Recombinant Mouse Janus kinase and microtubule-interacting protein 1 (Jakmip1)

What is Jakmip1 and what are its primary functions?

Jakmip1, or Janus Kinase and Microtubule Interacting Protein 1, is a protein-coding gene that enables GABA receptor binding activity and RNA binding activity. It is primarily involved in cognition and serves as an extrinsic component of the membrane within ribonucleoprotein complexes .

At the molecular level, Jakmip1 associates with microtubules and plays a significant role in the microtubule-dependent transport of the GABA-B receptor. Additionally, it may function in JAK1 signaling and regulate microtubule cytoskeleton rearrangements . Recent research has established its importance in neurodevelopmental processes through its interaction with the FMRP protein, which is disrupted in Fragile X syndrome .

Where is Jakmip1 expressed and localized within cells?

Jakmip1 demonstrates dual localization within cells, being present in both microtubules and the plasma membrane . This characteristic localization pattern is consistent with its function in microtubule-dependent transport processes and its role in JAK1 signaling pathways .

In terms of expression patterns during development, studies indicate that Jakmip1 has specific temporal expression profiles in the central nervous system that correlate with critical periods of neurodevelopment . This spatiotemporal expression pattern supports its proposed role in neurodevelopmental disorders such as autism.

What is the molecular weight of mouse Jakmip1 and how can I detect it?

Mouse Jakmip1 has a molecular weight of approximately 80 kDa as detected by Western blotting techniques . For detection purposes, researchers can use commercially available antibodies such as rabbit-derived antibodies that show cross-reactivity with human, mouse, and rat Jakmip1 .

When performing Western blotting for Jakmip1 detection, a dilution of 1:1000 is typically recommended for optimal results . It is important to note that detection sensitivity may vary based on expression levels, which can differ across tissue types and developmental stages.

How can I generate recombinant mouse Jakmip1 for in vitro studies?

To generate recombinant mouse Jakmip1 for in vitro studies, researchers can employ the following methodological approach:

• cDNA cloning: Isolate total RNA from mouse brain tissue, followed by reverse transcription to generate cDNA. The full-length Jakmip1 sequence can be amplified using PCR with gene-specific primers designed based on the mouse Jakmip1 sequence (NCBI Gene ID: 152789) .

• Expression vector construction: Clone the amplified Jakmip1 cDNA into an appropriate expression vector containing a strong promoter and an affinity tag (such as His-tag or GST-tag) to facilitate purification.

• Expression system selection: For mammalian protein production, transfect the construct into HEK293 or CHO cells. Alternatively, for higher yield but potentially different post-translational modifications, use bacterial (E. coli) or insect cell (Sf9) expression systems.

• Protein purification: Isolate the recombinant protein using affinity chromatography based on the chosen tag, followed by size exclusion chromatography to ensure high purity.

• Validation: Confirm the identity and integrity of the purified protein using Western blotting with Jakmip1-specific antibodies and mass spectrometry analysis.

What are the recommended protocols for studying Jakmip1-microtubule interactions?

When investigating Jakmip1-microtubule interactions, researchers should consider these methodological approaches:

• Co-sedimentation assays: Incubate purified recombinant Jakmip1 with polymerized microtubules (assembled from purified tubulin), followed by ultracentrifugation. Analyze the pellet and supernatant fractions by SDS-PAGE to determine binding.

• Immunofluorescence microscopy: In cultured neurons or cell lines expressing Jakmip1, perform double immunostaining with antibodies against Jakmip1 and tubulin to visualize co-localization patterns.

• Live-cell imaging: Generate fluorescently tagged Jakmip1 constructs (e.g., GFP-Jakmip1) for real-time visualization of Jakmip1 movement along microtubules in living cells.

• In vitro reconstitution: For advanced biophysical characterization, purify both Jakmip1 and tubulin to reconstitute the interaction in a controlled environment, allowing for detailed kinetic and structural analyses.

