ABSTRACT
His Domain Protein Tyrosine Phosphatase (HD-PTP) facilitates function of the endosomal sorting complexes required for transport (ESCRTs) during multivesicular body (MVB) formation. To uncover its role in physiological homeostasis, embryonic lethality caused by a complete lack of HD-PTP was bypassed through generation of hypomorphic mice expressing reduced protein, resulting in animals that are viable into adulthood. These mice exhibited marked lipodystrophy and decreased receptor-mediated signaling within white adipose tissue (WAT), involving multiple prominent pathways including RAS/MAPK, PI3K/AKT and RTKs such as EGFR. EGFR signaling was dissected in vitro to assess the nature of defective signaling, revealing decreased trans-autophosphorylation and downstream effector activation, despite normal EGF binding. This corresponds to decreased plasma membrane cholesterol and increased lysosomal cholesterol, likely resulting from defective endosomal maturation necessary for cholesterol trafficking and homeostasis. ESCRT components Vps4 and HRS have previously been implicated in cholesterol homeostasis, thus these findings expand knowledge on which ESCRT subunits are involved in cholesterol homeostasis and highlight a non-canonical role for HD-PTP in signal regulation and adipose tissue homeostasis.
ABSTRACT
Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)(2) tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)(2). We show that the Rtt106-(H3-H4)(2) interaction is important for gene silencing and the DNA damage response.
Subject(s)
Histones/chemistry , Histones/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Acetylation , Animals , Binding Sites , Crystallography, X-Ray , DNA Damage , Gene Silencing , Genomic Instability , Lysine/analogs & derivatives , Lysine/chemistry , Lysine/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Chaperones/genetics , Mutation/genetics , Pliability , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/genetics , Structure-Activity Relationship , Substrate Specificity , Xenopus laevisABSTRACT
Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal sorting complex required for transport III (ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a microtubule-interacting and trafficking domain-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction, in concert with a more open conformation of the Ist1 core, resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.
Subject(s)
Adenosine Triphosphatases/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/genetics , Mutagenesis, Site-Directed , Protein Conformation , Protein Folding , Protein Transport , Saccharomyces cerevisiae Proteins/chemistry , Vesicular Transport Proteins/chemistryABSTRACT
The endosomal sorting complexes required for transport (ESCRTs) function in a variety of membrane remodeling processes including multivesicular body sorting, abscission during cytokinesis, budding of enveloped viruses, and repair of the plasma membrane. Vps4 ATPase activity modulates ESCRT function and is itself modulated by its cofactor Vta1 and its substrate ESCRT-III. The carboxyl-terminal Vta1/SBP-1/Lip5 (VSL) domain of Vta1 binds to the Vps4 ß-domain to promote Vps4 oligomerization-dependent ATP hydrolysis. Additionally, the Vps4 stimulatory element (VSE) of Vta1 contributes to enhancing Vps4 oligomer ATP hydrolysis. The VSE is also required for Vta1-dependent stimulation of Vps4 by ESCRT-III subunits. However, the manner by which the Vta1 VSE contributes to Vps4 activation is unknown. Existing structural data were used to generate a model of the Vta1 VSE in complex with Vps4. This model implicated residues within the small ATPase associated with various activities (AAA) domain, specifically α-helices 7 and 9, as relevant contact sites. Rational generation of Vps4 mutants defective for VSE-mediated stimulation, as well as intergenic compensatory mutations, support the validity of this model. These findings have uncovered the Vps4 surface responsible for coordinating ESCRT-III-stimulated Vta1 input during ESCRT function and identified a novel mechanism of Vps4 stimulation.
