Recombinant Rat Protein Shroom2 (Shroom2), partial

What is Shroom2 and what are its primary functions in rat models?

Shroom2 is a protein involved in regulating proper endothelial morphogenesis through direct interaction with Rho kinase and the subsequent assembly of a cortical actomyosin network. This protein plays a crucial role in maintaining cellular contractility and cytoskeletal organization within endothelial cells. Research has demonstrated that Shroom2 facilitates the formation of a contractile network, which is essential for normal vascular development and endothelial cell behavior. Loss of Shroom2 results in decreased cell contractility, reduced stress fiber organization, and diminished cytoskeletal organization, ultimately leading to increased cell migration and enhanced angiogenesis .

What is the molecular structure of rat Shroom2 protein?

Rat Shroom2 contains a conserved SD2 motif that mediates direct interaction with Rho kinase (Rock). This SD2 domain is evolutionarily conserved and has been shown to facilitate similar interactions in other species, including the interaction between Shrm3 and Rock in mammals and between Drosophila Shroom and Drosophila Rho-associated kinase. The functional significance of this domain lies in its ability to facilitate apical constriction, a critical process in cellular morphogenesis . While the complete structure of recombinant rat Shroom2 is not fully detailed in the available research, understanding this SD2 domain is essential for comprehending the protein's functional mechanism.

How does Shroom2 regulate endothelial cell behavior?

Shroom2 regulates endothelial cell behavior through several interconnected mechanisms:

• Direct interaction with Rho kinase (Rock): Shroom2 binds to Rock through its SD2 domain

• Promotion of actomyosin network assembly: This interaction facilitates the formation of a cortical actomyosin network

• Regulation of cell contractility: The formed network maintains proper cellular tension and contractility

• Control of cytoskeletal organization: Shroom2 influences stress fiber formation and organization

• Modulation of cell-cell adhesion: Shroom2 knockdown results in the loss of Rock and activated myosin II from sites of cell-cell adhesion

Through these mechanisms, Shroom2 effectively controls endothelial cell sprouting, migration, and angiogenesis. When Shroom2 is depleted, endothelial cells exhibit hyperbranched networks with numerous filopodia-like extensions, indicating the protein's role in suppressing excessive angiogenic behavior .

What are effective methods for Shroom2 knockdown in experimental models?

Based on published research, effective Shroom2 knockdown can be achieved using RNA interference techniques, specifically small interfering RNAs (siRNAs). Two different siRNAs have been successfully employed:

• siRNA targeting the 3′ untranslated region (siShrm2–7)

• siRNA targeting the coding sequence (siShrm2–8) of the Shroom2 mRNA

Both approaches have demonstrated approximately 70% reduction in Shroom2 protein levels, as confirmed by both immunostaining and Western blotting analyses. Importantly, these siRNA treatments did not affect cellular proliferation rates, suggesting they specifically target Shroom2 without major off-target effects. For verification of experimental outcomes, it is recommended to use both siRNA approaches to confirm that observed phenotypes result from specific depletion of Shroom2 rather than off-target effects .

How can researchers effectively validate Shroom2 knockdown efficiency?

Researchers can validate Shroom2 knockdown efficiency through multiple complementary methods:

• Immunofluorescence staining: This technique allows visualization of Shroom2 protein levels and distribution within cells before and after knockdown

• Western blotting: Quantitative assessment of protein reduction, with reported knockdown efficiency of approximately 70% using established siRNAs

• Functional assays: Assessment of phenotypic changes consistent with Shroom2 depletion, such as altered endothelial network formation

• Long-term validation: For extended studies, confirm that knockdown remains efficient after differentiation in models such as embryoid bodies derived from stable ES cells

A comprehensive validation approach using multiple methods provides greater confidence in experimental results and helps distinguish specific Shroom2-related phenotypes from potential off-target effects .

How does Shroom2 interact with the Rho kinase pathway to regulate cell contractility?

Shroom2 regulates cell contractility through a sophisticated interaction with the Rho kinase (Rock) pathway. The conserved SD2 motif in Shroom2 mediates direct binding to Rock, which is analogous to the interaction between Shrm3 and Rock in other systems. This interaction facilitates apical constriction and is crucial for proper cytoskeletal organization. When Shroom2 is present, it localizes Rock and activated myosin II to sites of cell-cell adhesion, enabling the formation of a contractile network that maintains proper endothelial cell morphology and behavior .

