Biology subjects

Bottanelli, F.

Publications and source records attributed to Bottanelli, F..

14 recordsLinked to original sources

bioRxiv2026

Cargo levels control endosomal PI(3)P homeostasis through direct feedback between SNX17 and MTM1

The sorting of internalised cell surface proteins through the endosomal network is crucial for cellular function. Phosphatidylinositol-3-phosphate (PI(3)P) is a critical lipid identity cue for organising endosomal sorting but there is limited understanding of how PI(3)P homeostasis is regulated. Here, we show that free PI(3)P is segregated into distinct endosomal subdomains and that depletion of the disease-relevant lipid phosphatase Myotubularin1 (MTM1) leads to its accumulation and mis-segregation, impairing cargo recycling. Complementary imaging and reconstitution approaches show that MTM1 recruitment and activity are controlled by binding to the endosomal cargo adaptor SNX17. This interaction is mediated by sorting motifs in the C-Terminal tail of MTM1 and is in competition with the same sorting motifs in cargo that bind to SNX17. This competitive binding mechanistically links cargo sorting to PI(3)P depletion and because SNX17 is itself a PI(3)P effector, establishes negative feedback regulating local PI(3)P levels. Importantly, we demonstrate that this feedback couples endosomal cargo levels to PI(3)P depletion. We propose that cargo levels thereby modulate endosomal phosphoinositide identity through cargo sensitive feedback between SNX17 and MTM1.

cell biology↗

bioRxiv2026

The AP-1 clathrin adaptor has a conserved role in recycling resident proteins of the trans-Golgi network

Although the AP-1 clathrin adaptor has been studied for decades, its role in membrane trafficking has been ambiguous. Here, we systematically investigated the localization and function of mammalian AP-1. This adaptor associates with the trans-Golgi network (TGN) and with TGN-related recycling endosomes. Disruption of the 1A subunit of AP-1 has only a mild effect on the Golgi export of secretory cargoes, but it causes depletion from the Golgi of multiple transmembrane TGN proteins. Live-cell imaging and a cell surface labeling assay indicate that when TGN proteins are unable to bind AP-1, they exit the Golgi to the plasma membrane. Our interpretation is that the vesicles generated with AP-1 recycle transmembrane TGN proteins, thereby keeping those proteins in the Golgi during cisternal maturation. The clathrin adaptor epsinR seems to function as a partner for AP-1 in recycling a subset of TGN proteins. These results closely align with studies of AP-1 function in yeast, suggesting that the conserved core role of AP-1 is the intra-Golgi recycling of TGN proteins.

cell biology↗

bioRxiv2026

A general role for GGA adaptors in the modulation of AP-1-dependent trafficking

The Golgi-localized, {gamma}-ear containing, ADP-ribosylation factor binding proteins (GGAs) are a family of adaptor proteins that regulate transport of specific cargo receptors from the Golgi to endosomes. For many years it was assumed that GGAs transport cargo via interaction with the adaptor complex AP-1. However, recent findings suggest that GGA and AP-1 may have opposing roles, with GGAs facilitating forward transport between Golgi and endosomes, and AP-1 mediating the opposite trafficking step. To shed light on the functional connection of GGAs with AP-1, we combined CRISPR-Cas9 gene editing with live-cell imaging and TurboID-based proximity labelling. We find that GGAs localize not only to the Golgi apparatus but also, to a greater extent, to peripheral ARF1-positive compartments responsible for secretory trafficking and endocytic recycling. At both, the Golgi and peripheral sites, we observe distinct sorting domains containing either AP-1 or GGAs alone, as well as domains in which both adaptors are present. Interestingly, GGAs can recruit clathrin lattices independently of AP-1. Proximome mapping shows that AP-1 specific cargoes only localize to AP-1 domains in the absence of GGAs. These findings point to a regulatory role of GGAs in AP-1 transport. We speculate that GGAs prevent binding of AP-1 to its cargo clients to avoid premature retrieval and to modulate bi-directional trafficking between the Golgi and endosomes.

