Recombinant Xenopus tropicalis Charged multivesicular body protein 5 (chmp5)

What is CHMP5 and why is Xenopus tropicalis an advantageous model for studying this protein?

CHMP5 (Charged multivesicular body protein 5) is a component of the endosomal sorting required for transport complex III (ESCRT-III) involved in multivesicular bodies (MVBs) formation and sorting of endosomal cargo proteins. It participates in the invagination and scission processes that generate intraluminal vesicles (ILVs) for lysosomal degradation of membrane proteins, growth factor receptors, and lipids .

Xenopus tropicalis offers several distinct advantages as a model organism:

• Diploid genome (unlike the allotetraploid X. laevis), simplifying genetic studies

• High degree of synteny with mammalian genomes, often in stretches of hundreds of genes

• Well-established CRISPR/Cas9 protocols for efficient gene editing

• Ability to generate thousands of embryos per day via natural mating or in vitro fertilization

• Unilateral mutation technique (injecting only one cell at 2-cell stage) providing within-animal controls

• Rapid development with organ systems forming within 4 days

• Cost-effective maintenance compared to rodent models

These advantages make X. tropicalis particularly suitable for high-throughput, parallelized analysis of gene function relevant to human disease mechanisms .

How can I express and purify recombinant Xenopus tropicalis CHMP5 for functional studies?

Recombinant X. tropicalis CHMP5 (219 amino acids, 24.8 kDa) can be produced using several expression systems:

Expression hosts:

• E. coli (most common for basic structural studies)

• Yeast systems

• Baculovirus-infected insect cells

• Mammalian cell expression systems

Standard E. coli expression protocol:

• Clone the full CHMP5 coding sequence into an appropriate expression vector (pGEX for GST-tag or pET for His-tag)

• Transform into an expression strain (BL21(DE3) or similar)

• Grow cultures at 37°C to OD600 of 0.5-0.6

• Induce with IPTG (typically 0.2-0.5 mM)

• Shift to lower temperature (16-18°C) for overnight expression

• Harvest cells and lyse using appropriate buffer systems

• Purify using affinity chromatography based on the fusion tag

• Consider additional purification steps (ion exchange, size exclusion)

• Verify purity by SDS-PAGE and identity by Western blot or mass spectrometry

For Xenopus-specific applications, researchers may adapt protocols from established methods for purifying proteins from Xenopus egg extracts, which can yield more physiologically relevant protein forms with appropriate post-translational modifications .

What are the structural and sequence characteristics of Xenopus tropicalis CHMP5?

X. tropicalis CHMP5 has the following key characteristics:

Sequence information:

• 219 amino acids in length

• Molecular weight of 24.8 kDa

• Full sequence: MNRLFGKSKPKVPPSTLTDCISNVDSRSESIDKKISRLDAELVKYKDQMKKMREGPSKNMVKQKALRVLKQKRMYEQQRDNLNQQSFNMEQTNYAIQSLKDTKTTVDAMKVGAKEMKKAYKQVKIDQIEDLQDQLEDMMENANEIQEALSRSYGTPEIDEDDLEAELDALGDELLLDDDTSYLDEAASAPAIPEGVPNDSKNKDGVLVDEFGLPQIPAT

Structural features:

• Belongs to the SNF7 family of proteins

• Contains coiled-coil domains characteristic of ESCRT-III components

• Features a putative bipartite nuclear localization signal (NLS) in the N-terminus, which is present in jawed vertebrates but absent in invertebrate eukaryotes

Conservation:

• CHMP5 is highly conserved across vertebrate species

• The X. tropicalis sequence shares significant homology with human CHMP5 (also known as Vps60, CGI-34, PNAS-2, or SNF7DC2)

• The conservation of sequence and function makes X. tropicalis CHMP5 relevant for understanding human CHMP5 biology

What experimental techniques are most effective for studying CHMP5 function in Xenopus tropicalis?