• Domain mapping: Create truncated versions of Jakmip1 to identify specific domains responsible for microtubule binding through iterative binding assays.

How can I effectively knockdown or knockout Jakmip1 in mouse models or cell cultures?

For effective manipulation of Jakmip1 expression in experimental systems, consider the following methodological approaches:

• siRNA/shRNA approaches for knockdown:

  • Design 3-4 siRNA sequences targeting different regions of Jakmip1 mRNA

  • Transfect cells using lipid-based reagents or electroporation

  • Validate knockdown efficiency by Western blotting (targeting the 80 kDa band) and qRT-PCR

  • Optimal knockdown typically occurs 48-72 hours post-transfection

• CRISPR-Cas9 for knockout:

  • Design guide RNAs targeting early exons of the Jakmip1 gene

  • For cell culture: Transfect cells with CRISPR-Cas9 components and select clones

  • For mouse models: Perform pronuclear injection of CRISPR components

  • Verify knockout by sequencing, Western blotting, and immunofluorescence

• Conditional knockout strategies:

  • Generate floxed Jakmip1 mouse lines for tissue-specific or temporally controlled deletion

  • Cross with appropriate Cre-driver lines for neuron-specific deletion

  • Validate the conditional knockout using tissue-specific protein and RNA analyses

• Phenotypic validation:

  • Assess cytoskeletal organization using immunofluorescence for tubulin

  • Examine GABA-B receptor transport using trafficking assays

  • Evaluate JAK1 signaling pathway activity through phosphorylation status of downstream targets

How does Jakmip1 interact with FMRP and what are the implications for autism research?

Research has demonstrated that Jakmip1 physically interacts with FMRP (Fragile X Mental Retardation Protein), forming a functional complex involved in RNA regulation. This interaction has significant implications for autism spectrum disorder (ASD) research due to the following findings:

• Physical interaction: Co-immunoprecipitation studies show that Jakmip1 binds to FMRP protein in neuronal cells, suggesting a direct physical interaction between these proteins .

• mRNA target regulation: Jakmip1 associates with and regulates well-established FMRP mRNA targets, including PSD95, which is crucial for synaptic development and function .

• Convergent pathways: The Jakmip1-FMRP interaction represents a convergence point between two autism-related pathways: JAK signaling and RNA translation regulation .

• Translational control: Jakmip1 may participate in FMRP-dependent translational regulation at the synapse through its interaction with CYFIP1, a regulator of FMRP-dependent translation .

For researchers investigating this interaction, methodological approaches should include:

• RNA immunoprecipitation followed by sequencing (RIP-seq) to identify shared mRNA targets

• Polysome profiling to assess translational regulation

• Proximity ligation assays to visualize the interaction in situ within neurons

• Structure-function analyses to map the interaction domains

What signaling pathways involve Jakmip1 and how can they be experimentally interrogated?

Jakmip1 participates in several signaling pathways that are relevant to neurodevelopment and neuronal function:

• JAK1 signaling pathway:

  • Jakmip1 may play a role in JAK1 signaling and regulate microtubule cytoskeleton rearrangements in response to cytokine stimulation

  • Experimental approach: Measure JAK1 phosphorylation and downstream STAT activation in the presence and absence of Jakmip1

• GABA-B receptor signaling:

  • Jakmip1 is involved in microtubule-dependent transport of GABA-B receptors, potentially affecting inhibitory neurotransmission

  • Experimental approach: Monitor GABA-B receptor surface expression and electrophysiological responses in Jakmip1-deficient neurons

• RNA regulation pathways:

  • Through its RNA binding activity and association with ribonucleoprotein complexes, Jakmip1 may regulate neuronal mRNA translation

  • Experimental approach: Perform RNA-seq and ribosome profiling in control vs. Jakmip1-deficient samples

• Cytoskeletal organization:

  • Given its association with microtubules, Jakmip1 likely influences cytoskeletal dynamics

  • Experimental approach: Live-cell imaging of microtubule dynamics in the presence and absence of Jakmip1

Each pathway can be experimentally interrogated using selective inhibitors, genetic manipulation (knockdown/knockout), and kinase activity assays to determine the specific contribution of Jakmip1.

