bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.07.29.667513

Coiled-coil interactions drive ectopic condensation of overexpressed MAD1 to promote mitotic slippage in cancer cells

Tones, J.·Jeffrey, J.·Lackner, R. M.·Chenoweth, D. M.·Zhang, H.
View original source ↗Export metadata ↓

Abstract

The mitotic checkpoint protein MAD1 is significantly overexpressed in several cancers, weakening the checkpoint and promoting mitotic slippage. Overexpressed MAD1 forms ectopic foci in mitotic cells, yet the biophysical nature of these foci and their contributions to mitotic slippage remain unclear. Here, we report that MAD1 foci are phase-separated condensates that shorten the mitotic duration by sequestering checkpoint proteins. Our biophysical quantifications reveal that MAD1 ectopic foci in mitotic cells exhibit dynamic condensate properties rather than those of a solid aggregate. Using an inducible phase separation assay in live cells, we show that MAD1 phase separation is driven by interactions between its coiled-coil and disordered domains at the N-terminus. We decouple the contributions of condensation from concentration by inducing the formation of MAD1 ectopic condensates in mitotic cells with low levels of MAD1, demonstrating that the condensation process directly drives mitotic slippage. Mechanistically, the MAD1 ectopic condensate traps the diffusive pool of MAD2, an interaction partner of MAD1, thereby weakening the MAD2 conversion cycle necessary for a robust mitotic checkpoint. Our work illustrates a loss of function caused by ectopic condensates in MAD1-overexpressed cancer cells.

Source connections

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Tones, J., Jeffrey, J., Lackner, R. M., Chenoweth, D. M., Zhang, H.. 2025-07-31. Coiled-coil interactions drive ectopic condensation of overexpressed MAD1 to promote mitotic slippage in cancer cells. https://doi.org/10.1101/2025.07.29.667513

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

KEEP EXPLORING

Related preprints

bioRxiv2026

OPN3 mediates retinal-dependent lipofuscin accumulation and its loss sensitizes keratinocytes to blue-light-induced proteomic remodeling

Lipofuscin is a blue-light-absorbing pigment that contributes to oxidative damage. Whether all-trans retinal (atRAL) contributes to its formation in response to light remains unclear. We asked whether blue-light photosensitization of atRAL promotes lipofuscin accumulation in human keratinocytes and whether this depends on Opsin 3 (OPN3). Blue-light excitation of atRAL reduced mitochondrial and lysosomal viability, impaired autophagic flux, and increased lipofuscin. OPN3 knockdown significantly reduced this accumulation. Label-free data-independent acquisition (DIA) proteomics showed that OPN3 acts at three levels. In the dark, OPN3 loss altered proteome networks associated with autophagy, apoptosis, and interferon-related responses. Under blue light, control cells activated a stress-adaptive program spanning inflammatory regulation, lipid metabolism, and mitochondrial function, which atRAL strongly amplified. This molecular signature, including induction of cellular respiration and ATP-production proteins, was largely absent when OPN3 was silenced. Respirometry showed that blue light suppressed oxygen consumption in both lines over the first 24 h, but a faster recovery in OPN3 knockdown cells at 48 and 72 h was observed. Together, these data define three functions of OPN3: maintaining the basal proteome in a blue-light-independent manner, enabling the adaptive blue-light response, and enabling retinal-dependent lipofuscin formation in keratinocytes.

cell biology↗
bioRxiv2026

Uncoupling microtubule lifetime, stability and post-translational modifications.

Microtubules (MTs) undergo continuous cycles of growth and disassembly. Because the transitions between these states are stochastic, MT age varies widely within a population. As MTs age, they are thought to accumulate post-translational modifications (PTMs) that, directly or indirectly, enhance their stability and thereby extend their lifetime. The rare MTs that withstand prolonged exposure to destabilizing drugs such as nocodazole (NZ) are indeed enriched in PTMs; yet the relationships between MT age, PTMs and stability remain unclear. Using microinjection of labelled tubulin, we measured microtubule network turnover in immortalized mouse embryonic fibroblasts. Half of the network was renewed within 3 minutes and 80% within 10 minutes, while approximately 5% of microtubules persisted for more than 20 minutes. These dynamics were comparable in quiescent and senescent cells, although the fraction of slowly renewing or non-renewing microtubules rose to 20% in senescent cells. Unexpectedly, neither the amount of PTMs (acetylation and detyrosination) nor resistance to NZ increased with MT age, and resistance to NZ was independent of these PTMs. Degrees of acetylation and detyrosination should therefore not be taken as readouts of MT age or stability. Because these PTMs do not accumulate on MTs over time, the chemical modification of polymerized tubulin is likely more reversible and dynamic than previously assumed.

cell biology↗
bioRxiv2026

Evidence of complete myofibril remodeling after severe damage in adult Drosophila.

Muscle function depends on the ability of myofibrils to withstand and repair mechanical damage, yet how adult muscles remodel damaged myofibrils remains poorly understood. Here, we establish a Drosophila model that allows the induction and longitudinal visualization of extensive myofibril damage and recovery in intact adult femur muscles. Sustained muscle depolarization caused extensive disruption of myofibrillar organization, with severe damage characterized by near-complete loss of Z-disc structures. Remarkably, myofibril architecture and muscle function were largely restored within days, revealing a substantial capacity for myofibril reconstruction in adult femur muscles. We identified two distinct states of myofibril damage, mild and severe, with mild damage appearing before severe damage during aging, suggesting a progressive process of myofibril deterioration and repair failure. We further show that filamins mechanosignaling is required for efficient myofibril remodeling. Following damage, wild-type filamin redistributes from the Z-disc and accumulates outside the myofibrils, whereas constitutively open filamin remains Z-disc-associated and constitutively closed filamin redistributes but results in increased damage and impaired recovery. These findings suggest that effective repair requires dynamic transitions between filamin conformational states and that filamin redistribution is an active component of the damage response rather than simply a consequence of muscle injury. During aging, filamin progressively redistributes from the Z-disc and muscle damage accumulates, with severe damage increasing after the appearance of mild damage. Together, our findings reveal a previously unappreciated capacity of adult muscle to reassemble damaged myofibrils and identify filamin mechanosignaling as a key component of this repair process, providing a framework for understanding how defective mechanosensing may contribute to age-related muscle decline and muscle disease.

cell biology↗