Biology subjects

Sonnett, M.

Publications and source records attributed to Sonnett, M..

11 recordsLinked to original sources

bioRxiv2026

Quantitative imaging of native tissue architecture combined with deep multi-omics reveals the spatial biology of kidney aging

Aging alters tissue architecture and metabolism in spatially heterogeneous ways, yet the connections between these measurable structural and compositional phenotypes and underlying molecular changes are largely unknown. Here, we introduce NORIGAMI (NoRI-Guided AI for Multimodal Integration), a pipeline combining Normalized Raman Imaging (NoRI), deep learning, quantitative morphology, deep proteomics, and metabolomics to investigate renal aging. Unlike conventional histology, NoRI provides label-free, scattering-corrected measurements of absolute total protein and lipid mass concentrations (mg/mL). In 32 male rats aged 9, 18, 21 and 25 months, NORIGAMI predicted the chronological age of held-out rats from NoRI images (mean absolute error 0.78 months; R^2 = 0.97), and whole-tissue images outperformed models trained on isolated tubules. Quantitative imaging identified 41 age-associated morphological and compositional features (FDR < 0.05), revealing anatomically heterogeneous and non-linear changes in lipid droplet concentration, droplet size, cytoplasmic lipid concentration and tubular architecture. Deep molecular profiling quantified 11,485 proteins and 269 metabolites, revealing coordinated declines in differentiated proximal tubule (PT) identity and oxidative metabolism, selective increases in basement-membrane collagen-IV chains, and increasing molecular heterogeneity with age. A protein clock, built mainly from extracellular-matrix and heparan-sulfate proteins, predicted age with a mean absolute error of 1.6 months. Old kidneys diverged along a dominant injury-remodeling axis, seen separately in the proteome, in kidney RNA from 54 other rats and in the metabolome, in which tubular-injury and immune proteins, including the NAD+ consuming enzyme CD38, rise as differentiated-tubule proteins and NAD+ fall. NORIGAMI subsequently linked NAD+ associated molecular changes to quantitative features of tubular geometry and protein-lipid organization, while complementary KIM-1-centered analyses identified overlapping morphological associations involving epithelial injury and lipid handling and yielded additional molecular candidates for mechanistic validation. Collectively, these findings suggest that declining tubular metabolic function, altered lipid organization and injury-associated molecular programs represent interconnected features of renal aging. NORIGAMI establishes a framework for identifying the quantitative structural manifestations of molecular aging and generating spatially informed, experimentally testable hypotheses about tissue dysfunction.

physiology↗

bioRxiv2026

Protein accumulation upstream of Cdk1 activation regulates the competence and the timing of meiosis resumption

The capacity to resume meiosis is progressively acquired during oogenesis and is ultimately restricted to fully grown oocytes. Meiotic resumption is triggered by hormonal stimulation and requires activation of Cdk1, the universal driver of M-phase entry. Cdk1 activation occurs in two steps: an initial activation of Cdk1, followed by an amplification phase that drives cell cycle re-entry. The first step depends on the accumulation of proteins that promote Cdk1 activation, while the second step involves a regulatory network of kinases and phosphatases. Using TMT-based quantitative proteomics, we reveal that growing oocytes first acquire the ability to regulate protein homeostasis in response to hormonal stimulation, and only later gain the competence to amplify initial Cdk1 activity and enter meiosis. Notably, protein accumulation, occurring independently of Cdk1 activation, is controlled by both translational and non-translational mechanisms. Together, our findings show that the molecular competence to trigger Cdk1 activation is acquired in a stepwise manner during oocyte growth. The earliest regulatory layer is the acquisition of the ability to respond to hormonal stimulation by accumulating proteins that are required for efficient Cdk1 activation and meiotic resumption.

Developmental Biology↗

bioRxiv2025

Unbounded Cell Growth and Proteome Imbalance in Doxorubicin-Induced Senescent RPE-1 Cells

Cellular senescence is traditionally viewed as a terminal state of cell-cycle arrest accompanied by widespread molecular remodeling, yet its underlying regulatory logic and progression remain poorly understood. Here, we combined quantitative phase microscopy and normalized Raman imaging with quantitative proteomic and phosphoproteomic profiling to examine human RPE1 cells undergoing doxorubicin-induced senescence. Senescent cells did not reach a steady state but instead exhibited sustained, unbounded growth over a 12 day period, marked by a continuous rise in dry mass and volume coupled with declining mass density. Time resolved proteomics revealed extensive and asynchronous remodeling across organelles, with lysosomal, ER, and Golgi proteins increasing in abundance, whereas nuclear and mitochondrial proteins declined, indicating large scale reorganization of cellular composition. Phosphoproteomic inference linked these structural shifts to regulatory signaling, confirming the expected downregulation of CDK activity while revealing coordinated activation of stress and DNA damage responsive kinases such as CAMK2D, DNAPK, and MARK family members. Together, these integrated data depict senescence as a dynamic, actively regulated state, maintained through coordinated remodeling of proteome composition and signaling activity rather than passive arrest. Our findings highlight how combining quantitative biophysical measurements with multi-layered molecular profiling exposes the regulatory architecture that sustains the senescent phenotype and its loss of internal homeostasis.