What techniques can be used to study the role of Jakmip1 in neuronal RNA transport?

Given Jakmip1's RNA binding activity and association with microtubules, it likely plays a role in neuronal RNA transport. Researchers can investigate this function using these advanced techniques:

• Live RNA imaging techniques:

  • MS2-GFP system: Tag target mRNAs with MS2 binding sites and express MS2-GFP fusion protein to visualize RNA movement in real-time

  • FISH (Fluorescent In Situ Hybridization) combined with immunofluorescence for Jakmip1 to visualize RNA localization

  • Photoactivatable fluorescent protein-tagged Jakmip1 to track its movement with associated RNAs

• Biochemical RNA-protein interaction analyses:

  • RNA immunoprecipitation (RIP) with Jakmip1 antibodies followed by qRT-PCR or sequencing to identify bound transcripts

  • CLIP-seq (Cross-Linking Immunoprecipitation followed by sequencing) to map Jakmip1 binding sites on RNAs at nucleotide resolution

  • RNA electrophoretic mobility shift assays (EMSA) to characterize binding affinity and specificity

• Functional assays:

  • Local translation reporters in dendrites/axons to measure the impact of Jakmip1 manipulation

  • Subcellular fractionation to quantify RNA distribution in different neuronal compartments

  • Optogenetic control of Jakmip1 function to temporally regulate its activity during RNA transport

• Structural analyses:

  • Cryo-EM or X-ray crystallography of Jakmip1-RNA complexes to determine binding interfaces

  • NMR studies of the RNA-binding domains to characterize molecular interactions

How can mouse models of Jakmip1 dysfunction contribute to understanding autism spectrum disorders?

Mouse models with altered Jakmip1 expression or function provide valuable tools for understanding the molecular and behavioral aspects of autism spectrum disorders (ASD):

• Behavioral phenotyping:

  • Jakmip1 knockout or knockdown mouse models can be assessed for ASD-relevant behaviors including:

    • Social interaction deficits (three-chamber social approach test)

    • Repetitive behaviors (marble burying, self-grooming)

    • Communication abnormalities (ultrasonic vocalizations)

    • Cognitive flexibility (reversal learning tasks)

• Synaptic function analysis:

  • Electrophysiological studies (patch-clamp recordings) to measure excitatory/inhibitory balance

  • Long-term potentiation and depression (LTP/LTD) assessments to evaluate synaptic plasticity

  • Dendritic spine morphology analyses using Golgi staining or confocal microscopy

• Molecular pathway conservation:

  • Comparison of gene expression profiles between Jakmip1-deficient mice and human ASD samples

  • Assessment of convergence with other ASD risk genes through multi-omics approaches

  • Evaluation of FMRP-regulated mRNA translation in Jakmip1 model systems

• Developmental trajectory studies:

  • Time-course analyses of brain development in Jakmip1 models

  • Critical period investigations through conditional, temporally controlled Jakmip1 manipulation

  • Circuit-specific effects through targeted viral manipulations of Jakmip1 in specific brain regions

What are the considerations for developing specific antibodies against mouse Jakmip1?