Several sophisticated techniques can be employed to investigate CHMP5 function:

Genetic manipulation:

• CRISPR/Cas9 genome editing for knockout or knock-in studies

• Unilateral CRISPR injection at 2-cell stage creates embryos with one wild-type side and one mutant side for internal control

• Morpholino antisense oligonucleotides for transient knockdown

• mRNA overexpression for gain-of-function studies

Protein analysis:

• Immunoisolation using cryolysis for Xenopus tissues (limiting yolk protein contamination)

• Co-immunoprecipitation to identify interaction partners

• Western blotting for expression analysis

• Chromatin immunoprecipitation (ChIP) to study nuclear functions

Cellular analysis:

• Immunofluorescence microscopy for protein localization

• Transmission electron microscopy to examine MVB morphology

• Live imaging with fluorescently tagged proteins

• Flow cytometry for cell cycle analysis

Functional assays:

• Endosomal sorting and receptor trafficking assays

• Protein degradation assays using labeled substrates

• Gene expression analysis through RNA-seq

• Phenotypic analysis of embryonic development in CHMP5-depleted embryos

These methods can be combined to build a comprehensive understanding of both cytoplasmic and nuclear CHMP5 functions.

How does CHMP5 deficiency affect multivesicular body formation and endosomal function?

CHMP5 deficiency has profound effects on endosomal morphology and function, as demonstrated in studies of CHMP5 knockout cells:

Morphological effects:

• Enlarged late endosomal compartments

• MVBs become abnormally enlarged and heavily packed with internal vesicles

• Increased electron-dense content within MVBs

Molecular markers:

• Structures positive for CI-M6PR (cation-independent mannose-6-phosphate receptor)

• Positive for LBPA (lysobisphosphatidic acid) and LAMP1 (lysosomal-associated membrane protein 1)

• More pronounced colocalization of these markers compared to wild-type cells

Functional consequences:

• Severely reduced capacity to degrade internalized materials

• After a 1-hour pulse of HRP (horseradish peroxidase), wild-type cells degraded nearly all internalized protein after 9 hours, while CHMP5-deficient cells showed minimal degradation

• Immunogold labeling revealed approximately five-fold more internalized HRP accumulated within enlarged MVBs in CHMP5-deficient cells

These findings indicate that while CHMP5 is not required for MVB formation itself, it is essential for the normal function of late endosomes and lysosomes in protein degradation.

What role does CHMP5 play in embryonic development based on animal model studies?

Studies in mouse models provide insights into the developmental roles of CHMP5 that are likely relevant to Xenopus:

Developmental phenotypes in CHMP5-deficient mice:

• Early embryonic lethality around embryonic day 10 (E10)

• Severe developmental abnormalities in the ventral region after E7.5

• Abnormal neural tube formation

• Defects in allantois-chorion fusion

• Impaired somite segmentation

• Normal embryonic axes initially, followed by severe disorganization

Comparative phenotypes between CHMP5-/- and Hrs-/- mice:

This comparison highlights the specific nature of CHMP5 function in MVB formation compared to other ESCRT components .

What is the role of CHMP5 in receptor trafficking and signaling pathway regulation?

CHMP5 plays critical roles in receptor trafficking that impact multiple signaling pathways:

Receptor trafficking effects:

• Regulates turnover and down-regulation of receptors, including TGF-β receptors

• CHMP5 depletion leads to accumulation of receptors in endosomal compartments

• Affects receptor recycling versus degradation decisions

• Influences duration and intensity of receptor-mediated signaling

Impact on signaling pathways:

• TGF-β signaling: CHMP5 deficiency affects TGF-β receptor turnover

• Notch signaling: Potentially affects Notch receptor processing

• Growth factor signaling: Likely impacts signaling via receptor tyrosine kinases

Experimental approaches to study these functions:

• Receptor internalization and degradation assays

• Signaling reporter assays to measure pathway activation

• Biochemical analysis of receptor levels and modifications

• Genetic interaction studies between CHMP5 and pathway components

Understanding these functions is critical as receptor trafficking defects underlie many developmental disorders and diseases.

How can I investigate the newly discovered nuclear functions of CHMP5?

Recent studies have revealed unexpected nuclear roles for CHMP5 in gene regulation:

Nuclear functions of CHMP5:

• Associates with BRD4 (Bromodomain-containing protein 4) on chromatin

• Binds to specific gene loci, including enhancers and promoters

• Promotes H3K27 acetylation at enhancers and super-enhancers

• Facilitates RNA polymerase II pause release

Experimental approaches:

• Nuclear localization studies:

  • Create NLS-deletion mutants by removing the N-terminal nuclear localization signal

  • Compare cellular distribution of wild-type versus ΔNLS-CHMP5 by immunofluorescence

  • Perform nuclear/cytoplasmic fractionation followed by Western blotting

• Chromatin association:

  • ChIP-qPCR targeting known regulatory regions (e.g., MYC enhancer and promoter)

  • ChIP-seq to identify genome-wide binding patterns

  • Treatment with BET inhibitors like JQ1 to assess BRD4-dependency of binding

• Protein-protein interactions:

  • Co-immunoprecipitation with nuclear factors like BRD4

  • Proximity labeling methods (BioID, APEX)

  • Cell-free assays with recombinant proteins to test direct interactions

• Functional consequences:

  • RNA-seq to assess transcriptional changes upon CHMP5 depletion

  • Analysis of Pol II traveling ratios to quantify pause release defects

  • H3K27ac ChIP-seq to examine enhancer and super-enhancer modifications

These approaches can distinguish nuclear functions from cytoplasmic ESCRT-related roles.

