Recombinant Ashbya gossypii Inheritance of peroxisomes protein 2 (INP2)

How does Ashbya gossypii differ from Saccharomyces cerevisiae as a model organism?

Ashbya gossypii differs significantly from the budding yeast S. cerevisiae in its growth pattern and cellular organization. While S. cerevisiae divides by budding with transient polarization during each cell cycle, A. gossypii exhibits persistent highly polarized growth with multiple axes of polarity coexisting in a single cell. These growth characteristics make A. gossypii an excellent model for studying sustained polar growth and organelle inheritance across extended hyphal structures . Additionally, A. gossypii has industrial relevance as it is widely used for riboflavin (vitamin B2) production and can produce other valuable compounds such as folates, nucleosides, and biolipids . These distinctive features provide researchers with unique opportunities to study cellular processes like organelle inheritance in a filamentous fungal context.

How should recombinant A. gossypii INP2 protein be stored and handled in the laboratory?

Proper storage and handling of recombinant A. gossypii INP2 protein is essential for maintaining its stability and functionality in laboratory settings. The recombinant protein should be stored at -20°C for short-term storage or at -80°C for extended storage. The recommended storage buffer typically consists of a Tris-based buffer with 50% glycerol, optimized for this specific protein . Alternatively, a Tris/PBS-based buffer with 6% trehalose at pH 8.0 can be used .

For routine laboratory use, follow these guidelines:

• Avoid repeated freeze-thaw cycles as they can degrade the protein

• Store working aliquots at 4°C for up to one week

• When reconstituting lyophilized protein, briefly centrifuge the vial before opening

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% for long-term storage aliquots

These storage and handling protocols help maintain the structural integrity and functional activity of the recombinant INP2 protein for various experimental applications .

What expression systems are most effective for producing recombinant A. gossypii INP2?

Based on the available research data, E. coli has been successfully used as an expression system for recombinant A. gossypii INP2 protein production. The full-length INP2 protein (1-652 amino acids) has been expressed in E. coli with an N-terminal His-tag for purification purposes . This approach yields protein with greater than 90% purity as determined by SDS-PAGE analysis.

When designing an expression system for INP2, researchers should consider:

• Codon optimization: Adapting the A. gossypii gene sequence for efficient expression in the host organism

• Affinity tag selection: His-tags are commonly used, but other tags may be selected based on downstream applications

• Protein folding requirements: Adding solubility enhancers or chaperones if the protein shows tendency to form inclusion bodies

• Expression conditions: Optimizing temperature, induction time, and media composition

For researchers requiring higher yields or specific post-translational modifications, alternative expression systems such as yeast (Pichia pastoris) might be considered, although specific data for INP2 expression in these systems is not provided in the available search results .

How can CRISPR/Cas9 genetic editing be applied to study INP2 function in A. gossypii?

CRISPR/Cas9 technology offers a powerful approach for studying INP2 function in A. gossypii through precise genomic manipulations. A one-vector CRISPR/Cas9 editing system has been specifically adapted for A. gossypii that allows marker-free engineering strategies to be implemented . This system can be applied to INP2 research in several ways:

• Gene knockout studies:

  • Design guide RNAs (gRNAs) targeting the INP2 gene

  • Generate a double-strand break (DSB) at the target site using Cas9

  • Provide a donor DNA template that introduces a deletion or frameshift mutation

  • Select transformants and verify the knockout using PCR and sequencing

• Protein tagging for localization studies:

  • Design gRNAs targeting the C-terminus of the INP2 gene

  • Provide a donor DNA template containing a fluorescent protein sequence

  • Generate in-frame fusions to visualize INP2 localization in live cells

• Domain-specific mutations:

  • Create precise mutations in functional domains to assess their role in peroxisome inheritance

  • Design gRNAs targeting specific regions and provide donor templates with desired mutations

The CRISPR/Cas9 system for A. gossypii requires a 5′-NGG-3′ trinucleotide protospacer adjacent motif (PAM) to generate the double-strand break. After transformation, the genomic edits can be confirmed through sequencing, and phenotypic analyses can be performed to assess the impact on peroxisome inheritance and distribution .

