Recombinant Mouse Emerin (Emd)

What is mouse emerin and how does it compare structurally to human emerin?

Mouse emerin is an integral membrane protein of the inner nuclear membrane containing 259 amino acids, slightly larger than its human ortholog with four additional amino acids at the C-terminus. On immunoblotting, mouse emerin appears as a protein with an apparent molecular mass of ~38 kd, which is larger than the 34-kd human emerin. This size difference is attributed to posttranslational modifications, as emerin contains multiple phosphorylation sites and one glycosylation site .

Like human emerin, mouse emerin belongs to the LEM (LAP2, Emerin, MAN1) domain protein family and functions through interactions with critical nuclear partners including lamins, barrier-to-autointegration factor (BAF), β-catenin, actin, and tubulin .

The functional domains of mouse emerin include:

• LEM domain (residues 1-45): Mediates interaction with BAF

• Lamin-binding domain: Critical for nuclear envelope localization

• Tubulin-binding region: Facilitates association with the microtubule network

• Transmembrane domain: Required for membrane integration

What phenotypes are observed in emerin knockout mice compared to human EDMD1?

Emerin knockout mice exhibit surprisingly mild phenotypes compared to human patients with Emery-Dreifuss muscular dystrophy type 1 (EDMD1). Key observations in emerin-lacking mice include:

• Normal expression levels of mRNA for emerin in both skeletal and cardiac muscles despite protein absence

• Minor motor coordination problems that develop only after 40 weeks of age

• Slight atrioventricular conduction elongation in older mice

• Delayed muscle regeneration after myotrauma

• Normal distribution of lamin A and lamin C in skeletal muscle, cardiac muscle, and brain tissue

This mild phenotype contrasts with human EDMD1, which manifests as progressive muscle wasting, contractures of the elbow and Achilles tendons, and cardiac conduction defects . The discrepancy is potentially explained by:

• Compensatory upregulation of downstream muscle regeneration genes

• The sedentary lifestyle of laboratory mice compared to human activity levels

• Possible functional compensation by lamina-associated polypeptide-1 (LAP1), which interacts with emerin and shows higher expression levels in mouse than human skeletal muscle

• The presence of the protein Lmo7 in mice, which may compensate for emerin loss

What are the key protein interaction partners of mouse emerin and their functional significance?

Mouse emerin engages in multiple protein interactions that facilitate its diverse nuclear functions. Key interaction partners include:

Notably, the interaction with LAP1 is functionally significant as shown by FRET analysis, where energy transfer efficiency between GFP-LAP1 and RFP-emerin was significantly greater than control pairs, confirming their interaction in living cells . In LAP1 null fibroblasts, emerin mislocalizes to distinct foci along the nuclear envelope in approximately 30% of cells, with A-type lamins co-localizing with emerin in these abnormal foci .

How is emerin expression and localization regulated during development and in different mouse tissues?

Emerin expression varies across developmental stages and tissue types in mice:

Methodologically, these localization patterns can be visualized using:

• Indirect immunofluorescence with monoclonal or polyclonal antibodies specific to emerin

• Immuno-gold EM labeling for high-resolution localization studies

• Expression of EGFP-emerin fusion proteins for live-cell imaging

What methodologies are recommended for producing and purifying recombinant mouse emerin for experimental applications?

For researchers working with recombinant mouse emerin, the following methodological approaches have proven effective:

Expression Systems:

• Bacterial expression system: Emerin can be expressed in bacteria and subsequently inserted into microsomal membranes in an ATP- and TRC40-dependent manner .

• Cell-free lysate system: Emerin expressed in cell-free lysates can be effectively inserted into microsomal membranes, providing an alternative to cellular expression systems .

• Mammalian expression: For studies requiring proper post-translational modifications, expression in mammalian cells using retroviral delivery systems has been successful. The pQCXIB vector with a CMV promoter has been used for constitutive expression .

