Biology subjects

Girao, H.

Publications and source records attributed to Girao, H..

7 recordsLinked to original sources

bioRxiv2026

Elevated MPS1 converts the fibrous corona into a source of chromosomal instability in colon cancer

Chromosomal instability (CIN), characterised by recurrent chromosome mis-segregation, fuels intratumour heterogeneity and tumour evolution. Yet, its proximal molecular causes remain ill-defined. Tumour-scale genomic and transcriptomic analyses associate MPS1 overexpression with CIN in colon carcinomas. Consistently, CIN+ patient-derived colon cancer cells (PCCCs) display elevated MPS1, frequent merotelic kinetochore-microtubule attachments and lagging chromosomes that are suppressed by partial MPS1 inhibition. Conversely, ectopic MPS1 overexpression in otherwise stable near-diploid cells phenocopies these defects, inducing micronuclei formation and karyotypic divergence. Mechanistically, MPS1 overactivity maintains ROD phosphorylation at Thr13/Ser15, continuously sustaining fibrous corona assembly despite mature end-on attachments. 3D-STED microscopy reveals split-interface configurations in which microtubules from opposing poles engage the canonical outer-kinetochore surface and a spatially distinct corona domain, explaining how merotely forms and persists into anaphase. Disrupting corona assembly restores chromosome segregation fidelity in MPS1-overexpressing RPE-1 cells and in CIN+ PCCCs. Together, our findings establish MPS1 overexpression as a driver of CIN and uncover how oncogenic transcriptional rewiring of mitotic signalling converts a transient microtubule-capture structure into a pathological source of merotely and chromosome mis-segregation in colon cancer.

cell biology↗

bioRxiv2026

A room-temperature ⁸⁹Zr⁴⁺ radiolabelling strategy for small extracellular vesicles with enhanced plasma stability for PET Imaging

Both for diagnostic purposes and regenerative medicine, it is essential to develop advanced imaging platforms capable of tracking the biodistribution of small extracellular vesicles (sEVs), as current methods are limited by inadequate resolution and sensitivity. In this study, we introduce a novel labeling strategy utilizing the radioisotope zirconium-89 (89Zr), which boasts a half-life of 78.4 h and is cost-effective to produce. To achieve this, we designed a new chelator tailored for 89Zr4+ that offers enhanced stability compared to the conventional deferoxamine (DFO). This chelator forms a robust complex with 89Zr4+ at room temperature, suitable for sEV labeling for PET imaging applications. The radiolabeling process involved a two-step procedure: first, conjugation of the chelator to the sEVs, and second, radiolabeling with 89Zr4+. The resulting sEV-L1-Zr demonstrated a radiochemical yield of approximately 60% and maintained around 80% stability in plasma over seven days. Importantly, our modifications did not alter the morphology, surface protein composition, internal RNA content, or bioactivity of the sEVs. We successfully visualized sEVs at very low doses in the mouse heart following intravenous injection of sEV-L1-Zr. Additionally, ex vivo experiments using a Langendorff rat heart perfusion model confirmed targeted accumulation of the vesicles in cardiomyocytes as compared to other cells in the heart compartment. This approach provides a promising platform for sensitive and stable in vivo tracking of sEVs, advancing their application in both diagnostic imaging and regenerative therapies.

bioengineering↗

bioRxiv2024

Loss of the lysosomal protein CLN3 modifies the lipid content of the nuclear envelope leading to DNA damage and activation of YAP1 pro-apoptotic signaling

Batten disease is characterized by early-onset blindness, juvenile dementia and death during the second decade of life. The most common genetic causes are mutations in the CLN3 gene encoding a lysosomal protein. There are currently no therapies targeting the progression of the disease, mostly due to the lack of knowledge about the disease mechanisms. To gain insight into the impact of CLN3 loss on cellular signaling and organelle function, we generated CLN3 knock-out cells in a human cell line (CLN3-KO), and performed RNA sequencing to obtain the cellular transcriptome. Following a multi-dimensional transcriptome analysis, we identified the transcriptional regulator YAP1 as a major driver of the transcriptional changes observed in CLN3-KO cells. We further observed that YAP1 pro-apoptotic signaling is hyperactive as a consequence of CLN3 functional loss in retinal pigment epithelia cells, and in the hippocampus and thalamus of CLN3ex{Delta}7/8 mice, an established model of Batten disease. Loss of CLN3 activates YAP1 by a cascade of events that starts with the inability of releasing glycerophosphodiesthers from CLN3-KO lysosomes, which leads to perturbations in the lipid content of the nuclear envelope and nuclear dysmorphism. This results in increased number of DNA lesions, activating the kinase c-Abl, which phosphorylates YAP1, stimulating its pro-apoptotic signaling. Altogether, our results highlight a novel organelle crosstalk paradigm in which lysosomal metabolites regulate nuclear envelope content, nuclear shape and DNA homeostasis. This novel molecular mechanism underlying the loss of CLN3 in mammalian cells and tissues may open new c-Abl-centric therapeutic strategies to target Batten disease.