How does INP2 function in the context of A. gossypii's polarized growth pattern?

While the search results don't provide specific information about INP2's role in A. gossypii's polarized growth, we can draw insights by examining how organelle inheritance functions in this uniquely structured organism. A. gossypii exhibits persistent highly polarized growth with multiple axes of polarity coexisting in one cell, unlike the budding yeast S. cerevisiae . This growth pattern creates distinct challenges for organelle inheritance and distribution.

In the context of this growth pattern, INP2 likely functions to:

• Facilitate the movement of peroxisomes along the hyphal cytoskeleton

• Ensure proper distribution of peroxisomes between growing hyphal tips and the older parts of the mycelium

• Coordinate peroxisome inheritance with branch formation and extension

The polarized growth of A. gossypii, characterized by persistent hyphal elongation and branching, requires specialized mechanisms for organelle positioning. For comparison, studies of Axl2 (another protein in A. gossypii) show that it integrates polarity establishment, maintenance, and environmental stress response for optimal polarized growth . INP2 likely plays a complementary role specifically focused on peroxisome positioning during this polarized growth.

Future research using fluorescently tagged INP2 in combination with peroxisome markers could provide valuable insights into how this protein facilitates organelle inheritance in the context of A. gossypii's unique growth pattern.

What methods can be used to assess INP2-dependent peroxisome movement in A. gossypii?

Several sophisticated methodologies can be employed to study INP2-dependent peroxisome movement in A. gossypii:

• Live-cell fluorescence microscopy:

  • Express fluorescently tagged peroxisome markers (e.g., mRFP-SKL) in wild-type and INP2 knockout strains

  • Track peroxisome movement in real-time using time-lapse confocal microscopy

  • Quantify movement parameters such as velocity, direction, and distribution patterns

• Dual-color imaging:

  • Co-express fluorescently tagged INP2 (e.g., INP2-GFP) and peroxisome markers

  • Determine colocalization and potential interaction during peroxisome inheritance

• FRAP (Fluorescence Recovery After Photobleaching):

  • Photobleach peroxisomes in specific regions of the hypha

  • Measure the rate of fluorescence recovery to assess peroxisome mobility

  • Compare recovery rates between wild-type and INP2 mutant strains

• Electron microscopy:

  • Use immunogold labeling to visualize INP2 localization at ultrastructural level

  • Examine peroxisome morphology and distribution in wild-type versus INP2 knockout strains

• Peroxisome isolation and biochemical analysis:

  • Isolate peroxisomes from wild-type and INP2 mutant A. gossypii

  • Compare protein and lipid compositions

  • Identify potential interaction partners through co-immunoprecipitation experiments

By combining these approaches, researchers can comprehensively characterize how INP2 contributes to peroxisome movement and inheritance in the context of A. gossypii's filamentous growth pattern.

How might the function of INP2 differ between A. gossypii and other fungal species?

The function of INP2 likely differs between A. gossypii and other fungal species due to their distinct morphologies and growth patterns. These differences may be particularly pronounced when comparing A. gossypii to the well-studied budding yeast S. cerevisiae.

Potential differences in INP2 function:

• Spatial coordination requirements:

  • In A. gossypii, INP2 must coordinate peroxisome inheritance across extended hyphal structures and multiple branching points simultaneously

  • In contrast, S. cerevisiae INP2 only needs to manage inheritance between a mother cell and a single bud during each cell cycle

• Temporal dynamics:

  • A. gossypii has persistent polarized growth, requiring continuous peroxisome distribution

  • S. cerevisiae has cyclical polarization linked to the cell cycle, potentially requiring more periodic INP2 activity

• Interaction with the cytoskeleton:

  • The arrangement of the cytoskeleton differs between filamentous fungi and yeasts

