RHOD Human

What is RHOD and what cellular functions does it regulate?

RHOD (ras homolog gene family, member D) is a 210 amino acid protein with a molecular weight of approximately 23 kDa that belongs to the small GTPase superfamily, Rho family . It functions as a molecular switch that cycles between active GTP-bound and inactive GDP-bound states to regulate various cellular processes.

RHOD is primarily involved in:

• Endosome dynamics and vesicular trafficking

• Coordination of membrane transport with cytoskeletal function

• Internalization and trafficking of activated tyrosine kinase receptors such as PDGFRB

• Reorganization of actin cytoskeleton, particularly filopodia formation and actin filament bundling

• Modulation of DAPK3's effect in reorganizing actin cytoskeleton and focal adhesion dissolution

To study RHOD's cellular functions, researchers typically employ combinations of:

• Live-cell imaging with fluorescently tagged RHOD constructs

• Co-immunoprecipitation to identify binding partners

• siRNA knockdown or CRISPR/Cas9 knockout experiments

• Overexpression of wild-type, constitutively active, or dominant-negative RHOD mutants

How does RHOD differ functionally from other Rho GTPases?

RHOD has distinct roles compared to better-studied Rho members like Cdc42, RhoA, and Rac1. While most Rho GTPases promote stress fiber formation and focal adhesion assembly, RHOD exhibits several unique characteristics:

RHOD uniquely binds to WHAMM (WASp homologue associated with actin Golgi membranes and microtubules) and FILIP1, which distinguishes its signaling pathway from other Rho GTPases . This interaction establishes a distinct regulatory mechanism where RHOD coordinates Arp2/3-dependent and filamin A-dependent processes to control actin dynamics .

What experimental approaches can reveal the structure-function relationship of RHOD?

To investigate structure-function relationships in RHOD research, consider these methodological approaches:

• Site-directed mutagenesis: Create point mutations in conserved domains (e.g., GTP-binding pocket, effector binding regions) to analyze their impact on RHOD function. The recombinant human RHOD protein (aa 18-207) can serve as a reference for designing mutations .

• Domain swapping experiments: Exchange domains between RHOD and other Rho GTPases to identify regions responsible for specific functions.

• Structural biology techniques:

  • X-ray crystallography of RHOD alone or in complex with effectors

  • NMR spectroscopy for dynamic structural information

  • Cryo-EM for visualization of larger complexes

• Biophysical interaction analysis:

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Fluorescence resonance energy transfer (FRET) to study protein-protein interactions in living cells

• Advanced imaging:

  • Super-resolution microscopy to visualize RHOD localization with nanometer precision

  • Single-molecule tracking to follow individual RHOD molecules in living cells

How does RHOD regulate actin dynamics through interaction with WHAMM?

RHOD forms a functional complex with WHAMM that plays a critical role in actin nucleation and cytoskeletal organization. This interaction represents a distinct regulatory pathway compared to other Rho GTPases .

The mechanism follows these steps:

• RHOD binds directly to WHAMM, which acts as an effector protein

• WHAMM then binds to and activates the Arp2/3 complex

• The activated Arp2/3 complex promotes actin nucleation and branched filament formation

• This process contributes to filopodia formation and actin filament bundling

• WHAMM acts downstream of RHOD in regulating these cytoskeletal dynamics

Experimental evidence shows that cells with reduced levels of RHOD and WHAMM (through siRNA treatment) exhibit increased cell attachment and decreased cell migration, highlighting the physiological importance of this pathway .

Research approaches to study this interaction include:

• Co-immunoprecipitation to verify complex formation

• In vitro actin polymerization assays with purified components

• Live-cell imaging with fluorescently tagged proteins

• siRNA-mediated knockdown of either protein to establish functional relationships

What methods can be used to visualize and quantify RHOD-mediated cytoskeletal changes?

