Biology subjects

Schink, K. O.

Publications and source records attributed to Schink, K. O..

11 recordsLinked to original sources

bioRxiv2026

Structural basis for the ATP-dependence of omegasome biogenesis by DFCP1

Autophagosome formation begins at phosphatidylinositol 3-phosphate-enriched endoplasmic reticulum (ER) subdomains termed omegasomes. DFCP1/ZFYVE1 is recruited to omegasomes through its FYVE domains and has recently been shown to function as an ATPase involved in omegasome constriction and autophagosome biogenesis. However, the structural basis of DFCP1 ATPase activity and how nucleotide-dependent conformational states regulate omegasome dynamics remain unknown. Here, we determined the crystal structures of the DFCP1 ATPase domain in complex with either ADP or the non-hydrolyzable ATP analogue, AppNHp at near atomic resolution. We employed structure-guided mutagenesis to define residues required for DFCP1 function in cells. The structures reveal that the active site contains a trans-acting Arg271 finger coordinating the {psi}-phosphate and trans-acting His323 that stacks on the ATP adenine ring. ATP hydrolysis results in a conformational switch in the vicinity of Arg271, while contacts with His323 persist in the presence of ADP. Biochemical analyses of DFCP1 and its mutants show that DFCP1 forms ATP-dependent microdomains on membranes. Disruption of His323 and Arg271 uncouples nucleotide binding from ATP hydrolysis and alters the oligomeric state of the protein. Live-cell imaging of DFCP1 knockout cells reconstituted with wild-type or mutant DFCP1 further demonstrates that these biochemical defects translate into distinct omegasome phenotypes. Together, our data provides a structural-function framework for DFCP1 ATPase activity and reveals how distinct catalytic elements control omegasome dynamics in vivo. We propose that nucleotide-dependent assembly and hydrolysis-driven conformational changes enable DFCP1 to regulate omegasome formation and its progression toward autophagosome closure. Significance StatementDFCP1 organizes the ER subdomains known as omegasomes, which are sites of autophagosome biogenesis. DFCP1 is an ATPase whose catalytic activity is required for function, but the precise role of its ATPase activity is unknown. Crystal structures of the DFCP1 ATPase domain in the presence of a non-hydrolyzable ATP analogue and ADP reveal an Arg finger that operates in trans, such that dimerization is required for ATP hydrolysis, and ATP hydrolysis destabilizes dimers. Dimerization is also promoted by an adenine base-stacking interaction in trans with H323. These residues are important for DFCP1 clustering on membranes and omegasome constriction, clarifying that the role of ATP is to promote the dimerization of the ATPase domain and thereby control the organization of DFCP1 on membranes.

biochemistry↗

bioRxiv2022

ATPase activity of DFCP1 controls selective autophagy

Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 had a minor effect on bulk autophagic flux, DFCP1 was required to maintain the autophagic flux of p62 under both fed and starved conditions, and this was dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localized to forming omegasomes, these omegasomes failed to constrict. Consequently, the release of nascent autophagosomes from omegasomes was markedly delayed. DFCP1 was found to associate with ubiquitinated proteins, and degradation of ubiquitinated cargoes such as protein aggregates, mitochondria and micronuclei was strongly inhibited when DFCP1 was knocked out or mutated. Thus, DFCP1 mediates ATPase-driven constriction of omegasomes to release autophagosomes for selective autophagy.

cell biology↗

bioRxiv2021

A phosphoinositide and RAB switch controls early macropinocytosis

Macropinocytosis is a non-selective endocytic process by which cells take up large amounts of extracellular fluids into giant vesicles known as macropinosomes. This mechanism is used by immune cells to sample the surroundings for antigens and can be exploited by cancer cells for nutrient uptake. What determines the fate of macropinosomes after they have been internalized is largely unknown. Here we investigate the role of the phosphatidylinositol 3-kinase VPS34/PIK3C3 and its product phosphatidylinositol 3-phosphate (PtdIns3P) in macropinosome fate determination. Inhibition of VPS34 led to a decrease in macropinosome survival and fluid phase uptake as well as preventing recruitment of early endosomal factors, including the small GTPase RAB5 and its effectors, to the forming macropinosomes. Instead, forming macropinosomes under VPS34 inhibition accumulated regulators of endocytic recycling, including RAB8A, RAB10, RAB11A, and PtdIns4P, which led to fusion of macropinosomes with the plasma membrane. Whereas RAB5 was critical for macropinosome formation, macropinosome fusion with the plasma membrane depended on RAB8A. Thus, macropinosome maturation is regulated by a PtdIns3P-controlled switch that balances macropinosome fate between the default, endolysosomal maturation and an alternative, secretory route.