What phenotypes result from CHMP5 knockdown or knockout in cell-based models?

CHMP5 depletion produces distinct cellular phenotypes depending on the cell type and context:

In T-ALL cells (CUTLL1):

• Proliferation defect with cell cycle arrest at S phase

• Impaired G2/M progression

• Significant gene expression changes (1057 upregulated and 702 downregulated genes)

• Downregulation of MYC and other critical T-ALL genes

• Impaired RNA polymerase II pause release

• Decreased H3K27ac at enhancers and super-enhancers

In embryonic stem (ES) cells:

• Normal fluid phase endocytosis

• Greatly reduced capacity to degrade internalized material

• Accumulation of cargo within enlarged MVBs

• Approximately five-fold more internalized HRP accumulated compared to wild-type cells

In embryonic cells:

• Enlarged endosomal compartments positive for late endosomal markers

• Abnormal colocalization of endosomal and lysosomal markers

• Defective receptor degradation

• Altered signaling pathway activation

These phenotypes highlight the dual roles of CHMP5 in endosomal function and gene regulation.

How do CHMP5 functions differ between Xenopus tropicalis and other model organisms?

While CHMP5's core functions are conserved, there are important species-specific considerations:

Similarities across species:

• Basic ESCRT-III component functions in MVB formation

• Role in receptor trafficking and degradation

• Involvement in embryonic development

Unique aspects in Xenopus tropicalis:

• Diploid genome simplifies genetic studies compared to X. laevis

• High conservation with mammalian CHMP5 including the N-terminal NLS

• Developmental context offers insights into vertebrate-specific functions

• X. tropicalis has distinct experimental advantages for embryological studies

Comparative limitations:

• Some specialized structures in mammals (like the placenta) are absent in Xenopus

• Differences in immune system development and function

• Aquatic versus terrestrial adaptations

Complementary nature of different models:

• Mouse models provide mammalian-specific insights but are more costly

• Cell culture models offer mechanistic detail but lack developmental context

• Xenopus offers a middle ground with vertebrate relevance and experimental accessibility

Understanding these differences helps researchers select the appropriate model system and interpret results in the context of human biology.

What are the most effective methods for immunoisolating CHMP5-containing protein complexes from Xenopus tissues?

Isolating CHMP5 protein complexes from Xenopus tissues requires specialized protocols:

Sample preparation using cryolysis:

• Flash-freeze Xenopus tissues or embryos in liquid nitrogen

• Cryogenically grind samples using a mortar and pestle kept at liquid nitrogen temperature

• This approach limits contamination from abundant yolk proteins while preserving native protein complexes

Immunoprecipitation protocol:

• Extract proteins using a gentle lysis buffer containing appropriate protease inhibitors

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Incubate with anti-CHMP5 antibodies or antibodies against epitope tags if using tagged CHMP5

• Capture complexes using magnetic beads conjugated with appropriate secondary antibodies

• Wash thoroughly while maintaining conditions that preserve protein-protein interactions

• Elute complexes under native or denaturing conditions depending on downstream applications

Analysis methods:

• Western blotting to confirm presence of CHMP5 and suspected interaction partners

• Mass spectrometry for unbiased identification of complex components

• Functional reconstitution assays to test activity of isolated complexes

This methodology can be extended for proteomic analysis of CHMP5-associated complexes in different developmental contexts or subcellular compartments .

How can I use CRISPR/Cas9 to effectively study CHMP5 function in Xenopus tropicalis embryos?