Researchers investigating RHOD's impact on cytoskeletal dynamics can employ these methodological approaches:

• Immunofluorescence microscopy:

  • Fix cells and stain with antibodies against RHOD (available antibodies work well at 1:50-1:500 dilution for IHC and 1:10-1:100 for IF)

  • Co-stain with phalloidin (for F-actin) and antibodies against WHAMM or FILIP1

  • Quantify colocalization using Pearson's correlation coefficient

• Live-cell imaging:

  • Express fluorescently-tagged RHOD (e.g., GFP-RHOD) in cells

  • Use SiR-Actin for live F-actin visualization

  • Employ spinning disk confocal microscopy for rapid acquisition

  • Apply TIRF microscopy to focus on cytoskeletal events near the plasma membrane

• Quantitative analysis of cytoskeletal parameters:

  • Measure filopodia number, length, and dynamics

  • Quantify actin filament organization using FilamentTracker or similar tools

  • Analyze focal adhesion turnover in relation to RHOD activity

• Advanced biophysical approaches:

  • Atomic force microscopy to measure cell stiffness

  • Traction force microscopy to quantify cell-generated forces

  • Laser ablation to study tension in actin networks

• Biochemical assays:

  • Actin co-sedimentation to measure F-actin binding

  • Pyrene-actin assays to measure polymerization kinetics

  • Pull-down assays using GST-RHOD to identify new binding partners

How does the RHOD-FILIP1 pathway contribute to filamin A-dependent processes?

RHOD binds not only to WHAMM but also to FILIP1 (Filamin A-interacting protein 1), establishing a connection to filamin A (FLNa)-dependent mechanisms . This interaction represents an important pathway through which RHOD influences cytoskeletal architecture.

The mechanism involves:

• RHOD binding to FILIP1

• FILIP1 interacting with filamin A

• Filamin A functioning as an actin-crosslinking protein that organizes actin filaments into orthogonal networks

• This pathway influencing cell adhesion, spreading, and migration properties

Research evidence suggests that RHOD coordinates both Arp2/3-dependent mechanisms (via WHAMM) and FLNa-dependent processes (via FILIP1) to comprehensively control the actin filament system . This dual regulatory capability explains RHOD's distinctive impact on cell morphology and behavior.

To investigate this pathway, researchers can:

• Perform co-immunoprecipitation studies with RHOD, FILIP1, and filamin A

• Use proximity ligation assays to visualize protein interactions in situ

• Conduct FRET analysis to measure binding dynamics in living cells

• Employ filamin A-null cells to assess RHOD-dependent phenotypes

• Analyze filamin A phosphorylation status in response to RHOD activation

How does RHOD regulate endosomal trafficking pathways?

RHOD localizes to early endosomes and recycling endosomes, indicating its important role in endosomal trafficking . Its function in this context appears to be coordinating membrane transport with cytoskeletal dynamics.

The mechanism includes:

• RHOD association with endosomal membranes in its active GTP-bound state

• Recruitment of effector proteins that connect endosomes to the actin cytoskeleton

• Regulation of endosome motility and positioning within the cell

• Facilitation of internalization and trafficking of activated tyrosine kinase receptors, particularly PDGFRB

To study RHOD's role in endosomal trafficking, researchers can:

• Track fluorescently labeled cargo proteins (e.g., transferrin, EGF) in cells with modified RHOD expression

• Use live-cell imaging with dual-color labeling of RHOD and endosomal markers

• Employ super-resolution microscopy to resolve endosomal subdomains

• Conduct pulse-chase experiments to follow receptor trafficking pathways

• Analyze endosomal pH and maturation in RHOD-depleted cells

What experimental approaches can distinguish between direct and indirect effects of RHOD on vesicular transport?

Distinguishing direct from indirect effects of RHOD on vesicular transport requires sophisticated experimental designs:

• Acute inactivation/activation approaches:

  • Rapamycin-inducible dimerization to rapidly recruit or remove RHOD from membranes

  • Optogenetic control of RHOD activity with light-sensitive domains

  • Small molecule inhibitors specific to RHOD (though these are currently limited)

• In vitro reconstitution assays:

  • Purify endosomal membranes and add recombinant RHOD protein

  • Measure changes in vesicle dynamics or fusion in a controlled environment

  • Add back specific components to identify minimal requirements

• Mutational analysis:

  • Create RHOD mutants that selectively disrupt binding to specific effectors

  • Generate endosomal targeting mutants that maintain cytoskeletal function

  • Use these tools to dissect which interactions are essential for vesicular transport

• High-resolution temporal studies:

  • Perform time-resolved proteomics of RHOD-associated complexes

  • Conduct live-cell imaging with high temporal resolution

  • Correlate RHOD activation with vesicular movement events

• Selective inhibition of downstream pathways:

  • Use specific inhibitors of actin dynamics, microtubules, or motor proteins

  • Determine if RHOD effects persist when these pathways are blocked

  • Apply computational modeling to predict direct versus feedback effects

How can CRISPR/Cas9 genome editing be optimized for studying RHOD function?