cell biology↗

bioRxiv2026

ARF-1 Coordinates Cargo Sorting at FERARI Endosomal Recycling Hubs

Endosomal pathways are central to cellular organization, compartmentalization, and intercellular communication. Independent of the endocytic entry route, sorting endosomes function as critical junctions at which membrane proteins are sorted for recycling or degradation. The multicomponent tether FERARI orchestrates recycling by coordinating Rab GTPase activity with vesicle fusion and fission through a kiss-and-run mechanism. Here, we identify the small GTPase ARF-1 as an essential regulator of cargo loading at FERARI-dependent endosomal kiss-and-run (KAR) sites. Cycling of ARF-1 between its active and inactive states is required for efficient cargo capture into recycling vesicles. Our findings support a model in which cycling ARF-1 activity governs the temporal association of cargo with adaptor proteins and complexes, thereby controlling selective uptake into vesicles destined for the plasma membrane. These results uncover a previously unappreciated layer of regulation of endosomal recycling and highlight the interplay between ARF and Rab family GTPases in this process.

cell biology↗

bioRxiv2025

T cell protrusions enable fast, localised initiation of CAR signalling

Actin-rich protrusions densely cover the surface of T cells and are well characterised for their role in cell migration. However, recent studies have uncovered their role in antigen surveillance and immune signalling initiation. To investigate how membrane protrusions initiate and contribute to signalling, from the first cell-cell contact to immunological synapse formation, we performed dynamic imaging experiments of endogenously tagged signalling proteins in T cells. To quantitatively capture the early dynamics of cell-cell interactions, we employed HER2-CAR-expressing T cells targeting HER2+breast cancer cells. By harnessing live-cell imaging and super-resolution stimulated emission depletion (STED) microscopy we were able to capture topological membrane changes and their correlation with mesoscale protein rearrangements over time. Our findings indicate that, prior to activation, key molecular players in T cell activation, including the kinase Lck, the phosphatase CD45 and the adaptor LAT, as well as the exogenously expressed CAR, lack any enrichment in actin-rich protrusions. However, upon initial contact of the T cell with the target cell, a dynamic and fast rearrangement of the surface receptors, phosphatases, and kinases occurs within the protrusions, ensuring a rapid and effective initiation of the immune signalling cascade. The rapid clustering of the HER2-CAR occurs preferentially within protrusions rather than flat membrane regions and is accompanied by enhanced recruitment of the kinase ZAP-70 and LAT. While the localisation of the kinase Lck remained unchanged, protrusion-cell contacts trigger a pronounced exclusion of the phosphatase CD45, an effect observed both with and without the cytosolic signalling domain of the CAR. Overall, the signalling machinery rearranged more rapidly and efficiently at contacts mediated by protrusive structures compared to non-protrusive regions. Together, our data provide a quantitative framework illustrating how signalling proteins are dynamically reorganised to facilitate CAR-mediated activation within these specialised structures.

cell biology↗

bioRxiv2024

Induced degradation of SNAP-fusion proteins

Self-labeling protein tags are an efficient means to visualize, manipulate, and isolate engineered fusion proteins with suitable chemical probes. The SNAP-tag, which covalently conjugates to benzyl-guanine and -chloropyrimidine derivatives is used extensively in fluorescence microscopy, given the availability of suitable SNAP-ligand-based probes. Here, we extend the applicability of the SNAP-tag to targeted protein degradation. We developed a set of SNAP PROteolysis TArgeting Chimeras (SNAP-PROTACs), which recruit the VHL or CRBN-ubiquitin E3 ligases to induce the degradation of SNAP-fusion proteins. Endogenous tagging enabled the visualization and the selective depletion of a SNAP-clathrin light chain fusion protein using SNAP-PROTACs. The addition of PROTACs to the SNAP-tag reagent toolbox facilitates the comprehensive analysis of protein function with a single gene tagging event.

cell biology↗

bioRxiv2023

Nanoscale imaging reveals the mechanisms of ER-to-Golgi transport via a dynamic tubular-vesicular network

