Biology subjects

Cutiongco, M. F.

Publications and source records attributed to Cutiongco, M. F..

7 recordsLinked to original sources

bioRxiv2025

Acute chromatin decompaction stiffens the nucleus as revealed by nanopillar-induced nuclear deformation in cells

Chromatin architecture is critical in determining nuclear mechanics. Most studies focus on the mechanical rigidity conferred by chromatin compaction from densely packed heterochromatin, but less is known on how transient changes in chromatin decompaction state impinge on nucleus stiffness. Here, we used an array of vertically aligned nanopillars to study nuclear deformability in situ after chromatin decompaction in cells. The nucleus significantly stiffened within 4 hours of chromatin decompaction but softened at longer timescales. This acute nucleus stiffening was predominantly underlied by an increase in nucleus volume, nuclear import and partially enhanced by lamin protein recruitment to the nuclear periphery. The coupling between nucleus stiffening and acute chromatin decompaction was observed in cancer cell lines with lower malignancy (e.g. MCF7, PEO1, A549) but weakened in those with higher metastatic potential (e.g. MDA-MB-231, HEYA8, HT1080), which was found to be associated with the capacity to efficiently compact heterochromatin into foci that sustains nucleus deformability required for confined migration. Our work signals how a rapid chromatin remodeling is a physiologically relevant pathway to modulate nucleus mechanics and cell migration behavior. STATEMENT OF SIGNIFICANCEMany cell processes such as wound healing, immune activation and DNA damage repair require a decompact and accessible chromatin structure. Whether such short-term remodeling of the chromatin impacts nucleus mechanics and function is poorly defined. Using nanopillars that allow interrogation of nucleus rigidity within intact cells, we showed that contrary to conventional knowledge the nucleus becomes less deformable and more rigid when chromatin is acutely decompacted due to enhanced nuclear import and swelling of the nucleus. In cancer cells, the coupling of transient chromatin decompaction to nucleus rigidity is weakened and appears to be countered by heterochromatin formation and compaction. We demonstrate here how short-term chromatin remodeling can impact nucleus and cellular properties in a time-dependent and non-genetic manner.

bioengineering↗

bioRxiv2025

Macrophages promote collagen deposition through circadian regulation of fibroblasts

Collagen deposition in fibroblasts, the primary collagen-producing cells, is regulated by both macrophages and the circadian rhythm, although how these regulatory processes interact with one another is unknown. Here, we reveal that macrophage-fibroblast interaction enhances collagen deposition which requires a functional circadian rhythm. This response is also dependent on macrophage polarisation status, where M0 (naive) and M1 (pro-inflammatory) macrophages require direct cell-cell contact; whereas M2 (anti-inflammatory) macrophages utilise an additional mechanism through secreted soluble factors that strengthen circadian rhythms and significantly increase collagen deposition in fibroblasts, independent of cell-cell contact. Using mass spectrometry proteomics analysis, we identified PDGFA as a key factor in induction of circadian amplitude in fibroblasts. Crucially, macrophages lacking PDGFA showed diminished ability to modulate fibroblast circadian rhythms and collagen production. Further, in fibroblasts with impaired circadian rhythms, neither macrophages nor macrophage-secreted factors are capable of inducing collagen deposition. These results confirmed that the collagen secretory pathway is also under circadian clock control in lung fibroblasts, and demonstrated that macrophages influence collagen deposition via circadian clock mediated mechanisms. In conclusion, our findings highlight M2-derived PDGFA as a key macrophage-derived factor in extracellular matrix remodelling.

cell biology↗

bioRxiv2024

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↗

bioRxiv2022

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↗

bioRxiv2020

Nanotopography controls single-molecule mobility to determine overall cell fate

The addition of nanoscale distortion to ordered nanotopographies consistently determines an osteogenic fate in stem cells. Although disordered and ordered nanopit arrays have identical surface areas, array symmetry has opposite effects on cell fate. We aimed to understand how cells sense disorder at the nanoscale. We observed effects in the early formation of cell and focal adhesions that controlled long-term cell fate. Disordered nanopits consistently yielded larger focal adhesions at a faster rate, prompting us to investigate this at the molecular scale. Super-resolution microscopy revealed that the nanopits did not act as nucleation points, as previously thought. Rather, nanopit arrays altered the plasma membrane and acted as barriers that changed molecular diffusion. The local areas corralled by four nanopits were the smallest structures that exerted diverging effects between ordered and disordered arrays. Heterogeneity in the local area on disordered arrays increased the proportion of fastest and slowest diffusing molecules. This resulted in higher quantity, more frequent formation and clustered arrangement of nascent adhesions, i.e., the modular units on which focal adhesions are built. This work presents a new pathway to exploit nanoscale sensing to dictate cell fate.

bioengineering↗

bioRxiv2020

Interactive machine learning for fast and robust cell profiling

Automatic profiling of cell morphology is a powerful tool for inferring cell function. However, this technique retains a high barrier to entry. In particular, configuring image processing parameters for optimal cell profiling is susceptible to cognitive biases and dependent on user experience. Here, we use interactive machine learning to identify the optimum cell profiling configuration that maximises quality of the cell profiling outcome. The process is guided by the user, from whom a rating of the quality of a cell profiling configuration is obtained. We use Bayesian optimisation, an established machine learning algorithm, to learn from this information and automatically recommend the next configuration to examine with the aim to maximize the quality of the processing or analysis. Compared to existing interactive machine learning tools that require domain expertise for per-class or per-pixel annotations, we rely on users explicit assessment of output quality of the cell profiling task at hand. We validated our interactive approach against the standard human trial-and-error scheme to optimise an object segmentation task using the standard software CellProfiler. Our toolkit enabled rapid optimisation of an object segmentation pipeline, increasing the quality of object segmentation over a pipeline optimised through trial-and-error. Users also attested to the ease of use and reduced cognitive load enabled by our machine learning strategy over the standard approach. We envision that our interactive machine learning approach can enhance the quality and efficiency of pipeline optimisation to democratise image-based cell profiling.

bioinformatics↗

bioRxiv2018

Predicting phenotype using morphological cell responses to nanotopography

Cells respond in complex ways to topographies, making it challenging to identify a direct relationship between surface topography and cell response. A key problem is the lack of informative representations of topographical parameters that translate directly into biological properties. Here, we present a platform to relate the effects of nanotopography on morphology to function. This platform utilizes the morphome, a multivariate dataset containing single cell measures of focal adhesions, the cytoskeleton, and chromatin. We demonstrate that nanotopography-induced changes in cell phenotype are uniquely encoded by the morphome. The morphome was used to create a Bayesian linear regression model that robustly predicted changes in bone, cartilage, muscle and fibrous tissue gene expression induced by nanotopography. Furthermore, the morphome effectively predicted nanotopography-induced phenotype within a complex co-culture microenvironment. Thus, the morphome enables the cell function-oriented exploration of new topographies, with potential applications in the development of novel surface-patterned biomaterials for tissue implants.

bioengineering↗