bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.07.17.665448

Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Midbody Remnant Proteins Following Epithelial-Mesenchymal Transition

Shafiq, A.·Rai, A.·Xu, R.·Chen, M.·Suwakulsiri, W.·Greening, D.·Simpson, R.
View original source ↗Export metadata ↓

Abstract

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process that allows highly polarized, immotile epithelial cells to transform into motile mesenchymal cells: it is a fundamental cellular process involved in embryonic development, tumour cell metastasis, organ fibrosis and tissue regeneration. To assess the contribution of secreted midbody remnants (MBRs) - a new class of membranous extracellular vesicle (EV) molecularly distinct from exosomes/small EVs - to the EMT process, we conducted a proteomic analysis of MBRs released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). MBRs were harvested from cell culture media in milligram quantities using a continuous culture bioreactor device and purified using sequential centrifugation and buoyant density gradient centrifugation (OptiPrep). Gel-MS/MS protein profiling showed MDCK cell-MBRs reflect their epithelial origin (e.g., enriched CDH1, DSP, THBS1, OLCN, EPCAM proteins) and 21D1 cell-MBRs their mesenchymal phenotype (e.g., HRAS, VIM, MMP14, CDH2, WNT5A and enriched invasive and cell motility proteins). Prominent findings were the unique expression of the immune checkpoint protein NT5E/CD73 (ecto-5'-nucleotidase), and ser/thr kinases LIMK1/K2 in 21D1-MBRs (not present in MDCK cell-MBRs), and enrichment in Wnt signalling network proteins. Collectively, our findings suggest MBRs might play a previously unrecognized role in the EMT process. SignificanceO_LIEpithelial-to-mesenchymal transition (EMT) is a critical cell biological process that occurs during normal embryonic development and cancer progression. Our study describes, for the first time, the large-scale sequential purification of secreted midbody remnants (MBRs) and exosomes/sEVs from the in vitro cell line EMT model Madin-Darby canine kidney (MDCK) cells and MDCK cells transformed with oncogenic H-Ras (21D1 cells): GeLC-MS/MS protein profiling identified the repertoire of enriched MDCK-MBR proteins following EMT. C_LIO_LIMBRs display a proteome profile distinct from sEVs that is enriched with factors of the centralspindlin complex (KIF23.1, KIF4A, INCENP, CEP55, PLK1) and further include components of the mitochondrial network, cytokinesis, microtubule movement, and intercellular connection. C_LIO_LIIn the context of EMT, our data reveal simultaneous activation of EMT signalling pathways in MBRs including signalling receptor binding, regulation of cell differentiation, and Wnt, VEGF and PDGF signalling. C_LIO_LIWe identify several mesenchymal enriched networks in MBRs associated with focal adhesion, cell matrix, kinase activity, and cell shape/organisation, while epithelial derived MBRs are show enriched networks predominately associated with mitochondrial (processing/transport), midbody, and plasma membrane annotation. C_LIO_LIOur study sheds light on the signalling architecture of MBRs following oncogenic H- Ras-induced EMT: collectively, our data informs ongoing efforts to delineate oncogenic drivers of cancer initiation, progression, and metastasis. C_LI

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Shafiq, A., Rai, A., Xu, R., Chen, M., Suwakulsiri, W., Greening, D., Simpson, R.. 2025-07-21. Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Midbody Remnant Proteins Following Epithelial-Mesenchymal Transition. https://doi.org/10.1101/2025.07.17.665448

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

The nuclear membrane protein Samp1 links peripheral genome organization to the myogenic transcriptional program

