CHMP6 Human

How does N-myristoylation affect CHMP6 function?

N-myristoylation is a critical post-translational modification for CHMP6 function. Metabolic labeling experiments with HEK-293 cells have demonstrated the incorporation of [3H]myristate into CHMP6-GFP fusion proteins . This lipid modification likely facilitates CHMP6's association with endosomal membranes, as myristoylation typically enhances protein-membrane interactions. Research indicates that the first 10 amino acids of CHMP6, which contain the myristoylation site, are functionally important - deleting these amino acids from a truncated CHMP6-N construct does not prevent its localization to the intercellular bridge during cell division but significantly reduces its ability to inhibit abscission . This suggests that myristoylation may play a role beyond simple membrane targeting and could be involved in specific protein-protein interactions required for CHMP6's function.

What are the key protein interactions of CHMP6?

CHMP6 interacts with multiple proteins within the ESCRT machinery:

• CHMP4b/Shax1 (another ESCRT-III component): This interaction has been demonstrated through co-immunoprecipitation of epitope-tagged proteins in HEK-293 cells and through in vitro pull-down assays with recombinant proteins .

• EAP20 (component of ESCRT-II): EAP20 is the human counterpart of yeast Vps25. The interaction between CHMP6 and EAP20 has been confirmed through co-immunoprecipitation and in vitro pull-down assays . Specifically, a region within the first 52 amino acids of CHMP6 (amino acids 11-42) is critical for interaction with VPS25 (EAP20) .

Both of these interactions are mediated by the N-terminal basic half of CHMP6, highlighting the importance of this region for CHMP6's functional integration within the ESCRT machinery .

What approaches can be used to study CHMP6 localization in cells?

Several experimental approaches have proven effective for studying CHMP6 localization:

• Fluorescence microscopy with GFP-tagged CHMP6: Overexpressed CHMP6-GFP in HeLa cells exhibits a punctate distribution throughout the cytoplasm, particularly in the perinuclear area . When designing GFP fusion constructs, it's important to consider the potential impact on protein function. Studies have used a flexible oligopeptide linker (three tandem repeats of Ser-Gly-Gly) between CHMP6 and GFP to minimize interference with function .

• Structured Illumination Microscopy (SIM): This high-resolution imaging technique has been successfully used to visualize CHMP6 and ESCRT-II components at the intercellular bridge during cytokinetic abscission. SIM reveals that these proteins form highly ordered structures at the bridge membrane, providing insights into their spatial organization during this process .

• Live-cell video recording: This approach allows for dynamic tracking of CHMP6 recruitment to specific cellular structures over time, such as during cytokinetic abscission .

• Co-localization studies: CHMP6 localization can be examined in relation to other cellular markers. For example, accumulation of LBPA (lysobisphosphatidic acid), a major phospholipid in internal vesicles of multivesicular bodies, has been observed in CHMP6-GFP-localizing areas .

How can researchers manipulate CHMP6 function experimentally?

Several experimental strategies can be employed to manipulate CHMP6 function:

• Overexpression of wild-type or modified CHMP6: Overexpression of CHMP6-GFP has been shown to cause cellular phenotypes including reduction of transferrin receptors on the plasma membrane surface with accumulation in the cytoplasm, and accumulation of ubiquitinated proteins and endocytosed EGF . This approach can help reveal the consequences of CHMP6 hyperactivity.

• Expression of truncated versions: A truncated version of CHMP6 composed of its first 52 amino acids (CHMP6-N) can be used to block ESCRT-mediated processes. This construct localizes to the intercellular bridge, blocks abscission, and subsequently leads to cell death . This provides a valuable tool for upstream inhibition of the ESCRT pathway in live mammalian cells.

• Site-directed mutagenesis: Specific mutations can be introduced to disrupt particular functions:

  • Point mutation at amino acid 2 (G2A) to prevent N-myristoylation

  • Mutations that prevent CHMP6-N binding to its ESCRT-II partner VPS25

  • Deletion of the first 10 amino acids to assess the role of the myristoylation region

• Co-expression experiments: FLAG-tagged EAP20 (ESCRT-II component) distributes diffusely when expressed alone but exhibits a punctate distribution when co-expressed with CHMP6-GFP, allowing studies of how CHMP6 influences localization of interaction partners .

What biochemical assays are effective for studying CHMP6 interactions?

Several biochemical assays have proven valuable for characterizing CHMP6 interactions:

• Co-immunoprecipitation of epitope-tagged proteins: This approach has been used to detect interactions between CHMP6 and other proteins such as CHMP4b/Shax1 and EAP20 when expressed in HEK-293 cells .