When developing antibodies against mouse Jakmip1 for research applications, consider these technical aspects:

• Antigen selection strategies:

  • Use unique epitopes that distinguish Jakmip1 from related proteins (especially JAKMIP2, an important paralog )

  • Consider targeting:

    • N-terminal regions (amino acids 1-200)

    • C-terminal regions (last 100 amino acids)

    • Unique internal domains not shared with paralogs

  • Avoid highly conserved regions if species specificity is desired

• Antibody format considerations:

  • Polyclonal antibodies: Provide broad epitope recognition but higher background

  • Monoclonal antibodies: Offer high specificity but may be limited to single epitopes

  • Recombinant antibodies: Allow for consistent reproduction and modification

• Validation requirements:

  • Western blotting against recombinant protein and endogenous Jakmip1 (expected MW: 80 kDa)

  • Immunoprecipitation efficiency testing

  • Immunofluorescence with colocalization studies (microtubules and plasma membrane)

  • Testing in Jakmip1 knockout tissues as negative controls

  • Cross-reactivity assessment with human and rat Jakmip1 if multi-species reactivity is desired

• Application-specific optimizations:

  • For Western blotting: 1:1000 dilution is typically effective

  • For immunohistochemistry: Antigen retrieval methods should be optimized

  • For immunoprecipitation: Binding conditions (salt, detergent) must be calibrated

How can transcriptomic and proteomic approaches be integrated to understand Jakmip1 function in neurodevelopment?

To comprehensively understand Jakmip1's role in neurodevelopment, researchers should consider integrating transcriptomic and proteomic approaches:

• Multi-omics experimental design:

  • Parallel RNA-seq and proteomics from the same Jakmip1 knockout/knockdown samples

  • Developmental time-course analyses at key neurodevelopmental stages

  • Cell-type specific profiling using FACS-sorted neuronal populations

  • Subcellular fractionation to focus on synapse-enriched compartments

• RNA-focused approaches:

  • RNA immunoprecipitation sequencing (RIP-seq) to identify direct Jakmip1-bound mRNAs

  • Ribosome profiling to assess translational efficiency of target mRNAs

  • Single-cell RNA-seq to resolve cell-type specific effects

  • Spatial transcriptomics to map regional changes in brain tissue

• Protein-focused approaches:

  • Proximity labeling proteomics (BioID or APEX) to identify Jakmip1 interaction partners

  • Quantitative proteomics using TMT or SILAC labeling

  • Phosphoproteomics to assess signaling pathway alterations

  • Synaptic proteome analysis focusing on PSD95 and other postsynaptic targets

• Integrative data analysis strategies:

  • Correlation of transcript and protein abundance changes

  • Pathway enrichment analyses across both datasets

  • Network analysis to identify hub genes/proteins affected by Jakmip1 manipulation

  • Comparison with existing ASD-related multi-omics datasets

How conserved is Jakmip1 across species and what does this suggest about its function?

Jakmip1 shows notable conservation across mammalian species, providing insights into its evolutionary significance and functional importance:

• Cross-species homology:

  • Antibodies recognize Jakmip1 across human, mouse, and rat species, suggesting structural conservation

  • The molecular weight remains consistent at approximately 80 kDa across these species

  • Functional domains involved in microtubule binding and RNA interaction show high sequence identity

• Functional domain conservation:

  • The microtubule-binding regions show high conservation, supporting the importance of cytoskeletal interactions

  • RNA-binding motifs demonstrate evolutionary constraint, suggesting critical roles in RNA regulation

  • Regions mediating FMRP interaction are likely conserved based on functional studies

• Evolutionary implications:

  • High conservation suggests essential functions that have been maintained through selective pressure

  • The preservation of both cytoskeletal and RNA regulatory functions indicates the importance of these dual roles

  • Paralog development (such as JAKMIP2) suggests functional specialization through gene duplication events

Researchers comparing Jakmip1 functions across species should consider:

• Using cross-reactivity of antibodies as an advantage for comparative studies

• Designing functional rescue experiments across species

• Assessing whether disease-associated mutations occur in highly conserved regions

What is known about Jakmip1 paralogs and how do they differ functionally?

