Recombinant Drosophila melanogaster Transitional endoplasmic reticulum ATPase TER94 (TER94), partial

What is TER94 and what are its key cellular functions?

TER94 is an AAA+ ATPase (ATPases Associated with diverse cellular Activities) that plays essential roles in multiple cellular processes in Drosophila melanogaster. It participates in protein quality control, membrane fusion of the Golgi apparatus and endoplasmic reticulum network, nuclear envelope reformation, and DNA replication . TER94 is critically involved in wing development by regulating the Hippo signaling pathway, which controls cell proliferation and apoptosis .

The gene is essential for viability, as evidenced by the fact that P-element insertions in the TER94 locus result in lethality, and this lethal phenotype can be reverted by excision of the P-element . Functionally, TER94 exhibits ATPase activity that is necessary for its biological functions, including proper wing size regulation . Its conservation across species underscores its biological importance, with its human homolog (hVCP/p97) capable of functionally substituting for TER94 in Drosophila wing size modulation .

What experimental systems are available to study TER94 function?

Researchers have developed several experimental approaches to study TER94:

• Genetic manipulation: Various P-element insertions into the TER94 locus (e.g., l(2)03775 and l(2)k15502) have been identified and characterized as lethal mutations . Additionally, ethylmethane sulfonate-induced mutations mapped to the TER94 locus provide genetic tools for studying its function .

• FLP/FRT system: Researchers can use the FLP/FRT-mediated recombination system to induce mitotic clones, allowing for cell-level analysis of TER94 function in specific tissues .

• RNAi-mediated knockdown: The GAL4/UAS system can be employed to express double-stranded RNA against TER94 in specific tissues. This approach has been used with various drivers:

  • Ubiquitous knockdown using Actin5C-Gal4 (resulting in complete developmental lethality)

  • Neuronal knockdown using elav-Gal4 (causing reduced viability and early death after eclosion)

  • Cholinergic neuron-specific knockdown using Cha-Gal4 (leading to death 20-30 days after eclosion)

  • Eye-specific knockdown using GMR-Gal4 (resulting in progressive degeneration of the compound eye)

• Rescue experiments: Co-expression of wild-type or mutant TER94 with RNAi constructs can be used to validate knockdown specificity and to assess the functional consequences of disease-associated mutations .

How does TER94 interact with the Hippo signaling pathway?

TER94 plays a critical regulatory role in the Hippo signaling pathway, which is essential for proper wing development in Drosophila. Specifically:

• TER94 reciprocally binds to Merlin (Mer), which is a critical upstream component of the Hippo pathway .

• This interaction disrupts Mer's association with Expanded (Ex) and Kibra (Kib), preventing the formation of the Ex-Mer-Kib complex .

• The disruption of this complex leads to inactivation of the Hippo pathway, which ultimately promotes proper wing development .

• When TER94 is depleted, it results in reduced wing size and increased apoptosis, which can be rescued by inhibiting the Hippo pathway .

• Loss of TER94 enables the suppression of Hippo target genes, indicating that TER94 normally functions to modulate Hippo pathway activity .

This mechanism highlights TER94's role as a positive regulator of wing size through its interference with the Ex-Mer-Kib complex and consequent suppression of the Hippo pathway.

What are the molecular mechanisms distinguishing loss-of-function versus gain-of-function in TER94/VCP-related diseases?

The distinction between loss-of-function and gain-of-function mechanisms in TER94/VCP-related diseases has important implications for understanding pathogenesis and developing therapeutic approaches. Research in Drosophila models has provided valuable insights:

• Evidence supporting loss-of-function mechanism:

  • RNAi-mediated knockdown of TER94 in Drosophila causes neurodegeneration, premature lethality, reduction in brain volume, and alterations in mushroom body morphology .

  • These phenotypes can be rescued by co-expression of wild-type TER94, confirming they result from TER94 deficiency .

  • Importantly, disease-linked mutants (such as the human A229E mutation) show reduced rescue capability compared to wild-type TER94, suggesting that these mutations cause loss of function .

• Experimental approaches to distinguish mechanisms:

  • Genetic rescue experiments comparing wild-type and mutant TER94 provide the most direct evidence for loss-of-function versus gain-of-function .

  • When wild-type TER94 restores normal phenotypes but disease-associated mutants do not, this strongly suggests a loss-of-function mechanism .

  • Quantitative assessments of brain volume, mushroom body morphology, and lifespan after genetic rescue can provide measurable endpoints for comparative analysis .

• Molecular consequences of TER94 loss:

  • TER94 knockdown causes the disappearance of TBPH (the Drosophila ortholog of TDP43/TARDBP) from nuclei, linking TER94 dysfunction to TDP43 pathology seen in frontotemporal lobar degeneration (FTLD) .

  • Dysregulation of neuronal proliferation, potentially through mechanisms involving cell cycle control proteins like Mcm2, may contribute to the neurodegenerative phenotypes .

These findings from Drosophila models suggest that VCP-linked FTLD is caused by loss-of-function rather than gain-of-function mechanisms, contradicting some earlier hypotheses .

How can researchers effectively validate TER94 knockdown and rescue experiments?

These methodological approaches ensure robust validation of TER94 manipulation and provide a framework for interpreting experimental results.

What is the role of TER94's ATPase activity in its diverse cellular functions?