CRISPR/Cas9 provides powerful tools for investigating RHOD function through precise genetic manipulation:

• Knockout strategies:

  • Design gRNAs targeting early exons of the RHOD gene

  • Create complete knockout cell lines to observe loss-of-function phenotypes

  • Generate conditional knockouts using Cre-lox or similar systems for temporal control

• Knockin approaches:

  • Create endogenously tagged RHOD (e.g., RHOD-GFP) to observe native expression levels

  • Introduce specific point mutations to study structure-function relationships

  • Generate reporter lines where fluorescent proteins are expressed under the RHOD promoter

• Domain-specific editing:

  • Target specific functional domains (GTP-binding, effector interaction regions)

  • Create truncation mutants to identify minimal functional units

  • Perform precise base editing to introduce subtle modifications

• Screening applications:

  • Conduct CRISPR screens targeting potential RHOD regulators or effectors

  • Use CRISPRi/CRISPRa to modulate RHOD expression without genetic alterations

  • Perform synthetic lethality screens in RHOD-depleted backgrounds

• Optimization considerations:

  • Validate editing efficiency using sequencing and Western blotting

  • Confirm phenotypes with rescue experiments using wild-type RHOD

  • Create multiple independent clones to control for off-target effects

What proteomics approaches can identify novel RHOD-interacting proteins?

Discovering new RHOD binding partners requires specialized proteomics techniques:

• Proximity-based approaches:

  • BioID or TurboID: Fuse RHOD to a biotin ligase to label proximal proteins

  • APEX: Use RHOD-APEX fusion to biotinylate nearby proteins upon H₂O₂ addition

  • These methods identify both stable and transient interactions in living cells

• Traditional affinity purification:

  • Immunoprecipitate endogenous RHOD using validated antibodies

  • Express tagged RHOD (FLAG, HA, GFP) for pull-down experiments

  • Use crosslinking to stabilize weak or transient interactions

• Quantitative comparison strategies:

  • SILAC, TMT, or label-free quantification to compare:

    • Active vs. inactive RHOD mutants

    • Stimulated vs. basal conditions

    • Wild-type vs. RHOD knockout cells

• Protein array screening:

  • Screen purified recombinant RHOD against protein domain arrays

  • Test specific protein families (e.g., actin-binding proteins, GEFs, GAPs)

  • Validate hits with secondary biochemical assays

• Computational prediction and validation:

  • Use protein-protein interaction prediction algorithms

  • Validate top candidates with co-immunoprecipitation

  • Map interaction networks using systems biology approaches

How can patient-derived samples be used to study RHOD in human disease contexts?

Investigating RHOD in clinical contexts requires specialized approaches for patient samples:

• Tissue analysis techniques:

  • Immunohistochemistry using optimized RHOD antibodies (1:50-1:500 dilution)

  • Immunofluorescence for co-localization studies (1:10-1:100 dilution)

  • Validated in human lung cancer tissue and heart tissue

  • RNA in situ hybridization to detect RHOD mRNA expression

• Patient-derived cell models:

  • Primary cell cultures from patient samples

  • Patient-derived xenografts (PDX) in immunocompromised mice

  • Induced pluripotent stem cells (iPSCs) from patient fibroblasts

  • Differentiation into relevant cell types (e.g., cardiomyocytes if studying heart function)

• Organoid applications:

  • Generate organoids from patient tissues

  • Create heart-forming organoids (HFOs) similar to those described for cardiac studies

  • Compare RHOD expression and function between healthy and diseased organoids

  • Test drug responses in 3D tissue context

• Clinical correlation studies:

  • Measure RHOD expression/activity in patient cohorts

  • Correlate with clinical parameters (disease progression, treatment response)

  • Identify potential biomarker applications

  • Perform genetic association studies for RHOD variants

• Ethical and practical considerations:

  • Ensure proper IRB approval for human subjects research

  • Determine if your research meets the definition of human subjects research

  • Follow systematic investigation protocols with predetermined plans

  • Design studies to contribute to generalizable knowledge

How does RHOD influence cell migration differently from other Rho GTPases?

