Endosomal Sorting Complex Required For transport-III Research Articles

CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells.

The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis

SummaryCytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues—CHMP4C—in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.

CHMP6 and VPS4A mediate the recycling of Ras to the plasma membrane to promote growth factor signaling

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III, were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding-loop and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound N-Ras, but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked EGFR recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling.

Charged Multivesicular Body Protein 2B (CHMP2B) of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) Polymerizes into Helical Structures Deforming the Plasma Membrane

The endosomal sorting complexes required for transport (ESCRT-0-III) allow membrane budding and fission away from the cytosol. This machinery is used during multivesicular endosome biogenesis, cytokinesis, and budding of some enveloped viruses. Membrane fission is catalyzed by ESCRT-III complexes made of polymers of charged multivesicular body proteins (CHMPs) and by the AAA-type ATPase VPS4. How and which of the ESCRT-III subunits sustain membrane fission from the cytoplasmic surface remain uncertain. In vitro, CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission. However, this model awaits the demonstration that such structures exist and can deform membranes in cellulo. Here, we show that depletion of VPS4 induces specific accumulation of endogenous CHMP2B at the plasma membrane. Unlike other CHMPs, overexpressed full-length CHMP2B polymerizes into long, rigid tubes that protrude out of the cell. CHMP4s relocalize at the base of the tubes, the formation of which depends on VPS4. Cryo-EM of the CHMP2B membrane tubes demonstrates that CHMP2B polymerizes into a tightly packed helical lattice, in close association with the inner leaflet of the membrane tube. This association is tight enough to deform the lipid bilayer in cases where the tubular CHMP2B helix varies in diameter or is closed by domes. Thus, our observation that CHMP2B polymerization scaffolds membranes in vivo represents a first step toward demonstrating its structural role during outward membrane deformation.

Absence of FUS-immunoreactive pathology in frontotemporal dementia linked to chromosome 3 (FTD-3) caused by mutation in the CHMP2B gene

Frontotemporal dementia linked to chromosome 3 (FTD-3) is a very rare disease described in a large Danish family [1] and also in an unrelated Belgian patient with familial FTD [12]. A mutation has been identified in the CHMP2B gene that is likely responsible for causing the disease in the Danish family [9] and a different mutation was identified in the Belgian patient [12]. CHMP2B is a component of the endosomal sorting complex required for transport-III (ESCRT-III), which is involved in the degradation of proteins in the endocytic and autophagic pathways [10]. According to the recommended nomenclature, the neuropathology of frontotemporal lobar degeneration (FTLD) is subdivided into major groups on the basis of the protein abnormality that is presumed to be pathogenic or most characteristic [5]. The major groups are: FTLD-tau where the protein is the microtubule-associated protein tau, FTLD-TDP where the protein is the transactive response DNA binding protein with Mr 43 kD (TDP-43), FTLDUPS where inclusions can only be demonstrated with immunohistochemistry against proteins of the ubiquitin proteasome system (UPS), FTLD-IF where the inclusions are immunoreactive for class IV intermediate filaments, and FTLD-ni where no inclusions can be demonstrated. The neuropathology of FTD-3 is characterized by FTLD with neuronal cytoplasmic inclusions (NCI) that are immunoreactive for ubiquitin and p62, but not for tau, TDP-43 or intermediate filaments [2], and therefore, belongs to the FTLD-UPS group [6]. In addition to FTD-3, FTLD-UPS includes a group of sporadic FTD with unique clinical and pathological features, originally designated as atypical FTLD with ubiquitinated inclusions (aFTLD-U) because the NCI are immunoreactive for ubiquitin but negative for TDP-43 [4, 8]. Recently, mutations in the fused in sarcoma (FUS) gene have been identified as the cause of familial amyotrophic lateral sclerosis (ALS) type 6 [3, 11]. Because of the recognized clinical, genetic and pathological overlap between ALS and FTD and since the pathology in cases with FUS mutations was originally described as including NCI that were TDP-43 negative but FUS positive, one of us (I.M.) recently investigated whether FUS might also be the pathological protein in aFTLD-U. In all the aFTLD-U cases studied (N = 15), FUS immunohistochemistry (IHC) labeled all the ubiquitinated NCI and also identified abundant glial inclusions. Biochemical analysis of aFTLDU postmortem brain tissue demonstrated increased levels of insoluble FUS protein. No genetic abnormalities were identified in the FUS gene. These findings suggest that FUS is likely the pathological protein in the majority of sporadic FTLD-UPS cases and that these should now be designated as FTLD-FUS [6]. Furthermore, FUS was recently found in several types of inclusions in postmortem brains from patients with neuronal intermediate filament inclusion On behalf of the FReJA Consortium.

Structure/Function Analysis of Four Core ESCRT‐III Proteins Reveals Common Regulatory Role for Extreme C‐Terminal Domain

Endosomal sorting complex required for transport-III (ESCRT-III) is a large complex built from related ESCRT-III proteins involved in multivesicular body biogenesis. Little is known about the structure and function of this complex. Here, we compare four human ESCRT-III proteins - hVps2-1/CHMP2a, hVps24/CHMP3, hVps20/CHMP6, and hSnf7-1/CHMP4a - to each other, studying the effects of deleting predicted alpha-helical domains on their behavior in transfected cells. Surprisingly, removing approximately 40 amino acids from the C-terminus of each protein unmasks a common ability to associate with endosomal membranes and assemble into large polymeric complexes. Expressing these truncated ESCRT-III proteins in cultured cells causes ubiquitinated cargo to accumulate on enlarged endosomes and inhibits viral budding, while expressing full-length proteins does not. hVps2-1/CHMP2a lacking its C-terminal 42 amino acids further fails to bind to the AAA+ adenosine triphosphatase VPS4B/SKD1, indicating that C-terminal sequences are important for interaction of ESCRT-III proteins with VPS4. Overall, our study supports a model in which ESCRT-III proteins cycle between a default 'closed' state and an activated 'open' state under control of sequences at their C-terminus and associated factors.