusionThe protocol described in this work allowed for accurate, spatially resolved analysis of ECM in FFPE tissues, with performance comparable to analysis of snap-frozen tissue. While the focus for this work was on the regional ECM composition of glioblastoma, we found that the LCM-proteomics protocol is also applicable to the study of the wider proteome, and represents a valuable tool for investigating tumour/tissue heterogeneity. This protocol opens the possibility to apply LCM-proteomics to retrospective studies with the advantage of accessing clinical history and follow-up information, providing a valuable resource for translational research in glioblastoma.

cancer biology↗

Vimentin intermediate filaments provide structural stability to the mammalian Golgi apparatus

The Golgi apparatus comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region of most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi apparatus with intermediate filaments. In this study we show that the Golgi is in close physical proximity to vimentin intermediate filaments (IFs) in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with IFs. Although loss of vimentin and/or GORAB does not have major effects upon Golgi morphology at steady-state, the Golgi undergoes more rapid disassembly upon chemical disruption with the drug brefeldin A, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin causes reduced Golgi ribbon integrity when cells are cultured on high stiffness hydrogels, which is exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi apparatus, and suggest a role for GORAB in this process.

cell biology↗

Mmp14-controlled extracellular matrix homeostasis is required for circadian rhythm

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

cell biology↗

Senescence inhibits the chaperone response to thermal stress

Cells respond to stress by synthesising chaperone proteins that correct protein misfolding to maintain function. However, protein homeostasis is lost in ageing, leading to aggregates characteristic of protein-folding diseases. Whilst much is known about how these diseases progress, discovering what causes protein-folding to deteriorate could be key to their prevention. Here, we examined primary human mesenchymal stem cells (hMSCs), cultured to a point of replicative senescence and subjected to heat shock, as an in vitro model of the ageing stress response. We found through proteomic analysis that the maintenance of homeostasis deteriorated in senescent cells. Time-resolved analysis of factors regulating heat shock protein 70 kDa (HSPA1A) revealed a lack of capacities for protein turnover and translation to be key factors in limiting the stress response during senescence. A kinetic model predicted a consequence of these reduced capacities to be the accumulation of misfolded protein, a hypothesis supported by evidence of systematic changes to protein fold state. These results thus further our understanding of the underlying mechanistic links between ageing and loss of protein homeostasis.

cell biology↗

Increased microenvironment stiffness leads to altered aldehyde metabolism and DNA damage in mammary epithelial cells through a RhoA-dependent mechanism

Microenvironmental stiffness regulates the behaviour of both normal and cancer cells. In breast tissue, high mammographic density (HMD), which reflects greater organisation and stiffness of the periductal collagen, represents a significant risk factor for cancer. However, the mechanistic link between extracellular matrix (ECM) stiffness and increased risk of breast tumour initiation remains unclear. In particular, how increased ECM stiffness might promote genomic damage, leading to the acquisition of transforming mutations, remains to be determined. Here we determine that ECM stiffness induces changes in mammary epithelial cell (MEC) metabolism that drive genomic damage. Using a 3D-culture model, we demonstrate that genome-wide transcriptional changes in response to increased ECM stiffness impair the ability of MECs to remove reactive aldehyde species, resulting in greater accumulation of DNA damage in a RhoA-dependent manner. Together, our results provide a mechanistic link between increased ECM stiffness and the genomic damage required for breast cancer initiation.

developmental biology↗