RHOD shows distinctive effects on cell migration compared to canonical Rho GTPases:

• Unique migration phenotypes:

  • Cells with reduced RHOD show increased cell attachment and decreased migration

  • This contrasts with RhoA (typically enhances contractility) and Rac1 (promotes leading edge protrusion)

  • RHOD's effects appear to be mediated through its specific effectors WHAMM and FILIP1

• Mechanistic differences:

  • RHOD regulation of filopodia differs from Cdc42-mediated filopodia formation

  • RHOD coordinates both Arp2/3-dependent and filamin A-dependent mechanisms

  • This dual regulation creates a unique control system for cellular motility

• Context-dependent effects:

  • RHOD's impact may vary by cell type and microenvironment

  • The balance between RHOD and other Rho GTPases likely determines migration outcomes

  • RHOD's endosomal localization suggests it may regulate receptor recycling during migration

To study these distinctive effects, researchers can:

• Perform wound healing assays comparing RHOD, RhoA, Rac1, and Cdc42 manipulations

• Use microfluidic devices to measure directed migration in controlled gradients

• Conduct single-cell tracking to quantify migration parameters

• Employ traction force microscopy to measure mechanical forces during migration

What methodologies can assess RHOD's role in cancer cell invasion?

To investigate RHOD in cancer invasion contexts, researchers can employ these approaches:

• 3D invasion assays:

  • Spheroid invasion into collagen or Matrigel matrices

  • Transwell invasion assays with different extracellular matrix components

  • Organotypic cultures mimicking tissue-specific microenvironments

  • Real-time imaging of invasion dynamics with fluorescently labeled cells

• Molecular manipulation strategies:

  • Compare effects of RHOD knockdown, knockout, and overexpression

  • Express constitutively active or dominant negative RHOD mutants

  • Target specific RHOD effectors (WHAMM, FILIP1) to dissect pathways

  • Use inducible systems to control RHOD activity temporally

• Analysis of invasion mechanisms:

  • Assess invadopodia formation and matrix degradation

  • Quantify expression of matrix metalloproteinases

  • Evaluate changes in cell-cell and cell-matrix adhesions

  • Measure effects on epithelial-mesenchymal transition markers

• In vivo metastasis models:

  • Intravital imaging of RHOD-manipulated cancer cells

  • Orthotopic implantation to study tissue-specific invasion

  • Circulating tumor cell analysis after RHOD modification

  • Lung colonization assays following tail vein injection

• Clinical correlation:

  • Analyze RHOD expression in invasive tumor fronts

  • Compare RHOD levels between primary tumors and metastases

  • Correlate RHOD expression with patient outcomes

  • Investigate RHOD in therapy-resistant populations

How can RHOD activity be monitored in living cells during experiments?

Researchers have several sophisticated options for visualizing RHOD activity:

• FRET-based biosensors:

  • Design intramolecular FRET sensors with RHOD flanked by fluorescent proteins

  • The sensor changes conformation upon GTP binding, altering FRET efficiency

  • Allows real-time visualization of RHOD activation in specific subcellular locations

  • Can be combined with other fluorescent markers for multiplexed imaging

• Effector domain-based reporters:

  • Express the RHOD-binding domain of WHAMM fused to a fluorescent protein

  • This reporter will relocalize to sites of active RHOD

  • Provides spatial information about RHOD activation

  • Can be combined with optogenetic activation for precise temporal control

• Pull-down assays for biochemical quantification:

  • Use the RHOD-binding domain of effectors in GST-tagged form

  • Perform pull-downs at different timepoints after stimulation

  • Quantify active RHOD by Western blotting

  • Combine with phospho-specific antibodies to correlate with downstream signaling

• Advanced microscopy approaches:

  • Fluorescence correlation spectroscopy (FCS) to measure RHOD diffusion

  • Fluorescence recovery after photobleaching (FRAP) to assess membrane association

  • Single-molecule tracking to follow individual RHOD molecules

  • Super-resolution microscopy to visualize RHOD nanoclusters

• Proteomic readouts:

  • Proximity labeling at different activation states

  • Phosphoproteomic analysis of downstream signaling events

  • Targeted mass spectrometry to quantify RHOD PTMs

  • Correlation with functional readouts like cytoskeletal reorganization