cell biology↗

bioRxiv2023

Unveiling the role of osteosarcoma-derived secretome in premetastatic lung remodeling

Lung metastasis represents the leading cause of osteosarcoma-related death. Progress in preventing lung metastasis is pretty modest due to the inherent complexity of the metastatic process and the lack of suitable models. Herein, we provide mechanistic insights into how osteosarcoma systemically reprograms the lung microenvironment for metastatic outgrowth using metastatic mouse models and a multi-omics approach. We found that osteosarcoma-bearing mice or those preconditioned with cell-secretome harbour profound lung structural alteration with airways damage, inflammation, neutrophil infiltration, and remodelling of the extracellular matrix with deposition of fibronectin and collagen by stromal activated fibroblasts for tumour cell adhesion. These changes, supported by transcriptomic and histological data, promoted and accelerated the development of lung metastasis. Comparative proteome profiling of the cell secretome and mouse plasma identified a large number of proteins engaged in the extracellular-matrix organization, cell-matrix adhesion, neutrophil degranulation, and cytokine-mediated signalling, which were consistent with the observed lung microenvironmental changes. Moreover, we identified EFEMP1, a secreted extracellular matrix glycoprotein, as a potential risk factor for lung metastasis and a poor prognosis factor in osteosarcoma patients.

cancer biology↗

bioRxiv2021

Kazrin C entraps early endosomes at the pericentriolar region and facilitates endocytic recycling

Kazrin is a protein widely expressed in vertebrates whose depletion causes a myriad of developmental defects, in part derived from altered cell adhesion, impaired cell migration and failure to undergo Epidermal to Mesenchymal Transition (EMT). However, the primary molecular role of kazrin, which might contribute to all these functions, has not been elucidated yet. We previously identified one of its isoforms, kazrin C, as a protein that potently inhibits clathrin-mediated endocytosis when overexpressed. We now generated kazrin knock out Mouse Embryonic Fibroblasts (MEFs) to investigate its endocytic function. We found that kazrin depletion delays perinuclear enrichment of internalized material, indicating a role in endocytic traffic from Early (EE) to Recycling Endosomes (REs). Consistently, we found that the C-terminal domain of kazrin C, predicted to be an Intrinsically Disordered Region (IDR), directly interacts with several components of the EEs, and that kazrin depletion impairs centripetal motility of EEs. Further, we noticed that the N-terminus of kazrin C shares homology with dynein/dynactin adaptors and that it directly interacts with the dynactin complex and the dynein Light Intermediate Chain 1 (LIC1). Altogether, the data indicate that one of the primary kazrin functions is to facilitate endocytic recycling via the perinuclear endocytic compartment, by promoting microtubule and dynein/dynactin-dependent transport of EEs or EE-derived transport intermediates to the RE.

cell biology↗

bioRxiv2021

LAMP2A regulates the loading of proteins into exosomes.

Exosomes are extracellular vesicles of endosomal origin released by virtually all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs and proteins between different cells, tissues or organs. However, the mechanisms that regulate the selective loading of cytosolic proteins into these vesicles are still largely unknow. Here we describe a mechanism whereby proteins containing a pentapeptide sequence, biochemically related to the KFERQ-motif, are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, this mechanism is independent of the ESCRT machinery components TSG101 and VPS4b and dependent on HSC70, CD63, Alix, Syntenin-1, Rab31 and ceramides. The transcription factor and master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. Additionally, by tagging fluorescent proteins with KFERQ-like sequences we were able to follow inter-organ transfer of exosomes in zebrafish larvae. Our findings identify LAMP2A as a key component in exosome biogenesis while opening new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.

cell biology↗

bioRxiv2019

CLASP2 lattice-binding near microtubule plus ends stabilizes kinetochore attachments

The fine regulation of kinetochore microtubule dynamics during mitosis ensures proper chromosome segregation by promoting error correction and spindle assembly checkpoint (SAC) satisfaction. CLASPs are widely conserved microtubule plus-end-tracking proteins that regulate microtubule dynamics throughout the cell cycle and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore microtubule dynamics, but the underlying molecular mechanism remains unknown. Here we found that human CLASP2 can dimerize through its C-terminal kinetochore-targeting domain, but kinetochore localization was independent of dimerization. CLASP2 kinetochore localization, microtubule plus-end-tracking and microtubule lattice binding through TOG2 and TOG3 (but not TOG1) domains, independently sustained normal spindle length, timely SAC satisfaction, chromosome congression and faithful segregation. Measurements of kinetochore microtubule half-life in living cells expressing RNAi-resistant mutants revealed that CLASP2 kinetochore localization, microtubule plus-end-tracking and lattice binding cooperatively modulate kinetochore microtubule stability during mitosis. Thus, CLASP2 regulates kinetochore microtubule dynamics by integrating distinctive microtubule-binding properties at the kinetochore-microtubule interface to ensure chromosome segregation fidelity.

cell biology↗