Biology subjects

Ciani, B.

Publications and source records attributed to Ciani, B..

6 recordsLinked to original sources

bioRxiv2025

Mapping EGFR1 sorting domains in endosomes with a calibrated 3D expansion microscopy toolkit

Endosomes are nanoscale intracellular compartments that sort and recycle cell surface receptors such as epidermal growth factor receptor 1 (EGFR1). Nanometre-scale interactions and co-clustering of signalling proteins, cargo, and the membrane are critical to this process. Direct visualisation of these interactions has been hindered by the limited 3D resolution achievable with conventional and super-resolution microscopies. Here, we present the adaptation of expansion microscopy (ExM) to visualise, and quantify nanoclusters of endosomal proteins of human retinal pigment epithelial (RPE-1) cells. A 3D distortion analysis was developed leveraging the Farneback optical flow principle for detecting anisotropies in the hydrogel expansion. Analysis of pre- and post- ExM image volumes for 3D anisotropies revealed under-expansion of cytoplasmic regions within ExM hydrogels, often leading to over-estimation of size and distance measurements of small compartments such as endosomes. A self-assembling protein nanocage that reports the true local and nanoscale expansion factor was genetically introduced into the cells to calibrate ExM images of cytoplasmic regions containing endosomes. To stimulate and visualise the internalisation and sorting of EGFR1 in mammalian cells, a pulse-chase protocol was carried out with fluorescently-tagged EGF. The cells were subsequently fixed at 15- and 30- minute time points and subjected to 10-fold ExM and multiplexed 3D Airyscan microscopy to map cargo and EGFR1 vs other endosomal proteins. A volume tracing pipeline was developed to visualise the changes in the labelled EGF and EGFR1 densities at the limiting membrane of the endosomes. With multiplexed 3D ExM image volumes, we observed the enrichment of both EGF and EGFR1 in the endosomal interior and the accumulation of endosomal protein Rab5a near the limiting membrane during this early maturation of the endosomes. Taken together, the multiplexed 3D ExM toolkit offers a quantitative framework for visualising and measuring the intrinsic biology of small sub-cellular organelles like endosomes at true molecular-scale resolution.

biophysics↗

bioRxiv2023

CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway

ObjectivesEpithelial colonisation is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilise several host factors to associate with cells, including 5{beta}l integrin and heparan sulphate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process while integrins are only critical for post-adhesion entry into human epithelial cells. Methods and ResultsTreatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion whereas blockade of {beta}1 integrins did not affect adhesion but did reduce the subsequent internalisation of adhered bacteria. CD9 disruption or deletion increased {beta}1 integrin-mediated internalisation, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalisation. ConclusionsCD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organisation to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogues could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis.

microbiology↗

bioRxiv2020

Direct PA-binding by Chm7 is required for nuclear envelope surveillance at herniations

Mechanisms that control nuclear membrane remodeling are essential to maintain the integrity of the nucleus but remain to be fully defined. Here, we identify a phosphatidic acid (PA)-binding activity in the nuclear envelope-specific ESCRT, Chm7, in budding yeast. PA-binding is mediated through a conserved hydrophobic stretch of amino acids, which confers specific binding to the inner nuclear membrane (INM). This INM-binding is independent but nonetheless required for interaction with the LAP2-emerin-MAN1 (LEM) domain protein, Heh1 (LEM2). Consistent with the functional importance of PA-binding, mutation of this region inhibits recruitment of Chm7 to the INM and abolishes Chm7 function in the context of nuclear envelope herniations or "blebs" that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that PA accumulates at nuclear envelope herniations. We suggest that local control of PA metabolism is important for ensuring productive nuclear envelope remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

cell biology↗

bioRxiv2020

The influence of phosphatidylserine localisation and lipid phase on membrane remodelling by the ESCRT-II/ESCRT-III complex