The endoplasmic reticulum (ER) and the Golgi apparatus are the first sorting stations along the secretory pathway of mammalian cells and have a crucial role in protein quality control and cellular homeostasis. While machinery components mediating ER-to-Golgi transport have been mapped, it is unclear how exchange between the two closely juxtaposed organelles is coordinated in living cells. Here, using gene editing to tag machinery components, live-cell confocal and stimulated emission depletion (STED) super-resolution microscopy, we show that ER-to-Golgi transport occurs via a dynamic network of tubules positive for the small GTPase ARF4. swCOPI machinery is tightly associated to this network and moves with tubular-vesicular structures. Strikingly, the ARF4 network appears to be continuous with the ER and ARF4 tubules remodel around static ER exit sites (ERES) defined by COPII machinery. We were further able to dissect the steps of ER-to-Golgi transport with functional trafficking assays. A wave of cargo released from the ER percolates through peripheral and Golgi-tethered ARF4 structures before filling the cis-Golgi. Perturbation via acute degradation of ARF4 shows an active regulatory role for the GTPase and COPI in anterograde transport. Our data supports a model in which anterograde ER-to-Golgi transport occurs via an ARF4 tubular-vesicular network directly connecting the ER and Golgi-associated pre-cisternae.

cell biology↗

bioRxiv2023

Multi-functional ARF1 compartments serve as a hub for short-range cargo transfer to endosomes

Cellular membrane homeostasis is maintained via a tightly regulated membrane and cargo flow between organelles of the endocytic and secretory pathways. Adaptor protein complexes (APs), which are recruited to membranes by the small GTPase ARF1, facilitate cargo selection and incorporation into trafficking intermediates. According to the classical model, small vesicles would facilitate bi-directional long-range transport between the Golgi, endosomes and plasma membrane. Here we revisit the intracellular organization of the vesicular transport machinery using a combination of CRISPR-Cas9 gene editing, live-cell high temporal (fast-confocal) or spatial (stimulated emission depletion (STED)) microscopy as well as correlative light and electron microscopy. We characterize novel tubulo-vesicular ARF1 compartments that harbor clathrin and different APs. Our findings reveal two functionally different classes of ARF1 compartments, each decorated by a different combination of APs. Perinuclear ARF1 compartments facilitate Golgi export of secretory cargo, while peripheral ARF1 compartments are involved in endocytic recycling downstream of early endosomes. Contrary to the classical model of long-range vesicle shuttling, we observe that ARF1 compartments shed ARF1 and mature into recycling endosomes. This maturation process is impaired in the absence of AP-1 and results in trafficking defects. Collectively, these data highlight a crucial role for ARF1 compartments in post-Golgi sorting.

cell biology↗

bioRxiv2023

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

Phosphoinositides are a small family of phospholipids, acting as signalling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organisation and is most commonly done by over-expression of biosensors. However, this leads to perturbations of phosphoinositide signalling and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of all 8 phosphoinositides using a unifying staining approach for fixed cells and tissue, based on recombinant biosensors with self-labelling SNAP tags. These recombinant biosensors are highly specific, and reliably visualise the subcellular distributions of phosphoinositides across scales, ranging from 2D or 3D cell culture to Drosophila tissue. Using stimulated emission depletion (STED) microscopy, we reveal the nanoscale organisation of PI(3)P on endosomes and PI(4)P on the Golgi and confirm the preservation of subcellular membranes. Multiplex staining enables the investigation of phosphoinositide conversions and reveals an unexpected presence of residual PI(3,5)P2 positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualisation of multiple phosphoinositide species in an unprecedented manner.

cell biology↗

bioRxiv2023

Sub-membrane actin rings compartmentalize the plasma membrane

The compartmentalization of the plasma membrane is a fundamental feature of cells. The diffusivity of membrane proteins in the plane of the membrane is significantly lower in cells than observed in artificial membranes. This seems due to the sub-membranous actin cortex, but tractable assays to prove a direct dependence of membrane protein motion on actin filaments have been elusive. We recently showed that a periodic array of sub-membrane actin filaments that surround neuronal axons in ring-like structures in the axonal initial segment (AIS) confines membrane protein motion between them, but the local enrichment of ion channels may offer an alternative explanation for compartmentalization. Here we show using computational modeling that in contrast to actin rings, the dense array of ion channels in the AIS cannot mediate membrane protein confinement. We go on to show that indeed the actin rings are closely apposed to the plasma membrane, and that they confine membrane protein motion between them in a [~] 200 nm periodic pattern in progenitor-derived neuronal cells. We find that this compartmentalization is also detectable for inner-leaflet membrane proteins and multi-spanning receptors. Strikingly, several cell types with actin rings in their protrusions, like progenitor-derived astrocytes and oligodendrocytes, also exhibit lateral confinement of membrane proteins in a periodic pattern consistent with actin ring spacing. Actin ring-mediated membrane compartmentalization is thus not unique to neurons. Finally, we show that acute actin disruption in live progenitor-derived neuronal cells leads to a loss of membrane compartmentalization in areas that were compartmentalized before treatment. Taken together, we here develop a much-needed tractable experimental system for the investigation of membrane compartmentalization and show that actin rings compartmentalize the plasma membrane of cells.

