Actin Polymerization Dynamics Research Articles

Abstract 6992: Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma

Abstract Background:Oral squamous cell carcinoma (OSCC) stands as a prevalent malignancy worldwide. Profilin 2 (PFN2), an actin-binding protein regulating actin polymerization dynamics and associated with cell motility, has recently gained attention as a significant regulator in various cancers. However, the clinical significance and biological function of PFN2 in OSCC remain debatable. This study aims to elucidate the role of PFN2 in OSCC. Methods:We assessed PFN2 expression in OSCC and adjacent normal oral mucosa tissues through Q-RT-PCR and immunohistochemistry analysis. Associations between PFN2 expression, clinicopathological features, and OSCC prognosis were analyzed. Cell proliferation, invasion, and migration effects were examined via MTT and Transwell assays. Western blotting assessed the protein expression related to epithelial–mesenchymal transition (EMT). Additionally, we established OSCC xenografts to determine tumorigenicity in vivo using female Nude mice. Results:PFN2 RNA and protein expression were significantly increased in OSCC compared to normal oral mucosa. PFN2 expression positively correlated with lymphovascular invasion and cervical lymph node metastasis. MTT and Transwell assays demonstrated that PFN2 promoted OSCC cell proliferation, migration, invasion, and endothelial invasion. Downregulation of PFN2 induced an EMT phenotype, including an increase in the expression of the epithelial marker E-cadherin and a decrease in the expression of mesenchymal markers Vimentin, Snail, and Slug in OSCC cells in vitro. Similarly, OSCC cells with reduced PFN2 expression exhibited weaker tumorigenicity in vivo. Conclusions:Our findings unveil a novel role for PFN2 in promoting OSCC progression and metastasis, suggesting its potential as an early biomarker for high-risk populations. Targeting PFN2 may offer a promising therapeutic strategy for OSCC treatment. Citation Format: Chung Ji LIu, Li-Han Lin, Kuo-Wei Chang, Hui-Wen Cheng. Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6992.

PIM1 Point Mutations Increase Migration of Diffuse Large B-Cell Lymphoma (DLBCL) Cells

PIM1 serine-threonine kinase is known proto-oncogene, highly expressed in lymphoid malignancies. It regulates crucial processes for the cancer cell, like proliferation, apoptosis, metabolism or migration. In DLBCLs, PIM1 gene is a target of somatic hypermutation and one of the most frequently mutated genes in DLBCL patients. PIM1 mutations are more frequent in ABC-type tumors, which have worse survival prognosis than GCB-type, and often co-occur with mutations in MYD88 and CD79B. Analyses of the available clinical data show that the most significant differences between the groups of DLBCL patients with or without PIM1 mutations are tumor mutational burden, age at diagnosis and extranodal (testicular) involvement. In line with this finding, PIM1, MYD88 and CD79B mutations have also been reported to be highly prevalent in extranodal B cell lymphomas, localized in central nervous system (PCNSL), testicles (PTL) or intravascular space (IVLBCL). Our goal was to assess the role of the most common PIM1 point mutations in the biology and pathogenesis of DLBCL cells. The HBL-1 cell line was chosen as a model of MYD88 L265P-positive ABC-DLBCL subtype. PIM1 knock-out was performed using CRISPR/Cas9 method. Sequences of wild type PIM1, kinase-dead PIM1K67M, serving as a an inactive kinase control, and 7 most frequent mutants (PIM1G28D, PIM1E70K, PIM1P81L, PIM1S97N, PIM1P125A, PIM1L164F, PIM1L184F) were reintroduced to the HBL-1 PIM1 KO cells using lentiviral vectors. PIM1 mutations did not affect the proliferation of DLBCL cells and did not increase resistance to doxorubicin, excluding the proliferation rate or treatment resistance as a cause of the poor prognosis associated with PIM1 mutations. We performed RNA-seq analysis of the HBL-1-PIM1-WT and HBL-1-PIM1-mutants. The most upregulated pathways in PIM1 mutants (including PIM1K67M) were those related to Interferon gamma, IL-15, STAT3, cMET and semaphorin-plexin signaling (involved in proliferation, actin dynamics and migration of cancer cells). We also found that PIM1 mutants universally overexpress cMET oncogene. Moreover, gene set and pathway enrichment analyses indicated that the PIM1-mutant cells exhibit features associated with altered actin polymerization, RHO GTPases and cell membrane dynamics, suggesting the increased migratory potential of the cells and their propensity to spread to extranodal sites. Higher surface level of cMET in PIM1 mutant cells was confirmed using FACS analysis. Since cMET and CXCR4 cooperate in signaling and trigger AKT/mTOR, which is required for CXCR4-mediated cell motility, we evaluated whether PIM1 mutant cells would exhibit increased phosphorylation of AKT. In line with this hypothesis, we noted higher pAKT and pERK1/2 in PIM1 mutant cells than in PIM1 WT cells. Finally, in transwell assays, PIM1 mutant cells exhibited markedly increased migratory potential. Taken together, these studies indicate that PIM1 mutants are not likely associated with differential drug responses, but rather facilitate tumor dissemination and are thus associated with inferior prognosis in DLBCL patients harboring PIM1 mutations.