Samp1 is an inner nuclear membrane protein required for myogenic differentiation and involved in chromatin organization at the nuclear periphery. Here, we investigated whether these functions are connected by studying the effects of Samp1 depletion during C2C12 myogenic differentiation using immunofluorescence microscopy, RNA sequencing, FRIC, and chromosome-positioning analysis. Samp1-depleted cells showed strongly reduced MyHC expression and virtually abrogated multinucleated fiber formation. Although cell-cycle withdrawal was not prevented, the transcriptional program driving differentiation was drastically perturbed, with reduced muscle-associated transcripts and incomplete repression of genes normally downregulated during myogenesis. Samp1 depletion also disrupted peripheral chromatin organization and prevented the accumulation of peripheral heterochromatin typically seen during differentiation. In addition, radial chromosome distribution was disrupted, evidenced by the failure of chromosome 8 to reposition to the nuclear periphery during differentiation. Together, these findings link the requirement for Samp1 in myogenic differentiation to its role in genome organization at the nuclear periphery.

cell biology↗
bioRxiv2026

Unraveling the metabolic landscape of alkaptonuria through a human-relevant in vitro liver disease model

Alkaptonuria (AKU) is a rare inherited metabolic disorder of tyrosine catabolism caused by a deficient homogentisate 1,2-dioxygenase (HGD) enzyme. This results in the accumulation of homogentisic acid (HGA), driving a progressive multisystem pathology characterized by debilitating early-onset osteoarthritis due to connective tissue degeneration. While previous in vitro studies have primarily relied on exogenous HGA exposure in osteoarticular cell models, the direct metabolic consequences of endogenous HGD deficiency within its native hepatic context remain poorly understood. Here, we established the first human-relevant HGD knockout hepatic in vitro model using a universal in-house-developed homology-directed repair approach. Integrative multi-omic analysis revealed that HGD deficiency induces widespread metabolic rewiring extending beyond disrupted tyrosine catabolism. HGD-deficient hepatocytes exhibited elevated oxidative stress accompanied by impaired mitochondrial respiration and a pseudohypoxic metabolic adaptation toward increased glycolytic dependency. Despite this glycolytic shift, the cells displayed reduced anabolic and translational activity alongside attenuated proliferation, consistent with a chronic stress-adaptive survival state rather than a proliferative metabolic phenotype. This study provides systems-level insights into the pathophysiology of AKU and establishes a versatile platform for mechanistic and therapeutic investigation.

cell biology↗
bioRxiv2026

The circadian clock regulates KCNH2 (hERG) promoter activity through daily temperature rhythms.

Background: KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (IKr), which is critical for cardiac repolarization. KCNH2 encodes two functional isoforms, Kv11.1a and Kv11.1b, via alternative transcription start sites. Kv11.1a is the principal determinant of cardiac IKr and ventricular repolarization. The circadian clock, a transcriptional-translational feedback loop that cycles with a period of ~24 hours and drives the circadian expression of many genes, including Kcnh2 in the mouse heart. Because daily body temperature rhythms provide a systemic signal that synchronizes cardiac circadian clocks, we tested whether physiological temperature cycles drive the circadian promoter activity of the cloned human KCNH2 (hKCNH2) promoter. Hypothesis: hKCNH2 is a direct transcriptional target of the circadian clock, with temperature driving its promoter activity through BMAL1:CLOCK acting at a conserved tandem E-box. Methods: We cloned the conserved proximal promoter of KCNH2 (-1631 bp upstream of Kv11.1a exon 1) to generate hKCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37{degrees}C) or temperature cycling (36.5-38.5{degrees}C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCK{Delta}19 co-expression. Results: Temperature cycling did not exhibit the rapid damping characteristic of serum-shock-synchronized oscillations, consistent with continuous entrainment by an external zeitgeber rather than a free-running oscillator. Deletion analysis identified a conserved tandem E-box required for oscillation under both serum shock and temperature cycling, and for BMAL1:CLOCK-dependent transactivation (1.75 {+/-} 0.21 vs. 0.86 {+/-} 0.06 RLU, p = 0.0038). CLOCK{Delta}19 reduced hKCNH2 promoter amplitude under temperature cycling without altering period. Conclusion: The circadian clock regulates KCNH2 promoter activity through daily temperature rhythms.

cell biology↗