Transport of Lysosome-Related Organelles

Many intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is largely unclear how these motor proteins are targeted to specific compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments and therefore are ideal candidates for controlling motor protein recruitment. This work shows that dynein-dynactin motor recruitment to lysosomal compartments requires activation of the GTPase Rab7 that subsequently associates with its effector protein, RILP (for Rab7-Interacting Lysosomal Protein). RILP maintains Rab7 in the vesicle-bound, activated state and transmits a signal for specific recruitment of the dynein-dynactin motor. As a consequence, lysosomes are transported towards the minus-end of microtubules. This signalling cascade thus regulates lysosomal transport. In addition, we showed that this pathway also regulates transport of several other lysosomal compartments, including Salmonella-containing vacuoles and melanosomes.The work descripted in this thesis was performed at the Division of Tumor Biology of the Netherlands Cancer Institute, Amsterdam, The Netherlands and sponsored by the Koningin Wilhelmina Fonds and the Netherlands Organisation for Scientific Research NWO. KWF; NWOUBL - phd migration 201

Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex

AbstractBackground: The Rho GTPases Rho, Rac, and Cdc42 regulate the organization of the actin cytoskeleton by interacting with multiple, distinct downstream effector proteins. Cdc42 controls the formation of actin bundle-containing filopodia at the cellular periphery. The molecular mechanism for this remains as yet unclear.Results: We report here that Cdc42 interacts with IRSp53/BAP2α, an SH3 domain-containing scaffold protein, at a partial CRIB motif and that an N-terminal fragment of IRSp53 binds, via an intramolecular interaction, to the CRIB motif-containing central region. Overexpression of IRSp53 in fibroblasts leads to the formation of filopodia, and both this and Cdc42-induced filopodia are inhibited by expression of the N-terminal IRSp53 fragment. Using affinity chromatography, we have identified Mena, an Ena/VASP family member, as interacting with the SH3 domain of IRSp53. Mena and IRSp53 act synergistically to promote filopodia formation.Conclusion: We conclude that the interaction of Cdc42 with the partial CRIB motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. This IRSp53:Mena complex initiates actin filament assembly into filopodia

Activation of endosomal dynein motors by stepwise assembly of Rab7–RILP–p150Glued, ORP1L, and the receptor βlll spectrin

The small GTPase Rab7 controls late endocytic transport by the minus end–directed motor protein complex dynein–dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP–Rab7–ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150Glued, which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150Glued recruitment by Rab7–RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as βIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150Glued dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7–RILP is transferred by ORP1L to βIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with βIII spectrin, which requires the activities of Rab7–RILP and ORP1L

Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt signaling by Daam1/2

The mammalian intestine is one of the most rapidly self-renewing tissues, driven by stem cells residing at the crypt bottom. Paneth cells form a major element of the niche microenvironment providing various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt3. Different Wnt ligands can selectively activate β-catenin-dependent (canonical) or -independent (noncanonical) signaling. Here, we report that the Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and noncanonical Wnt (Wnt/PCP) signaling. Daam1/2 interacts with the Wnt inhibitor RNF43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing RNF43-dependent degradation of the Wnt receptor, Frizzled (Fzd). Single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion because of defective Wnt/PCP signaling. Together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and noncanonical Wnt, which is fundamental for specifying an adequate number of Paneth cells

E3 ligases RNF43 and ZNRF3 display differential specificity for endocytosis of Frizzled receptors

The transmembrane E3 ligases RNF43 and ZNRF3 perform key tumour suppressor roles by inducing endocytosis of members of the Frizzled (FZD) family, the primary receptors for WNT. Loss-of-function mutations in RNF43 and ZNRF3 mediate FZD stabilisation and a WNT-hypersensitive growth state in various cancer types. Strikingly, RNF43 and ZNRF3 mutations are differentially distributed across cancer types, raising questions about their functional redundancy. Here, we compare the efficacy of RNF43 and ZNRF3 of targeting different FZDs for endocytosis. We find that RNF43 preferentially down-regulates FZD1/FZD5/FZD7, whereas ZNRF3 displays a preference towards FZD6. We show that the RNF43 transmembrane domain (TMD) is a key molecular determinant for inducing FZD5 endocytosis. Furthermore, a TMD swap between RNF43 and ZNRF3 re-directs their preference for FZD5 down-regulation. We conclude that RNF43 and ZNRF3 preferentially down-regulate specific FZDs, in part by a TMD-dependent mechanism. In accordance, tissue-specific expression patterns of FZD homologues correlate with the incidence of RNF43 or ZNRF3 cancer mutations in those tissues. Consequently, our data point to druggable vulnerabilities of specific FZD receptors in RNF43- or ZNRF3-mutant human cancers

RSPO3 Furin domain-conjugated liposomes for selective drug delivery to LGR5-high cells