Research has shown that Shroom2 depletion results in:

• Decreased stress fiber organization

• Reduced collagen contraction

• Increased cellular migration

• Loss of Rock and activated myosin II from cell-cell adhesion sites

These findings suggest that Shroom2 functions as a critical regulator of the Rho kinase pathway in endothelial cells, influencing contractility through modulation of myosin II activation and localization. Understanding this interaction provides insights into the molecular mechanisms controlling endothelial morphogenesis and vascular development .

What phenotypic changes occur in vascular networks following Shroom2 depletion?

Depletion of Shroom2 leads to distinct and consistent phenotypic changes in vascular networks. Research using multiple experimental models, including C166 cells, HUVECs, and embryoid bodies derived from ES cells, has demonstrated the following alterations:

• Hyperbranched endothelial networks: Shroom2-deficient vasculature exhibits significantly increased branching compared to control vasculature

• Aberrant cell morphology: Endothelial cells show irregular cellular borders rather than the uniform borders observed in control conditions

• Increased filopodial extensions: Shroom2-depleted vasculature displays numerous filopodia-like protrusions, in contrast to the limited filopodial extensions seen in control vasculature

• Enhanced sprouting and migration: Loss of Shroom2 promotes increased endothelial sprouting and cellular migration

These phenotypic changes are consistent across different experimental systems and can be observed through immunostaining to visualize the vasculature. The hyperbranched phenotype is particularly notable and suggests that Shroom2 normally functions to restrain excessive angiogenic behavior .

What controls should be included when studying Shroom2 function in endothelial cells?

When designing experiments to study Shroom2 function in endothelial cells, researchers should include several critical controls:

• Multiple siRNA controls:

  • Use at least two different siRNAs targeting distinct regions of the Shroom2 transcript (e.g., one targeting the 3′ UTR and another targeting the coding sequence)

  • Include a non-targeting siRNA control

  • Consider a rescue experiment by reintroducing Shroom2 resistant to siRNA

• Protein expression validation:

  • Perform Western blotting to quantify knockdown efficiency

  • Conduct immunofluorescence staining to confirm reduction of the protein and examine its localization

• Phenotypic controls:

  • Compare phenotypes across multiple cell types (e.g., C166 cells, HUVECs, ES cell-derived endothelial cells)

  • Assess proliferation rates to ensure they remain unaffected by siRNA treatment

• Functional pathway controls:

  • Include assays for Rho kinase activity

  • Monitor myosin II activation and localization

  • Examine stress fiber organization using appropriate cytoskeletal markers

These comprehensive controls help ensure that observed phenotypes are specifically attributable to Shroom2 depletion rather than experimental artifacts or off-target effects .

How can researchers effectively quantify changes in endothelial morphogenesis following Shroom2 manipulation?

Quantifying changes in endothelial morphogenesis following Shroom2 manipulation requires multiple analytical approaches:

• Network analysis:

  • Measure branch points per unit area

  • Calculate average vessel length

  • Determine network complexity using fractal dimension analysis

  • Assess network density and coverage area

• Cellular morphology assessment:

  • Count filopodial extensions per cell

  • Measure cell border regularity

  • Analyze cell shape parameters (aspect ratio, roundness)

  • Evaluate cell-cell junction integrity

• Migration and sprouting quantification:

  • Perform time-lapse imaging to track migration speed and persistence

  • Measure sprouting frequency and extension rate

  • Assess directional migration in response to gradients

  • Quantify collective versus single-cell migration behaviors

• Contractility measurements:

  • Perform collagen gel contraction assays

  • Measure traction forces using deformable substrates

  • Analyze stress fiber organization and orientation

  • Quantify myosin II activation and localization

Standardized image acquisition parameters and blinded analysis are essential for obtaining reliable quantitative data. Advanced image analysis software can facilitate high-throughput analysis of these parameters across multiple experimental conditions .

What are common challenges in producing recombinant Shroom2 protein and how can they be addressed?

