Recombinant Rat Coiled-coil and C2 domain-containing protein 1B (Cc2d1b), partial

What is the basic molecular structure of rat Cc2d1b protein?

Rat Cc2d1b is a protein of approximately 120 kD that contains coiled-coil domains and a C2 domain. Unlike some membrane-associated proteins, Cc2d1b has no transmembrane segment but features four coiled-coil domains and a putative C-terminal consensus motif that enables binding to PDZ domains . This structural arrangement facilitates protein-protein interactions that are critical to its function. The protein's C2 domain is evolutionarily conserved and appears to have significant functional importance, as mutations in related C2 domain-containing proteins are associated with neurological disorders .

The structural features of Cc2d1b enable it to participate in multiple cellular processes, particularly those involving the coordination of protein complexes. These domains allow Cc2d1b to function in diverse cellular contexts, from synaptic organization to nuclear envelope reformation during cell division.

How do the expression patterns of Cc2d1b differ from its homolog Cc2d1a?

While Cc2d1a and Cc2d1b share structural similarities, they display distinct tissue expression patterns that suggest non-redundant physiological roles. Cc2d1a is highly expressed in neurons and has been implicated in intellectual disability and autism spectrum disorders . In contrast, Cc2d1b is predominantly expressed in myelinating glial cells and peripheral nerves .

What role does Cc2d1b play in nuclear envelope reformation?

Cc2d1b serves as an essential regulatory factor in the reformation of the nuclear envelope during mitosis. Research has demonstrated that Cc2d1b coordinates endoplasmic reticulum-derived membrane deposition and ESCRT-III polymerization to ensure proper nuclear envelope resealing after mitotic division .

Specifically, Cc2d1b acts as a regulator of ESCRT-III polymerization, localizing at the intersection between the nuclear envelope and microtubules at the time of nuclear resealing. It effectively "licenses" the formation of ESCRT-III polymers, ensuring the orderly reformation of the nuclear envelope . This coordination is critical for maintaining the proper compartmentalization of genetic material following cell division, as defects in this process can lead to genomic instability.

How is Cc2d1b involved in synaptic signaling and organization?

Cc2d1b appears to function as part of the cytomatrix at the active zone (CAZ) of synapses, a specialized region critical for calcium-dependent exocytosis of neurotransmitters. Studies have shown that Cc2d1b may interact with other CAZ proteins, including RIM1, bassoon, and potentially Munc13-1 . These interactions suggest Cc2d1b participates in the complex protein networks that regulate synaptic vesicle release and organization.

In co-immunoprecipitation experiments, Cc2d1b was found to associate with RIM1 and bassoon, components of the presynaptic active zone known to regulate neurotransmitter release . The PDZ domain-binding motif at the C-terminus of Cc2d1b appears essential for its binding to RIM1, suggesting a direct interaction between these proteins that may influence synaptic function .

What cognitive phenotypes are observed in Cc2d1b knockout mouse models?

Cc2d1b knockout (1b-KO) mice exhibit several cognitive deficits that highlight the protein's importance in learning and memory processes:

• Object memory deficits: In novel object recognition tests (NORT), 1b-KO mice show no preference for novel objects compared to familiar ones, indicating impaired object memory formation or recall .

• Spatial memory impairment: When tested in the Morris Water Maze, 1b-KO male mice demonstrated significant delays in learning the location of the hidden platform. Furthermore, they showed impaired spatial memory retention, spending less time searching in the target quadrant during probe trials .

• Sex-specific effects: Interestingly, some of these cognitive deficits display male-specific patterns. While both male and female 1b-KO mice showed delays in learning the platform location, only males exhibited significant spatial memory retention deficits .

These findings collectively suggest that Cc2d1b plays an important role in multiple aspects of memory formation and retention, with potentially sex-specific effects on certain cognitive processes.

How do phenotypes of Cc2d1a/Cc2d1b double heterozygous mice compare to single knockouts?

Double heterozygous (1a/1b-dHET) mice carrying heterozygous mutations in both Cc2d1a and Cc2d1b display phenotypes that partially overlap with, but are not identical to, the Cc2d1b single knockout:

These findings suggest partial redundancy between Cc2d1a and Cc2d1b in certain cognitive functions, but also indicate distinct roles. The compound heterozygous model reveals complex interactions between these related proteins in regulating cognitive processes.

What protein-protein interactions has Cc2d1b been shown to participate in?

Cc2d1b engages in several protein-protein interactions that are critical to its cellular functions:

• Interactions at the CAZ: Cc2d1b has been shown to associate with other proteins at the cytomatrix of the active zone, including RIM1 and bassoon. These interactions were demonstrated through co-immunoprecipitation experiments and cosedimentation assays .

• PDZ domain interactions: The C-terminal consensus motif of Cc2d1b enables binding to PDZ domains of other proteins, particularly RIM1. This interaction was confirmed through pull-down assays where RIM1 stoichiometrically bound to GST-CAST-4 containing the C-terminal consensus motif .

• ESCRT-III regulation: Cc2d1b interacts with components of the ESCRT-III machinery, inhibiting ESCRT-III polymerization in vitro. This regulatory function is essential for coordinating nuclear envelope reformation during mitosis .

These diverse interactions position Cc2d1b as a multifunctional adaptor protein that participates in various cellular processes through direct and indirect protein associations.

What is the relationship between Cc2d1b and the endosomal pathway?

Cc2d1b belongs to the evolutionarily conserved Lgd protein family, which plays important roles in regulating signaling receptor degradation via the endosomal pathway . The functional significance of this relationship is highlighted by studies of Lgd function, which show that its loss results in ectopic and ligand-independent activation of the Notch pathway .