Subject(s)
Adenosine Triphosphate/metabolism , Coenzymes/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Protein Subunits/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , ATPases Associated with Diverse Cellular Activities , Amino Acid Sequence , Animals , Coenzymes/chemistry , Coenzymes/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/genetics , Gene Expression Regulation , Humans , Hydrolysis , Kinetics , Mice , Models, Molecular , Molecular Sequence Data , Mutation , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Vacuolar Proton-Translocating ATPases/chemistry , Vacuolar Proton-Translocating ATPases/geneticsABSTRACT
The AAA-type ATPase Vps4 functions with components of the ESCRT (endosomal sorting complex required for transport) machinery in membrane fission events that are essential for endosomal maturation, cytokinesis, and the formation of retroviruses. A key step in these events is the assembly of monomeric Vps4 into the active ATPase complex, which is aided in part by binding of Vps4 via its N-terminal MIT (microtubule interacting and trafficking) domain to its substrate ESCRT-III. We found that the 40-amino acid linker region between the MIT and the ATPase domain of Vps4 is not required for proper function but plays a role in regulating Vps4 assembly and ATPase activity. Deletion of the linker is expected to bring the MIT domains into close proximity to the central pore of the Vps4 complex. We propose that this localization of the MIT domain in linker-deleted Vps4 mimics a repositioning of the MIT domain normally caused by binding of Vps4 to ESCRT-III. This structure would allow the Vps4 complex to engage ESCRT-III subunits with both the pore and the MIT domain simultaneously, which might be essential for the ATP-driven disassembly of ESCRT-III.
Subject(s)
Adenosine Triphosphatases/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Gene Expression Regulation, Fungal , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Cytokinesis , DNA Mutational Analysis , Endosomes/metabolism , Microtubules/metabolism , Molecular Sequence Data , Mutation , Plasmids/metabolism , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Homology, Amino AcidABSTRACT
The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.
Subject(s)
Adenosine Triphosphatases/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Protein Multimerization/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Enzyme Activation/physiology , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Multivesicular body (MVB) formation is the result of invagination and budding of the endosomal limiting membrane into its intralumenal space. These intralumenal vesicles (ILVs) contain a subset of endosomal transmembrane cargoes destined for degradation within the lysosome, the result of active selection during MVB sorting. Membrane bending and scission during ILV formation is topologically similar to cytokinesis in that both events require the abscission of a membrane neck that is oriented away from the cytoplasm. The endosomal sorting complexes required for transport (ESCRTs) represent cellular machinery whose function makes essential contributions to both of these processes. In particular, the AAA-ATPase Vps4 and its substrate ESCRT-III are key components that seem to execute the membrane abscission reaction. This review summarizes current knowledge about the Vps4-ESCRT-III system and discusses a model for how the recruitment of Vps4 to the different sites of function might be regulated.
Subject(s)
Cytokinesis/physiology , Endosomal Sorting Complexes Required for Transport/physiology , Multivesicular Bodies/metabolism , Vacuolar Proton-Translocating ATPases/physiology , ATPases Associated with Diverse Cellular Activities , Animals , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Humans , Models, Biological , Multivesicular Bodies/physiology , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.
Subject(s)
Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Vesicular Transport Proteins/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Conserved Sequence , Crystallography, X-Ray , Endosomal Sorting Complexes Required for Transport , Humans , Mice , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Conformation , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Species Specificity , Vesicular Transport Proteins/metabolismABSTRACT
The AAA-ATPase Vps4 is critical for function of the MVB sorting pathway, which in turn impacts cellular phenomena ranging from receptor downregulation to viral budding to cytokinesis. Vps4 dissociates ESCRTs from endosomal membranes during MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. Vta1 potentiates Vps4 activity and interacts with ESCRT-III family members. We have investigated the impact of Vta1 and ESCRT-III family members on Vps4 ATPase activity. Two distinct mechanisms of Vps4 stimulation are described: Vps2 can directly stimulate Vps4 via its MIT domain, whereas Vps60 stimulates via Vta1. Moreover, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. Recent structural determination of the ESCRT-III-binding region of Vta1 unexpectedly revealed a MIT-like region. These data support a model wherein a network of MIT and MIT-like domain interactions with ESCRT-III subunits contributes to the regulation of Vps4 activity during MVB sorting.
