Biology subjects

Caswell, P. T.

Publications and source records attributed to Caswell, P. T..

8 recordsLinked to original sources

bioRxiv2026

Stromal COL12A1 defines a pro-invasive extracellular matrix niche in appendiceal mucinous neoplasms and pseudomyxoma peritonei

Pseudomyxoma peritonei (PMP) is an uncommon peritoneal malignancy, most often arising from appendiceal mucinous neoplasms (AMNs), in which tumour progression is accompanied by extensive mucus accumulation. Although PMP is clinically dominated by peritoneal dissemination, the stromal matrix cues that support invasion and metastatic niche formation remain poorly defined. Here, we profile extracellular matrix niche from PMP patient samples, and show that AMNs and peritoneal metastases (PMs) exhibit marked ECM remodelling, including enrichment of mucins, fibrillar matrix components and stromal regulators of collagen architecture. Three-dimensional matrix models demonstrate that PMP cell invasion is strongly influenced by ECM composition, with collagen/fibronectin-rich environments promoting invasion and selected small leucine-rich proteoglycans (SLRPs) attenuating this response. Collagen organisation analysis reveals increased matrix deposition, density and alignment in metastatic lesions, consistent with development of a remodelled pro-invasive stromal niche. Among altered matrix proteins, COL12A1 is prominently upregulated in both primary and peritoneal disease and is enriched within the stromal compartment. Functional co-culture experiments further show that COL12A1-expressing cancer-associated fibroblasts (CAFs) enhance PMP cell invasion, whereas COL12A1 silencing in CAFs reduces this pro-invasive effect. These findings identify stromal COL12A1 as a key component of the PMP matrix niche and suggest that collagen XII-dependent remodelling of the tumour microenvironment may contribute may support dissemination and establishment of the metastatic disease. Together, our data indicate that human AMN and PMP have a compositionally and architecturally remodelled extracellular matrix, within which stromal COL12A1 supports CAF-driven tumour-cell invasion.

cancer biology↗

bioRxiv2026

Cell-matrix mechanosensing and cellular metabolic demand are linked through SKT and mTORC2

Integrin-based adhesion complexes mediate cell adhesion to the extracellular matrix and enable the cell to interpret and respond to both biochemical and mechanical cues. Such cues can affect a cells metabolic rate; however, how mechanical signals are converted into metabolic rate changes is not clear. We identified an uncharacterised protein; Sickle Tail Protein Homolog (KIAA1217), SKT to be enriched in cell-matrix adhesion complexes in stiff microenvironments. Low SKT expression correlates with an improved prognosis in pancreatic ductal adenocarcinoma (PDAC), suggesting an important role for SKT in extracellular matrix dependent tumour progression. Here, we show that SKT interacts with the mechanistic target of rapamycin complex 2 (mTORC2), a pivotal signalling complex in glucose metabolism, cell growth, and survival. SKT recruits mTORC2 to cell-matrix adhesions in a mechanoresponsive manner. SKT mediated mTORC2 signalling from adhesions is required for maintaining glycolytic flux and control of adhesion dynamics. Our findings show that SKT serves as a rheostat that controls metabolic adaptation of cells to their matrix microenvironment. Collectively, our research provides insights into the molecular mechanisms and interplay between cell adhesion and metabolic signalling in complex and stiff tumour microenvironments. The novel functions identified for SKT in cell-matrix adhesions, mTORC2 signalling and glycolysis unveils a signalling axis between the tumour microenvironment and cellular metabolism that are required for PDAC growth and invasion.

cell biology↗

bioRxiv2024

Biofunctional coating of synthetic magnetic nanoparticles enables magnetogenetic control of protein functions inside cells

Remote control of cellular functions via magnetic forces offers unique opportunities in fundamental research and biomedical application. Intracellular delivery of functionalized magnetic nanoparticles (MNP) provides versatile opportunities to assemble signalling platforms for spatiotemporal control by magnetic forces. Such magnetogenetic application, however, has remained highly challenging due to a lack of MNP providing suitable biological, physicochemical and magnetic properties. Here, we achieved single-step surface coating of synthetic maghemite core nanoparticles with green fluo-rescent protein fused to the iron binding site of Mms6 from magnetotactic bacteria. We yielded MNP with intracellular stealth properties (syMagIcS), which could be readily biofunctionalized in situ and translocated within cells via magnetic field gradients. We successfully exploited syMagIcS for spati-otemporal control of Rac1 signalling at the plasma membrane via its guanine nucleotide exchange factor protein TIAM1 and for spatial control of liquid-liquid phase separation using the intrinsically disordered domain of the protein DDX4.