• In vitro pull-down assays: Using recombinant proteins purified from E. coli, direct physical interactions between CHMP6 and its partners can be demonstrated. These assays have confirmed that the N-terminal basic half of CHMP6 mediates interactions with both CHMP4b and EAP20 .

• Metabolic labeling: To confirm N-myristoylation, metabolic labeling of cells with [3H]myristate followed by immunoprecipitation of CHMP6 and detection of incorporated radioactivity has been employed .

• Protein domain mapping: By creating truncated versions or specific mutations of CHMP6, the domains responsible for particular interactions can be identified. For example, this approach has revealed that the region comprising amino acids 11-42 of CHMP6 is critical for binding to VPS25 .

What is the role of CHMP6 in endosomal sorting?

CHMP6 plays a critical role in endosomal sorting as a component of the ESCRT-III complex:

• It acts as an acceptor for ESCRT-II on endosomal membranes, helping to recruit and position the ESCRT machinery .

• CHMP6 regulates cargo sorting in multivesicular bodies (MVBs). Overexpression of CHMP6-GFP causes:

  • Reduction of transferrin receptors on the plasma membrane surface with accumulation in the cytoplasm

  • Continuous accumulation of ubiquitinated proteins in cells

  • Accumulation of endocytosed EGF

• CHMP6 localization correlates with LBPA (lysobisphosphatidic acid) accumulation, a major phospholipid in internal vesicles of MVBs, suggesting its involvement in the formation or maturation of these structures .

The N-myristoylation of CHMP6 likely facilitates its association with endosomal membranes, positioning it to function effectively in the ESCRT-mediated sorting of proteins into MVBs for degradation or recycling .

How does CHMP6 contribute to cytokinetic abscission?

CHMP6 plays a key role in cytokinetic abscission, the final stage of cell division where the intercellular membrane bridge connecting daughter cells is cleaved:

• Together with ESCRT-II components, CHMP6 forms highly ordered structures at the intercellular bridge during abscission progression, as revealed by high-resolution imaging .

• CHMP6 appears to have a similar localization pattern to other ESCRT-III components at the intercellular bridge, distinct from the pattern observed for ESCRT-II components like VPS36 .

• The interaction between CHMP6 and its ESCRT-II partner VPS25 is critical for CHMP6 localization to the intercellular bridge, suggesting a recruitment hierarchy .

• A truncated version of CHMP6 (CHMP6-N, comprising the first 52 amino acids) can act as a dominant negative, arriving at the intercellular bridge, blocking abscission, and subsequently leading to cell death . This phenotype is abolished in a mutated version designed to prevent CHMP6-N binding to VPS25, confirming the importance of this interaction for CHMP6's role in abscission .

• Both the VPS25 interaction domain (aa 11-42) and the myristoylation region (first 10 aa) are crucial for CHMP6-N-induced inhibition of ESCRT-driven abscission .

How do we reconcile contradictory findings about ESCRT-II and CHMP6 roles in abscission?

Some studies have suggested that ESCRT-II and CHMP6 may be dispensable for certain ESCRT-mediated processes, particularly in cytokinetic abscission, creating apparent contradictions in the literature . Several methodological approaches can help address these contradictions:

• Validation across multiple cell types: The requirement for ESCRT-II and CHMP6 may vary between cell types or physiological conditions. Systematic studies across diverse cell lineages can help identify context-specific dependencies.

• Functional redundancy analysis: Investigate whether alternative pathways or compensatory mechanisms exist that can substitute for ESCRT-II-CHMP6 interactions under certain conditions.

• Quantitative assessment of protein depletion: Ensure that knockdown or knockout approaches achieve sufficient depletion to reveal functional requirements, as residual protein may support essential functions.

• Acute vs. chronic depletion: Compare the effects of acute inhibition (e.g., using CHMP6-N dominant negative) with long-term depletion approaches to distinguish immediate requirements from adaptive responses.

• Development of highly specific inhibitors: Tools like the CHMP6-N construct that specifically block ESCRT-II-CHMP6 interactions provide powerful approaches to dissect the precise role of these proteins in abscission .

Recent research has provided strong evidence for an active role of ESCRT-II and CHMP6 in ESCRT-mediated abscission , suggesting that earlier contradictory findings may reflect methodological limitations or context-specific variations.

What are the structural determinants of CHMP6's interaction specificity?

Understanding the structural basis for CHMP6's specific interactions presents several research challenges:

• Protein domain analysis: The N-terminal basic half of CHMP6 mediates interactions with both CHMP4b and EAP20, suggesting potential overlapping or distinct binding sites within this region . Precise mapping through mutagenesis studies can help delineate these sites.