  • INP2 likely has adapted to interact with the specific cytoskeletal organization in A. gossypii

• Stress response integration:

  • Similar to how Axl2 in A. gossypii integrates polarity maintenance and stress response , INP2 might have evolved additional functions related to environmental adaptation

  • These functions may be less prominent in non-filamentous fungi

Comparative studies examining INP2 sequence conservation, domain structure, and interacting partners across fungal species would help elucidate these functional differences. Additionally, heterologous expression experiments, where INP2 from different fungi is expressed in A. gossypii INP2 knockout strains, could provide insights into functional conservation and specialization.

What are common challenges when working with recombinant A. gossypii INP2 and how can they be addressed?

Researchers working with recombinant A. gossypii INP2 may encounter several challenges that can impact experimental outcomes. Here are common issues and recommended solutions:

Table 4.1: Challenges and Solutions for Working with Recombinant INP2

Addressing these challenges requires careful optimization of expression, purification, and storage conditions specific to INP2. Preliminary small-scale experiments to determine optimal conditions before scaling up can save time and resources in the long run.

How can researchers design experiments to determine INP2 binding partners in A. gossypii?

Identifying INP2 binding partners is crucial for understanding its function in peroxisome inheritance. Researchers can design comprehensive experiments using these approaches:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express His-tagged INP2 in A. gossypii

  • Perform affinity purification under gentle conditions to maintain protein-protein interactions

  • Analyze co-purified proteins by mass spectrometry

  • Validate interactions using reciprocal pulldowns

• Yeast two-hybrid screening:

  • Use INP2 as bait against an A. gossypii cDNA library

  • Screen for positive interactions

  • Validate promising candidates through secondary assays

• Proximity-dependent biotin identification (BioID):

  • Create a fusion protein of INP2 with a biotin ligase (BirA*)

  • Express this construct in A. gossypii

  • Proteins in close proximity to INP2 will be biotinylated

  • Purify biotinylated proteins and identify by mass spectrometry

• Co-immunoprecipitation (Co-IP) with specific antibodies:

  • Generate antibodies against INP2 or use antibodies against the recombinant tag

  • Perform immunoprecipitation from A. gossypii cell lysates

  • Identify co-precipitated proteins by western blot or mass spectrometry

• FRET/BRET analysis for in vivo interactions:

  • Create fluorescent protein fusions of INP2 and candidate interactors

  • Measure energy transfer as an indicator of protein proximity

  • Quantify interaction strengths in different cellular compartments

Control experiments should include:

• Parallel experiments with unrelated proteins to identify non-specific interactions

• Competition assays with purified recombinant proteins

• Domain mapping to identify specific interaction regions

The CRISPR/Cas9 system adapted for A. gossypii could be particularly useful for creating tagged versions of INP2 at its endogenous locus to ensure physiological expression levels during interaction studies.

What considerations are important when using antibodies against recombinant A. gossypii INP2 in research applications?

When using antibodies against recombinant A. gossypii INP2 in research applications, several important considerations must be addressed to ensure reliable and reproducible results:

• Antibody specificity:

  • Validate antibodies using western blot against both recombinant INP2 and A. gossypii cell lysates

  • Compare wild-type and INP2 knockout strains to confirm specificity

  • Consider using epitope-tagged INP2 and tag-specific antibodies as alternatives

• Epitope selection:

  • Choose unique regions of INP2 that have low homology with other A. gossypii proteins

  • Avoid hydrophobic regions that may be inaccessible in the native protein

  • Consider using multiple antibodies targeting different epitopes for confirmation

• Cross-reactivity assessment:

  • Test for cross-reactivity with related proteins, especially if studying homologs across species

  • Perform peptide competition assays to demonstrate specificity

  • Consider pre-absorbing antibodies against lysates from INP2 knockout strains

• Application-specific optimization:

  • For western blotting: Determine optimal antibody dilution, blocking conditions, and detection methods

  • For immunofluorescence: Optimize fixation methods that preserve INP2 epitopes while maintaining cellular structure

  • For immunoprecipitation: Test different lysis buffers and antibody concentrations

• Storage and handling:

  • Store antibodies according to manufacturer recommendations (typically aliquoted at -20°C)

  • Avoid repeated freeze-thaw cycles

  • Include proper controls in each experiment (positive, negative, and isotype controls)

By carefully considering these factors, researchers can maximize the reliability of antibody-based detection methods for studying A. gossypii INP2 in various experimental contexts.