biophysics↗

bioRxiv2024

Differential roles of cyclin-CDK1 complexes in cell migration and invasion

Two key hallmarks of cancer are dysregulated proliferation and metastasis, which are co- ordinated at the single cell level by regulation of cell-cycle progression and invasive cell migration respectively. We have previously described a central role for CDK1 at the nexus of adhesion signalling and cell cycle progression, demonstrating that CDK1 has a non-canonical role in regulating integrin adhesion complexes and in the migration of cancer cells in 3D interstitial matrix. Here we show that the CDK1 binding partners cyclinB1 and cyclinA2 also have roles in cell migration and invasion in both cancer and non-transformed cells. CyclinB1 plays a key role in RhoA activation to promote rear retraction in a membrane tension dependent manner, while cyclinA2 has a general role in promoting motility. Knockdown of either cyclin significantly perturbs migration with contrasting phenotypes, while knockdown of both together has an additive effect which arrests both migration and division. We find that the cell migration specific role of CDK1 is independent of cell-cycle phase, with inhibition or knockdown of CDK1 perturbing migration in G0/G1 arrested cells, while CDK1-cyclin expression correlates strongly with invasive potential of bladder cancer cell lines. Our findings therefore describe how cyclin-CDK1 complexes orchestrate migration as well as division of cells and that cyclinA2-CDK1 and cyclinB1-CDK1 complexes play distinct roles in motility. Furthermore, these findings suggest that targeting CDK1 signalling in aggressive and invasive tumours may have an unexpected dual potential to combat metastasis in addition to proliferation.

cell biology↗

bioRxiv2023

Growth factor-induced desialylation for the fast control of endocytosis

It is commonly assumed that the glycan makeup of glycoproteins that reach the cell surface is final and static. Here, we challenge this notion by the discovery of a molecular switch that induces acute and reversible changes of glycans on the plasma membrane. We demonstrate that within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process, mediated by the Na+/H+ antiporter NHE-1 and the neuraminidases Neu1/3, requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. Desialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan makeup is reset, and their function is repurposed to regulate EGF-dependent invasive cell migration. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

cell biology↗

bioRxiv2022

Proximity labelling identifies pro-migratory endocytic recycling cargo and machinery of the Rab4 and Rab11 families

Endocytic recycling controls the return of internalised cargos to the plasma membrane to coordinate their positioning, availability and downstream signalling. The Rab4 and Rab11 small GTPase families regulate distinct recycling routes, broadly classified as fast recycling from early endosomes (Rab4) and slow recycling from perinuclear recycling endosomes (Rab11), and both routes handle a broad range of overlapping cargos to regulate cell behaviour. We adopted a proximity labelling approach, BioID, to identify and compare the protein complexes recruited by Rab4a, Rab11a and Rab25 (a Rab11 family member implicated in cancer aggressiveness), revealing statistically robust protein-protein interaction networks of both new and well characterised cargos and trafficking machinery in migratory cancer cells. Gene ontological analysis of these interconnected networks revealed that these endocytic recycling pathways are intrinsically connected to cell motility and cell adhesion. Using a knock sideways relocalisation approach we were further able to confirm novel links between Rab11/25 and the ESCPE-1 and retromer multiprotein sorting complexes and identify new endocytic recycling machinery associated with Rab4, Rab11 and Rab25 that regulates cancer cell migration in 3D-matrix.

cell biology↗

bioRxiv2021

DExCon, DExogron, LUXon: on-demand expression control of endogenous genes reveals differential dynamics of Rab11 family members

CRISPR technology has made generation of gene knockouts widely achievable in cells. However, once inactivated, their reactivation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation) and LUXon (light responsive DExCon), approaches which combine one-step CRISPR-Cas9 mediated targeted knock-in of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene transcription with the ability to reactivate transcription on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knockout/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon) or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, expression kinetics and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in cells. GRAPHICAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY IN BRIEFWe describe development of DExCon, LUXon and DExogron approaches, where a single CRIPR/Cas9-mediated gene editing event can block endogenous gene expression, with the ability to reactivate expression encoded such that even silent genes can be expressed. Expression can be controlled systematically using doxycycline, or spatiotemporally by light, allowing fluorescent tagging of endogenous proteins and quantification of expression kinetics, protein dynamics and stability for highly similar genes such as members of the Rab11 family.

molecular biology↗

bioRxiv2020

Intracellular nanovesicles mediate integrin trafficking during cell migration

Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs), are a recently identified class of transport vesicle that are involved in multiple membrane trafficking steps including the recycling pathway. The only known marker for INVs is Tumor Protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype which suggests cell migration may be altered under these conditions. Here we show that TPD54 associates with INVs by directly binding high curvature membrane via a conserved positively charged motif in its C-terminus. We describe how other members of the TPD52-like family are also associated with INVs and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion; and we show that this is likely due to altered integrin recycling. Our study highlights the involvement of INVs in the trafficking of cell surface proteins to generate biologically important outputs in health and disease.

cell biology↗