biophysics↗

bioRxiv2022

Gene editing and super-resolution microscopy reveal multiple distinct roles for ARF GTPases in cellular membrane organization

ADP-ribosylation factor (ARF) GTPases are major regulators of cellular membrane homeostasis. High sequence similarity and multiple, possible redundant functions of the five human ARFs make investigating their function a challenging task. To shed light on the roles of the different Golgi-localized ARF members in membrane trafficking, we generated CRISPR-Cas9 knock ins (KIs) of type I (ARF1 and ARF3) and type II ARFs (ARF4 and ARF5) and mapped their nanoscale localization with stimulated emission depletion (STED) super-resolution microscopy. We find ARF1, ARF4 and ARF5 on segregated nano-domains on the cis-Golgi and ER-Golgi intermediate compartments (ERGIC), revealing distinct roles in COPI recruitment on early secretory membranes. Interestingly, ARF4 and ARF5 define Golgi-tethered ERGIC elements decorated by COPI and devoid of ARF1. Differential localization of ARF1 and ARF4 on distal ERGICs suggests the presence of functionally different classes of intermediate compartments that could regulate bi-directional transport between the ER and the Golgi. Furthermore, ARF1 and ARF3 localize to segregated nano-domains on the trans-Golgi network (TGN) and TGN-derived post-Golgi tubules, strengthening the idea of distinct roles in post-Golgi sorting. This work provides the first map of the nanoscale organization of human ARF GTPases on cellular membranes and sets the stage to dissect their numerous cellular roles.

cell biology↗

bioRxiv2022

A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

The TGF{beta} superfamily of secreted growth factors comprises more than 30 members including TGF{beta}s, BMPs and Activins. While all TGF{beta} superfamily members signal through heteromeric receptor complexes to regulate a plethora of developmental and homeostatic processes, each ligand possesses a unique affinity towards a subset of BMP and TGF{beta} type I and type II receptors. Whereas the Activin and TGF{beta} class display a higher affinity towards type II receptors, BMPs and GDFs preferentially bind to type I receptors. Sofar, the lack of specific antibodies and chemical biology tools hampered simultaneous testing of ligand binding towards all BMP and TGF{beta} receptors. Here we present a N-terminally Halo- and SNAP-tagged TGF{beta}/BMP receptor library to visualize the receptor complexes in dual color. In combination with novel fluorescently labeled TGF{beta} superfamily ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent growth factor binding for Activin A, TGF{beta}1 and BMP9 in a cellular context. We confirm ligand-receptor interface specificity by identifying BMPR2- or ALK2-mutants that switch from a low-affinity Activin A- or BMP9-receptor to a high-affinity receptor, respectively.

cell biology↗

bioRxiv2021

When less is more - Endogenous tagging with TurboID increases the sensitivity of proximity labelling-based experiments

In recent years, proximity labelling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. While physiological expression of the labelling enzyme is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for the rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labelling enzymes when endogenously expressed. Endogenous tagging of the subunit of the AP-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labelling enzyme fusion protein could not reveal. We used the KI-strategy to compare the interactome of the different adaptor protein (AP) complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labelling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month.

cell biology↗

bioRxiv2020

3D Adaptive Optical Nanoscopy for Thick Specimen Imaging at sub-50 nm Resolution

Understanding cellular organization demands the best possible spatial resolution in all three dimensions (3D). In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here, we have developed a nanoscope that, by utilizing an advanced adaptive optics strategy, achieves sub-50 nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue.

biophysics↗