How does studying INP2 in A. gossypii contribute to our understanding of peroxisome inheritance in other systems?

Studying INP2 in A. gossypii provides unique insights into peroxisome inheritance mechanisms that complement studies in other organisms for several key reasons:

• Evolutionary perspective:

  • A. gossypii represents a filamentous fungal lineage with different growth patterns than the well-studied S. cerevisiae

  • Comparative analysis of INP2 function across these species reveals conserved and divergent mechanisms of peroxisome inheritance

• Complex spatial organization:

  • A. gossypii's multinucleate hyphae with multiple growth points creates a more complex cellular environment than unicellular yeasts

  • This complexity may reveal mechanisms of organelle distribution that are more relevant to higher eukaryotes

• Persistent polarity versus transient polarity:

  • A. gossypii maintains polarized growth for extended periods, unlike the transient polarization in budding yeast

  • This allows researchers to study how peroxisome inheritance mechanisms operate under conditions of continuous polarized growth

• Industrial relevance:

  • A. gossypii is used industrially for riboflavin production

  • Understanding peroxisome function and inheritance in this organism has practical applications for metabolic engineering and biotechnology

The findings from A. gossypii INP2 studies could provide valuable comparative data for understanding peroxisome inheritance in more complex eukaryotic systems, particularly in cells with polarized structures like neurons and epithelial cells. Additionally, these studies may reveal fundamental principles of organelle positioning during polarized growth that apply across diverse biological systems.

What advanced imaging techniques can reveal INP2's role in peroxisome dynamics?

Advanced imaging techniques can provide unprecedented insights into INP2's role in peroxisome dynamics in A. gossypii. These techniques go beyond conventional microscopy to reveal functional and mechanistic details:

• Super-resolution microscopy:

  • Techniques such as STORM, PALM, or STED microscopy

  • Resolution below the diffraction limit (20-50 nm)

  • Can resolve individual peroxisomes and potential INP2-mediated tethering structures

  • Applications: Visualizing INP2 distribution on peroxisome membranes and at contact sites with other cellular structures

• Single-particle tracking:

  • Track individual peroxisomes labeled with photoconvertible fluorescent proteins

  • Measure directional movement, velocity, and pausing frequency

  • Compare movement parameters between wild-type and INP2 mutant strains

  • Applications: Quantitative analysis of how INP2 affects peroxisome motility along hyphal structures

• Lattice light-sheet microscopy:

  • Reduced phototoxicity for long-term imaging

  • High temporal resolution for capturing rapid events

  • Applications: Following peroxisome inheritance during hyphal growth and branching over extended periods

• Fluorescence correlation spectroscopy (FCS):

  • Measure diffusion coefficients of INP2-GFP fusion proteins

  • Determine if INP2 exists in different mobile fractions

  • Applications: Characterizing INP2 dynamics in different cellular compartments

• FRET-based tension sensors:

  • Engineer INP2 with internal FRET-based tension sensors

  • Measure mechanical forces experienced during peroxisome movement

  • Applications: Understanding the biophysical aspects of peroxisome transport

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence localization of INP2 with ultrastructural context

  • Visualize INP2-mediated peroxisome interactions at nanometer resolution

  • Applications: Detailed structural analysis of peroxisome-cytoskeleton interactions

Implementation of these advanced imaging approaches, particularly using the CRISPR/Cas9 system to create endogenously tagged INP2 , would significantly advance our understanding of how this protein coordinates peroxisome inheritance in the context of A. gossypii's polarized growth.