While the provided information doesn't specifically address recombinant Shroom2 production, we can draw insights from recombinant protein production principles applicable to Shroom2:

• Expression system selection:

  • Bacterial systems (E. coli): May be suitable for partial Shroom2 domains but might not provide proper folding for full-length protein

  • Mammalian expression systems: Consider for full-length Shroom2 where post-translational modifications may be important

  • Insect cell systems: Baculovirus expression systems offer a balance between proper folding and yield

• Solubility challenges:

  • Use solubility tags (e.g., MBP, SUMO, GST) for difficult-to-express domains

  • Optimize expression conditions (temperature, induction time, media composition)

  • Consider expressing functional domains separately if full-length protein proves difficult

• Purification optimization:

  • Design constructs with appropriate affinity tags for purification

  • Include protease cleavage sites for tag removal

  • Develop multi-step purification strategies to achieve high purity

• Stability considerations:

  • Determine optimal buffer conditions for long-term storage

  • Consider adding stabilizing agents similar to BSA used in other recombinant proteins

  • Test freeze-thaw stability and develop appropriate aliquoting strategies

Researchers should validate protein functionality after purification through binding assays with known interaction partners like Rock, as the SD2 domain-Rock interaction is critical for Shroom2 function .

How should researchers interpret contradictory results in Shroom2 functional studies?

When encountering contradictory results in Shroom2 functional studies, researchers should consider:

• Experimental model variations:

  • Different cell types may express varying levels of Shroom2 interacting partners

  • Primary cells versus cell lines might show different responses to Shroom2 manipulation

  • In vitro versus in vivo models may reveal context-dependent functions

• Knockdown efficiency considerations:

  • Incomplete knockdown might lead to partial phenotypes

  • Different siRNAs may achieve varying degrees of protein reduction

  • Temporal aspects of knockdown (acute versus chronic) may influence results

• Pathway redundancy:

  • Related proteins (Shrm family members) may compensate for Shroom2 loss

  • Alternative pathways regulating contractility might be differentially activated

  • Cell-type specific expression of compensatory factors could mask phenotypes

• Technical approach to resolving contradictions:

  • Use multiple methodological approaches to confirm findings

  • Perform rescue experiments with wild-type and mutant Shroom2 constructs

  • Conduct epistasis experiments to position Shroom2 in signaling pathways

  • Consider dose-dependent effects through titration of siRNA or expression constructs

• Data integration:

  • Develop a model incorporating seemingly contradictory results

  • Consider that Shroom2 may have multiple functions depending on cellular context

  • Use computational approaches to integrate diverse datasets

Careful consideration of these factors can help researchers develop a more comprehensive understanding of Shroom2 function across different experimental contexts .

How might Shroom2 manipulation be utilized in vascular tissue engineering?

The role of Shroom2 in regulating endothelial morphogenesis suggests several potential applications in vascular tissue engineering:

• Controlled angiogenic patterning:

  • Temporary Shroom2 knockdown could promote initial vascular network formation

  • Subsequent restoration of Shroom2 expression might stabilize formed networks

  • Spatial control of Shroom2 expression could guide vessel patterning in engineered tissues

• Modulation of vessel permeability:

  • Shroom2's role in maintaining endothelial cell junctions suggests it may influence barrier function

  • Controlled Shroom2 expression might help regulate vessel permeability in engineered tissues

  • This could be particularly relevant for modeling blood-brain barrier or tumor vasculature

• Engineered vessel maturation:

  • Timed manipulation of Shroom2 could help transition from sprouting angiogenesis to vessel stabilization

  • Co-regulation with pericyte recruitment factors might enhance vessel maturation

  • Integration with extracellular matrix engineering could optimize vessel functionality

• Disease modeling applications:

  • Creation of pathological vascular networks through Shroom2 manipulation

  • Development of high-throughput screening platforms for compounds affecting vascular morphogenesis

  • Generation of patient-specific vascular models for personalized medicine applications

These applications would require precise temporal and spatial control of Shroom2 expression, potentially through inducible expression systems or localized delivery of siRNAs or expression constructs .

What are the most promising directions for future research on Shroom2 in vascular biology?