cell biology↗

bioRxiv2020

JIP4 is recruited by the phosphoinositide-binding protein Phafin2 to promote recycling tubules on macropinosomes

Macropinocytosis allows cells to take up extracellular material in a non-selective manner. The molecular mechanisms that mediate recycling of membranes and transmembrane proteins from macropinosomes still need to be defined. Here we report that JIP4, a coiled-coil containing protein previously described to bind to microtubule motors, is recruited to retromer- and actin-containing tubulating subdomains on macropinosomes by binding to the PH domain of the phosphatidylinositol 3-phosphate (PtdIns3P)-binding protein Phafin2. This recruitment is not shared by the closely related isoforms JIP3 and Phafin1. Disruption of Phafin2 or PtdIns3P impairs JIP4 recruitment to macropinosomes whereas forced localization of Phafin2 to mitochondria causes mitochondrial targeting of JIP4. While knockout of JIP4 suppresses tubulation, overexpression enhances tubulation from macropinosomes. JIP4 knockout cells display increased retention of macropinocytic cargo in both early and late macropinosomes, consistent with a recycling defect. Collectively, these data identify JIP4 and Phafin2 as components of a tubular recycling pathway that operates from macropinosomes.

cell biology↗

bioRxiv2020

Nanoscopy on the Chea(i)p

Super-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.

microbiology↗

bioRxiv2020

GAS6-AXL signaling triggers actin remodeling and macropinocytosis that drive cancer cell invasion

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand GAS6 are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion (FA) turnover, cell spreading and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL but not TYRO3, and downstream activation of PI3K. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements and macropinocytosis that jointly contribute to cancer cell invasion.

cell biology↗

bioRxiv2018

Critical role for Piccolo in Synaptic Vesicle Retrieval

Loss of function of the presynaptic active zone protein Piccolo has recently been linked to a devastating disease causing brain atrophy. Here, we address how Piccolo inactivation adversely affects synaptic function and thus may contributes to neuronal loss. Our analysis shows that Piccolo is critical for the activity dependent recycling and maintenance of synaptic vesicles (SVs). Specifically, we find that boutons lacking Piccolo have deficits in the Rab5/EEA1 dependent formation of early endosomes and thus the recycling of SVs. Mechanistically, impaired Rab5 function was caused by the reduced synaptic recruitment of Pra1, known to interact selectively with the zinc fingers of Piccolo. Importantly, over-expression of GTPase deficient Rab5 or the Znf1 domain of Piccolo restores the size and recycling of SV pools. These data provide a molecular link between the active zone and endosome sorting at synapses providing hints to how Piccolo contributes to both developmental and psychiatric disorders.\n\nImpact StatementThe efficient recycling of synaptic vesicle proteins is critical for the integrity and reliability of synaptic transmission. Increasingly genetic and environmental insults have been shown to affect this recycling pathway, resulting in both cognitive impairment in humans and neurodegenerative diseases, yet the underlying mechanisms are poorly understood. Here we could show that the presynaptic active zone protein Piccolo regulates efficient recycling of synaptic vesicles via Pra1 and Rab5, perhaps explaining why Piccolo loss of function contributes to Pontocerebellar Hypoplasia and major depressive disorders.

neuroscience↗

bioRxiv2018

ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival

Although lysosomes perform a number of essential cellular functions, damaged lysosomes represent a potential hazard to the cell. Such lysosomes are therefore engulfed by autophagic membranes in the process known as lysophagy, which is initiated by recognition of luminal glycoprotein domains by cytosolic lectins such as Galectin-3. Here we show that, under various conditions that cause injury to the lysosome membrane, components of the endosomal sorting complex required for transport (ESCRT) machinery are recruited. This recruitment occurs before that of Galectin-3 and the lysophagy machinery. Subunits of the ESCRT-III complex show a particularly prominent recruitment, which depends on the ESCRT-I component TSG101 and the TSG101- and ESCRT-III-binding protein ALIX. Interference with ESCRT recruitment abolishes lysosome repair and causes otherwise reversible lysosome damage to become cell lethal. Vacuoles containing the intracellular pathogen Coxiella burnetii show reversible ESCRT recruitment, and interference with this recruitment reduces intravacuolar bacterial replication. We conclude that the cell is equipped with an endogenous mechanism for lysosome repair which protects against lysosomal damage-induced cell death but which also provides a potential advantage for intracellular pathogens.

