PFN2 Human

What is PFN2 and what is its primary function in human cells?

PFN2 (Profilin 2) is a small G-actin-binding protein and a well-characterized regulator of actin polymerization. It functions primarily as an actin cytoskeleton regulator and plays important roles in cell motility. PFN2 was previously considered to be expressed mainly in neurons of vertebrates, but research has demonstrated that its expression is altered in different types of cancer, indicating broader tissue expression . The protein has at least two isoforms: PFN2a and PFN2b, both of which have been identified in human cell lines such as HeLa and HAP1 .

PFN2 participates in cytoskeletal reorganization, which directly influences cellular processes like migration and invasion. Its interaction with actin monomers helps regulate the polymerization process, thereby affecting cellular structure and movement. Additionally, PFN2 interacts with NAA80, an actin N-terminal acetyltransferase, suggesting a role in actin acetylation pathways .

How does PFN2 differ from other profilin family members?

While PFN1 (Profilin 1) is ubiquitously expressed in almost all cell and tissue types throughout embryonic development and adulthood, PFN2 shows a more tissue-specific expression pattern. PFN2 is expressed primarily in the developing nervous system and in adult differentiated neurons, making it a neuronal tissue-specific isoform .

A critical functional difference lies in protein interactions. The search results indicate that PFN2 is a specific and direct interactor of NAA80 (an actin N-terminal acetyltransferase), whereas PFN1 does not interact with this protein . This specificity suggests unique roles for PFN2 in actin modification pathways not shared by other profilin family members.

The tissue specificity of PFN2 may make it a potentially better candidate for targeted cancer therapies with fewer side effects on other tissues compared to more ubiquitously expressed family members like PFN1 .

What methodologies are commonly used to study PFN2 expression?

Several robust experimental methods are employed to quantify and characterize PFN2 expression:

• Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for mRNA expression analysis, which allows precise quantification of PFN2 transcript levels .

• Western blotting for protein expression analysis, enabling researchers to determine PFN2 protein levels across different tissues and cell lines .

• Immunohistochemical analysis for evaluating tissue expression patterns, which provides spatial information about PFN2 distribution within tissues .

• Database analysis (e.g., TCGA database) for correlating expression with clinical outcomes, allowing researchers to assess the relationship between PFN2 expression and patient survival .

For cell-based functional studies, researchers commonly employ:

• Cell counting kit-8 (CCK8) assays to investigate effects on cell proliferation

• Transwell assays to evaluate migration and invasion capabilities

• Wound-healing assays to assess cell migration dynamics

What cell lines are appropriate for studying PFN2 function?

Based on the research literature, several cell line models have proven valuable for PFN2 studies:

• For colorectal cancer research:

  • SW620 cells express low levels of endogenous PFN2, making them suitable for overexpression studies

  • HCT116 cells exhibit high levels of endogenous PFN2, making them appropriate for knockdown experiments

• For breast cancer studies, particularly triple negative breast cancer (TNBC) cell lines, which have been used to demonstrate PFN2's role in promoting proliferation, migration, and invasion .

• HeLa and HAP1 cells for studying PFN2 interactions with other proteins like NAA80 .

When selecting appropriate cell lines, researchers typically perform preliminary western blotting experiments to examine baseline expression of PFN2 across multiple cell lines and select those with expression levels suitable for the experimental design . This approach helps ensure that the cells will provide meaningful data about PFN2 function in the relevant biological context.

How does PFN2 contribute to cancer progression, and why does it appear to have opposing roles in different cancer types?

One of the most intriguing aspects of PFN2 research is its apparently contradictory roles across cancer types:

In Triple Negative Breast Cancer (TNBC):

In Colorectal Cancer (CRC):

• PFN2 expression is downregulated, particularly in metastatic CRC

• Low PFN2 expression is related to enhanced EMT and increased migratory capabilities

• PFN2 overexpression inhibits the EMT process and associated migration

• PFN2 appears to suppress cancer metastasis by regulating cytoskeletal reorganization

• PFN2 increases myosin light chain (MLC) phosphorylation, which supports the formation of contractile actin bundles that suppress cancer cell migration

These opposing roles likely reflect context-dependent functions influenced by:

• Tissue-specific signaling networks and protein interactions

• Different upstream regulators controlling PFN2 expression

• Varying dependency on cytoskeletal reorganization between cancer types

• Cancer-specific post-translational modifications of PFN2

These findings underscore the importance of context-specific research when studying PFN2, as findings from one cancer type cannot be automatically extrapolated to others.