cell biology↗

bioRxiv2025

Using regularized regression and biological covariation to impute missing values in quantitative proteomics

Proteomics studies analyzing many samples typically generate datasets with missing values where many protein abundances are only quantified in a subset of assayed conditions. While multiplexing with isobaric tags can address this by combining multiple samples into a single injection, missing values are unavoidable when the sample count exceeds the number of available isobaric tags (currently >35). Such missing data complicates the interpretation of large-scale studies across diverse experimental conditions. Here, we introduce a method to impute missing values of relative protein abundance by leveraging measurements from other proteins in the dataset through regularized regression. Our technique, which is applicable to diverse datasets including different cell lines, animals, or biochemical perturbations, capitalizes on the hitherto overlooked biological covariation among protein abundance changes. Our analysis of eight published proteomics datasets reveals a robust imputation capability, achieving a median R2 of 0.55 to 0.8 between imputed and measured data. We demonstrate a similar imputation efficacy in multiple measurement modalities: TMT, DIA, label free, and TMT phosphoproteomics. When examining regression coefficients that were pivotal for accurate data imputation we found that those often mirror known biology. We propose that previously overlooked biological covariation might lead to the generation of novel hypotheses and ultimately advance our understanding of systems level protein organization.

biochemistry↗

bioRxiv2025

Diverse microtubule-destabilizing drugs induce equivalent molecular pathway responses in endothelial cells

Drugs that modulate microtubule (MT) dynamics are well-characterized at the molecular level, yet the mechanisms linking these molecular effects to their distinct clinical outcomes remain unclear. Several MT-destabilizing drugs, including vinblastine, combretastatin A4, and plinabulin, are widely used, or are under evaluation for cancer treatment. Although all three depolymerize MTs, they do so through distinct biochemical mechanisms. Furthermore, their clinical profiles and therapeutic uses differ considerably. To investigate whether differential modulation of molecular pathways might account for clinical differences, we compared gene expression and signaling pathway responses in human pulmonary microvascular endothelial cells (HPMECs), alongside the MT-stabilizing drug docetaxel and the pro-inflammatory cytokine TNF-. RNA-sequencing and phosphoproteomics revealed that all three MT destabilizers triggered equivalent molecular responses. The substantial changes in gene expression caused by MT destabilization were completely dependent on Rho family GTPase activation. These findings suggest that the distinct clinical profiles of the destabilizing drugs depend on differences in pharmacokinetics (PK) and tissue distribution rather than molecular actions. The washout rate of the three drugs differed, which likely translates to PK differences. Our data provide insights into how MT destabilization triggers signaling changes, potentially explaining how these drugs induce cell cycle re-entry in quiescent cells and how plinabulin ameliorates chemotherapy-induced neutropenia. Significance StatementMicrotubule (MT)-destabilizing drugs, despite their potential for toxicity, are used to treat a surprising range of diseases, including cancers, inflammatory disorders, and parasitic infections. To investigate how drugs with apparently similar actions on MTs achieve diverse clinical effects, we compared the molecular pathways that are activated by three drugs with different clinical profiles, vinblastine, combretastatin A4, and plinabulin. All three elicited similar gene expression responses via Rho GTPase activation. This finding suggests that their distinct clinical effects are not caused by different effects on MTs, but rather by differences in drug transport, pharmacokinetics or tubulin isotype affinity. Our findings provide insights into how plinabulin might protect the bone marrow and may help medicinal chemists design MT drugs for new applications.

pharmacology and toxicology↗

bioRxiv2024

Decoding protein phosphorylation during oocyte meiotic divisions using phosphoproteomics

Oocyte meiotic divisions represent a critical process in sexual reproduction, as a diploid non-dividing oocyte is transformed into a haploid fertilizable egg, as a prelude for the subsequent embryonic divisions and differentiation. Although cell differentiation and proliferation are governed by transcription, oocyte maturation and early embryonic divisions depend entirely on changes in protein abundance and post- translational modifications. Here, we analyze the abundance and phosphorylation of proteins during Xenopus oocyte meiotic maturation. We reveal significant shifts in protein stability, related to spindle assembly, DNA replication and RNA-binding. Our analysis pinpoints broad changes in phosphorylation correlating with key cytological meiotic milestones, noteworthy changes in membrane trafficking, nuclear envelope disassembly and modifications in microtubule dynamics. Additionally, specific phosphorylation events target regulators of protein translation, Cdk1 and the Mos/MAPK pathway, thereby providing insight into the dynamics of Cdk1 activity, as related to the meiotic cell cycle. This study sheds light on the orchestration of protein dynamics and phosphorylation events during oocyte meiotic divisions, providing a rich resource for understanding the molecular pathways orchestrating meiotic progression in the frog, and most likely applicable to other vertebrate species.

developmental biology↗

bioRxiv2024

Synergy Between TNFα and Proteostatic Stress Drives Cell Death and Guard Immunity