TER94's ATPase activity is central to its diverse cellular functions. Research has provided several insights into this critical aspect:

• Wing development regulation:

  • TER94's regulation of wing size is dependent on its ATPase activity

  • This dependency suggests that the protein's ability to hydrolyze ATP is mechanistically linked to its interaction with the Hippo pathway components

• Viral infection processes:

  • As an AAA+ ATPase, TER94 functions as an energy-supplying chaperone during baculovirus infection

  • It participates in multiple steps of baculovirus infection, including viral DNA replication and virion formation

  • The membrane fission/fusion function of TER94, which requires ATPase activity, is likely exploited by baculoviruses for virion morphogenesis

  • TER94 interacts with viral early proteins LEF3 and helicase to transport and recruit viral replication-related proteins to establish viral replication factories

• Neuronal maintenance:

  • The ATPase activity of TER94/VCP appears critical for neuronal survival, as loss of this function leads to neurodegeneration

  • Disease-associated mutations that affect ATPase activity show reduced ability to rescue knockdown phenotypes, highlighting the importance of this enzymatic function

• Experimental approaches to study ATPase function:

  • Site-directed mutagenesis of key residues in the ATPase domain

  • Biochemical assays to measure ATPase activity of wild-type versus mutant proteins

  • Rescue experiments comparing wild-type TER94 with ATPase-deficient mutants

  • Analysis of protein interactions using co-immunoprecipitation to identify ATPase-dependent binding partners

These findings underscore the critical importance of TER94's ATPase activity across its diverse cellular functions, from development to disease processes.

What are the implications of TER94 research for understanding human VCP-related diseases?

Research on Drosophila TER94 has significant translational relevance for understanding human VCP-related diseases:

• Functional conservation:

  • The human homolog of TER94 (hVCP) can substitute for TER94 in modulating Drosophila wing size, demonstrating functional conservation across species

  • This conservation validates Drosophila as a model system for studying human VCP-related disorders

• Disease mechanisms:

  • The findings that TER94 loss-of-function causes neurodegeneration in Drosophila suggest that human VCP-linked frontotemporal lobar degeneration (FTLD) may similarly result from loss of VCP function

  • This challenges some previous hypotheses that suggested disease mutations might cause a toxic gain of function

  • Specific disease-associated mutations (like A229E) show reduced rescue capability compared to wild-type protein, indicating functional impairment

• TDP43 pathology connection:

  • TER94 knockdown causes mislocalization of TBPH (the Drosophila ortholog of TDP43) from nuclei

  • This connects TER94/VCP dysfunction to TDP43 pathology, which is a hallmark of several neurodegenerative disorders including FTLD

  • Understanding this mechanism may provide insights into the pathogenesis of TDP43 proteinopathies

• Potential therapeutic implications:

  • If VCP-related diseases result from loss of function, therapeutic strategies aimed at enhancing remaining VCP activity or compensating for its loss might be beneficial

  • The rescue of TER94 knockdown phenotypes by wild-type protein expression provides proof-of-concept for gene therapy or protein replacement approaches

  • Understanding the specific molecular pathways disrupted by VCP/TER94 dysfunction may reveal additional therapeutic targets

• Experimental models for drug screening:

  • The quantifiable phenotypes in Drosophila TER94 models (brain volume, mushroom body morphology, lifespan, eye degeneration) provide valuable endpoints for evaluating potential therapeutic compounds

  • High-throughput screening approaches using Drosophila could identify compounds that rescue TER94/VCP-related phenotypes

These translational implications highlight the value of Drosophila TER94 research for advancing our understanding of human VCP-related diseases and developing potential therapeutic strategies.

What tissue-specific approaches can be used to study TER94 function?

Tissue-specific studies of TER94 function can provide insights that might be obscured in whole-organism analyses. Several methodological approaches have been successfully employed:

These methodological approaches provide a comprehensive toolkit for investigating TER94 function in specific tissues, allowing researchers to dissect its diverse roles in different cellular contexts.

How can researchers effectively study TER94's interactions with the Hippo pathway?

Studying TER94's interactions with the Hippo pathway requires specialized approaches to elucidate the molecular mechanisms involved:

• Biochemical interaction studies:

  • Co-immunoprecipitation assays to detect binding between TER94 and Hippo pathway components (particularly Mer, Ex, and Kib)

  • Reciprocal pull-down experiments to confirm direct interactions

  • Mapping of interaction domains through truncation or deletion mutants

  • Analysis of how TER94's ATPase activity affects these interactions

• Genetic interaction experiments:

  • Double knockdown/mutation of TER94 and Hippo pathway components

  • Rescue experiments testing whether Hippo pathway inhibition can rescue TER94 knockdown phenotypes

  • Epistasis analysis to determine the genetic hierarchy between TER94 and Hippo pathway components

• Transcriptional readouts:

  • Analysis of Hippo pathway target gene expression (e.g., using qPCR or reporter constructs)

  • Comparison of target gene expression in TER94 knockdown versus control tissues

  • Assessment of how TER94 manipulation affects Yorkie (Yki) nuclear localization and activity

• Cell biological approaches:

  • Immunofluorescence microscopy to analyze co-localization of TER94 with Hippo pathway components

  • Live imaging to track dynamics of protein interactions

  • Analysis of how TER94 affects the formation of the Ex-Mer-Kib complex

• Structural studies:

  • Determination of crystal structures of TER94 in complex with Hippo pathway components

  • Molecular modeling to predict interaction interfaces

  • Design of mutants to disrupt specific interactions for functional validation

By combining these approaches, researchers can comprehensively characterize the molecular mechanisms by which TER94 regulates the Hippo pathway, particularly focusing on its disruption of the Ex-Mer-Kib complex and the consequent effects on pathway activity and wing development.