What is the relationship between PFN2 and epithelial-to-mesenchymal transition (EMT) in cancer?

PFN2 exhibits complex and cancer type-specific relationships with EMT:

In TNBC:

• PFN2 promotes the EMT process

• Smad2 and Smad3 are upregulated in PFN2 overexpressing TNBC cells

• This upregulation further induces EMT, enhancing migratory and invasive properties

• PFN2 overexpression correlates with increased tumorigenicity

In CRC:

• PFN2 inhibits the EMT process

• Low PFN2 expression is associated with enhanced EMT

• PFN2 overexpression in SW620 cells (which have low endogenous PFN2) inhibits EMT

• This inhibition is associated with increased myosin light chain (MLC) phosphorylation

• Inhibition of MLC phosphorylation (using Y27632) attenuates the inhibitory effects of PFN2 on EMT

These findings suggest that PFN2's effect on EMT is mediated through cytoskeletal reorganization, particularly through regulation of contractile actin bundles. In CRC, PFN2 triggers MLC phosphorylation, which promotes the formation of contractile actin bundles that suppress cancer cell migration and invasion .

The differing effects on EMT may explain why PFN2 serves as a poor prognostic factor in breast cancer but potentially a favorable factor in colorectal cancer.

How does PFN2 regulate cytoskeletal reorganization in the context of cancer cell migration?

PFN2's regulation of cytoskeletal reorganization appears central to its role in cancer progression:

• Actin dynamics regulation: As an actin-binding protein, PFN2 directly influences actin polymerization, affecting cellular architecture and motility .

• MLC phosphorylation: In CRC cells, PFN2 expression significantly increases phosphorylated myosin light chain (pMLC) levels. pMLC is a marker of myosin motor contractions and promotes the formation of contractile actin bundles .

• Contractile actin bundles: These structures act as suppressors of cancer protrusive activity, migration, and invasion. In normal colon tissues and non-metastatic CRC tissues, pMLC is expressed at higher levels compared to metastatic CRC tissues, suggesting a suppressive role for these structures in cancer progression .

• Experimental evidence: The inhibition of MLC phosphorylation (using Y27632) reverses the effects of PFN2 overexpression in SW620 cells, resulting in decreased E-cadherin (epithelial marker) and increased vimentin (mesenchymal marker) expression, and enhanced migration capacity. This confirms that PFN2's effects on migration are mediated through cytoskeletal contractility .

• Actin acetylation pathway: PFN2 interacts with NAA80, an actin N-terminal acetyltransferase, suggesting a potential role in regulating actin acetylation, which could further influence cytoskeletal dynamics .

These findings collectively indicate that PFN2 regulates cancer cell migration through its effects on the cytoskeleton, particularly through modulation of contractile structures that influence cell motility.

What techniques should be used to investigate the interaction between PFN2 and NAA80?

The emerging interaction between PFN2 and NAA80 (an actin N-terminal acetyltransferase) represents an important area of research. Based on successful approaches documented in the literature, researchers should consider:

• Affinity-enrichment LC-MS approach:

  • Express V5-tagged NAA80 in appropriate cell lines (HeLa cells have been used successfully)

  • Perform immunoprecipitation (IP)

  • Analyze and quantify the IPs by LC-MS and label-free quantification (LFQ)

  • Include appropriate controls to validate specificity

• Co-immunoprecipitation (co-IP) validation:

  • Conduct reciprocal co-IP experiments pulling down either protein and probing for the other

  • Include specificity controls testing interaction with related proteins (e.g., PFN1)

  • Verify results across multiple cell lines

• Isoform-specific analysis:

  • Design experiments to distinguish between PFN2a and PFN2b isoforms

  • Use mass spectrometry peptide identification to differentiate isoforms

  • Determine if both isoforms interact equally with NAA80

• Functional interaction studies:

  • Investigate whether PFN2 affects NAA80's acetyltransferase activity

  • Examine if the interaction influences actin acetylation patterns

  • Study potential consequences for cytoskeletal dynamics and cell motility

How can researchers reconcile the contradictory findings about PFN2 function across different cancer types?