The endosomal sorting complex required for transport (ESCRT) organises in supramolecular structures on the surface of lipid bilayers to drive membrane invagination and scission of intraluminal vesicles (ILVs), a process also controlled by membrane mechanics. However, ESCRT association with the membrane is also mediated by electrostatic interactions with anionic phospholipids. Phospholipid distribution within natural biomembranes is inhomogeneous due to, for example, the formation of lipid rafts and curvature-driven lipid sorting. Here, we have used phase-separated giant unilamellar vesicles (GUVs) to investigate the link between phosphatidylserine (PS)-rich lipid domains and ESCRT activity. We employ GUVs composed of phase separating lipid mixtures, where unsaturated DOPS and saturated DPPS lipids are incorporated individually or simultaneously to enhance PS localisation in liquid disordered (Ld) and/or liquid ordered (Lo) domains, respectively. PS partitioning between the coexisting phases is confirmed by a fluorescent Annexin V probe. Ultimately, we find that ILV generation promoted by ESCRTs is significantly enhanced when PS lipids localise within Ld domains. However, the ILVs that form are rich in Lo lipids. We interpret this surprising observation as preferential recruitment of the Lo phase beneath the ESCRT complex due to its increased rigidity, where the Ld phase is favoured in the neck of the resultant buds to facilitate the high membrane curvature in these regions of the membrane during the ILV formation process. Ld domains offer lower resistance to membrane bending, demonstrating a mechanism by which the composition and mechanics of membranes can be coupled to regulate the location and efficiency of ESCRT activity.

biophysics↗

bioRxiv2018

ESCRT-III accumulates in micronuclei with ruptured nuclear envelopes

1.Micronuclei represent the cellular attempt to compartmentalize DNA to maintain genomic integrity threatened by mitotic errors and genotoxic events. Micronuclei show aberrant nuclear envelopes that collapse, generating damaged DNA and promoting complex genome alterations. However, ruptured micronuclei also provide a pool of cytosolic DNA that stimulates anti-tumour immunity, revealing the complexity of micronuclei impact on tumour progression.\n\nThe ESCRT-III complex ensures nuclear envelope (NE) resealing during late mitosis and NE repair in interphase. Therefore, ESCRT-III activity maybe crucial for maintaining the integrity of other genomic structures enclosed by a nuclear envelope. ESCRT-III activity at the nuclear envelope is coordinated by the subunit CHMP7.\n\nWe show that CHMP7 and ESCRT-III protects against the genomic instability associated with micronuclei formation. Loss of ESCRT-III activity increases the population of micronuclei with ruptured nuclear envelopes, in interphase cells. Surprisingly, ESCRT-III is retained at acentric micronuclei suggesting that ESCRT-III cannot repair these structures. Depletion of CHMP7 expression removes ESCRT-III accumulations at ruptured micronuclei, and removes the population of micronuclei with damaged DNA also containing a sensor for cytosolic DNA.\n\nThus, ESCRT-III activity appears to protect from the consequence of genomic instability in a dichotomous fashion. Membrane repair activity prevents the occurrence of MN with weak envelopes; conversely, aberrant membrane remodelling at micronuclei generates a steady state pool of cytosolic DNA that may contribute to sustaining pro-inflammatory pathways in cancer cells.

cancer biology↗

bioRxiv2018

In vitro membrane remodelling by ESCRT-II/ESCRT-III is regulated by negative feedback from membrane tension

Artificial cells can shed new light on the molecular basis for life and hold potential for new chemical technologies. Inspired by how nature dynamically regulates its membrane compartments, we aim to repurpose the endosomal sorting complex required for transport (ESCRT) to generate complex membrane architectures as suitable scaffolds for artificial cells. Purified ESCRT-III components perform topological transformations on giant unilamellar vesicles (GUVs) to create complex \"vesicles-within-a-vesicle\" architectures resembling the compartmentalisation in eukaryotic cells. Thus far, the proposed mechanisms for this activity are based on how assembly and disassembly of ESCRT-III on the membrane drives deformation. Here we demonstrate the existence of a negative feedback mechanism from membrane mechanics that regulates ESCRT-III activity. ILV formation removes excess membrane area, increasing tension, which in turn suppresses downstream ILV formation. This mechanism for in vitro regulation of ESCRT-III activity may also have important implications for its in vivo functions.

synthetic biology↗