Defining Signaling Interactomes Involved in Non-Canonical Wnt Signaling By ROR1 or ROR2 in Hairy Cell Leukemia That Can Influence Cell Morphology and Migration

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) and 2 (ROR2) are highly conserved oncoembryonic surface proteins expressed on neoplastic cells of patients with a variety of human cancers, but not on most normal postpartum tissues. ROR1 and ROR2 are each a receptor for non-canonical Wnt factors that direct migration and planar cell polarity, which is essential for organ development. We developed humanized monoclonal antibodies (mAbs) each specific for ROR1 (UC-961) or ROR2 (6E6-70) and found the leukemia cells of patients with classic hairy cell leukemia (cHCL) or variant HCL (vHCL) express both ROR1 and ROR2 and that these receptors function in HCL non-canonical Wnt signaling (Widhopf, G. et al). We hypothesized that ROR1 and ROR2 each may associate with overlapping and distinctive sets of proteins, which may contribute to ROR1/2, ROR1, or ROR2 signaling in HCL. To test this hypothesis, we optimized conditions for generating immune precipitates (i.p.) with either UC-961 or 6E6-70, and examined the proteins of each i.p. via data dependent acquisition mass spectrometry. Consistent with the assumed capacity of each of these mAb to disrupt ROR1/2 heteroligomerization, we identified ROR1, but not ROR2, in i.p. generated using UC-961 and ROR2, but not ROR1, in i.p. generated using 6E6-70 on cell lysates of leukemia cells isolated from a patient with cHCL. In the anti-ROR1 i.p. we identified 1,414 proteins, 849 (60%) of which also were found in the anti-ROR2 i.p.. In the anti-ROR2 i.p. we identified 2,523 proteins, a third of which also were found in the ROR1 i.p.. Among proteins common to both anti-ROR1 and anti-ROR2 i.p. we noted hematopoietic cell-specific protein-1 (HS1 or HCLS1), which we found associated a SH3-binding motif that was dependent on a proline residue at position 841 of activated ROR1 in chronic lymphocytic leukemia (CLL); we found this SH3-binding motif was critical for ROR1-dependent Wnt5a-induced chemokine-directed migration ( Leukemia 12:2615, 2017). Also found common to both ROR1 and ROR2 in HCL were proteins that drive actin polymerization and cytoskeletal dynamics that influence cell morphology and migration. To validate that ROR2 itself was involved in binding HS1 and in enhancing migration, we generated mutant forms of ROR2, each harboring a proline-to-alanine mutation in one of several proline residues found in the proline-rich domain of ROR2. We transduced MEC1 cells, which lack ROR1 or ROR2, with lentivirus vectors encoding wild-type ROR2 or each one of several P-A mutants of ROR2. Flow cytometry confirmed that each of the transduced cells had comparable expression of surface ROR2. We generated anti-ROR2 i.p. using lysates of each and found that all but one of the mutant forms of ROR2 could bind to HS1 by immunoblot analysis. In contrast to wild-type ROR2, or other mutant forms of ROR2, expression of this mutant form of ROR2 could not enhance chemokine-directed migration relative to that of wild-type MEC1 cells. Collectively, our studies define signaling interactomes involved in non-canonical Wnt signaling by ROR1 and/or ROR2 in hairy cell leukemia and define a residue within ROR2 that is necessary for binding HS1 and facilitating cellular migration.

Effect of Porous Substrate Topographies on Cell Dynamics: A Computational Study.

Controlling cell-substrate interactions via the microstructural characteristics of biomaterials offers an advantageous path for modulating cell dynamics, mechanosensing, and migration, as well as for designing immune-modulating implants, all without the drawbacks of chemical-based triggers. Specifically, recent in vivo studies have suggested that a porous implant's microscale curvature landscape can significantly impact cell behavior and ultimately the immune response. To investigate such cell-substrate interactions, we utilized a 3D computational model incorporating the minimum necessary physics of cell migration and cell-substrate interactions needed to replicate known in vitro behaviors. This model specifically incorporates the effect of membrane tension, which was found to be necessary to replicate in vitro cell behavior on curved surfaces. Our simulated substrates represent two classes of porous materials recently used in implant studies, which have markedly different microscale curvature distributions and pore geometries. We found distinct differences between the overall migration behaviors, shapes, and actin polymerization dynamics of cells interacting with the two substrates. These differences were correlated to the shape energy of the cells as they interacted with the porous substrates, in effect interpreting substrate topography as an energetic landscape interrogated by cells. Our results demonstrate that microscale curvature directly influences cell shape and migration and, therefore, is likely to influence cell behavior. This supports further investigation of the relationship between the surface topography of implanted materials and the characteristic immune response, a complete understanding of which would broadly advance principles of biomaterial design.