Future research on Shroom2 in vascular biology should explore several promising directions:

• Mechanistic investigations:

  • Detailed structural analysis of the Shroom2-Rock interaction

  • Identification of additional Shroom2 binding partners in endothelial cells

  • Elucidation of regulatory mechanisms controlling Shroom2 expression and activity

  • Investigation of potential post-translational modifications affecting Shroom2 function

• Physiological relevance:

  • Development of conditional Shroom2 knockout mouse models

  • Analysis of Shroom2 expression and function during embryonic vascular development

  • Examination of Shroom2's role in pathological angiogenesis (tumor, diabetic retinopathy)

  • Investigation of potential links between Shroom2 variants and human vascular disorders

• Therapeutic potential:

  • Development of small molecules targeting the Shroom2-Rock interaction

  • Exploration of Shroom2 as a target for controlling pathological angiogenesis

  • Assessment of Shroom2 manipulation in ischemic disease models

  • Investigation of Shroom2's role in vascular response to existing therapeutics

• Advanced methodological approaches:

  • Application of live-cell imaging to monitor Shroom2 dynamics during endothelial morphogenesis

  • Use of optogenetic tools to achieve precise spatiotemporal control of Shroom2 activity

  • Development of tension sensors to monitor Shroom2-dependent contractility in real-time

  • Integration of multi-omics approaches to place Shroom2 in broader signaling networks

These research directions would significantly advance our understanding of Shroom2's role in vascular biology and potentially reveal new therapeutic targets for vascular disorders .

What are the broader implications of Shroom2 research for understanding vascular development and disease?

Research on Shroom2 has significant implications for our understanding of vascular development and disease:

• Developmental vascular biology:

  • Shroom2's role in regulating endothelial morphogenesis provides insights into the molecular mechanisms controlling vascular network formation

  • Understanding how cytoskeletal regulation influences vascular patterning may inform developmental models

  • The balance between sprouting and stabilization phases of angiogenesis appears to involve Shroom2-mediated contractility

• Pathological angiogenesis:

  • Aberrant Shroom2 function might contribute to dysregulated angiogenesis in conditions like cancer or diabetic retinopathy

  • The hyperbranched phenotype observed with Shroom2 depletion resembles aspects of tumor vasculature

  • Therapeutic strategies targeting endothelial contractility could emerge from Shroom2 research

• Vascular barrier function:

  • Shroom2's interaction with the actomyosin network at cell-cell junctions suggests potential roles in regulating vascular permeability

  • This has implications for conditions involving vascular leakage, such as inflammatory disorders or stroke

• Mechanotransduction pathways:

  • Shroom2 represents an important component of cellular machinery translating mechanical forces into biochemical signals

  • This connects to broader questions about how physical forces shape vascular morphogenesis

By advancing our understanding of these fundamental processes, Shroom2 research contributes to the foundation for developing new therapeutic approaches for vascular disorders with significant clinical impact .

How does the current understanding of Shroom2 compare with knowledge about other cytoskeletal regulators in endothelial cells?

Shroom2 functions within a complex network of cytoskeletal regulators in endothelial cells, with both shared and unique characteristics:

• Comparative mechanism:

  • Like other actin-binding proteins, Shroom2 influences cytoskeletal organization, but its specific interaction with Rock provides a distinctive regulatory mechanism

  • While many cytoskeletal regulators respond to Rho GTPase signaling, Shroom2 appears to function as a direct Rock effector

  • Unlike dynamic regulators of actin polymerization (e.g., WAVE complex, formins), Shroom2 appears more involved in stabilizing contractile structures

• Functional overlap and specificity:

  • Shroom2's control of endothelial sprouting parallels functions of other regulators like VEGF-regulated cytoskeletal effectors

  • The specific combination of increased sprouting with decreased contractility distinguishes Shroom2 from regulators that affect these processes in parallel

  • Shroom2's conserved role across multiple cell types suggests fundamentally important cytoskeletal regulatory functions

• Integration in signaling networks:

  • Shroom2 represents one component of the broader Rho-Rock-myosin II pathway controlling endothelial contractility

  • How Shroom2 coordinates with other Rock substrates and interacting proteins remains an important area for investigation

  • The relationship between Shroom2 and other members of the Shroom family in endothelial cells requires further exploration

• Evolutionary context:

  • The conservation of the SD2 domain across species highlights the fundamental importance of Shroom2's cytoskeletal regulatory mechanism

  • This evolutionary conservation supports the notion that Shroom2 performs essential functions that complement other cytoskeletal regulators