Biology subjects

Cavarischia-Rega, C.

Publications and source records attributed to Cavarischia-Rega, C..

5 recordsLinked to original sources

bioRxiv2026

Characterization of Vlf1 as a regulator of lipophagy.

Lipid droplets (LDs) are unique organelles, surrounded by a phospholipid monolayer. They are present in most eukaryotic cells including the unicellular model organism S. cerevisiae. LDs store neutral lipids which serve as precursors for amphipathic membrane lipids and as an energy reserve. Loss of LDs in S. cerevisiae results in multiple cellular defects impairing lipid homeostasis and the biogenesis and function of other organelles. Here, we find that the expression levels of many proteins in isolated mitochondrial fractions are altered in cells that cannot synthesize neutral lipids and therefore lack LDs. In addition, among several downregulated proteins, we identified the previously uncharacterized Ylr001c (which we name Vlf1 for Vacuolar Lipophagy Factor 1). We show that Vlf1 is glycosylated and, in contrast to some previous reports, is actually localized to the vacuole. Furthermore, we demonstrate that changes in Vlf1 expression alter growth sensitivity to rapamycin, and detected a physical interaction of Vlf1 with Atg15, a lipase involved in autophagy. Additionally, we observe higher levels of autophagy/lipophagy in the absence of Vlf1 and a reduction upon overexpression of the protein. Taken together, the effects on lipohagy by Vlf1 makes it, according to our knowledge, the first vacuolar lipophagy regulator identified in S. cerevisiae.

cell biology↗

bioRxiv2026

Proximity Labelling Reveals Spatial Organisation of Mitochondrial Protein Import Complexes

Mitochondrial protein import and local translation at the mitochondrial outer membrane (MOM) require coordinated interactions between protein translocases and RNA-associated factors, yet the molecular organisation of these interactions remains largely unresolved. Here, we used APEX2-mediated proximity labelling to define the proximal proteomes associated with the cytosolic face of the TOM and SAM complexes by fusing APEX2 to TOMM22 and MTX2, respectively. Quantitative mass spectrometry identified known TOM/SAM-associated proteins together with multiple RNA-binding proteins (RBPs), supporting the emerging role of the MOM in localised translation and other RNA-related processes. Among identified RBPs, the exonuclease EXD2 was consistently enriched in APEX2-TOMM22 and MTX2-APEX2 datasets and remained associated upon puromycin treatment, indicating a translation-independent interaction. Together, our findings provide insights into the molecular organisation of mitochondrial import sites and identify EXD2 as a TOM/SAM-associated factor at the MOM.

biochemistry↗

bioRxiv2026

Spheroid culture remodels mitosis and the proteome in tumor cells

Mitosis depends on precise spindle assembly and positioning, processes influenced by cell shape, size, and microenvironment. Most mechanistic insights into mitosis come from two-dimensional (2D) monolayer cultures, which lack the spatial constraints and extracellular matrix found in tissues, leaving the influence of the tissue environment on mitosis poorly understood. Here, we combine high-resolution imaging and quantitative proteomics to compare mitosis in three-dimensional (3D) multicellular spheroids, generated by magnetic levitation, with that in 2D monolayers. Using a non-transformed cell line and three cancer cell lines from breast, bone, and ovary, we show that 3D culture reshapes mitotic cells and their spindles. Tumor spheroids exhibited a prometaphase delay together with minor chromosome alignment defects, yet chromosome segregation remained largely accurate. Cells in spheroids were rounder, and their spindles were smaller, with increased multipolarity and defects in orientation and position, which varied by cell line. Proteomic profiling revealed broad downregulation of mitotic regulators in spheroids, including kinesins (KIF11, KIF4A), spindle checkpoint proteins, and APC/C components, accompanied by enrichment of metabolic and mitochondrial pathways. Together, our results reveal both shared and cell line-specific modes of mitotic restructuring and establish a framework that connects proteome state to mitotic architecture in 3D environments.

cell biology↗

bioRxiv2025

PINK1 regulates cholesterol homeostasis via SCAP phosphorylation in human dopaminergic neurons

Cholesterol is a key lipid enriched in neuronal membranes and essential for signaling and synaptic transmission. An imbalance in cholesterol levels may affect synaptic plasticity and contribute to neurodegeneration. Here, we identify in human dopaminergic neurons a mechanism linking loss of function of the Parkinsons disease (PD) gene PINK1 to altered cholesterol homeostasis. Loss of functional PINK1 impaired SCAP phosphorylation at Ser822 and Ser838, stabilizing SCAP and driving excess cholesterol biosynthesis. Cholesterol accumulated at the plasma membrane and in flotillin-rich lipid rafts, causing reduced neurotransmitter uptake and altering the distribution of dopamine transporter (DAT). Restoring PINK1 expression normalized cholesterol biosynthesis and levels. Moreover, the cholesterol-lowering drugs simvastatin and {beta}-cyclodextrin rescued DAT distribution and neurotransmitter uptake defects. These findings demonstrate that PINK1 influences cholesterol homeostasis through SCAP phosphorylation at Ser822 and Ser838 and that restoring cholesterol levels mitigates phenotypes observed in PINK1 PD neurons. These findings further highlight the cross-talk between mitochondria and lipid homeostasis in PD models, underscoring the relevance of cholesterol levels to dopaminergic functions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/672574v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@165b4d4org.highwire.dtl.DTLVardef@42e3a2org.highwire.dtl.DTLVardef@16ea6f7org.highwire.dtl.DTLVardef@193e170_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

neuroscience↗

bioRxiv2023

Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1

In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 has been linked to neurodegenerative diseases including Parkinsons as well as cardiovascular diseases and cancer. Here, we developed nanobodies (Nbs) for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs as capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a "turnover-accelerated" format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.

cell biology↗