Recombinant Candida glabrata Cytoplasmic dynein intermediate light chain DYN3 (DYN3)

What is the cytoplasmic dynein intermediate light chain DYN3 in Candida glabrata?

Cytoplasmic dynein intermediate light chain DYN3 in Candida glabrata is a component of the dynein motor complex involved in intracellular transport and cellular motility. While specific information about DYN3 in C. glabrata is limited in the current literature, research on homologous proteins in related species suggests its role in vesicular transport, nuclear migration, and potentially in pathogenesis. Similar to other dynein components, DYN3 likely interacts with microtubules and contributes to various cellular processes that may influence C. glabrata virulence and stress responses .

How does DYN3 differ structurally from other dynein components in C. glabrata?

DYN3, as an intermediate light chain of the dynein complex, possesses specific structural domains that distinguish it from heavy and light chains. The protein likely contains conserved regions for protein-protein interactions with other dynein subunits and cargo-binding domains. When studying recombinant DYN3, researchers should examine its primary sequence for motifs associated with ATP binding, microtubule interaction, and regulatory phosphorylation sites that may influence its function. Comparative analysis with DYN3 homologs in related Candida species can provide insights into conserved structural elements and C. glabrata-specific adaptations that might correlate with its unique pathogenic properties .

What is the genetic organization of the DYN3 gene in C. glabrata genome?

The DYN3 gene in C. glabrata is part of the eukaryotic dynein gene family. When studying this gene, researchers should examine its promoter regions for stress-response elements similar to those found in other C. glabrata virulence factors. Analogous to other C. glabrata genes involved in pathogenesis (such as CgDTR1), the expression of DYN3 may be regulated in response to environmental stresses encountered during infection. Understanding the genetic organization includes mapping intron-exon boundaries, identifying regulatory elements, and determining if the gene exists in a cluster with other cytoskeletal or transport-related genes, which could indicate coordinated expression during specific cellular processes .

What are the most effective methods for expressing recombinant C. glabrata DYN3 protein?

For effective recombinant expression of C. glabrata DYN3, researchers should consider several expression systems with their respective advantages:

Expression System Options:

• E. coli expression systems: Using pET vectors with T7 promoters provides high yield but may lack post-translational modifications

• Yeast expression systems: S. cerevisiae or Pichia pastoris systems offer appropriate eukaryotic post-translational modifications

• Baculovirus-insect cell systems: Provide higher eukaryotic processing capabilities for complex proteins

Optimization Parameters:

• Codon optimization based on the expression host

• Addition of solubility tags (MBP, SUMO, GST) to improve protein folding

• Expression temperature modulation (typically lower temperatures of 16-20°C improve folding)

• Induction conditions optimization (IPTG concentration for bacterial systems; carbon source for yeast systems)

When working with DYN3 specifically, researchers should be mindful that, as a component of a multi-protein complex, the recombinant protein may require co-expression with interaction partners to achieve proper folding and stability. A stepwise purification protocol including affinity chromatography followed by size exclusion chromatography is recommended to ensure high purity for subsequent functional studies .

How can I design CRISPR-Cas9 experiments to investigate DYN3 function in C. glabrata?

Designing effective CRISPR-Cas9 experiments to investigate DYN3 function in C. glabrata requires careful consideration of several factors:

Guide RNA Design:

• Target sequences within the DYN3 coding region that have minimal off-target effects

• Preferentially target the 5' portion of the gene to ensure functional disruption

• Design at least 3-4 different sgRNAs to increase success probability

Delivery Methods:

• Electroporation of Cas9-sgRNA ribonucleoprotein complexes

• Plasmid-based delivery systems adapted for C. glabrata

Verification Approaches:

• PCR amplification and sequencing of the target region

• Western blot analysis to confirm protein disruption

• RT-qPCR to measure transcript levels

Phenotypic Analysis:

• Growth rate comparison under various stress conditions

• Virulence assessment using infection models such as Galleria mellonella

• Cell motility and intracellular transport assays to assess dynein-related functions

When analyzing CRISPR-edited strains, it's crucial to include appropriate controls, such as wild-type strains and strains edited with non-targeting sgRNAs, to account for potential off-target effects or stress responses induced by the CRISPR system itself. Additionally, complementation experiments with wild-type DYN3 should be performed to confirm phenotype specificity .

What infection models are most appropriate for studying the role of DYN3 in C. glabrata virulence?

Several infection models can be employed to investigate DYN3's potential role in C. glabrata virulence:

In Vitro Models:

• Macrophage infection assays to assess survival and replication within phagocytes

• Epithelial cell adhesion and invasion assays

• Biofilm formation assays on various substrates

In Vivo Models:

• Galleria mellonella: This invertebrate model offers several advantages for initial virulence studies. G. mellonella larvae provide a relatively ethical and economical system with innate immune responses that parallel aspects of mammalian immunity. The model has been successfully used to assess C. glabrata virulence factors, such as CgDtr1 . Hemocytes in G. mellonella function similarly to mammalian macrophages, allowing assessment of C. glabrata survival within phagocytic cells.

