Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR

Ligand binding to the EGF receptor (EGFR) triggers multiple signal transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its crosstalk with signaling are poorly understood. Here, we combined peroxidase-catalyzed proximity labeling, isobaric peptide tagging and quantitative mass-spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identified a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We showed that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, was enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes, and demonstrated that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases.

Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer’s disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aβ peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aβ secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.

Nuclear-capture of endosomes depletes nuclear G-actin to promote SRF/MRTF activation and cancer cell invasion

Signals are relayed from receptor tyrosine kinases (RTKs) at the cell surface to effector systems in the cytoplasm and nucleus, and coordination of this process is important for the execution of migratory phenotypes, such as cell scattering and invasion. The endosomal system influences how RTK signalling is coded, but the ways in which it transmits these signals to the nucleus to influence gene expression are not yet clear. Here we show that hepatocyte growth factor, an activator of MET (an RTK), promotes Rab17- and clathrin-dependent endocytosis of EphA2, another RTK, followed by centripetal transport of EphA2-positive endosomes. EphA2 then mediates physical capture of endosomes on the outer surface of the nucleus; a process involving interaction between the nuclear import machinery and a nuclear localisation sequence in EphA2’s cytodomain. Nuclear capture of EphA2 promotes RhoG-dependent phosphorylation of the actin-binding protein, cofilin to oppose nuclear import of G-actin. The resulting depletion of nuclear G-actin drives transcription of Myocardin-related transcription factor (MRTF)/serum-response factor (SRF)-target genes to implement cell scattering and the invasive behaviour of cancer cells.

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural–functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.

Retromer dependent changes in cellular homeostasis and Parkinson’s disease

To date, mechanistic treatments targeting the initial cause of Parkinson’s disease (PD) are limited due to the underlying biological cause(s) been unclear. Endosomes and their associated cellular homeostasis processes have emerged to have a significant role in the pathophysiology associated with PD. Several variants within retromer complex have been identified and characterised within familial PD patients. The retromer complex represents a key sorting platform within the endosomal system that regulates cargo sorting that maintains cellular homeostasis. In this review, we summarise the current understandings of how PD-associated retromer variants disrupt cellular trafficking and how the retromer complex can interact with other PD-associated genes to contribute to the disease progression.

Loss of PIKfyve Causes Transdifferentiation of Dictyostelium Spores Into Basal Disc Cells

The 1-phosphatidylinositol-3-phosphate 5-kinase PIKfyve generates PtdIns3,5P2 on late phagolysosomes, which by recruiting the scission protein Atg18, results in their fragmentation in the normal course of endosome processing. Loss of PIKfyve function causes cellular hypervacuolization in eukaryotes and organ failure in humans. We identified pikfyve as the defective gene in a Dictyostelium mutant that failed to form spores. The amoebas normally differentiated into prespore cells and initiated spore coat protein synthesis in Golgi-derived prespore vesicles. However, instead of exocytosing, the prespore vesicles fused into the single vacuole that typifies the stalk and basal disc cells that support the spores. This process was accompanied by stalk wall biosynthesis, loss of spore gene expression and overexpression of ecmB, a basal disc and stalk-specific gene, but not of the stalk-specific genes DDB_G0278745 and DDB_G0277757. Transdifferentiation of prespore into stalk-like cells was previously observed in mutants that lack early autophagy genes, like atg5, atg7, and atg9. However, while autophagy mutants specifically lacked cAMP induction of prespore gene expression, pikfyve− showed normal early autophagy and prespore induction, but increased in vitro induction of ecmB. Combined, the data suggest that the Dictyostelium endosomal system influences cell fate by acting on cell type specific gene expression.

The Golgi-associated retrograde protein (GARP) complex plays an essential role in the maintenance of the Golgi glycosylation machinery

The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans, and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-KO cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text]

The role of the lipid kinase PIKfyve in the endosomal and phagosomal systems in macrophages

Macrophages engulf pathogens into phagosomes for degradation through a process known as phagocytosis. Nascent phagosomes progressively mature and fuse with early and late endosomes and lysosomes to form phagolysosomes, where pathogens are degraded by hydrolytic enzymes. Phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] are signaling lipids that recruit a unique set of effector proteins involved in distinct stages of membrane traffic to govern the function of early and late endosomes, respectively. Phagosome maturation requires the transient expression of PtdIns(3)P on early phagosomal membranes. Subsequently, PtdIns(3)P can be converted to PtdIns(3,5)P2 by the lipid kinase PIKfyve. Thus, it remains unclear if the role of PtdIns(3)P in phagosome maturation is direct and/or indirect, through the synthesis of PtdIns(3,5)P2. The role of PtdIns(3,5)P2 in the endosomal system in macrophages also requires further investigation. My thesis employs a pharmacological approach to address the role of PIKfyve in macrophage biology. In general, PtdIns(3,5)P2 appears to principally coordinate the later stages of endosome and phagosome maturation.

The role of the lipid kinase PIKfyve in the endosomal and phagosomal systems in macrophages

Macrophages engulf pathogens into phagosomes for degradation through a process known as phagocytosis. Nascent phagosomes progressively mature and fuse with early and late endosomes and lysosomes to form phagolysosomes, where pathogens are degraded by hydrolytic enzymes. Phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] are signaling lipids that recruit a unique set of effector proteins involved in distinct stages of membrane traffic to govern the function of early and late endosomes, respectively. Phagosome maturation requires the transient expression of PtdIns(3)P on early phagosomal membranes. Subsequently, PtdIns(3)P can be converted to PtdIns(3,5)P2 by the lipid kinase PIKfyve. Thus, it remains unclear if the role of PtdIns(3)P in phagosome maturation is direct and/or indirect, through the synthesis of PtdIns(3,5)P2. The role of PtdIns(3,5)P2 in the endosomal system in macrophages also requires further investigation. My thesis employs a pharmacological approach to address the role of PIKfyve in macrophage biology. In general, PtdIns(3,5)P2 appears to principally coordinate the later stages of endosome and phagosome maturation.

Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) can infer molecular, functional and dynamic information to ultrastructure by linking information of different imaging modalities. One of the main challenges, especially in 3D-CLEM, is the accurate registration of fluorescent signals to electron microscopy (EM). Here, we present fluorescent BSA-gold (fBSA-Au), a bimodal endocytic tracer as fiducial marker for 2D and 3D CLEM applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent bovine serum albumin (BSA). The conjugate is efficiently endocytosed and distributed throughout the 3D endo-lysosomal network of the cells, and has an excellent visibility both in fluorescence microscopy (FM) and EM. We demonstrate the use of fBSA-Au in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryo-EM. As a fiducial marker, fBSA-Au facilitates rapid registration of regions of interest between FM and EM modalities and enables accurate (50-150 nm) correlation of fluorescence to EM data. Endocytosed fBSA-Au benefits from a homogenous 3D distribution throughout the endosomal system within the cell, and does not obscure any cellular ultrastructure. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo-)CLEM applications.