The transmembrane receptor LGR5 potentiates Wnt/β-catenin signaling by binding both secreted R-spondin (RSPOs) and the Wnt tumor suppressors RNF43/ZNRF3, directing clearance of RNF43/ZNRF3 from the cell surface. Besides being widely used as a stem cell marker in various tissues, LGR5 is overexpressed in many types of malignancies, including colorectal cancer. Its expression characterizes a subpopulation of cancer cells that play a crucial role in tumor initiation, progression and cancer relapse, known as cancer stem cells (CSCs). For this reason, ongoing efforts are aimed at eradicating LGR5-positive CSCs. Here, we engineered liposomes decorated with different RSPO proteins to specifically detect and target LGR5-positive cells. Using fluorescence-loaded liposomes, we show that conjugation of full-length RSPO1 to the liposomal surface mediates aspecific, LGR5-independent cellular uptake, largely mediated by heparan sulfate proteoglycan binding. By contrast, liposomes decorated only with the Furin (FuFu) domains of RSPO3 are taken up by cells in a highly specific, LGR5-dependent manner. Moreover, encapsulating doxorubicin in FuFuRSPO3 liposomes allowed us to selectively inhibit the growth of LGR5-high cells. Thus, FuFuRSPO3-coated liposomes allow for the selective detection and ablation of LGR5-high cells, providing a potential drug delivery system for LGR5-targeted anti-cancer strategies

RSPO3 Furin domain-conjugated liposomes for selective drug delivery to LGR5-high cells

The transmembrane receptor LGR5 potentiates Wnt/β-catenin signaling by binding both secreted R-spondin (RSPOs) and the Wnt tumor suppressors RNF43/ZNRF3, directing clearance of RNF43/ZNRF3 from the cell surface. Besides being widely used as a stem cell marker in various tissues, LGR5 is overexpressed in many types of malignancies, including colorectal cancer. Its expression characterizes a subpopulation of cancer cells that play a crucial role in tumor initiation, progression and cancer relapse, known as cancer stem cells (CSCs). For this reason, ongoing efforts are aimed at eradicating LGR5-positive CSCs. Here, we engineered liposomes decorated with different RSPO proteins to specifically detect and target LGR5-positive cells. Using fluorescence-loaded liposomes, we show that conjugation of full-length RSPO1 to the liposomal surface mediates aspecific, LGR5-independent cellular uptake, largely mediated by heparan sulfate proteoglycan binding. By contrast, liposomes decorated only with the Furin (FuFu) domains of RSPO3 are taken up by cells in a highly specific, LGR5-dependent manner. Moreover, encapsulating doxorubicin in FuFuRSPO3 liposomes allowed us to selectively inhibit the growth of LGR5-high cells. Thus, FuFuRSPO3-coated liposomes allow for the selective detection and ablation of LGR5-high cells, providing a potential drug delivery system for LGR5-targeted anti-cancer strategies

Generation of Active Protein Phosphatase 2A Is Coupled to Holoenzyme Assembly

Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Δ strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit

RNF43 mutations facilitate colorectal cancer metastasis via formation of a tumour-intrinsic niche

In colorectal cancer (CRC), RNF43 mutations are linked to BRAF V600E-initiated serrated adenomas that advance into mucinous adenocarcinomas with poor prognosis upon metastasis. How RNF43 mutations facilitate a metastasis-prone growth state remains unknown. Here, we addressed this issue by repairing mutant RNF43 in patient-derived BRAF-mutant CRC organoids using gene editing. Upon RNF43 correction, CRC organoids exhibit strongly decreased mucus production and, moreover, display loss of niche factor independence and metastatic capacity upon orthotopic transplantation in mice. Mechanistically, we show that mutant RNF43 promotes cancer cell lineage specification towards a non-dividing niche population that secretes essential growth factors, providing a state of self-sufficiency to the cancer epithelium. We show that phenotypic diversification into tumour-intrinsic niche cells (TINCs) and proliferative cancer stem cells depends on tuneable WNT levels enabled by mutations in RNF43, but not APC. In patient samples, enhanced TINC profiles correlate with RNF43-mutant CRC, mucinous histology and metastatic disease, thus representing a general cellular mechanism by which tumours acquire a self-sufficient, pro-metastatic growth state

Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt signaling by Daam1/2

The mammalian intestine is one of the most rapidly self-renewing tissues, driven by stem cells residing at the crypt bottom. Paneth cells form a major element of the niche microenvironment providing various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt3. Different Wnt ligands can selectively activate β-catenin-dependent (canonical) or -independent (noncanonical) signaling. Here, we report that the Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and noncanonical Wnt (Wnt/PCP) signaling. Daam1/2 interacts with the Wnt inhibitor RNF43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing RNF43-dependent degradation of the Wnt receptor, Frizzled (Fzd). Single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion because of defective Wnt/PCP signaling. Together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and noncanonical Wnt, which is fundamental for specifying an adequate number of Paneth cells