How might genetic engineering of INP2 be used to optimize industrial applications of A. gossypii?

A. gossypii is an industrially important filamentous fungus used for the production of riboflavin (vitamin B2), folates, nucleosides, and biolipids . Genetic engineering of INP2 could potentially optimize these industrial applications through several strategic approaches:

• Enhanced metabolic efficiency:

  • Peroxisomes house critical metabolic pathways, including fatty acid β-oxidation and detoxification of reactive oxygen species

  • Optimizing peroxisome distribution through INP2 engineering could improve metabolic efficiency

  • Potential approach: Creating INP2 variants that increase peroxisome density in metabolically active hyphal regions

• Stress tolerance improvement:

  • Similar to how Axl2 integrates polarity establishment and stress response in A. gossypii , INP2 might be engineered to enhance stress tolerance

  • Industrial fermentation conditions often involve oxidative and osmotic stresses

  • Potential approach: Creating stress-responsive INP2 variants that optimize peroxisome function under industrial conditions

• Growth optimization:

  • Proper organelle inheritance is crucial for normal growth and development

  • Engineered INP2 variants could potentially enhance growth characteristics

  • Potential approach: Fine-tuning INP2 expression levels to optimize hyphal extension and branching patterns

• Biosynthetic pathway enhancement:

  • For products that involve peroxisome-localized biosynthetic steps

  • Engineering INP2 to alter peroxisome distribution could increase production yields

  • Potential approach: Creating chimeric INP2 proteins that can recruit additional biosynthetic enzymes to peroxisomes

Table 5.3: Potential INP2 Engineering Strategies for Industrial Applications

The one-vector CRISPR/Cas9 system adapted for A. gossypii would be particularly valuable for implementing these engineering strategies, allowing precise, marker-free genomic modifications . Additionally, ongoing research into A. gossypii as a platform for producing compounds like sabinene from agro-industrial wastes demonstrates the potential for metabolic engineering approaches in this organism .

What are the most promising directions for future research on A. gossypii INP2?

The most promising directions for future research on A. gossypii INP2 span fundamental biology to applied biotechnology. Based on the available information and current gaps in knowledge, several key research directions emerge:

• Mechanistic studies of peroxisome inheritance:

  • Detailed investigation of how INP2 coordinates peroxisome movement along the cytoskeleton

  • Identification and characterization of INP2 binding partners in A. gossypii

  • Elucidation of regulatory mechanisms controlling INP2 activity during hyphal growth

• Comparative analysis across fungal species:

  • Systematic comparison of INP2 function between A. gossypii and other fungi

  • Investigation of how INP2 has evolved to support different growth patterns

  • Assessment of functional conservation through cross-species complementation studies

• Integration with cellular stress responses:

  • Similar to Axl2's role in integrating polarity establishment and stress response , investigation of how INP2 may coordinate peroxisome function with environmental adaptation

  • Examination of INP2 regulation under different stress conditions

• Applied biotechnology applications:

  • Development of INP2 engineering strategies to enhance industrial production of riboflavin and other valuable compounds

  • Integration of peroxisome optimization into broader metabolic engineering approaches for A. gossypii

• Advanced visualization and modeling:

  • Implementation of cutting-edge imaging techniques to track peroxisome dynamics in real-time

  • Development of mathematical models to predict peroxisome distribution based on INP2 activity

  • Systems biology approaches to understand peroxisome inheritance in the context of whole-cell physiology

These research directions would benefit from the application of the CRISPR/Cas9 system adapted for A. gossypii , which enables precise genetic manipulations for functional studies. Additionally, leveraging the knowledge of A. gossypii as a versatile platform for producing valuable compounds could inform applied aspects of INP2 research.

By pursuing these directions, researchers can advance our understanding of fundamental cellular processes while potentially developing biotechnological applications that leverage A. gossypii's unique capabilities.