Recombinant Mouse MORN repeat-containing protein 2 (Morn2)

What is the molecular composition of MORN2 and how does it relate to other MORN repeat proteins?

MORN2 belongs to the MORN (Membrane Occupation and Recognition Nexus) repeat protein family, which has a wide taxonomic distribution across both prokaryotes and eukaryotes . Mouse MORN2 is encoded by a gene located on chromosome 17E3, spanning approximately 7 kb. The gene contains a 669 nucleotide open reading frame encoding a relatively small protein of 79 amino acids .

Unlike some larger MORN proteins (such as TgMORN1 from Trypanosoma brucei or TgMORN2 from Toxoplasma gondii), mouse MORN2 represents a more compact MORN-motif containing protein. For comparison, TgMORN2 contains nine MORN repeats , while the consensus MORN repeat itself has been classically defined as approximately 14 amino acids in length, with a sequence approximating YEGEWxNGKxHGYG . To study MORN2's structure, researchers typically use a combination of techniques including X-ray crystallography, small-angle X-ray scattering, and electron microscopy - similar to approaches used for other MORN proteins .

How can researchers effectively generate and verify MORN2 knockout models?

Creating MORN2 knockout models requires careful targeting strategies for gene disruption. For effective MORN2 knockout generation, researchers have successfully employed CRISPR/Cas9-mediated genome editing in C57BL/6 mice . The procedure involves:

• Strategic selection of target sites (e.g., exons 4-5 in mouse Morn2)

• Co-injection of Cas9 and gRNA into fertilized C57BL/6 mouse eggs

• Transplantation of injected eggs to generate F0 founder mice

• Verification of targeted deletions (e.g., 1841 bp deletion in successful models)

• Breeding with wild-type mice to establish stable F1 generation models

• Genotyping via PCR followed by DNA sequencing analysis

Verification of knockout should include multiple methods:

• PCR and sequencing to confirm genetic modification

• RT-PCR to confirm absence of mRNA expression

• Western blotting to verify protein absence

• Phenotypic analysis to confirm functional consequences

When conducting RT-PCR verification, researchers typically use gene-specific primers (e.g., Forward: 5-CGTCTCCATCAACTTCGTC-3 and Reverse: 5-CTGTCCCGTTTCTCTCACA-3), with housekeeping genes like Gapdh as controls . All verification experiments should be replicated at least three times with distinct biological samples to ensure reproducibility .

What specific fertility defects are observed in MORN2 knockout mice and how can they be assessed?

MORN2 knockout (Morn2-/-) male mice exhibit complete infertility with severe sperm motility defects . Comprehensive assessment of these defects requires multiple analytical approaches:

Fertility Assessment:

• In vivo breeding tests with wild-type females

• In vitro fertilization assays comparing Morn2-/- with Morn2+/- sperm

• Quantification of fertilization rates by counting two-cell embryos 24 hours post-fertilization

Sperm Analysis:

• Computer-assisted sperm analysis (CASA) for quantitative motility parameters

• Light microscopy for basic morphological examination

• Immunofluorescence assays for protein localization

• TUNEL assay for detecting apoptotic sperm cells

• Transmission electron microscopy (TEM) for ultrastructural analysis of sperm components

Sperm from Morn2-/- mice display distinct morphological abnormalities, particularly bent heads and aberrant mitochondrial sheath formation in the mid-piece region of the flagellum . TEM analysis is especially valuable for detailed ultrastructural examination, requiring:

• Fixation in 2.5% glutaraldehyde

• Post-fixation in 1% OsO4

• Dehydration through graded acetone

• Embedding in resin

• Ultrathin sectioning

• Double staining with uranyl acetate and lead citrate

• Imaging with TEM (e.g., JEM-1400)

How does MORN2 regulate mitochondrial function in spermatozoa?

MORN2 plays a critical role in regulating mitochondrial function in spermatozoa, with knockout mice exhibiting several mitochondrial defects that contribute to asthenospermia (poor sperm motility) . To investigate MORN2's role in mitochondrial regulation, researchers employ multiple complementary approaches:

Mitochondrial Membrane Potential (MMP) Analysis:

• JC-1 staining of sperm cells

• Flow cytometry to quantify changes in membrane potential

• Comparative analysis between knockout and control samples

Reactive Oxygen Species (ROS) Assessment:

• DCFH-DA staining for detecting intracellular ROS

• Flow cytometry quantification of fluorescence intensity

• Microscopic visualization of ROS distribution patterns

ATP Content Measurement:

• Luciferin-luciferase based assays

• Normalization to sperm count or protein concentration

• Comparative analysis across genotypes

Research findings demonstrate that Morn2-/- sperm exhibit significantly lower mitochondrial membrane potential, higher ROS levels, and decreased ATP content compared to wild-type controls . These parameters collectively indicate severely impaired energy metabolism in knockout sperm, explaining their motility defects. Analysis of mitochondrial respiration using techniques like Seahorse XF analyzers can provide additional insights into specific respiration parameters (basal respiration, maximal respiration, spare capacity) affected by MORN2 deficiency.

What protein interaction methodologies are optimal for identifying MORN2 binding partners?

