Recombinant Mouse Nuclear pore complex protein Nup88 (Nup88)

What is Nup88 and what is its structural organization?

Nup88 is a 741 amino acid nucleoporin that forms a critical component of the nuclear pore complex (NPC), which regulates macromolecular transport between the nucleus and cytoplasm in eukaryotic cells. The protein contains distinct functional domains, with the N-terminal domain (residues 1-550) mediating interactions with nuclear lamins, while the C-terminal domain (residues 551-741) is involved in other protein-protein interactions . Structurally, Nup88 contains a β-propeller domain that interacts with the NPC-targeting domain of nucleoporin Nup98, which localizes to both nuclear and cytoplasmic sides of the NPC .

How does Nup88 function in nuclear transport?

Nup88 functions as part of a regulatory network controlling nucleocytoplasmic transport. The Nup88/Nup214 complex is not directly required for nuclear protein import but plays a crucial role in CRM1-mediated nuclear export . Research has shown that Nup88 depletion in both Drosophila and human cells causes nuclear accumulation of NF-κB transcription factors, which are CRM1 targets .

Beyond transport, Nup88 participates in cell cycle regulation through its interactions with other nuclear proteins. It forms part of a regulatory axis with NUP98-RAE1 that ensures accurate chromosome segregation by controlling premitotic anaphase-promoting complex/cyclosome (APC/C) activity against select substrates . This demonstrates how individual NPC components and subcomplexes maintain genomic integrity by regulating specific cell cycle processes in addition to their transport roles.

What detection methods are available for studying mouse Nup88?

Several robust methodologies are available for detecting and studying mouse Nup88:

• Antibody-based detection: Commercial antibodies like Nup88 Antibody (H-7), a mouse monoclonal IgG2b kappa light chain antibody, can detect Nup88 protein from mouse, rat, and human origins using multiple techniques :

  • Western blotting (WB)

  • Immunoprecipitation (IP)

  • Immunofluorescence (IF)

  • Immunohistochemistry with paraffin-embedded sections (IHCP)

  • Enzyme-linked immunosorbent assay (ELISA)

• Epitope-specific antibodies: Researchers can utilize antibodies directed against specific regions of Nup88, including:

  • N-terminus (residues 27-45)

  • Central region (residues 314-425)

  • C-terminus (residues 509-741)

• Fluorescence tagging: Experimental approaches often employ GFP-tagged versions of Nup88 for live cell imaging and localization studies .

• Immunoelectron microscopy: For high-resolution localization studies, gold-conjugated antibodies against different Nup88 domains can be used with electron microscopy .

How does Nup88 interact with the nuclear lamina?

Nup88 exhibits a specific interaction with A-type lamins that is not observed with B-type lamins. In vitro and in vivo studies have demonstrated that:

• The N-terminus of Nup88 (residues 1-550) mediates binding to lamin A .

• Nup88 specifically binds to the tail domain of lamin A but not to lamins B1 and B2 .

• This interaction can be visualized in cellular contexts, as expression of GFP-tagged lamin A causes masking of binding sites for Nup88 antibodies in immunofluorescence assays .

The interaction between Nup88 and lamin A appears to be physiologically significant, as it is disrupted in cells expressing lamin A mutants associated with laminopathic diseases . This suggests that Nup88-lamin A binding may play a role in maintaining nuclear structure and function, and disruption of this interaction could contribute to disease pathogenesis.

The localization of a pool of Nup88 on the nuclear side of the NPC provides an unexpected binding site for nuclear lamin A, potentially forming a link between the nuclear pore complex and the nuclear lamina .

What is the role of Nup88 in chromosomal instability and cancer?

Nup88 overexpression is a significant driver of tumorigenesis through mechanisms leading to chromosomal instability (CIN) and aneuploidy. Research utilizing transgenic mice has revealed the following key mechanisms:

• Sequestration of NUP98-RAE1: When overexpressed, NUP88 sequesters NUP98-RAE1 away from APC/C CDH1, triggering premature proteolysis of polo-like kinase 1 (PLK1), a tumor suppressor and multitasking mitotic kinase .

• Disruption of mitotic processes: The premature destruction of PLK1 disrupts centrosome separation, causing:

  • Mitotic spindle asymmetry

  • Merotelic microtubule-kinetochore attachments

  • Lagging chromosomes

  • Aneuploidy

• Cancer predisposition: Mice engineered with doxycycline-inducible expression of Nup88 showed:

  • 56% of transgenic mice developed at least one neoplastic lesion compared to 21% of control mice

  • Particular predisposition to lung tumors, which often express high NUP88 levels in human patients

• Pre-tumorigenic aneuploidy: Lung tissues from Nup88 transgenic mice showed significant aneuploidy before overt tumor formation, as assessed by fluorescence in situ hybridization (FISH) and karyotypic analysis .