CRISPR/Cas9 mutagenesis in X. tropicalis provides powerful approaches to study CHMP5 function:

Comprehensive protocol:

• Design stage:

  • Design 2-3 sgRNAs targeting early exons of the CHMP5 gene

  • Verify target specificity using Xenbase (https://www.xenbase.org) resources

  • Consider targeting conserved functional domains

• sgRNA and Cas9 preparation:

  • Synthesize sgRNAs using in vitro transcription

  • Prepare Cas9 protein or mRNA (protein often gives higher efficiency)

• Microinjection:

  • For complete knockout: inject both cells at 2-cell stage

  • For unilateral knockout (preferred): inject only one cell at 2-cell stage to create within-animal control

  • Typical injection mix: 500 pg Cas9 protein + 300 pg sgRNA

• Validation:

  • Collect samples at early stages to verify editing efficiency

  • Use T7 endonuclease assay or direct sequencing of PCR products

  • Verify protein loss by Western blot if antibodies are available

• Phenotype analysis:

  • Examine morphological development at appropriate stages

  • Compare injected versus uninjected sides in unilateral injections

  • Perform molecular and cellular analyses to characterize effects

The unilateral CRISPR approach is particularly valuable as it allows direct comparison between wild-type and mutant tissues within the same embryo, controlling for any variation between individuals .

What are the critical differences between CHMP5 and other ESCRT-III components in MVB formation?

CHMP5 has distinctive roles within the ESCRT-III complex compared to other components:

Unique aspects of CHMP5 function:

• Acts as a peripherally associated component of ESCRT-III

• CHMP5 deficiency results in enlarged MVBs with abundant internal vesicles, unlike other ESCRT-III mutants which typically show defects in MVB formation

• This phenotypic difference suggests CHMP5 functions after internal vesicle formation, potentially in cargo sorting or processing

Comparative functional analysis:

Hierarchical assembly:

• ESCRT-III complex assembly is sequential

• CHMP5 appears to function at late stages of the process

• May be involved in ESCRT-III disassembly or recycling

Understanding these functional differences is crucial for interpreting phenotypes and designing targeted experiments to study specific aspects of MVB biology.

How does CHMP5 contribute to gene expression regulation in development and disease?

Recent studies reveal that CHMP5 has unexpected nuclear roles in gene regulation:

Mechanisms of CHMP5-mediated gene regulation:

• Association with chromatin regulators:

  • Directly interacts with BRD4 on chromatin

  • Promotes recruitment of the co-activator BRD4 by the histone acetyltransferase p300

  • This interaction potentiates H3K27 acetylation at regulatory enhancers

• Effects on transcriptional machinery:

  • Enhances RNA polymerase II pause release

  • CHMP5-depleted cells show decreased Pol II occupancy at transcriptional end sites (TES)

  • Increased Pol II binding at promoters indicates impaired pause-release

• Target genes and pathways:

  • Promotes expression of MYC and other critical T-ALL genes

  • Affects genes involved in mitochondrial oxidation and endoplasmic reticulum homeostasis

  • Influences transcriptional programs driven by ICN1 (intracellular NOTCH1)

Disease relevance:

• Promotes T-cell acute lymphoblastic leukemia (T-ALL) by controlling gene expression programs

• May contribute to other cancers through effects on receptor signaling and transcription

• Developmental disorders could potentially arise from CHMP5 dysfunction

These findings reveal CHMP5 as a dual-function protein with both cytoplasmic ESCRT-related roles and nuclear gene regulatory functions that impact development and disease.

What approaches can be used to study post-translational modifications of CHMP5 in Xenopus tropicalis?

Investigating post-translational modifications (PTMs) of CHMP5 requires specialized techniques:

Sample preparation considerations:

• Rapid tissue processing to preserve labile PTMs

• Use of appropriate inhibitors (phosphatase inhibitors, deubiquitinase inhibitors)

• Extraction buffers optimized to maintain native modifications

Analytical techniques:

• Mass spectrometry-based approaches:

  • Enrichment of modified peptides (e.g., TiO2 for phosphopeptides)

  • Tandem mass spectrometry to identify specific modification sites

  • Quantitative proteomics to compare modification levels between conditions

• Biochemical methods:

  • Phos-tag SDS-PAGE for phosphorylation analysis

  • 2D gel electrophoresis to separate modified protein forms

  • Western blotting with modification-specific antibodies

• Functional analysis:

  • Site-directed mutagenesis of modified residues

  • Phosphomimetic mutations (S/T to D/E) or phospho-null mutations (S/T to A)

  • Structure-function studies comparing wild-type and mutant proteins

Xenopus-specific considerations:

• Use established protocols for protein extraction from Xenopus tissues

• Consider developmental stage-specific and tissue-specific PTM patterns

• Combine with immunoisolation techniques optimized for Xenopus samples