• Murine models: For more complex immunity assessment, including adaptive immune responses.

• Ex vivo organ cultures: To study tissue-specific interactions.

When using these models to study DYN3 function, researchers should compare wild-type, DYN3 deletion mutants, and complemented strains to assess:

• Survival rates of host organisms

• Fungal burden in tissues

• Inflammatory responses

• Ability to proliferate within host cells

The selection of an appropriate model should be based on the specific aspect of virulence being investigated, with G. mellonella serving as an excellent initial screening model before proceeding to more complex mammalian systems .

How does DYN3 contribute to C. glabrata stress responses and virulence mechanisms?

While specific data on DYN3's role in C. glabrata virulence is not fully characterized, research on related proteins suggests several potential mechanisms:

DYN3, as part of the dynein complex, may contribute to stress responses and virulence through:

• Intracellular transport regulation: DYN3 likely participates in vesicular trafficking that could influence the secretion of virulence factors or the distribution of stress response proteins within the cell.

• Phagosome escape or survival: Similar to how CgDtr1 helps C. glabrata survive within macrophages by conferring resistance to oxidative and acidic stress , DYN3 might facilitate intracellular survival through:

  • Positioning of membrane transporters like CgDtr1

  • Trafficking of detoxification enzymes to sites of reactive oxygen species (ROS) production

  • Vacuolar positioning to counteract phagosomal acidification

• Stress granule formation and dynamics: During stress conditions, DYN3 may contribute to the formation or transport of stress granules, which help cells withstand adverse conditions.

Experimental approaches to investigate these functions should include:

• Fluorescence microscopy to track DYN3 localization during various stress conditions

• Co-immunoprecipitation studies to identify DYN3 interaction partners

• Transcriptomic analysis to determine if DYN3 expression changes in response to host-relevant stresses

• Comparative survival assays of wild-type and DYN3 mutants under oxidative, acidic, or nitrosative stress conditions that mimic the phagosomal environment

What is the interactome of DYN3 in C. glabrata and how does it differ from related species?

Understanding the DYN3 interactome is crucial for elucidating its functional role in C. glabrata pathogenesis. To map this interactome, researchers should employ multiple complementary approaches:

Experimental Techniques for Interactome Mapping:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Tag DYN3 with epitopes like FLAG or HA

  • Perform pull-downs under various conditions (normal growth, oxidative stress, acid stress)

  • Identify co-precipitating proteins through mass spectrometry

• Proximity-dependent Biotin Identification (BioID):

  • Fuse DYN3 to a biotin ligase

  • Identify proteins in close proximity through streptavidin purification and MS

• Yeast Two-Hybrid Screening:

  • Use DYN3 as bait against a C. glabrata cDNA library

  • Validate interactions through secondary assays

Comparative Analysis:
When analyzing the C. glabrata DYN3 interactome, compare it with:

• Interactomes of DYN3 homologs in S. cerevisiae

• Interactomes of DYN3 homologs in C. albicans

• Interactomes of other virulence-associated proteins in C. glabrata like CgDtr1

This comparative approach may reveal C. glabrata-specific interactions that contribute to its unique pathogenic properties, such as enhanced ability to survive within macrophages or resist antifungal treatments. The interactome data should be visualized using protein-protein interaction networks and analyzed for enrichment of specific biological processes or cellular components.

How do post-translational modifications regulate DYN3 function during infection?

Post-translational modifications (PTMs) likely play crucial roles in regulating DYN3 function during the infection process:

Key PTMs to Investigate:

• Phosphorylation:

  • Identify potential phosphorylation sites using bioinformatic predictions

  • Perform phosphoproteomic analysis of DYN3 under various infection-relevant conditions

  • Create phosphomimetic and phospho-deficient mutants to assess functional consequences

• Ubiquitination:

  • Determine if DYN3 undergoes stress-induced ubiquitination

  • Identify E3 ligases responsible for DYN3 ubiquitination

  • Assess how ubiquitination affects DYN3 stability and function

• Acetylation and Methylation:

  • Investigate these modifications as potential regulators of DYN3 interaction with binding partners

Methodological Approaches:

To study these PTMs effectively, researchers should:

• Use mass spectrometry-based approaches to map modification sites

• Develop modification-specific antibodies for immunoblotting

• Employ CRISPR-Cas9 to mutate key residues and assess phenotypic consequences

• Compare modification patterns between in vitro culture and in vivo infection conditions

When investigating PTMs, consider that stress conditions encountered during infection, such as oxidative stress within macrophages, may trigger specific modifications that alter DYN3 function. These modifications could potentially shift DYN3's role from normal cellular functions to pathogenesis-related activities, similar to how stress conditions induce the up-regulation of CgDTR1 when C. glabrata is internalized in hemocytes .