This connection to Notch signaling is particularly relevant in the context of myelination, as Notch promotes early stages of Schwann cell development but inhibits myelination . Given that Cc2d1b is expressed in myelinating glial cells, it may modulate myelination through the recruitment of specific signaling complexes that regulate endosomal trafficking and Notch pathway activity.

What are effective methods for genotyping Cc2d1b knockout and heterozygous mouse models?

Effective genotyping of Cc2d1b mouse models can be performed using polymerase chain reaction (PCR) on DNA samples:

• Sample preparation: Extract DNA from tail samples and digest with proteinase K (such as from New England Biolabs) .

• PCR reaction composition: Set up 50 μL reactions containing:

  • GoTaq Flexi buffer

  • 100 μM dNTPs

  • 50 μM each of forward and reverse primers

  • 1 mM MgCl₂

  • 1.25 U GoTaq Flexi DNA polymerase

• Analysis: Analyze PCR products by gel electrophoresis on a 1.0% agarose gel to detect the presence of target bands .

For Cc2d1b null mice, it's important to note that they carry an engrailed 2 splice acceptor (En2SA) gene-trap allele with bicistronic expression of β-galactosidase and a neomycin resistance cassette, flanked by FRT recombination sites, in the genomic region between exons 2 and 3 of Cc2d1b . Primer design should account for this genetic modification.

What behavioral tests are most effective for assessing Cc2d1b function in cognition?

Several behavioral paradigms have proven effective for evaluating the cognitive functions affected by Cc2d1b:

• Novel Object Recognition Test (NORT):

  • This test evaluates object memory by measuring preference for novel versus familiar objects

  • Design: In the training phase, mice explore two identical objects; in the test phase (after a 15-minute interval), one familiar object is replaced with a novel object

  • Analysis: Calculate preference for the novel object as a ratio of exploration time

• Morris Water Maze:

  • This test assesses hippocampus-dependent spatial learning and memory

  • Multiple phases include visible platform (VP), hidden platform (HP), and reversal (RV) tests

  • Analysis: Measure latency to escape in each phase, and assess memory retention through probe trials that quantify time spent in the target quadrant

• Data analysis approaches:

  • For Morris Water Maze hidden platform phases: Two-way ANOVA with repeated measures

  • For individual timepoints and probe analysis: Multiple t-tests with equal variance

These behavioral paradigms can effectively capture the cognitive deficits associated with Cc2d1b dysfunction, particularly in memory formation and retention.

How might Cc2d1b contribute to myelination processes?

Several lines of evidence suggest potential roles for Cc2d1b in myelination:

• Expression pattern: Cc2d1b is expressed in myelinating glial cells and peripheral nerves, suggesting direct involvement in glial cell function .

• Notch pathway regulation: As a member of the Lgd protein family, Cc2d1b may regulate the endosomal pathway and Notch signaling. Since Notch promotes early Schwann cell development but inhibits myelination, Cc2d1b could influence myelination through modulation of this pathway .

• Learning and memory connection: There is growing evidence that myelin plays a role in learning and memory. Given that Cc2d1b knockout mice show delayed memory acquisition and retention, it is possible that these deficits are partially related to altered myelination processes .

Further investigation is warranted to determine the specific mechanisms by which Cc2d1b influences myelination, particularly in central nervous system white matter development and maintenance.

What is the potential relationship between Cc2d1b and YAP/TAZ signaling in Schwann cells?

YAP and TAZ have been identified as regulators of Cc2d1b in Schwann cells, suggesting a potential role for Cc2d1b in the mechanotransduction pathways that govern Schwann cell differentiation and myelination . Ablation of YAP/TAZ has been shown to alter the expression of transcription regulators known to regulate Schwann cell myelin gene transcription and differentiation, including Cc2d1b .

This regulatory relationship places Cc2d1b within the broader context of the signaling networks that control Schwann cell development and myelination. Since Schwann cells are exquisitely sensitive to environmental elasticity, with their differentiation and myelination capacity depending on mechanical stimuli transduction by YAP/TAZ, Cc2d1b may play a role in linking these mechanical cues to cellular responses.

What is the controversy regarding the transcriptional role of Cc2d1b?

The potential role of Cc2d1b as a transcriptional regulator remains subject to debate in the scientific literature:

This controversy raises several important research questions:

• Does Cc2d1b translocate from the cytoplasm to the nucleus under specific conditions?

• Can Cc2d1b directly control transcription in vivo?

• If Cc2d1b does influence transcription, does it do so through direct DNA binding or through interactions with other transcriptional regulators?

Resolution of these questions will require careful subcellular localization studies under various conditions, as well as chromatin immunoprecipitation experiments to assess potential direct interactions with DNA.

How might the functions of Cc2d1b in nuclear envelope reformation and synaptic organization be interconnected?

Cc2d1b's dual roles in nuclear envelope reformation and potential synaptic organization raise intriguing questions about whether these functions might be mechanistically related:

• Protein-protein interaction networks: Both functions involve Cc2d1b in coordinating complex protein assemblies (ESCRT-III at the nuclear envelope; potentially RIM1/bassoon/Munc13-1 at synapses) .

• Membrane organization: Both processes involve precise spatial and temporal regulation of membrane structures.

• Functional implications: Disruption of nuclear envelope reformation could potentially impact neuronal function and synaptic organization through effects on gene expression or nuclear-cytoplasmic transport.

Future research should investigate whether common molecular mechanisms underlie these seemingly distinct functions, particularly in the context of neuronal development and differentiation where both nuclear division and synaptic organization are critical processes.