Jakmip1 has paralogous proteins, most notably JAKMIP2, which share structural similarities but may have distinct functional roles:

• Paralog identification:

  • JAKMIP2 is identified as an important paralog of JAKMIP1

  • Both belong to the JAKMIP family of proteins that share structural features including microtubule and JAK kinase interaction domains

• Structural comparisons:

  • Both paralogs contain coiled-coil domains important for protein-protein interactions

  • They share RNA-binding motifs but may have different RNA target specificities

  • Microtubule interaction domains show significant homology

• Functional distinctions:

  • While JAKMIP1 is strongly associated with autism and neurodevelopmental functions , JAKMIP2 may have more diverse roles

  • Tissue expression patterns likely differ, with JAKMIP1 showing stronger neuronal expression

  • Differential regulation by signaling pathways may lead to context-specific functions

• Experimental approaches to distinguish paralogs:

  • Paralog-specific antibodies can be developed targeting unique epitopes

  • Selective knockdown/knockout studies to identify non-redundant functions

  • Comparative interactome analyses to map distinct protein interaction networks

  • Replacement studies where one paralog is expressed in the absence of the other to test functional substitution

What are the most promising research directions for Jakmip1 in neurodevelopmental disorders?

Based on current knowledge, several high-priority research directions emerge for Jakmip1 in neurodevelopmental disorders:

• Mechanistic understanding of the Jakmip1-FMRP interaction:

  • Detailed structural characterization of the interaction interface

  • Identification of the complete set of co-regulated mRNAs

  • Development of small molecules that could modulate this interaction

  • Investigation of how this interaction is dysregulated in autism spectrum disorders

• Circuit-level effects of Jakmip1 dysfunction:

  • Region-specific and cell-type-specific manipulation of Jakmip1 expression

  • Assessment of local protein synthesis in dendrites and its impact on synaptic function

  • Evaluation of excitatory/inhibitory balance in relevant neural circuits

  • Developmental trajectory studies to identify critical periods for Jakmip1 function

• Translation to human neurodevelopmental disorders:

  • Screening for JAKMIP1 variants in larger autism cohorts

  • Development of patient-derived models (iPSCs, organoids) with JAKMIP1 mutations

  • Correlation of JAKMIP1 expression levels with clinical phenotypes

  • Exploration of potential biomarker applications

• Therapeutic targeting strategies:

  • Identification of druggable nodes in Jakmip1-associated pathways

  • Investigation of RNA-binding small molecules that could modulate Jakmip1 function

  • Development of peptide inhibitors that disrupt specific protein-protein interactions

  • Gene therapy approaches for cases with loss-of-function mutations

What are the key methodological challenges in Jakmip1 research and how might they be addressed?

Researchers working with Jakmip1 face several methodological challenges that require innovative solutions:

• Protein purification difficulties:

  • Challenge: Recombinant Jakmip1 may be prone to aggregation or misfolding due to its size (80 kDa) and multiple functional domains

  • Solutions:

    • Use of solubility tags (SUMO, MBP) to improve expression

    • Expression of functional domains separately

    • Optimization of buffer conditions to maintain native structure

    • Co-expression with binding partners to stabilize the protein

• Visualizing dynamic interactions:

  • Challenge: Capturing the dynamic association of Jakmip1 with microtubules and RNA in living neurons

  • Solutions:

    • Development of split fluorescent protein systems for specific interactions

    • Super-resolution microscopy techniques (STORM, PALM)

    • FRET-based biosensors to detect conformational changes

    • Lattice light-sheet microscopy for reduced phototoxicity during long imaging sessions

• Distinguishing direct vs. indirect effects:

  • Challenge: Separating primary effects of Jakmip1 manipulation from secondary consequences

  • Solutions:

    • Acute vs. chronic manipulation comparisons

    • Temporally controlled expression systems

    • Direct target identification through proximity labeling

    • Rescue experiments with specific domain mutants

• Translating findings across species:

  • Challenge: Ensuring that mouse model findings are relevant to human conditions

  • Solutions:

    • Parallel studies in human cellular models (neurons derived from iPSCs)

    • Comparative genomics approaches to identify conserved regulatory mechanisms

    • Focus on evolutionarily conserved interacting partners and pathways

    • Development of humanized mouse models for critical domains