The apparently opposing roles of PFN2 in different cancers pose a significant challenge for researchers. To reconcile these findings, consider these methodological approaches:

• Comparative molecular profiling:

  • Conduct comprehensive signaling pathway analyses across cancer types

  • Identify cancer-specific PFN2 interactors through proteomic approaches

  • Compare post-translational modifications of PFN2 between cancer types

  • Analyze transcriptional and epigenetic regulation of PFN2

• Parallel experimental design:

  • Study PFN2 function in multiple cancer cell lines simultaneously

  • Use consistent methodologies and readouts across cancer types

  • Perform gain- and loss-of-function studies in each model

  • Control for cell-specific factors that might influence results

• Context-dependent analysis:

  • Investigate tissue-specific factors that could modify PFN2 function

  • Study how the tumor microenvironment influences PFN2 activity

  • Examine how genetic background (common mutations in each cancer) affects PFN2 function

  • Consider differences in baseline cytoskeletal organization between tissue types

• Systems biology approach:

  • Construct network models of PFN2 interactions in each cancer type

  • Identify nodes where regulation diverges between cancers

  • Use computational modeling to predict context-specific behaviors

  • Validate predictions experimentally

Understanding the molecular basis for these contradictory roles could lead to more effective targeting of PFN2 or its regulated pathways in a cancer type-specific manner. The different effects on MLC phosphorylation and cytoskeletal organization may provide a starting point for investigating these differences .

How should researchers design PFN2 overexpression experiments?

Based on published methodologies, here are detailed recommendations for PFN2 overexpression experiments:

• Vector construction:

  • Amplify full-length human PFN2 cDNA by RT-PCR using specific primers (e.g., forward: 5′-GCGGCCGCATGGCCGGTTGGCAGAGCTACG-3′ and reverse: 5′-GGATCCTTACACATCAGACCTCCTCAG-3′)

  • Clone the PFN2 coding sequence into an appropriate expression vector (e.g., pQCXIH)

  • Verify the construct by sequencing before use

• Transfection protocol:

  • Seed cells (e.g., 2×10^5 cells/well in a 6-well plate) 24 hours prior to transfection

  • Culture to approximately 80% confluency

  • Transfect with appropriate amount of plasmid DNA (e.g., 1.5 μg) using Lipofectamine 2000 (2.5 μl)

  • Include empty vector transfection as a control

  • Harvest cells 24 hours post-transfection for further experiments

• Validation steps:

  • Confirm PFN2 overexpression by western blotting

  • Assess functional consequences using appropriate assays (proliferation, migration, invasion)

  • Examine effects on downstream pathways (e.g., EMT markers, MLC phosphorylation)

  • Include appropriate time points to capture both early and late effects

• Cell line selection:

  • Choose cell lines with low endogenous PFN2 expression (e.g., SW620 for CRC studies)

  • Perform preliminary western blotting to confirm expression levels

  • Consider multiple cell lines to ensure reproducibility of findings

These approaches have been successfully applied in published studies and provide a robust foundation for investigating PFN2 function in cancer cells.

What assays are most effective for studying PFN2's impact on cell migration and invasion?

To comprehensively assess PFN2's effects on migration and invasion, researchers should employ multiple complementary assays:

• Wound healing (scratch) assay:

  • Create a scratch in a confluent monolayer of cells

  • Monitor and quantify wound closure over time

  • Compare migration rates between PFN2-manipulated cells and controls

  • This assay has successfully demonstrated differences in migratory capacity following PFN2 overexpression in SW620 cells

• Transwell migration assay:

  • Place cells in serum-free medium in the upper chamber

  • Add chemoattractant in the lower chamber

  • Quantify cells that migrate through the membrane

  • This approach has shown that PFN2 promotes migration of TNBC cells

• Transwell invasion assay:

  • Coat transwell membranes with Matrigel to mimic extracellular matrix

  • Assess the ability of cells to invade through this barrier

  • This method has demonstrated that PFN2 enhances invasive capabilities of TNBC cells

• 3D spheroid invasion assay:

  • Grow cells as spheroids embedded in a matrix

  • Monitor invasion into surrounding matrix

  • Provides more physiologically relevant context than 2D assays

• Live-cell imaging:

  • Track individual cell movements over time

  • Analyze parameters like velocity, directionality, and persistence

  • Provides detailed information about migration patterns

• EMT marker analysis:

  • Assess expression of epithelial markers (E-cadherin) and mesenchymal markers (vimentin)

  • Correlate changes with PFN2 expression and migratory behavior

  • Both TNBC and CRC studies have used this approach to connect PFN2 to EMT status

When performing these assays, researchers should:

• Include appropriate controls (empty vector, scrambled siRNA)

• Conduct time-course experiments to capture dynamic effects

• Normalize results to account for potential proliferation differences

• Consider the impact of cell density on migration behavior

How can researchers effectively analyze PFN2's effect on cytoskeletal reorganization?

Studying PFN2's impact on the cytoskeleton requires specialized techniques:

• Immunofluorescence microscopy:

  • Fix cells and stain with phalloidin for F-actin visualization

  • Co-stain for PFN2 and other cytoskeletal components

  • Analyze stress fiber formation, focal adhesions, and cell morphology

  • Use quantitative image analysis to measure parameters like fiber alignment and density

• Western blotting for cytoskeletal markers:

  • Analyze levels of phosphorylated myosin light chain (pMLC), a marker of contractile activity

  • Compare pMLC levels between PFN2-manipulated cells and controls

  • This approach has successfully demonstrated that PFN2 overexpression increases pMLC levels in CRC cells

• Pharmacological intervention:

  • Use inhibitors like Y27632 (3 μmol/l) to block specific cytoskeletal pathways

  • Determine if inhibition reverses PFN2-induced phenotypes

  • This strategy has shown that inhibiting MLC phosphorylation attenuates the effects of PFN2 on EMT and migration in CRC cells

• G-actin/F-actin ratio measurement:

  • Separate and quantify globular and filamentous actin fractions

  • Determine how PFN2 manipulation affects this balance

  • Correlate with changes in cell behavior

• Quantitative analysis approaches:

  • Develop metrics for cytoskeletal organization (e.g., stress fiber density, orientation)

  • Use automated image analysis to reduce bias

  • Perform statistical comparisons across multiple fields and experiments

By combining these approaches, researchers can gain comprehensive insights into how PFN2 regulates cytoskeletal dynamics and how these changes impact cancer cell behavior.

What are the best practices for correlating PFN2 expression with clinical outcomes?

To robustly analyze relationships between PFN2 expression and patient outcomes, researchers should follow these methodological guidelines:

Following these practices will yield more reliable and clinically meaningful insights into PFN2's prognostic significance.

What controls should be included when studying PFN2 in different experimental systems?

Robust experimental design requires careful consideration of controls. When studying PFN2, researchers should include:

• Expression manipulation controls:

  • For overexpression: Empty vector transfection to control for transfection effects

  • For knockdown: Non-targeting siRNA/shRNA to control for non-specific effects

  • Rescue experiments: Re-expressing PFN2 in knockdown cells to confirm specificity

• Cell-specific controls:

  • Multiple cell lines within each cancer type to ensure reproducibility

  • Matched normal and cancer cell lines to understand disease-specific effects

  • Preliminary expression analysis to establish baseline PFN2 levels

• Pathway-specific controls:

  • Pharmacological inhibitors (e.g., Y27632 for ROCK inhibition) to validate mechanistic findings

  • Positive controls for assays (known inducers of migration, EMT, etc.)

  • Analysis of related family members (e.g., PFN1) to assess specificity

• Technical controls:

  • Loading controls for western blotting (e.g., β-actin, GAPDH)

  • Internal controls for RT-qPCR (reference genes)

  • Vehicle controls for drug treatments

  • Time-matched controls for time-course experiments

• Validation controls:

  • Orthogonal methods to confirm key findings

  • Independent experimental replication

  • Blinded analysis of results when possible

The CRC studies in the search results exemplify good control practices, including the use of both vector-transfected and untransfected control groups for overexpression experiments, and the inclusion of pharmacological inhibitors to validate mechanistic findings .

How might PFN2 serve as a therapeutic target, and what approaches show promise?