cell biology↗

bioRxiv2018

ERM proteins: The missing actin linkers in clathrin-mediated endocytosis

Canonical clathrin-coated pits (CCPs) are nucleated by the coordinated arrival of clathrin triskelia and AP2 adaptor proteins at phosphatidylinositol 4,5-bisphosphate (PIP2) enriched plasma membrane domains (1, 2). Subsequent propagation of the clathrin lattice helps to deform the membrane into a sharply curved pit (3). A large proportion of the initiated pits fall apart as abortive endocytic events within about 20 s, possibly due to insufficient cargo capture (4). Successful clathrin-mediated endocytosis (CME) is concluded when a clathrin-coated vesicle is released into the cell by dynamin-mediated fission of the CCP membrane neck (5, 6). A vast array of accessory proteins important for successful CME has been identified. Among these is actin, which has been shown to be required for the maturation and internalization of a subset of CCPs in human cells (7, 8). Actin dependency during CME correlates with elevated membrane tension, and CCP maturation requires actin polymerization during mitosis, in microvilli at the apical surface of polarized cells and in cells upon global mechanical stretching or hypotonic treatment (9-11). The Arp2/3 complex is thought to trigger an acute actin burst coinciding with CCP internalization, but how actin is recruited to CCPs in the first place is not known (12, 13). Here we show that the ERM (ezrin, radixin, moesin) protein family of membrane-actin linkers associates with CCPs, and that functional perturbation of ERM proteins impedes CCP maturation and reduces the rate of transferrin uptake. By total internal reflection fluorescence (TIRF) microscopy and unbiased object detection and tracking, we show that ezrin localizes to nascent CCPs and that these pits subsequently recruit actin. Based on these data, we propose a model in which activated ERM proteins recruit the initial actin filament during CME.

cell biology↗

bioRxiv2018

WDFY2 restrains matrix metalloprotease secretion and cell invasion by retention of VAMP3 in endosomal tubules

The endosomal FYVE- and WD40-domain-containing protein WDFY2 has been assigned a function as tumour suppressor, but its functional mechanism has remained elusive. Here we have used confocal, widefield and super-resolution fluorescence microscopy to show that WDFY2 localizes to the base of retromer-containing endosomal tubules by a mechanism that involves recognition of a specific pool of phosphatidylinositol 3-phosphate (PtdIns3P) by the WDFY2 FYVE domain. Affinity purification and mass spectrometry identified the v-SNARE VAMP3 as an interaction partner of WDFY2, and cellular knockout of WDFY2 caused a strong redistribution of VAMP3 into small vesicles near the plasma membrane. This was accompanied by increased secretion of the matrix metalloprotease MT1-MMP, enhanced degradation of extracellular matrix, and increased cell invasion. WDFY2 is frequently lost in metastatic cancers, most predominantly in ovarian and prostate cancer. We propose that WDFY2 acts as a tumor suppressor by serving as a gatekeeper for VAMP3 recycling.

cell biology↗

bioRxiv2017

The PtdIns3P-binding protein Phafin2 escorts macropinosomes through the cortical actin cytoskeleton

Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis plays important roles in infection, immunity and cancer development. A key question is how large macropinosomes are able to squeeze through the dense actin network underlying the plasma membrane in order to move towards the cell centre for maturation. Here we show that, immediately after macropinosomes have been sealed off from the plasma membrane, the PH-and FYVE domain-containing protein Phafin2 is recruited by a mechanism that involves binding to phosphatidylinositol 3-phosphate (PtdIns3P) generated in a non-canonical manner. Phafin2 in turn regulates the actin cross-linking protein Filamin A to promote entry of macropinosomes through the subcortical actin matrix and subsequent maturation. Depletion of Phafin2 inhibits macropinocytic internalization and maturation. We conclude that PtdIns3P and its effector Phafin2 are key components of a system that allows nascent macropinosomes to navigate through the dense subcortical actin network.

cell biology↗