How can I troubleshoot poor expression or solubility issues with recombinant DYN3?

When facing expression or solubility challenges with recombinant C. glabrata DYN3, consider implementing this systematic troubleshooting approach:

Expression Optimization:

Solubility Enhancement:

• Fusion partners: Test multiple solubility-enhancing tags:

  • MBP (maltose-binding protein)

  • SUMO

  • Thioredoxin

  • GST (glutathione S-transferase)

• Buffer optimization:

  • Screen various pH conditions (pH 6.0-8.5)

  • Test different salt concentrations (100-500 mM NaCl)

  • Include stabilizing agents (5-10% glycerol, 1-5 mM DTT)

  • Add detergents for membrane-associated proteins (0.05-0.1% Triton X-100)

• Co-expression strategies:

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Co-express with known binding partners from the dynein complex

For particularly challenging constructs, consider expressing individual domains rather than the full-length protein, as domains often fold independently and may retain specific functions for analysis .

What are the best approaches for analyzing DYN3 interactions with other dynein components?

To effectively analyze DYN3 interactions with other dynein components in C. glabrata, researchers should employ multiple complementary approaches:

In Vitro Interaction Analysis:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified DYN3 on a sensor chip

  • Flow other dynein components as analytes

  • Measure binding kinetics (ka, kd) and affinity constants (KD)

• Isothermal Titration Calorimetry (ITC):

  • Provides thermodynamic parameters of binding (ΔH, ΔS, ΔG)

  • Requires no protein labeling or immobilization

• Microscale Thermophoresis (MST):

  • Requires minimal protein amounts

  • Works well with complex biological samples

In Vivo Interaction Analysis:

• Förster Resonance Energy Transfer (FRET):

  • Tag DYN3 and potential partners with appropriate fluorophores

  • Measure energy transfer as indication of proximity

  • Can be performed in living cells

• Bimolecular Fluorescence Complementation (BiFC):

  • Split fluorescent protein fragments fused to potential interacting partners

  • Fluorescence occurs only upon interaction

• Proximity Ligation Assay (PLA):

  • Detects protein interactions with high sensitivity

  • Provides spatial information about interactions

Structural Analysis:

• X-ray crystallography of co-crystals of DYN3 with binding partners

• Cryo-electron microscopy of assembled dynein complexes

• Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

When investigating DYN3 interactions, researchers should consider that these interactions may be dynamically regulated during infection processes, potentially responding to stress conditions similar to how CgDtr1 expression is induced during interaction with host immune cells .

How can I validate DYN3 function in vivo without affecting cell viability?

Validating DYN3 function in vivo while maintaining C. glabrata viability requires careful experimental design:

Conditional Expression/Repression Systems:

• Tetracycline-regulatable promoter systems:

  • Replace the native DYN3 promoter with a tet-ON or tet-OFF promoter

  • Allows temporal control of DYN3 expression

  • Titrate tetracycline to achieve partial repression

• Auxin-inducible degron (AID) system:

  • Tag DYN3 with an auxin-responsive degron

  • Addition of auxin induces rapid protein degradation

  • Can be reversed by auxin removal

Domain-Specific Mutagenesis:

• Instead of complete gene deletion, introduce point mutations in specific functional domains

• Target conserved residues identified through sequence alignment

• Create a panel of mutants with varying degrees of functional impairment

Proximity-based Perturbation:

• Optogenetic approaches:

  • Fuse light-sensitive domains to DYN3

  • Use light to induce conformational changes or protein interactions

  • Allows spatial and temporal control

• Chemical-induced dimerization:

  • Engineer DYN3 to respond to small molecules that induce interactions

  • Can rapidly alter protein localization or interactions

Partial Knockdown Approaches:

• Use RNA interference or CRISPRi to achieve partial repression

• Titrate the degree of knockdown to avoid lethal effects

When implementing these approaches, monitor multiple cellular parameters including growth rate, morphology, and stress responses to ensure the interventions themselves are not causing general cellular dysfunction. Additionally, include appropriate controls such as wild-type strains and strains with non-functional modifications to differentiate specific DYN3-related phenotypes from general stress responses, similar to the methodological approaches used in studying other C. glabrata virulence factors .

How might DYN3 contribute to antifungal resistance mechanisms in C. glabrata?