Identifying MORN2 binding partners requires robust experimental approaches that can detect both strong and transient interactions. Based on successful studies with related MORN proteins, researchers should consider these methodologies:

Proximity Labeling Techniques:

• TurboID fusion protein construction (e.g., MORN2-TurboID-HA)

• Expression verification by PCR and Western blotting

• Biotin supplementation for proximity labeling

• Streptavidin-based pulldown of biotinylated proteins

• Mass spectrometry identification of interacting proteins

GST Pull-down Assays:

• Production of GST-MORN2 fusion proteins

• Incubation with candidate interacting proteins

• Precipitation and washing of protein complexes

• Western blot detection of co-precipitated proteins

• GST-only controls to exclude non-specific binding

Co-Immunoprecipitation:

• Co-transfection of tagged constructs (e.g., pEGFP-MORN2 and pCMV-Flag-candidate)

• Cell lysis under non-denaturing conditions

• Immunoprecipitation with antibodies against one tag

• Western blot detection of co-precipitated proteins using antibodies against the other tag

These approaches have successfully identified interaction partners for MORN proteins. For example, TgMORN2 interactions with TgRTN (an ER protein) and β-Tubulin were confirmed using both GST pull-down and Co-IP approaches . When designing these experiments, researchers should include appropriate controls and perform at least three biological replicates to ensure result reliability .

How can researchers effectively express and purify recombinant MORN2 for structural and functional studies?

Production of high-quality recombinant MORN2 presents several challenges due to its structural properties. Based on experiences with related MORN proteins, researchers should consider these methodological approaches:

Expression System Selection:

• Bacterial systems (E. coli) for basic structural studies

  • BL21(DE3) or Rosetta strains often provide good yields

  • Consider codon optimization for mouse sequences

• Mammalian expression for functional studies requiring post-translational modifications

  • HEK293T cells have been successfully used for MORN protein expression

• Insect cell systems for difficult-to-express constructs

  • Baculovirus expression systems may improve folding

Vector Design Considerations:

• Affinity tags for purification (His10, GST, or OneStrep tags)

  • N-terminal His10-tag with 3C protease cleavage site works well for MORN proteins

  • C-terminal tags as alternative if N-terminal tagging affects function

• Fusion partners to enhance solubility (MBP, SUMO, etc.)

• Fluorescent tags (GFP, mCherry) for localization studies

Purification Strategy:

• Initial capture with affinity chromatography

• Tag removal with specific proteases (e.g., 3C protease)

• Secondary purification steps (ion exchange, size exclusion)

• Quality assessment by SDS-PAGE and Western blotting

• Functional verification through activity or binding assays

For difficult constructs, researchers might consider truncation approaches similar to those used for TgMORN1 and PfMORN1 using ligase-independent cloning methods . Solubility and stability testing under various buffer conditions (pH, salt concentration, additives) should be performed to optimize conditions for downstream applications.

How can MORN2 knowledge be applied to reproductive biology and fertility research?

MORN2's essential role in male fertility makes it a valuable target for both basic and applied reproductive biology research. Several key applications include:

Diagnostic Applications:

• Development of molecular diagnostic tools for asthenospermia evaluation

  • PCR-based screening for MORN2 mutations

  • Immunostaining protocols for MORN2 localization in sperm samples

  • Functional assays to assess MORN2-dependent mitochondrial function

Therapeutic Target Exploration:

• Structure-based design of compounds that enhance MORN2 functionality

• Gene therapy approaches for MORN2-related infertility

• Mitochondrial support strategies to compensate for MORN2 deficiency

Contraceptive Development:

• MORN2 as a potential target for male contraception

  • Temporary inhibition of MORN2 function

  • Reversible approaches that don't cause permanent damage

Research Applications:

• MORN2 as a tool for studying mitochondrial sheath formation

• Model systems for investigating mitochondrial energy metabolism in specialized cells

• Comparative studies across species to understand evolutionary conservation of function

The identification of MORN2 as a causal gene for asthenospermia provides researchers with a new perspective on male infertility mechanisms . Research in this area should focus on translational approaches that can bridge the gap between basic MORN2 biology and clinical applications in reproductive medicine.

How can researchers address contradictions in MORN protein functional assignments?

MORN proteins have historically been associated with membrane binding functions, yet recent research has challenged this assumption, revealing potential contradictions that require careful experimental design to resolve:

Experimental Approaches to Resolve Contradictions:

• Direct membrane binding assays

  • Liposome sedimentation assays

  • Surface plasmon resonance with lipid bilayers

  • Fluorescence-based membrane association assays

• Protein-protein interaction mapping

  • Comprehensive interactome studies using proximity labeling

  • Yeast two-hybrid screening

  • Domain-specific interaction mapping

• In vivo localization studies

  • Super-resolution microscopy

  • Correlative light and electron microscopy

  • Live-cell imaging with photoactivatable tags

Data Interpretation Considerations:

• Distinguish direct vs. indirect effects

  • Use of domain deletion/mutation analysis

  • Temporal studies to establish causality

  • In vitro reconstitution of minimal systems

• Context-dependent function assessment

  • Cell-type specific analyses

  • Developmental stage considerations

  • Stress response vs. normal conditions

Research on junctophilins provides an instructive example of such contradictions - while N-terminal regions containing MORN repeats mediate plasma membrane targeting, it has not been definitively demonstrated whether this targeting results from direct lipid binding or protein-protein interactions . Similar contradictions might exist for MORN2, where its role in mitochondrial function could be direct or mediated through interactions with other proteins such as β-Tubulin .