Recombinant Chicken Kinesin-like protein KIF2A (KIF2A), partial

What is KIF2A and how does it function at the molecular level?

KIF2A belongs to the kinesin-13 family (M-kinesin) of proteins characterized by an internal motor domain. Unlike conventional kinesins that function as molecular motors, KIF2A destabilizes microtubules in an ATP-dependent manner . The protein binds to microtubule ends and catalyzes depolymerization, which is essential for regulating microtubule dynamics during neuronal development and cell division. KIF2A's activity regulates critical cellular processes including cell migration, axon elongation, and pruning in the developing nervous system . Recent studies have demonstrated that KIF2A can also function as a microtubule stabilizer under specific cellular contexts, particularly during cytokinesis in embryonic stem cells, indicating a dual role that depends on cellular context and developmental stage .

How is KIF2A expression regulated during neural development?

KIF2A expression follows a specific temporal and spatial pattern during development. In early postnatal development, KIF2A is broadly expressed throughout the brain, reaching peak expression in the hippocampus during early postnatal weeks . After this peak period, expression becomes gradually restricted to specific brain regions, particularly the hippocampus, where neurogenesis continues throughout life . This expression pattern suggests a critical role in postnatal neuronal development, particularly in regions undergoing continuous neurogenesis and circuit refinement . Studies using tamoxifen-inducible conditional knockout mice have demonstrated that postnatal KIF2A expression is essential for proper dentate granule cell development and hippocampal wiring .

What are the optimal methods for assessing KIF2A-mediated microtubule dynamics in neurons?

Assessment of KIF2A-mediated microtubule dynamics in neurons requires specialized techniques that can capture real-time changes in the cytoskeleton. The following methods have proven most effective:

• EB3 comet analysis: This technique involves time-lapse imaging of neurons expressing EB3-GFP to visualize growing microtubule plus-ends. KIF2A activity influences the velocity, direction, and persistence of EB3 comets. Studies have shown that overexpression of mutant KIF2A induces microtubule stability and halts microtubule depolymerization, which can be quantitatively measured through EB3 comet analysis .

• Immunostaining for post-translational modifications: Acetylated and detyrosinated tubulin are markers for stable microtubules. During cytokinesis, KIF2A controls microtubule acetylation, suggesting a role in maintaining microtubule stability. Quantitative immunofluorescence can be used to assess these modifications in neurons with manipulated KIF2A expression .

• Live imaging of fluorescently tagged microtubules: This approach allows direct visualization of microtubule dynamics in living neurons. In KIF2A knockout or mutant neurons, altered patterns of microtubule growth, shrinkage, and stability can be observed, particularly at growth cones and branch points .

How does KIF2A regulate neuronal migration and positioning?

KIF2A plays distinct roles in both radial migration of glutamatergic neurons and tangential migration of GABAergic interneurons during cortical development . The mechanisms involve:

• Regulation of leading-edge dynamics: KIF2A controls microtubule remodeling at the leading edge of migrating neurons, affecting their motility and directionality. Conditional deletion of KIF2A in GABAergic interneurons impairs their migration velocity, net displacement, and directionality, similar to the phenotype observed in KIF2A-deficient neuroblasts migrating along the rostral migratory stream .

• Multipolar-to-bipolar transition: During radial migration, KIF2A is crucial for the transition from multipolar to bipolar morphology. Manipulation of KIF2A expression increases the number of multipolar cells in the upper intermediate zone and delays radial migration .

• Nucleokinesis and centrosome positioning: KIF2A regulates microtubule dynamics around the centrosome, which is essential for proper nucleokinesis during neuronal migration. Disruption of this function leads to migration defects and abnormal neuronal positioning in the developing brain .

What is the mechanism of KIF2A-mediated dendro-axonal conversion in dentate granule cells?

The dendro-axonal conversion observed in KIF2A knockout dentate granule cells represents a fundamental disruption of neuronal polarity that occurs through several mechanisms:

• Failure of neurite length regulation: KIF2A normally suppresses the elongation of future dendrites by destabilizing microtubules during early developmental stages. In KIF2A-deficient neurons, dendrites become abnormally extended, eventually acquiring axonal properties .

• Altered microtubule stability: Loss of KIF2A's depolymerizing activity leads to increased microtubule stability in dendrites, which typically display more dynamic microtubules than axons. This shift in stability may trigger molecular changes that promote axonal specification in dendrites .

• Molecular reprogramming: As dendrites extend beyond their normal length in KIF2A-deficient neurons, they undergo molecular changes, including the expression of axonal markers. This process resembles what occurs in PTEN knockout neurons, suggesting potential overlap in the signaling pathways regulated by KIF2A and PTEN .

The consequences of dendro-axonal conversion include the formation of aberrant connections, mossy fiber sprouting, and the establishment of recurrent excitatory circuits that contribute to hyperexcitability and epilepsy in KIF2A-deficient mice .

How do different KIF2A mutations affect protein function and cellular phenotypes?

KIF2A mutations associated with human disorders show distinct molecular and cellular effects, primarily related to their impact on microtubule dynamics:

Research using conditional knock-in mouse models has demonstrated that the p.His321Asp mutation exerts pathogenicity through a dominant negative mechanism, affecting both neuronal migration and circuit formation . When expressed in mouse models, these mutations lead to hyperactivity, impaired novel object recognition, and epileptic seizures, recapitulating aspects of human neurological disorders associated with KIF2A mutations .