Subject(s)
Adenosine Triphosphatases/metabolism , Biomarkers, Tumor/metabolism , Nerve Tissue Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Vesicular Transport Proteins/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Binding Sites , Biomarkers, Tumor/chemistry , Endosomal Sorting Complexes Required for Transport , Kinetics , Models, Molecular , Nerve Tissue Proteins/chemistry , Plasmids , Protein Subunits/chemistry , Protein Subunits/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/geneticsABSTRACT
Endocytosis of cell surface receptors plays an important role in regulating cell signaling cascades. In some cases, internalization of an activated receptor attenuates the signaling process, while in other cases the clustering of activated receptors on early endosomal structures has been proposed to be essential for fully activating signaling cascades. Regulating the movement of receptors and other signaling proteins through the endocytic pathway, therefore, has a direct impact on cellular homeostasis. The small GTPase Rab5 is a crucial regulatory component of the endocytic pathway. Activation of Rab5 is mediated by GDP-GTP exchange factors (GEFs) that generate the Rab5-GTP complex. A large number of proteins have been identified that contain a specific, highly conserved domain (Vps9) that catalyzes nucleotide exchange on Rab5, linking the regulation of cell signaling cascades with intracellular receptor trafficking through the endocytic pathway.
Subject(s)
Endocytosis , Neurons/physiology , Protein Structure, Tertiary , Vesicular Transport Proteins/physiology , Yeasts/physiology , rab5 GTP-Binding Proteins/physiology , Animals , Carrier Proteins/chemistry , Carrier Proteins/physiology , Enzyme Activation , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/physiology , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/physiology , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/physiology , Signal Transduction , Ubiquitin/physiology , Vesicular Transport Proteins/chemistry , rab5 GTP-Binding Proteins/chemistryABSTRACT
Mutation of ciliopathy protein HYLS1 causes the perinatal lethal hydrolethalus syndrome (HLS), yet the underlying molecular etiology and pathogenesis remain elusive. Here, we reveal unexpected mechanistic insights into the role of mammalian HYLS1 in regulating primary cilia. HYLS1 is recruited to the ciliary base via a direct interaction with the type Iγ phosphatidylinositol 4-phosphate [PI(4)P] 5-kinase (PIPKIγ). HYLS1 activates PIPKIγ by interrupting the autoinhibitory dimerization of PIPKIγ, which thereby expedites depletion of centrosomal PI(4)P to allow axoneme nucleation. HYLS1 deficiency interrupts the assembly of ciliary NPHP module and agonist-induced ciliary exit of ß-arrestin, which, in turn, disturbs the removal of ciliary Gpr161 and activation of hedgehog (Hh) signaling. Consistent with this model of pathogenesis, the HLS mutant HYLS1D211G supports ciliogenesis but not activation of Hh signaling. These results implicate mammalian HYLS1 as a multitasking protein that facilitates ciliogenesis and ciliary signaling by coordinating with the ciliary lipid kinase PIPKIγ.
Subject(s)
Cilia , Ciliopathies , Animals , Ciliopathies/genetics , Ciliopathies/metabolism , Female , Hand Deformities, Congenital , Heart Defects, Congenital , Hedgehog Proteins/metabolism , Hydrocephalus , Mammals/metabolism , Pregnancy , Signal TransductionABSTRACT
Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1-Vps4-ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.
