Recombinant Danio rerio Vacuolar protein-sorting-associated protein 36 (vps36)

What is the biological role of vacuolar protein-sorting-associated protein 36 (vps36) in zebrafish?

Vacuolar protein-sorting-associated protein 36 (vps36) is a component of the endosomal sorting complex required for transport II (ESCRT-II), which plays a critical role in the sorting of ubiquitinated membrane proteins into multivesicular bodies (MVBs) for lysosomal degradation. In zebrafish (Danio rerio), this protein is involved in cellular processes such as membrane trafficking, signal transduction, and maintaining cellular homeostasis . Studies have shown that vps36 is evolutionarily conserved across species, underscoring its fundamental biological importance.

In zebrafish, vps36 is encoded by the gene symbolized as vps36 or its synonyms such as fj33d07 and zgc:55965 . Functional studies often utilize genetic models like morpholino knockdowns or CRISPR/Cas9-mediated knockouts to elucidate its role during early development and organogenesis .

How is recombinant vps36 from zebrafish expressed and purified?

Recombinant vps36 from zebrafish is typically expressed in bacterial systems such as Escherichia coli. For example, full-length vps36 proteins fused with N-terminal His-tags are expressed to facilitate purification via affinity chromatography. The protein is often lyophilized and stored in Tris/PBS-based buffers supplemented with stabilizers like trehalose .

Purity levels exceeding 90% are achieved using SDS-PAGE analysis, ensuring the suitability of the recombinant protein for downstream applications such as structural studies or functional assays . Researchers should avoid repeated freeze-thaw cycles to maintain protein integrity.

What are the key experimental conditions for studying vps36 in zebrafish embryos?

Zebrafish embryos provide an excellent model for studying vps36 due to their optical transparency and rapid development. Experimental conditions include maintaining embryos at 28°C under a controlled photoperiod (e.g., 14 hours light/10 hours dark) . Morpholino oligonucleotides or CRISPR/Cas9 genome editing can be used to manipulate vps36 expression levels.

For microinjection experiments, embryos are typically collected at the one-cell stage and injected with reagents such as morpholinos or plasmids encoding CRISPR components. Phenotypic analyses, including imaging of embryonic structures or functional assays like heart rate measurement, are conducted at specific developmental stages (e.g., 72 hours post-fertilization) .

What are the known phenotypic effects of vps36 disruption in zebrafish?

Disruption of vps36 function in zebrafish can lead to developmental abnormalities due to impaired endosomal sorting and lysosomal degradation pathways. Phenotypes may include defects in organogenesis, altered cellular signaling, and disrupted homeostasis . For example, studies using morpholino knockdowns have reported changes in heart rate and ventricular function during early development .

Advanced imaging techniques such as confocal microscopy or electron microscopy are often employed to analyze cellular phenotypes resulting from vps36 disruption. These methods provide insights into subcellular structures like multivesicular bodies and lysosomes.

How does vps36 interact with other components of the ESCRT machinery?

Vps36 interacts with other components of the ESCRT-II complex, including vps22 and vps25, as well as upstream ESCRT-I proteins and downstream ESCRT-III proteins. These interactions facilitate the sequential assembly of ESCRT complexes on endosomal membranes .

Biochemical studies using recombinant proteins have demonstrated that vps36 binds ubiquitinated cargo proteins via its GLUE domain (GRAM-like ubiquitin-binding in EAP45). This interaction is essential for cargo recognition and sorting into MVBs . Functional assays using co-immunoprecipitation or pull-down experiments can be employed to study these interactions.

What experimental approaches can be used to study post-translational modifications (PTMs) of recombinant vps36?

Post-translational modifications (PTMs) such as phosphorylation or ubiquitination can regulate the activity and interactions of vps36. To study PTMs, researchers can use mass spectrometry-based proteomics following enrichment of modified peptides via immunoprecipitation or affinity chromatography.

For example, phospho-specific antibodies can be used to detect phosphorylation sites on recombinant vps36 in Western blot assays. Additionally, site-directed mutagenesis can be employed to generate non-modifiable mutants (e.g., replacing serine/threonine residues with alanine) to assess the functional significance of specific PTMs.

How can CRISPR/Cas9 technology be applied to investigate the function of vps36 in zebrafish?

CRISPR/Cas9 genome editing allows precise manipulation of the vps36 gene in zebrafish by introducing targeted mutations. Researchers design guide RNAs (gRNAs) complementary to specific regions of the vps36 coding sequence or regulatory elements . Co-injection of gRNAs with Cas9 mRNA or protein into one-cell stage embryos results in indel mutations that disrupt gene function.

To validate successful editing, PCR amplification followed by sequencing is performed on genomic DNA extracted from edited embryos. Functional analyses may include phenotypic characterization, transcriptomic profiling, or proteomic studies to assess downstream effects of vps36 disruption.

What are the challenges associated with studying genetic compensation mechanisms following vps36 knockout?

Genetic compensation refers to upregulation of related genes or pathways that mitigate phenotypic effects following gene knockout. In zebrafish, this phenomenon has been observed for various genes but remains poorly understood for vps36 .

To address this challenge, researchers can perform transcriptomic analyses using RNA sequencing (RNA-seq) to identify compensatory changes in gene expression following vps36 knockout. Comparative studies using knockdown models (e.g., morpholinos) versus knockout models (e.g., CRISPR/Cas9) can also provide insights into compensation mechanisms.

How does vps36 contribute to intracellular signaling pathways beyond its role in endosomal sorting?

Emerging evidence suggests that vps36 may influence intracellular signaling pathways by regulating receptor turnover and degradation. For example, dysregulation of ESCRT components has been linked to altered signaling through pathways such as Wnt/β-catenin or Notch .

Functional assays using reporter constructs for specific signaling pathways can be employed to investigate how loss of vps36 affects pathway activity. Additionally, proteomic approaches can identify changes in receptor abundance or downstream effectors following vps36 disruption.

What are the implications of studying zebrafish vps36 for understanding human diseases?

Studying zebrafish vps36 provides valuable insights into conserved mechanisms underlying human diseases associated with ESCRT dysfunction, such as neurodegeneration, cancer, and lysosomal storage disorders . Zebrafish models offer unique advantages due to their genetic tractability and suitability for high-throughput drug screening.

Comparative studies between zebrafish and mammalian systems can reveal species-specific differences in ESCRT function while highlighting conserved pathways relevant to human health. For example, investigating how mutations in human VPS36 orthologs affect disease phenotypes may inform therapeutic strategies targeting ESCRT-related pathways.