• Structural studies: X-ray crystallography or cryo-electron microscopy of CHMP6 in complex with its binding partners could reveal the atomic details of these interactions. Focus on the region comprising amino acids 11-42, which has been identified as critical for VPS25 binding .

• Binding kinetics and affinities: Quantitative analysis of binding affinities between CHMP6 and its various partners under different conditions (e.g., in the presence or absence of membranes) could provide insights into the hierarchy of interactions and potential regulatory mechanisms.

• Role of post-translational modifications: Investigate how N-myristoylation and potentially other modifications affect binding specificity. The observation that deleting the first 10 amino acids of CHMP6-N significantly reduces its inhibitory effect on abscission without preventing localization suggests complex roles for this region .

• Membrane-dependent conformational changes: Consider how membrane association might alter CHMP6 conformation and thereby its interaction preferences, potentially using techniques like hydrogen-deuterium exchange mass spectrometry to detect conformational dynamics.

How might targeted CHMP6 modulation be used as a research tool?

The CHMP6-N construct (first 52 amino acids of CHMP6) has proven to be a valuable tool for inhibiting ESCRT-mediated processes, particularly cytokinetic abscission . This tool and related approaches could be extended in several ways:

• Inducible expression systems: Develop cell lines with inducible CHMP6-N expression to allow temporal control over ESCRT inhibition, enabling studies of ESCRT requirements at specific stages of cellular processes.

• Cell-specific targeting: Design viral vectors or other delivery methods to express CHMP6-N in specific cell types within tissues or organisms to study cell-autonomous effects of ESCRT inhibition.

• Structure-based design of specific inhibitors: Using insights from structure-function studies, develop more specific inhibitors targeting particular CHMP6 interactions rather than globally disrupting CHMP6 function.

• Combination with live imaging: Pair CHMP6-N expression with high-resolution live imaging of cellular structures or other ESCRT components to dissect the temporal sequence of events following ESCRT-II-CHMP6 disruption.

• Application to disease models: Explore how CHMP6-N-mediated inhibition affects cellular processes relevant to diseases where ESCRT function is implicated, such as neurodegeneration or cancer.

It's worth noting that CHMP6-N inhibition ultimately leads to cell death , limiting its long-term applications but potentially making it valuable for studying acute cellular responses or as a basis for developing targeted cell death approaches.

What are the main technical barriers to studying native CHMP6 function?

Several technical challenges complicate the study of native CHMP6 function:

• Transient and dynamic nature of ESCRT assemblies: ESCRT components typically form transient complexes during their normal function, making it difficult to capture and study these interactions under native conditions. Advanced imaging techniques with high temporal resolution are needed to track these dynamic processes.

• Functional redundancy: Potential compensation by other ESCRT components may mask phenotypes in knockdown or knockout studies. Combining multiple interventions or using acute inhibition approaches like CHMP6-N expression may help overcome this challenge .

• Low abundance of native protein: ESCRT components including CHMP6 may be expressed at relatively low levels, making detection of the endogenous protein challenging. Development of highly sensitive antibodies or gene tagging approaches is needed for reliable detection of native CHMP6.

• Complexity of inter-ESCRT interactions: CHMP6 functions within a network of ESCRT interactions, making it difficult to isolate its specific contributions. Systematic approaches combining genetic, biochemical, and imaging methods are needed to unravel this complexity.

• Context-dependent functions: CHMP6 may have different roles or requirements depending on the specific ESCRT-mediated process (e.g., MVB formation vs. cytokinetic abscission), necessitating process-specific experimental approaches.

How can we develop more selective tools to modulate CHMP6 function?

More selective tools for modulating CHMP6 function could be developed through several approaches:

• Structure-guided mutagenesis: Based on detailed structural information about CHMP6's interactions with different partners, design mutations that selectively disrupt specific interactions while preserving others. This could help dissect the relative importance of CHMP6's interactions with ESCRT-II versus ESCRT-III components.

• Interaction-specific inhibitory peptides or small molecules: Develop peptides or small molecules that target specific binding interfaces between CHMP6 and its partners. The CHMP6-N construct provides a starting point for this approach, but more selective derivatives could be engineered.

• Optogenetic or chemically-inducible approaches: Create systems where CHMP6 function or interactions can be modulated with light or small molecules, allowing precise temporal control over inhibition or activation.

• Targeted degradation approaches: Adapt technologies like PROTACs (proteolysis targeting chimeras) to achieve rapid, inducible degradation of CHMP6 or its interaction partners.

• Domain-specific antibodies or nanobodies: Develop antibodies or nanobodies that recognize and potentially inhibit specific domains of CHMP6, allowing more precise functional perturbation than global protein depletion.