Purification Strategies:

• For bacterial expression: Affinity purification using His-tagged constructs followed by size exclusion chromatography

• For studies requiring the transmembrane domain: Consider using detergent solubilization or amphipol stabilization

• For functional studies: Purification in complex with TRC40 has been shown to maintain functional integrity of emerin

Construction of Expression Vectors:
Mouse emerin sequences can be based on Uniprot ID O08579. Several approaches have been employed:

• C-terminal tagging with GFP using stitching PCR, where the open reading frame is amplified with the start codon included and stop codon omitted

• For domain studies, deletion constructs can be generated by Quickchange mutagenesis

• Gateway cloning can be used for introducing ORFs into expression vectors

Quality Control Considerations:
Verify proper folding and functionality of purified recombinant emerin through:

• Binding assays with known interactors (lamins, BAF)

• Circular dichroism to assess secondary structure

• Membrane integration assays to confirm functionality of the transmembrane domain

How can researchers experimentally investigate emerin's role in nuclear envelope reassembly after mitosis?

Investigating emerin's role in nuclear envelope reassembly requires multi-faceted approaches:

1. Live-cell imaging techniques:

• Express fluorescently-tagged emerin (EGFP-emerin) to track its dynamics during mitosis and nuclear reassembly

• Combine with markers for the nuclear envelope, chromatin, and mitotic spindle to correlate emerin localization with specific mitotic events

• Use high-resolution time-lapse microscopy to capture the temporal sequence of emerin recruitment during nuclear reformation

2. Cell-free nuclear assembly systems:

• The Xenopus egg extract system provides an excellent model as it naturally lacks emerin

• Add recombinant emerin or emerin mutants to Xenopus egg extracts containing demembranated sperm chromatin to observe effects on nuclear formation

• This system allows biochemical manipulation and isolation of specific steps in nuclear assembly

3. Domain mapping experiments:

• Express emerin deletion mutants to identify domains required for proper nuclear reassembly

• Key constructs to test include:

  • LEM domain deletions (involved in BAF and chromatin binding)

  • Tubulin-binding region deletions

  • Transmembrane domain mutations

4. Interaction disruption approaches:

• Use dominant-negative fragments of TRC40-receptor proteins WRB and CAML to inhibit membrane insertion of emerin

• Apply small molecules or peptides that disrupt specific emerin interactions

• Employ rapamycin-based dimerization assays to assess transport of wild-type and mutant emerin to the INM

5. Quantitative assessments of nuclear envelope integrity:

• Measure nuclear envelope permeability using fluorescent dextrans of varying sizes

• Assess nuclear morphology abnormalities and chromatin organization defects

• Quantify the rate and completeness of nuclear envelope closure

Research has demonstrated that emerin deletion mutants produce different phenotypes, including aberrant nuclear shape, tubulin network mislocalization, aberrant mitosis, and mislocalization of centrosomes . These phenotypes correlate with the mutants' chromatin binding capacities in in vitro nuclear assembly assays .

What techniques can be used to visualize and analyze emerin nanodomains at the nuclear membrane?

Recent research has revealed that emerin self-assembles into nanometer-size domains at the inner nuclear membrane, with size and occupancy changes observed under mechanical stress and in EDMD-associated mutations . To study these nanodomains, researchers can employ:

1. Super-resolution microscopy techniques:

• Stochastic Optical Reconstruction Microscopy (STORM)

• Photoactivated Localization Microscopy (PALM)

• Stimulated Emission Depletion (STED) microscopy
  These approaches overcome the diffraction limit of conventional microscopy to resolve structures at the nanometer scale.

2. Fluorescence Resonance Energy Transfer (FRET):

• This technique has successfully demonstrated emerin interactions with partners like LAP1

• For nanodomain studies, FRET can detect proximities between emerin molecules within domains

• Using acceptor photobleaching methodology: increases in donor fluorescence after acceptor photobleaching measures efficiency of energy transfer between proteins

3. Atomic Force Microscopy (AFM):

• Provides topographical imaging of membrane proteins at nanometer resolution

• Can be combined with force measurements to study mechanical properties of nanodomains

4. Reaction-diffusion modeling approaches:
A quantitative reaction-diffusion model has been developed that explains emerin nanodomain self-assembly . This model provides:

• Quantitative agreement with experimental observations on nanodomain size and occupancy

• Explanatory framework for differences between wild-type and EDMD-associated mutations

• Predictive capability for emerin diffusion coefficients based on nanodomain properties

5. Correlative light and electron microscopy (CLEM):

• Combines fluorescence microscopy with electron microscopy

• Allows correlation of nanodomain fluorescence with ultrastructural features

6. Single-particle tracking:

• Labels individual emerin molecules with quantum dots or photoactivatable fluorophores

• Tracks their movement to determine diffusion characteristics and confinement within nanodomains

7. Nanodomain isolation techniques:

• Detergent-resistant membrane fraction isolation

• Proximity labeling approaches (BioID, APEX)

• Mass spectrometry analysis of nanodomain composition

The reaction-diffusion model developed by Alas et al. (2025) provides a physical understanding of EDMD-associated defects in emerin organization in terms of changes in key reaction and diffusion properties of emerin and its nuclear binding partners , offering valuable insights for researchers investigating nanodomain dynamics.

How can one experimentally validate the involvement of the TRC40/GET pathway in emerin trafficking and membrane integration?

The TRC40/GET pathway has been implicated in post-translational insertion of emerin into membranes . To experimentally validate this involvement, researchers can employ several approaches:

1. Proximity ligation assays (PLA):

• This technique has successfully demonstrated emerin interaction with TRC40 in situ

• PLA detects protein-protein interactions at endogenous expression levels with high specificity

• The method generates fluorescent signals only when target proteins are in close proximity (<40 nm)

2. Membrane insertion assays:

• Express emerin in bacteria or cell-free lysates

• Test insertion into microsomal membranes under varying conditions:

  • With and without ATP (insertion is ATP-dependent)

  • With and without TRC40 (insertion is TRC40-dependent)

  • In the presence of dominant-negative fragments of TRC40-receptor proteins WRB and CAML

3. Rapamycin-based dimerization assay:

• This method has revealed correct transport of wild-type emerin to the INM

• TRC40-binding, membrane integration, and INM-targeting of EDMD-associated emerin mutants were disturbed in this assay

• The approach uses rapamycin-induced dimerization of FKBP and FRB domains to detect proper targeting

4. In vitro transcription/translation systems:

• Program with emerin mRNA in the presence of microsomes

• Compare membrane integration efficiency with and without TRC40 immunodepletion

• Rescue experiments by adding back purified TRC40

5. Knockdown/knockout approaches:

• siRNA or CRISPR-based depletion of TRC40 pathway components

• Assess effects on emerin localization and membrane integration

• Complementation with wild-type or mutant TRC40 pathway components

6. TRC40 binding assays:

• Express and purify recombinant emerin and TRC40

• Perform pull-down assays to demonstrate direct binding

• Use surface plasmon resonance (BIAcore) to measure binding kinetics

7. Structural studies of the emerin-TRC40 complex:

• Cryo-EM or X-ray crystallography of the complex

• Map interaction interfaces and design mutations that specifically disrupt binding

These methodological approaches collectively provide strong evidence for the involvement of the TRC40/GET pathway in emerin trafficking. Research has shown that proper membrane integration contributes to correct targeting of emerin to the INM, and disruption of this pathway affects the localization of emerin mutant proteins associated with EDMD .

What are the best methods to investigate emerin's newly discovered role in neuronal plasticity and protein synthesis regulation?

Recent research has revealed a novel function of emerin in neuronal systems, where activity-induced neuronal emerin is abundant in the endoplasmic reticulum and broadly inhibits protein synthesis . To investigate this role, researchers can employ:

1. Metabolic labeling approaches:

• Optimized metabolic labeling to characterize the visual experience-dependent nascent proteome within defined time windows after stimulation

• This approach revealed cell type-specific and age-dependent alterations in the nascent proteome

• Identified emerin as the top activity-induced candidate plasticity protein (CPP)

2. Transcriptional dependence analysis:

• Pharmacological inhibition of transcription (e.g., actinomycin D)

• Demonstration that rapid activity-induced synthesis of emerin is transcription-independent

