Biology subjects

Campsteijn, C.

Publications and source records attributed to Campsteijn, C..

4 recordsLinked to original sources

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↗

bioRxiv2019

Unrestrained ESCRT-III drives chromosome fragmentation and micronuclear catastrophe

The ESCRT-III membrane fission machinery1,2 restores nuclear envelope integrity during mitotic exit3,4 and interphase5,6. Whereas primary nuclei resealing takes minutes, micronuclear envelope ruptures appear irreversible and result in catastrophic collapse associated with chromosome fragmentation and rearrangements (chromothripsis), thought to be a major driving force in cancer development7-10. Despite its importance11-13, the mechanistic underpinnings of nuclear envelope sealing in primary nuclei and the defects observed in micronuclei remain largely unknown. Here we show that CHMP7, the nucleator of ESCRT-III filaments at the nuclear envelope3,14, and the inner nuclear membrane protein LEMD215 act as a compartmentalization sensor detecting the loss of nuclear integrity. In cells with intact nuclear envelope, CHMP7 is actively excluded from the nucleus to preclude its binding to LEMD2. Nuclear influx of CHMP7 results in stable association with LEMD2 at the inner nuclear membrane that licenses local polymerization of ESCRT-III. Tight control of nuclear CHMP7 levels is critical, as induction of nuclear CHMP7 mutants is sufficient to induce unrestrained growth of ESCRT-III foci at the nuclear envelope, causing dramatic membrane deformation, local DNA torsional stress, single-stranded DNA formation and fragmentation of the underlying chromosomes. At micronuclei, membrane rupture is not associated with repair despite timely recruitment of ESCRT-III. Instead, micronuclei inherently lack the capacity to restrict accumulation of CHMP7 and LEMD2. This drives unrestrained ESCRT-III recruitment, membrane deformation and DNA defects that strikingly resemble those at primary nuclei upon induction of nuclear CHMP7 mutants. Preventing ESCRT-III recruitment suppresses membrane deformation and DNA damage, without restoring nucleocytoplasmic compartmentalization. We propose that the ESCRT-III nuclear integrity surveillance machinery is a double-edged sword, as its exquisite sensitivity ensures rapid repair at primary nuclei while causing unrestrained polymerization at micronuclei, with catastrophic consequences for genome stability16-18.

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↗