What cellular processes are disrupted in inhibitory interneurons lacking KIF2A?

Conditional ablation of KIF2A specifically in forebrain inhibitory neurons reveals several critical cellular processes that are disrupted, contributing to circuit dysfunction and epilepsy:

These disruptions collectively lead to functional alterations in neuronal circuitries, contributing to epileptic susceptibility in mice with interneuron-specific KIF2A deletion . This provides insight into how KIF2A mutations might contribute to epilepsy in human patients.

How can the role of KIF2A in spindle assembly and chromosome segregation be investigated?

Investigating KIF2A's role in spindle assembly and chromosome segregation requires specialized techniques to visualize and quantify its dynamic behavior during cell division:

• Genome-engineered cell lines with fluorescent KIF2A: CRISPR-Cas9 technology can be used to create knock-in cell lines expressing fluorescently tagged KIF2A from its endogenous locus. This approach has revealed that KIF2A primarily localizes to spindle poles during metaphase and regulates spindle length consistent with its role as a microtubule minus-end depolymerase .

• Live-cell imaging of microtubule dynamics: Time-lapse microscopy of dividing cells with labeled tubulin and KIF2A enables visualization of dynamic interactions during spindle assembly. This technique has shown that KIF2A depolymerizes microtubule minus-ends, in contrast to its paralog MCAK, which acts on plus-ends .

• In vitro reconstitution assays: Combining purified components including γ-tubulin ring complex (γTuRC), KIF2A, and microtubule severing enzymes has revealed that KIF2A still allows microtubule nucleation by plus-end growth from γTuRC, which in turn protects the minus end against KIF2A binding. Efficient γTuRC-uncapping requires the combined action of KIF2A and a microtubule severing enzyme .

These approaches have provided insights into how KIF2A coordinates with other factors to regulate spindle assembly and function, with implications for understanding developmental disorders associated with KIF2A mutations.

What are the emerging concepts regarding KIF2A's role transition from depolymerase to stabilizer?

Recent research has uncovered an unexpected dual role of KIF2A as both a microtubule depolymerase and stabilizer, particularly during cytokinesis in embryonic stem cells. The mechanisms underlying this functional transition include:

This functional duality has significant implications for understanding KIF2A's roles in different cellular contexts and may explain some of the complex phenotypes observed in KIF2A-deficient systems, including effects on pluripotency and mRNA homeostasis in embryonic stem cells .

What are the critical unanswered questions regarding KIF2A function in neurological disorders?

Despite significant advances in understanding KIF2A's roles in neuronal development and function, several critical questions remain unanswered:

• Temporal specificity of KIF2A requirements: How do the requirements for KIF2A function change across developmental stages and in mature neurons? Research suggests distinct roles during embryonic development, early postnatal periods, and in adult neurons, but the molecular mechanisms underlying these stage-specific functions remain unclear .

• Interaction with other cytoskeletal regulators: How does KIF2A coordinate with other microtubule-regulating proteins in neurons? Potential interactions with pathways involving PTEN have been suggested, but comprehensive mapping of the KIF2A interactome in different neuronal contexts is needed .

• Relevance of animal models to human pathology: How well do the hippocampal phenotypes observed in KIF2A knockout mice translate to human patients with KIF2A mutations? Future research should explore the hippocampal features of patients with KIF2A-related malformations of cortical development .

• Therapeutic targets: What downstream pathways affected by KIF2A dysfunction might represent viable therapeutic targets? Identifying key molecular pathways that mediate the effects of KIF2A loss on neuronal development and circuit function could reveal new approaches for treating KIF2A-related disorders .

Addressing these questions will require integrated approaches combining structural biology, advanced imaging, genetic models, and clinical studies to fully understand how KIF2A dysfunction contributes to neurological disorders and to develop potential therapeutic strategies.

What methodological advances could improve research on recombinant KIF2A?

Advancing research on recombinant KIF2A will require methodological innovations in protein engineering, structural analysis, and functional assays:

• Optimization of expression systems: Current approaches for producing recombinant KIF2A often yield proteins with suboptimal activity due to folding issues or lack of post-translational modifications. Development of expression systems that better recapitulate the native conditions, such as mammalian cell-based systems with appropriate chaperones, could improve protein quality .

• Single-molecule techniques: Applying single-molecule imaging and force measurements to study KIF2A interactions with microtubules could provide insights into the mechanisms of depolymerization and the transition to stabilizing functions. These approaches could reveal how nucleotide binding and hydrolysis regulate KIF2A activity at the molecular level .

• Cryo-electron microscopy: High-resolution structural analysis of KIF2A bound to microtubules in different nucleotide states could elucidate the conformational changes that drive microtubule depolymerization or stabilization. This information would be valuable for designing specific modulators of KIF2A activity .

• Optogenetic control of KIF2A activity: Development of light-controllable KIF2A variants would enable precise temporal and spatial manipulation of its activity in living cells, allowing researchers to dissect its functions in specific subcellular compartments and at different stages of neuronal development .

These methodological advances would significantly enhance our ability to study KIF2A's diverse functions and potentially lead to new therapeutic approaches for disorders associated with KIF2A dysfunction.