Subject(s)
Adenosine Triphosphatases/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Multivesicular Bodies/enzymology , Organelle Biogenesis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adenosine Triphosphatases/genetics , Binding Sites , Endosomal Sorting Complexes Required for Transport/genetics , Enzyme Activation , Microscopy, Fluorescence , Models, Molecular , Multivesicular Bodies/genetics , Mutation , Protein Domains , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin/metabolism , UbiquitinationABSTRACT
The multivesicular body (MVB) sorting pathway impacts a variety of cellular functions in eukaryotic cells. Perhaps the best understood role for the MVB pathway is the degradation of transmembrane proteins within the lysosome. Regulation of cargo selection by this pathway is critically important for normal cell physiology, and recent advances in our understanding of this process have highlighted the endosomal sorting complexes required for transport (ESCRTs) as pivotal players in this reaction. To better understand the mechanisms of cargo selection during MVB sorting, we performed a genetic screen to identify novel factors required for cargo-specific selection by this pathway and identified the Mvb12 protein. Loss of Mvb12 function results in differential defects in the selection of MVB cargoes. A variety of analyses indicate that Mvb12 is a stable member of ESCRT-I, a heterologous complex involved in cargo selection by the MVB pathway. Phenotypes displayed upon loss of Mvb12 are distinct from those displayed by the previously described ESCRT-I subunits (vacuolar protein sorting 23, -28, and -37), suggesting a distinct function than these core subunits. These data support a model in which Mvb12 impacts the selection of MVB cargoes by modulating the cargo recognition capabilities of ESCRT-I.
Subject(s)
Endosomes/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Endosomal Sorting Complexes Required for Transport , Endosomes/chemistry , Phenotype , Protein Transport , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins/analysis , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
A subset of proteins that transit the endosomal system are directed into the intralumenal vesicles of multivesicular bodies (MVBs). MVB formation is critical for a variety of cellular functions including receptor down-regulation, viral budding, antigen presentation, and the generation of lysosome-related organelles. Entry of transmembrane proteins into the intralumenal vesicles of a MVB is a highly regulated process that is positively modulated by covalent modification of cargoes with ubiquitin. To identify additional MVB sorting signals, we examined the previously described ubiquitination-independent MVB cargo Sna3. Although Sna3 ubiquitination is not essential, Sna3 MVB sorting is positively modulated by its ubiquitination. Examination of MVB sorting determinants within a form of Sna3 lacking all lysine residues identified two critical regions: an amino-terminal tyrosine-containing region and a carboxyl-terminal PPAY motif. This PPAY motif interacts with the WW domains of the ubiquitin ligase Rsp5, and mutations in either the WW or, surprisingly, the HECT domains of Rsp5 negatively impacted MVB targeting of lysine-minus Sna3. These data indicate that Rsp5 function is required for MVB targeting of Sna3 in a capacity beyond cargo ubiquitination. These results uncover a series of determinants impacting Sna3 MVB sorting, including unexpected roles for Rsp5.
Subject(s)
Membrane Proteins/metabolism , Protein Sorting Signals , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Transport Vesicles/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Endosomal Sorting Complexes Required for Transport , Membrane Proteins/analysis , Membrane Proteins/genetics , Molecular Sequence Data , Mutation , Protein Interaction Mapping , Protein Sorting Signals/genetics , Protein Transport , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/genetics , Transport Vesicles/chemistry , Ubiquitin-Protein Ligase Complexes/analysis , Ubiquitin-Protein Ligase Complexes/geneticsABSTRACT
Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2-interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.
Subject(s)
Bile Ducts/metabolism , Epithelial Cells/metabolism , Extracellular Vesicles/metabolism , Biological Transport , Cell Line , Cell Movement , Cell Polarity/physiology , Endocytosis , Endosomes/metabolism , Epithelium/metabolism , Exosomes/metabolism , Humans , Signal TransductionABSTRACT
FoxM1 activates genes that regulate S-G2-M cell-cycle progression and, when overexpressed, is associated with poor clinical outcome in multiple cancers. Here we identify FoxM1 as a tumor suppressor in mice that, through its N-terminal domain, binds to and inhibits Ect2 to limit the activity of RhoA GTPase and its effector mDia1, a catalyst of cortical actin nucleation. FoxM1 insufficiency impedes centrosome movement through excessive cortical actin polymerization, thereby causing the formation of non-perpendicular mitotic spindles that missegregate chromosomes and drive tumorigenesis in mice. Importantly, low FOXM1 expression correlates with RhoA GTPase hyperactivity in multiple human cancer types, indicating that suppression of the newly discovered Ect2-RhoAmDia1 oncogenic axis by FoxM1 is clinically relevant. Furthermore, by dissecting the domain requirements through which FoxM1 inhibits Ect2 GEF activity, we provide mechanistic insight for the development of pharmacological approaches that target protumorigenic RhoA activity.
