Biology subjects

Ciossani, G.

Publications and source records attributed to Ciossani, G..

7 recordsLinked to original sources

bioRxiv2026

Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

Aberrant endocytosis has long been associated with epithelial plasticity and tumorigenesis, but direct in vivo evidence of its causal role in tumor progression and metastasis has been lacking. Here, we identify and molecularly characterize a previously unrecognized form of E-cadherin (ECAD) internalization in mammary epithelial cells. This process is mediated by the endocytic adaptor Epsin 3 (EPN3) through glycolipid-lectin (GL-Lect) driven endocytosis requiring galectin-3 and Eps15-family adaptors. Leveraging an EPN3 knock-in mouse model, we show that dysregulation of GL-Lect driven endocytosis disrupts mammary gland morphogenesis and activates epithelial-to-mesenchymal plasticity (EMP), synergizing with the ERBB2/Neu breast oncogene to drive metastasis. Pharmacologic inhibition of the GL-Lect mechanism suppresses morphogenetic and invasive phenotypes ex vivo, providing proof-of-concept for therapeutic targeting. These findings establish the GL-Lect mechanism as a driver of metastatic plasticity and uncover a tractable vulnerability in BC.

cancer biology↗

bioRxiv2025

Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

NUMB is an endocytic protein with tumor suppressor activity, largely mediated by its ability to inhibit p53 degradation. This function depends on the inclusion of a short alternatively spliced exon (Ex3) in NUMB, although the mechanistic link between endocytosis and p53 regulation remains unclear. Here, we show that the Ex3-encoded sequence directs NUMB to the plasma membrane, where it forms a complex with the endocytic adaptor SNX9. This complex recruits p53 in a SNX9-dependent manner and is internalized and trafficked to multivesicular bodies, culminating in exosomal secretion, in a process requiring both SNX9 and NUMB. Exosomal p53 is taken up by recipient cells and translocated to the nucleus, where it activates p53-dependent transcriptional and phenotypic programs. These findings suggest that exosome-mediated p53 transfer may contribute to the establishment of a tumor-suppressive microenvironment.

cancer biology↗

bioRxiv2023

A novel, RAS-independent role for NF1 in microtubular dynamics and damage repair dictates sensitivity to T-DM1 in HER2-positive breast cancer

Antibody-Drug Conjugates (ADC) have revolutionized the treatment of several tumors, and extensive research is being devoted to the identification of predictive biomarkers. These are particularly sought after in fields, like breast cancer, in which multiple ADCs with identical target but different payloads have been approved. NF1 is a tumor suppressor widely mutated across several cancers, best characterized as an inhibitor of RAS signaling. Additional functions have been proposed but not deeply investigated, due to its large size and complex domain structure. Whether somatic NF1 mutations can be used to guide clinical decisions is not known. Here, combining patient data, in vitro/in vivo models and protein biochemistry, we show that NF1 loss sensitizes cancer cells to T-DM1, the first approved ADC in breast cancer, through a novel, RAS-independent function on microtubular dynamics and repair. NF1 exhibits all biochemical properties of a bona fide Microtubule-Associated Protein (MAP) and specifically enhances intratubular repair, a recently discovered phenomenon whose regulation remains poorly characterized. NF1 loss results in mitotic defects and low-grade aneuploidy in cell lines and patients. Increased sensitivity to T-DM1 upon NF1 loss is confirmed in breast cancer patients analysed across institutions in Europe and USA. Our results define NF1 as a key regulatory factor for microtubular repair and the first ADC payload-associated predictive biomarker identified to date.

cancer biology↗

bioRxiv2023

A mechanism that integrates microtubule motors of opposite polarity at the kinetochore corona

Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin 7) and Dynein-Dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The coronas building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we report near-complete depletion of RZZS and DD from kinetochores after depletion of CENP-E and the outer kinetochore protein KNL1. With inhibited MPS1, CENP-E, which we show binds directly to RZZS, is required to retain kinetochore RZZS. An RZZS phosphomimetic mutant bypasses this requirement. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.

cell biology↗

bioRxiv2021

Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores

In metazoans, a {asymp}1 megadalton (MDa) super-complex comprising the Dynein-Dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.

biochemistry↗

bioRxiv2019

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging, and chemical biology

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of kinetochores, a spatially confined and well-studied subcellular structures. After electroporation in human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes displayed native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation reconstituted robust kinetochore localization of Spindly. Thus, electroporation is uniquely versatile for delivering synthetic and, as required, chemically modified functional mimics of endogenous proteins, and is therefore a promising tool for chemical and synthetic biology.

cell biology↗

bioRxiv2018

Kinetochore recruitment of CENP-F illustrates how paralog divergence shapes kinetochore composition and function

The metazoan proteins CENP-E and CENP-F are components of a fibrous layer of mitotic kinetochores named the corona. Several features suggest that CENP-E and CENP-F are paralogs: they are very large (approximately 2700 and 3200 residues, respectively), rich in predicted coiled-coil structure, C-terminally prenylated, and endowed with microtubule-binding sites at their termini. In addition, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus-end directed motion. Here, we show that CENP-E and CENP- F are recruited to mitotic kinetochores independently of the Rod-Zwilch-ZW10 (RZZ) complex, the main corona constituent. We identify selective interactions of CENP-E and CENP-F respectively with BubR1 and Bub1, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. While BubR1 is dispensable for kinetochore localization of CENP-E, Bub1 is stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrate that the CENP-E:BubR1 and CENP-F:Bub1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BubR1 or Bub1. Our findings are consistent with the existence of pseudo-symmetric, paralogous Bub1:CENP-F and BubR1:CENP-E axes, supporting evolutionary relatedness of CENP-E and CENP-F.

cell biology↗