• qRT-PCR and RNA sequencing to monitor changes in transcript levels

3. Subcellular localization studies:

• Immunocytochemistry with compartment-specific markers

• Subcellular fractionation followed by Western blotting

• These approaches revealed that activity-induced neuronal emerin localizes to the ER rather than the nuclear envelope

4. Protein synthesis measurement techniques:

• Surface sensing of translation (SUnSET) to measure global protein synthesis rates

• Ribosome profiling to identify specific mRNAs affected by emerin

• FUNCAT (fluorescent non-canonical amino acid tagging) for visualization of newly synthesized proteins

5. Dendritic spine morphology analysis:

• Downregulating emerin shifts dendritic spines from mature mushroom morphology to immature filopodial morphology

• High-resolution imaging and quantitative morphometric analysis

• Time-lapse imaging to track spine dynamics

6. Network connectivity assessment:

• Calcium imaging to measure neuronal activity

• Electrophysiological recordings (patch-clamp)

• These approaches revealed that decreasing emerin reduces network connectivity

7. In vivo functional studies:

• Visual response testing in mice with decreased emerin

• This revealed reduced magnitude of visually evoked responses and impaired visual information processing

• Behavioral assays to assess learning and memory

8. Temporal manipulation of emerin expression:

• Optogenetic or chemogenetic approaches for precise temporal control

• Investigation of the "temporal gating" hypothesis for neuronal plasticity

The methodological approaches above address emerin's feed-forward role in temporally gating neuronal plasticity through negative regulation of translation , representing an exciting new direction in emerin research distinct from its well-established roles in muscle and cardiac tissues.

How can researchers investigate the potential therapeutic applications of recombinant emerin for EDMD1-related conditions?

Investigating therapeutic applications for recombinant emerin requires approaches that address both the cellular defects and potential delivery methods:

1. Structure-function analyses of EDMD1-associated mutations:

• Studies of Polish patients with EDMD1 revealed that for several mutations thought to be null for emerin protein, truncated emerin protein was present

• This suggests the EDMD1 phenotype may be strengthened by toxicity of truncated emerin expressed in patients

• Screening therapeutic compounds should target both loss of function and toxic gain of function mechanisms

2. Cell-based rescue experiments:

• Patient-derived fibroblasts or induced pluripotent stem cells (iPSCs)

• Tests of recombinant emerin delivery using:

  • Cell-penetrating peptides

  • Lipid nanoparticles

  • Viral vectors

• Assessment of nuclear envelope structure, gene expression normalization, and cellular function

3. Mouse model testing:

• Conditional knockout or knockin models of EDMD1

• Studies of LAP1/emerin double mutants, which show more severe phenotypes than emerin knockout alone

• Delivery methods including:

  • Systemic injection of recombinant protein with targeting moieties

  • AAV-mediated gene therapy

  • Antisense oligonucleotides to modulate splicing of mutant emerin

4. Phenotypic rescue assessment:

• Cardiac conduction testing using electrocardiography

• Muscle strength and coordination testing

• Histological analysis of muscle structure and regeneration

• Molecular markers of nuclear envelope integrity

5. Alternative therapeutic approaches:

• Small molecules that stabilize truncated emerin or enhance its proper localization

• Compounds that upregulate compensatory proteins like LAP1

• CRISPR-based gene editing to correct mutations in patient-derived cells

6. Biomarker development:

• Identification of circulating biomarkers of disease progression

• Use of emerin antibodies for diagnostic applications

• Development of imaging biomarkers for monitoring therapy response

7. Combined therapeutic approaches:

• Target multiple aspects of disease pathophysiology:

  • Nuclear envelope integrity

  • Mechanotransduction

  • Gene expression dysregulation

  • Cardiac conduction abnormalities

Research in LAP1/emerin interaction suggests promising therapeutic avenues, as LAP1 levels are significantly higher in mouse than human skeletal muscle, potentially explaining the milder phenotype in emerin-null mice . Targeting upregulation of compensatory factors like LAP1 could be a viable therapeutic approach alongside direct emerin replacement strategies.