Subject(s)
Actins , Forkhead Box Protein M1/metabolism , Neoplasms , Actins/metabolism , Animals , GTP Phosphohydrolases , Mice , Neoplasms/genetics , Signal TransductionABSTRACT
MVB (multivesicular body) formation occurs when the limiting membrane of an endosome invaginates into the intraluminal space and buds into the lumen, bringing with it a subset of transmembrane cargoes. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis, wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena. The AAA (ATPase associated with various cellular activities) Vps4 (vacuolar protein sorting 4) performs a critical function in the MVB sorting pathway. Vps4 appears to dissociate the ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes during the course of MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. We have investigated the mechanisms by which ESCRT components stimulate the ATPase activity of Vps4. These studies support a model wherein Vps4 activity is subject to spatial and temporal regulation via distinct mechanisms during MVB sorting.
Subject(s)
Adenosine Triphosphatases/metabolism , Endosomes/metabolism , Multiprotein Complexes/metabolism , Adenosine Triphosphatases/chemistry , Animals , Enzyme Activation , Protein Structure, Quaternary , Protein TransportABSTRACT
A homozygous truncating frameshift mutation in CEP57 (CEP57T/T) has been identified in a subset of mosaic-variegated aneuploidy (MVA) patients; however, the physiological roles of the centrosome-associated protein CEP57 that contribute to disease are unknown. To investigate these, we have generated a mouse model mimicking this disease mutation. Cep57T/T mice died within 24 hours after birth with short, curly tails and severely impaired vertebral ossification. Osteoblasts in lumbosacral vertebrae of Cep57T/T mice were deficient for Fgf2, a Cep57 binding partner implicated in diverse biological processes, including bone formation. Furthermore, a broad spectrum of tissues of Cep57T/T mice had severe aneuploidy at birth, consistent with the MVA patient phenotype. Cep57T/T mouse embryonic fibroblasts and patient-derived skin fibroblasts failed to undergo centrosome maturation in G2 phase, causing premature centriole disjunction, centrosome amplification, aberrant spindle formation, and high rates of chromosome missegregation. Mice heterozygous for the truncating frameshift mutation or a Cep57-null allele were overtly indistinguishable from WT mice despite reduced Cep57 protein levels, yet prone to aneuploidization and cancer, with tumors lacking evidence for loss of heterozygosity. This study identifies Cep57 as a haploinsufficient tumor suppressor with biologically diverse roles in centrosome maturation and Fgf2-mediated bone formation.
Subject(s)
Carrier Proteins/metabolism , Chromosome Disorders/metabolism , Frameshift Mutation , Haploinsufficiency , Neoplasms/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Carrier Proteins/genetics , Cell Cycle Proteins , Centrosome/metabolism , Centrosome/pathology , Chromosome Disorders/genetics , Chromosome Disorders/pathology , Chromosomes, Mammalian/genetics , Chromosomes, Mammalian/metabolism , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/metabolism , Humans , Mice , Mice, Mutant Strains , Mosaicism , Neoplasms/genetics , Neoplasms/pathology , Tumor Suppressor Proteins/geneticsABSTRACT
Study of the lysosomal protein transport system has been facilitated through dissection of the analogous vacuolar protein sorting (VPS) pathway in Saccharomyces cerevisiae. Resident enzymes of the yeast vacuole are synthesized as inactive precursors and are cleaved to their mature forms upon delivery to this compartment. Quantitative assessment of this delivery can be achieved through the use of pulse-chase experiments monitoring the cleavage of zymogens to their mature forms. The experimental procedures for analysis of carboxypeptidase Y (CPY) and carboxypeptidase S (CPS) maturation are described.