• Decreased tumor suppressor levels: Both PLK1 and securin protein levels were lower in transgenic Nup88 lungs compared to controls, consistent with untimely APC/C CDH1-mediated degradation .

These findings establish Nup88 as part of a critical regulatory network that safeguards against merotely-induced genomic instability, with deregulation of this axis potentially contributing to the initiating stages of a broad spectrum of human cancers.

How can Nup88 function be modulated in experimental models?

Researchers have developed several approaches to experimentally modulate Nup88 function:

• Transgenic overexpression models:

  • Doxycycline-inducible Nup88 expression systems in mice allow for temporal control of overexpression

  • These models express substantially higher levels of NUP88 than control mice in a broad spectrum of tissues

  • Transgene expression is tightly controlled in vivo, with no expression in the absence of doxycycline

• Gene inactivation approaches:

  • TALEN-mediated gene inactivation has been used to generate Nup88+/- mice

  • These can be crossed with other genetic backgrounds (e.g., Nup98+/- Rae1+/-) to study genetic interactions

• In vitro binding assays:

  • Using purified recombinant his-tagged lamin A immobilized on Ni-sepharose beads incubated with in vitro synthesized 35S-labeled Nup88 fragments

  • This approach allows for domain-specific interaction studies between Nup88 and its binding partners

• Cell culture models:

  • Mouse embryonic fibroblasts (MEFs) derived from transgenic animals provide a controlled system for studying Nup88 function

  • Expression of GFP-tagged lamin A in cells can be used to study Nup88-lamin interactions

What evidence supports the role of Nup88 in the NUP88-NUP98-RAE1-APC/C axis?

Several lines of experimental evidence support the role of Nup88 in regulating the NUP98-RAE1-APC/C axis:

• Genetic interaction studies: Reducing NUP88 protein levels in Nup98+/- Rae1+/- MEFs restored G2- and M-phase securin protein levels, demonstrating genetic interaction between these components .

• Chromosome segregation errors: These errors were reduced from 37% in Nup98+/- Rae1+/- MEFs to 18% in Nup88+/- Nup98+/- Rae1+/- triple-mutant MEFs, and the mitotic checkpoint was fully restored in triple-mutant MEFs .

• Substrate protection: The data suggest that cytoplasmic NUP98-RAE1 normally inhibits APC/C CDH1 but is sequestered away by NUP88 when overexpressed, promoting the ubiquitin ligase activity of APC/C CDH1 .

• In vivo validation: Analysis of non-transformed lung tissue from 5-month-old Nup88 transgenic mice showed:

  • Significantly higher aneuploidy rates compared to control lung tissues

  • Lower PLK1 and securin protein levels compared to controls

These findings establish that NUP88, NUP98, and RAE1 comprise a regulatory network that inhibits premitotic activity of the anaphase-promoting complex/cyclosome (APC/C), with dysregulation of this network contributing to chromosomal instability.

What are the best practices for working with recombinant mouse Nup88?

When working with recombinant mouse Nup88, researchers should consider the following best practices:

• Domain-specific approaches: For interaction studies, consider that:

  • The N-terminal domain (residues 1-550) mediates interactions with lamin A

  • The β-propeller domain interacts with the NPC-targeting domain of Nup98

  • Different domains may require different buffer conditions for optimal activity

• Antibody selection: Choose antibodies strategically based on experimental needs:

  • Nup88 Antibody (H-7) is available in multiple formats:

    • Non-conjugated: For general detection (sc-365868)

    • Agarose-conjugated: For immunoprecipitation (sc-365868 AC)

    • HRP-conjugated: For direct detection in Western blots

    • Fluorophore-conjugated: For immunofluorescence (PE, FITC, and various Alexa Fluor® conjugates)

• Epitope considerations: When designing experiments, be aware that:

  • GFP-tagged lamin A expression can mask binding sites for Nup88 antibodies

  • Lamin A mutants associated with laminopathic diseases may disrupt interactions with Nup88

• Expression systems: For controlled expression:

  • Doxycycline-inducible systems provide tight regulation in vivo

  • Some leakiness may occur in cultured MEFs even without doxycycline induction

How can researchers distinguish between nuclear and cytoplasmic pools of Nup88?

Distinguishing between nuclear and cytoplasmic pools of Nup88 requires specialized techniques:

• Immunoelectron microscopy: Gold-conjugated antibodies against different Nup88 domains can precisely localize the protein at the ultrastructural level. Studies with Xenopus laevis oocyte nuclei showed:

  • Antibodies against the N-terminus of Nup88 recognized epitopes on both nuclear and cytoplasmic sides of the NPC

  • Approximately 40% of gold particles were associated with the nuclear face at a mean distance of -53.8 nm ± 22.6 nm from the central plane

  • The mean radial distance was 37.1 nm ± 13.9 nm

• Domain-specific antibodies: Using antibodies against different regions:

  • N-terminus (residues 27-45)

  • Central region (residues 314-425)

  • C-terminus (residues 509-741)

• Cell fractionation: Biochemical separation of nuclear and cytoplasmic fractions followed by Western blotting can quantify the distribution of Nup88 between compartments.