The production and sensing of type I interferons (IFN-I) are critical for antiviral defense, yet most virus-infected cells do not produce IFN-I or upregulate IFN-stimulated genes. Using quantitative proteomics and global protein synthesis measurements, we show that productive viral infection globally down-regulates protein synthesis, restricting the IFN response. Guard immunity, which responds to disruptions in essential cellular processes, might compensate for the lack of IFN-I response by rapidly killing infected cells. However, non-pathological stressors can also disrupt proteostasis, making it unclear how cells decide to trigger guard immunity. We hypothesized that TNF, produced by macrophages, provides a contextual signal allowing specificity. Using live-cell fluorescence microscopy and mathematical modeling, we showed that TNF synergizes with the rapid decay of the anti-apoptotic protein c-FLIP to induce cell death and prevent viral spread. Our findings demonstrate that TNF contextualizes proteostasis loss as non-sterile, enabling the activation of guard immunity to counteract viral infection.

immunology↗

bioRxiv2021

Glycogen-dependent demixing of frog egg cytoplasm at increased crowding

Crowding increases the tendency of macromolecules to aggregate and phase separate, and high crowding can induce glass-like states of cytoplasm. To explore the effect of crowding in a well-characterized model cytoplasm we developed methods to selectively concentrate components larger than 25 kDa from Xenopus egg extracts. When crowding was increased 1.4x, the egg cytoplasm demixed into two liquid phases of approximately equal volume. One of the phases was highly enriched in glycogen while the other had a higher protein concentration. Glycogen hydrolysis blocked or reversed demixing. Quantitative proteomics showed that the glycogen phase was enriched in proteins that bind glycogen, participate in carbohydrate metabolism, or are in complexes with especially high native molecular weight. The glycogen phase was depleted of ribosomes, ER and mitochondria. These results inform on the physical nature of a glycogen-rich cytoplasm and suggest a role of demixing in the localization of glycogen particles in tissue cells.

cell biology↗

bioRxiv2020

Mammalian Cell Proliferation Requires Noncatalytic Functions of O-GlcNAc Transferase

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGTs myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGTs enzymatic activities for cell viability. Using genetic complementation, we found that OGTs glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically-inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGTs catalytic and noncatalytic activities affect protein abundance, we found that OGTs noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology. SignificanceMammalian cells contain only one glycosyltransferase, OGT, that operates in the nucleus and cytoplasm rather than the secretory pathway. OGT is required for cell proliferation, but a basic unanswered question is what OGT functions are essential. This question is challenging to address because OGT has thousands of glycosylation substrates, two different enzymatic activities, and a large number of binding partners. Here, by establishing genetic tools to replace endogenous OGT with variants that preserve only a subset of its activities, we show that only a low level of glycosylation activity is required to maintain cell viability; however, cell proliferation requires noncatalytic OGT function(s). The ability to replace OGT with variants provides a path to identifying its essential substrates and binding partners.

biochemistry↗

bioRxiv2020

Co-movement of astral microtubules, organelles and F-actin suggests aster positioning by surface forces in frog eggs

How bulk cytoplasm generates forces to separate post-anaphase microtubule (MT) asters in Xenopus laevis and other large eggs remains unclear. Previous models proposed dynein-based organelle transport generates length-dependent forces on astral MTs that pull centrosomes through the cytoplasm, away from the midplane. In Xenopus egg extracts, we co-imaged MTs, endoplasmic reticulum (ER), mitochondria, acidic organelles, F-actin, keratin, and fluorescein in moving and stationary asters. In asters that were moving in response to dynein and actomyosin forces, we observed that all cytoplasmic components moved together, i.e., as a continuum. Dynein-mediated organelle transport was restricted by interior MTs and F-actin. Organelles exhibited a burst of dynein-dependent inward movement at the growing aster surface, then mostly halted inside the aster. Dynein-coated beads were slowed by F-actin, but in contrast to organelles, beads did not halt inside asters. These observations call for new models of aster positioning based on surface forces and internal stresses.

cell biology↗

bioRxiv2017

Accurate, Sensitive, and Precise Multiplexed Proteomics using the Complement Reporter Ion Cluster

Quantitative analysis of proteomes across multiple time points, organelles, and perturbations is essential for understanding both fundamental biology and disease states. The development of isobaric tags (e.g. TMT) have enabled the simultaneous measurement of peptide abundances across several different conditions. These multiplexed approaches are promising in principle because of advantages in throughput and measurement quality. However, in practice existing multiplexing approaches suffer from key limitations. In its simple implementation (TMT-MS2), measurements are distorted by chemical noise leading to poor measurement accuracy. The current state-of- the-art (TMT-MS3) addresses this, but requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter ion approach (TMTc) produces high accuracy measurements and is compatible with many more instruments, like quadrupole-Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2- isolation step into the deconvolution algorithm. The resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty cycle, and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative label-free proteomics of total HeLa lysate and find that TMTc+ quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time the median coefficient of variation improves from 13% to 4%. Thus, TMTc+ advances quantitative proteomics by enabling accurate, sensitive, and precise multiplexed experiments on more commonly used instruments.

systems biology↗