Given PFN2's divergent roles in different cancers, therapeutic targeting strategies would need to be cancer-specific:

• For cancers where PFN2 promotes progression (e.g., TNBC):

  • Small molecule inhibitors targeting PFN2-actin interaction

  • RNA interference approaches to downregulate PFN2 expression

  • Targeting downstream effectors in the TGF-β/Smad pathway

  • Blocking PFN2-mediated EMT through complementary approaches

• For cancers where PFN2 suppresses progression (e.g., CRC):

  • Gene therapy approaches to restore PFN2 expression

  • Small molecules that enhance PFN2 stability or activity

  • Compounds that promote MLC phosphorylation to compensate for PFN2 loss

  • Targeting pathways activated by PFN2 loss

• Developing targeted delivery systems:

  • Exploit the tissue-specific expression pattern of PFN2 (primarily neuronal)

  • Design therapeutic agents that selectively target cancer cells

  • Consider local delivery strategies to minimize off-target effects

• Biomarker applications:

  • Use PFN2 expression levels to guide treatment selection

  • Develop companion diagnostics for PFN2-targeted therapies

  • Monitor changes in PFN2 expression as an indicator of treatment response

The neuronal tissue-specific expression pattern of PFN2 may provide a therapeutic window that could be exploited to minimize off-target effects, potentially making it a more attractive target than the ubiquitously expressed PFN1 .

What questions remain unanswered about PFN2 isoforms in cancer biology?

Several critical questions about PFN2 isoforms require further investigation:

• Isoform-specific expression patterns:

  • How do the expression levels of PFN2a and PFN2b vary across cancer types?

  • Is the ratio between isoforms altered during cancer progression?

  • Are there tissue-specific patterns of isoform expression?

• Functional differences:

  • Do PFN2a and PFN2b have distinct roles in cytoskeletal regulation?

  • How do they differentially affect cancer cell behaviors like migration and invasion?

  • Do they interact with different protein partners?

• Regulation mechanisms:

  • What controls the alternative splicing of PFN2 isoforms?

  • Are there cancer-specific splicing factors that regulate this process?

  • How is isoform expression affected by tumor microenvironment signals?

• Clinical relevance:

  • Do specific isoforms correlate with different clinical outcomes?

  • Could isoform ratios serve as more precise biomarkers than total PFN2?

  • Would isoform-specific targeting provide therapeutic advantages?

How does the PFN2-NAA80 interaction influence actin dynamics in cancer cells?

The interaction between PFN2 and NAA80 (an actin N-terminal acetyltransferase) represents an exciting research frontier:

• Biochemical mechanisms to investigate:

  • Does PFN2 modulate NAA80's acetyltransferase activity?

  • Does this interaction affect specificity for actin modification?

  • Is the interaction regulated during cancer progression?

• Functional consequences to explore:

  • How does actin acetylation affect polymerization dynamics?

  • Does altered acetylation influence cellular motility?

  • Are metastatic properties affected by this interaction?

• Cancer type variations to consider:

  • Is the PFN2-NAA80 interaction consistent across cancer types?

  • Does it contribute to the divergent roles of PFN2 in different cancers?

  • Are there cancer-specific modulators of this interaction?

• Technical approaches needed:

  • Develop acetylatisn assays to measure NAA80 activity in the presence/absence of PFN2

  • Create interaction-deficient mutants to test functional importance

  • Use live-cell imaging to track actin dynamics with altered PFN2-NAA80 interaction

The search results show that PFN2 (but not PFN1) interacts with NAA80, suggesting isoform specificity in this interaction . Understanding how this specific interaction affects actin dynamics could provide crucial insights into PFN2's role in cancer progression.

What is the relationship between PFN2 and the tumor microenvironment?

While the search results focus primarily on cell-autonomous effects of PFN2, its role in cytoskeletal regulation likely mediates interactions with the tumor microenvironment:

• Cell-matrix interactions:

  • How does PFN2 affect adhesion to different extracellular matrix components?

  • Does PFN2 influence matrix remodeling through contractility regulation?

  • Is mechano-sensing altered by PFN2 expression?

• Cancer cell-stromal cell interactions:

  • Does PFN2 modulate cancer cell interactions with fibroblasts, immune cells, or endothelial cells?

  • Are paracrine signaling pathways affected by PFN2-mediated cytoskeletal changes?

  • How do stromal cells influence PFN2 expression or function in cancer cells?

• Migration in complex environments:

  • How does PFN2 affect migration through 3D matrices versus 2D surfaces?

  • Does it influence collective versus single-cell migration?