DYN3, as part of the cytoplasmic dynein complex involved in intracellular transport, may contribute to antifungal resistance through several potential mechanisms:

• Efflux Pump Trafficking and Positioning:

  • DYN3 might facilitate the transport of efflux pumps to the plasma membrane

  • Similar to how CgDtr1 functions as a plasma membrane exporter that expels acetic acid , DYN3 could be involved in the correct localization of drug transporters

  • This would enhance the ability of C. glabrata to expel antifungal compounds from the cell

• Cell Wall Remodeling:

  • Dynein-mediated transport may be crucial for delivering cell wall synthesis enzymes

  • Could contribute to the thickening or altered composition of the cell wall observed in resistant strains

  • May affect the accessibility of antifungal drugs to their targets

• Stress Response Coordination:

  • DYN3 might facilitate the nuclear import of transcription factors involved in stress responses

  • Could coordinate the transport of proteins involved in ergosterol biosynthesis, which is targeted by azole antifungals

Future research directions should include:

• Comparative analysis of DYN3 expression in drug-sensitive versus resistant clinical isolates

• Evaluation of antifungal susceptibility profiles in DYN3 mutants

• Investigation of potential interactions between DYN3 and known resistance mediators like efflux pumps

• Assessment of how stress conditions similar to those encountered during antifungal treatment affect DYN3 function and localization

What are the emerging technologies that could advance our understanding of DYN3 dynamics in live C. glabrata cells?

Several cutting-edge technologies are poised to revolutionize our understanding of DYN3 dynamics in live C. glabrata cells:

Super-Resolution Microscopy Techniques:

• Structured Illumination Microscopy (SIM):

  • Achieves ~100 nm resolution

  • Compatible with live cell imaging

  • Can visualize dynamic processes of DYN3 trafficking

• Stimulated Emission Depletion (STED) Microscopy:

  • Reaches ~30-80 nm resolution

  • Allows visualization of individual dynein complexes

  • Can be combined with live cell imaging

• Single-Molecule Localization Microscopy (PALM/STORM):

  • Achieves ~20-30 nm resolution

  • Ideal for quantifying DYN3 molecule numbers and distribution

Advanced Fluorescent Protein Technologies:

• Split Fluorescent Proteins:

  • Monitor protein-protein interactions in real-time

  • Assess DYN3 assembly with other dynein components

• Fluorescent Timers:

  • Track protein age and turnover rates

  • Distinguish newly synthesized from mature DYN3 proteins

Biosensors and Optogenetic Tools:

• FRET-based tension sensors:

  • Measure mechanical forces experienced by DYN3 during transport

  • Provide insights into motor function

• Optogenetic control of DYN3 activity:

  • Light-inducible dimerization or conformational changes

  • Spatiotemporal control of DYN3 function

Microfluidic Approaches:

• Microfluidic devices coupled with live imaging:

  • Subject cells to controlled environmental changes

  • Monitor dynamic responses of DYN3 to stress conditions similar to those encountered during infection

• Single-cell analysis platforms:

  • Correlate DYN3 dynamics with cell-to-cell variability in stress responses

  • Identify subpopulations with distinct phenotypes

These technologies, when applied to studying DYN3, will help elucidate its dynamic behavior during normal cellular processes and under infection-relevant stress conditions, potentially revealing new therapeutic targets.

How can systems biology approaches integrate DYN3 function into broader models of C. glabrata pathogenicity?

Systems biology approaches offer powerful frameworks to contextualize DYN3 function within the broader landscape of C. glabrata pathogenicity:

Multi-omics Integration:

• Integrative analysis of transcriptomics, proteomics, and metabolomics data:

  • Map how DYN3 expression correlates with other virulence factors

  • Identify potential co-regulation patterns similar to those observed with CgDtr1

  • Construct regulatory networks activated during host interaction

• Temporal multi-omics during infection:

  • Track changes in DYN3 expression and modification status during different infection stages

  • Correlate with global adaptation responses

Network Biology Approaches:

• Protein-protein interaction network analysis:

  • Position DYN3 within the context of virulence-associated protein networks

  • Identify network hubs that connect DYN3 to established virulence mechanisms

• Gene regulatory network reconstruction:

  • Identify transcription factors controlling DYN3 expression

  • Map how these regulators respond to host-relevant stresses

Computational Modeling:

• Agent-based models of host-pathogen interactions:

  • Simulate how DYN3-dependent processes affect C. glabrata behavior within host cells

  • Model various infection scenarios with different DYN3 activity levels

• Flux balance analysis of metabolic networks:

  • Determine how DYN3-mediated transport processes affect metabolic capabilities

  • Predict metabolic adaptations in DYN3 mutants

Comparative Genomics and Phylogenetics:

• Cross-species analysis of dynein components:

  • Compare DYN3 sequence and function across Candida species with varying virulence

  • Identify C. glabrata-specific adaptations that may contribute to its unique pathogenic properties

The integration of these systems biology approaches will help position DYN3 within the complex networks that drive C. glabrata pathogenicity, potentially revealing unexpected connections to established virulence mechanisms like those observed with transporters such as CgDtr1, which significantly affects C. glabrata's ability to proliferate in host environments .