• Super-resolution microscopy: Techniques like STORM or PALM can resolve the localization of Nup88 with nanometer precision, helping to distinguish nuclear from cytoplasmic pools.

What are key controls for Nup88 overexpression studies?

When designing Nup88 overexpression experiments, researchers should implement these critical controls:

• Expression control: For doxycycline-inducible systems, include:

  • Non-induced transgenic controls (no doxycycline) to assess leakiness

  • Wild-type controls with doxycycline to control for drug effects

  • Expression level verification across different tissues

• Genetic background controls: In mouse models, use:

  • Transgenic activator (TA) mice without the Nup88 transgene as controls for in vitro experiments

  • Consistent genetic backgrounds for meaningful comparisons

• Age-matched cohorts: For cancer studies:

  • Establish age-matched cohorts of Nup88 transgenic and control mice

  • Monitor tumor development over consistent time periods (e.g., 14 months)

• Molecular verification: Confirm mechanism by:

  • Assessing levels of predicted downstream targets (PLK1, securin)

  • Validating protein-protein interactions under overexpression conditions

  • Measuring aneuploidy rates in pre-tumorigenic tissues

How can researchers identify and validate novel Nup88 interaction partners?

To identify and validate novel Nup88 interaction partners, researchers can employ these approaches:

• Domain-specific binding assays:

  • In vitro binding with purified recombinant his-tagged proteins immobilized on Ni-sepharose beads

  • Testing interactions with in vitro synthesized 35S-labeled Nup88 fragments (full-length, N-terminal domain, C-terminal domain)

  • Separating bound and unbound fractions by SDS-PAGE followed by autoradiography

• Co-immunoprecipitation strategies:

  • Using agarose-conjugated Nup88 antibodies (such as sc-365868 AC)

  • Performing reciprocal IPs with antibodies against suspected interaction partners

  • Including RNase/DNase treatments to exclude nucleic acid-mediated interactions

• Protein localization:

  • Immunofluorescence co-localization studies using antibodies or fluorescently tagged proteins

  • FRET or BiFC approaches to validate direct interactions in living cells

  • Assessing epitope masking when potential partners are overexpressed

• Genetic interaction studies:

  • Generating single, double, and triple mutants (e.g., Nup88+/-, Nup98+/- Rae1+/-, and Nup88+/- Nup98+/- Rae1+/- MEFs)

  • Determining if phenotypes of double mutants are exacerbated or rescued in triple mutants

What therapeutic implications arise from understanding Nup88's role in cancer?

Understanding Nup88's role in cancer suggests several therapeutic strategies:

• Targeting the NUP88-NUP98-RAE1 axis:

  • Developing inhibitors that prevent NUP88 from sequestering NUP98-RAE1

  • Small molecules that restore normal PLK1 levels in cells overexpressing NUP88

  • Peptide-based approaches to disrupt specific protein-protein interactions

• Biomarker development:

  • Using NUP88 overexpression as a diagnostic or prognostic marker

  • Combining NUP88 with PLK1 and securin levels for improved cancer detection

  • Monitoring chromosome instability in tissues with elevated NUP88

• Synthetic lethality approaches:

  • Identifying genes that, when inhibited, cause selective death in cells overexpressing NUP88

  • Targeting the CIN phenotype that arises from NUP88 overexpression

• Prevention strategies:

  • Screening for compounds that normalize NUP88 expression

  • Interventions that protect PLK1 from premature degradation

These approaches could be particularly relevant for lung cancer and other tumor types that frequently overexpress NUP88 .

What questions remain unanswered about Nup88 biology?

Despite significant advances, several key questions about Nup88 remain unanswered:

• Developmental roles: How does Nup88 function during embryonic development and tissue differentiation?

• Tissue-specific effects: Why does Nup88 overexpression predominantly lead to lung tumors in mouse models when the protein is expressed broadly?

• Post-translational modifications: How is Nup88 itself regulated by phosphorylation or other modifications? Evidence suggests Nup88 undergoes phosphorylation by ATM or ATR .

• Species conservation: To what extent are Nup88's functions conserved across species, and what can model organisms tell us about its fundamental roles?

• Non-cancer functions: What roles does Nup88 play in normal tissue homeostasis, aging, or immune function?

• Therapeutic targeting: Can Nup88 or its interactions be safely targeted therapeutically without disrupting essential nuclear transport functions?

• Structural biology: What is the complete three-dimensional structure of Nup88, and how do conformational changes affect its various functions?