  • Are invasion-associated structures (invadopodia, podosomes) regulated by PFN2?

• Methodological approaches needed:

  • 3D culture systems to better recapitulate tissue architecture

  • Co-culture experiments with stromal components

  • In vivo imaging to observe cytoskeletal dynamics in physiological contexts

Understanding these interactions could help explain the context-dependent effects of PFN2 observed in different cancer types .

How might PFN2 contribute to therapy resistance in cancer?

Although not directly addressed in the search results, PFN2's role in cytoskeletal regulation suggests potential impacts on therapy response:

• Chemotherapy resistance mechanisms to investigate:

  • Does PFN2-mediated cytoskeletal reorganization affect drug uptake or efflux?

  • Are apoptotic pathways influenced by PFN2 expression?

  • Does EMT regulation by PFN2 contribute to chemoresistance?

• Targeted therapy considerations:

  • How does PFN2 interact with commonly targeted signaling pathways?

  • Could PFN2 serve as a bypass mechanism for kinase inhibition?

  • Are there synthetic lethal interactions that could be exploited?

• Radiotherapy response:

  • Does PFN2 affect DNA damage repair through nuclear architecture regulation?

  • Is radiation-induced migration influenced by PFN2 expression?

  • Could targeting PFN2 enhance radiosensitivity?

• Biomarker potential:

  • Could PFN2 expression predict response to specific therapies?

  • Would monitoring PFN2 during treatment provide insights into resistance development?

  • Is the PFN2 isoform ratio relevant to therapy response?

Given the opposing roles of PFN2 in different cancer types, its contribution to therapy resistance likely varies by context. The relationship between PFN2, EMT, and MLC phosphorylation observed in the search results provides a foundation for exploring these questions .

What is the current consensus on PFN2's role in human cancer?

Based on the available research, the current consensus on PFN2 in cancer can be summarized as follows:

• PFN2 plays important but context-dependent roles in cancer progression, with functions that vary significantly between cancer types.

• In triple negative breast cancer (TNBC), PFN2 acts as a promoter of malignancy:

  • It is upregulated in TNBC tissues

  • Higher expression correlates with worse survival

  • It promotes proliferation, migration, and invasion

  • PFN2 enhances EMT through the TGF-β/Smad pathway

• In colorectal cancer (CRC), PFN2 appears to function as a tumor suppressor:

  • Its expression is downregulated in metastatic CRC

  • Loss of PFN2 contributes to enhanced EMT and metastasis

  • It inhibits migration by promoting MLC phosphorylation and contractile actin bundle formation

  • Low expression is associated with poor prognosis

• PFN2 interacts with NAA80, an actin N-terminal acetyltransferase, suggesting a role in regulating actin acetylation, which may further influence cytoskeletal dynamics and cell motility .

• Multiple isoforms of PFN2 (PFN2a and PFN2b) exist and may have distinct functions, though both interact with NAA80 .

This consensus highlights the complexity of PFN2 biology and emphasizes the need for cancer type-specific research when investigating its functions and therapeutic potential.

What methodological advances are needed to better understand PFN2 biology?

To advance our understanding of PFN2 biology, several methodological improvements are needed:

• Isoform-specific tools:

  • Development of antibodies that specifically recognize PFN2a versus PFN2b

  • Creation of isoform-specific knockout and knockin models

  • Methods to selectively modulate each isoform

• Advanced imaging techniques:

  • Improved live-cell imaging approaches to visualize PFN2-actin interactions in real-time

  • Super-resolution microscopy to better resolve cytoskeletal structures

  • Correlative light and electron microscopy to link functional and structural data

• Biochemical and proteomic approaches:

  • More sensitive techniques to detect actin post-translational modifications

  • Improved methods for analyzing PFN2 interaction networks

  • Assays to measure the functional consequences of the PFN2-NAA80 interaction

• In vivo cancer models:

  • Development of genetic models with conditional PFN2 manipulation

  • Patient-derived xenografts to better represent human disease

  • Intravital imaging to observe cytoskeletal dynamics in the tumor microenvironment

• Computational and systems biology:

  • Better algorithms for analyzing cytoskeletal organization from imaging data

  • Network analysis tools to integrate PFN2 into broader signaling contexts

  • Predictive models of how PFN2 alterations affect cell behavior