Abstract P2015: A Human Flii Gene Variant Shortens Sarcomeric Actin Thin Filament Length And Predisposes To Cardiomyopathy

Objective: We identified a human variant in the Flightless-I homolog ( FLII ) gene that generates a R1243H missense change associated with cardiomyopathy in human genome-wide association studies. However, the causality and its molecular mechanism remain unclear. Methods and Results: To examine how the human variant resulting in R1243H amino acid substitution might be pathogenic in vivo, we generated a mouse model harboring the R1245H mutation in Flii locus using CRISPR/Cas9 technology. The Flii R1245H/R1245H homozygotes showed reduced cardiac function by 16 weeks of age compared with control ( P =0.01). Eight-week-old Flii R1245H/R1245H mice subjected to 8 weeks of TAC showed significantly worse cardiac function with greater cardiac hypertrophy and premature lethality compared with control ( P <0.01). Here we show that Flii functions in the heart to regulate sarcomeric actin thin filament length. Indeed, hearts from Flii R1245H/R1245H mice at 16 weeks of age showed reduced thin filament length compared with controls ( P =0.009). To further investigate the mechanistic effects of the R1245H mutation in mouse Flii , we examined actin polymerization dynamics in vitro between wild-type (WT) Flii and the R1245H mutant proteins. The data showed that WT Flii augmented G-actin polymerization into F-actin in a concentration dependent manner, while Flii-R1245H mutant protein antagonized polymerization ( P <0.001). Since LMOD2 is reported to elongate sarcomeric actin thin filaments, we overexpressed LMOD2 using AAV9 system in Flii R1245H/R1245H mice and conducted TAC surgeries at 8 weeks of age. AAV9-LMOD2 rescued the shortening of thin filament length in Flii R1245H/R1245H mice and rescued the exacerbated TAC induced pathology associated with this homozygous mutation (P<0.05). Conclusions: A human FLII variant, R1243H, shortens the sarcomeric actin thin filament, which predisposes to cardiomyopathy. Elongation of thin filament length by LMOD2 overexpression is a possible therapeutic strategy in humans harboring this R1243H mutation.

ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells.

CD4 T cell activation induces nuclear and cytoplasmic actin polymerization via the Arp2/3 complex to activate cytokine expression and strengthen T cell receptor (TCR) signaling. Actin polymerization dynamics and filament morphology differ between nucleus and cytoplasm. However, it is unclear how the Arp2/3 complex mediates distinct nuclear and cytoplasmic actin polymerization in response to a common stimulus. In humans, the ARP3, ARPC1, and ARPC5 subunits of the Arp2/3 complex exist as two different isoforms, resulting in complexes with different properties. Here, we show that the Arp2/3 subunit isoforms ARPC5 and ARPC5L play a central role in coordinating distinct actin polymerization events in CD4 T cells. While ARPC5L is heterogeneously expressed in individual CD4 T cells, it specifically drives nuclear actin polymerization upon T cell activation. In contrast, ARPC5 is evenly expressed in CD4 T cell populations and is required for cytoplasmic actin dynamics. Interestingly, nuclear actin polymerization triggered by a different stimulus, DNA replication stress, specifically requires ARPC5 but not ARPC5L. TCR signaling but not DNA replication stress induces nuclear actin polymerization via nuclear calcium-calmodulin signaling and N-WASP. Diversity in the molecular properties and individual expression patterns of ARPC5 subunit isoforms thus tailors Arp2/3-mediated actin polymerization to different physiological stimuli.

G Protein-coupled Receptor-mediated Membrane Targeting of PLCγ2 is Essential for Neutrophil Chemotaxis.

The current dogma is that chemoattractants G protein-coupled receptors (GPCRs) activate β phospholipase C (PLCβ) while receptor tyrosine kinases (RTKs) activate γ phospholipase C (PLCγ). Here, we show that chemoattractant/GPCR-mediated membrane recruitment of PLCγ2 constitutes GPCR-mediated phospholipase C (PLC) signaling and is essential for neutrophil polarization and migration during chemotaxis. In response to a chemoattractant stimulation, cells lacking PLCγ2 (plcg2kd) displayed altered dynamics of diacylglycerol (DAG) production and calcium response; increased Ras/PI3K/Akt activation; elevated GSK3 phosphorylation and cofilin activation; impaired dynamics of actin polymerization; and consequently, defects in cell polarization and migration during chemotaxis. The study reveals a molecular mechanism of membrane targeting of PLCγ2 and the signaling pathways by which PLCγ2 plays an essential role in neutrophil chemotaxis.

Modeling cellular adhesion.

Cellular adhesion strength, described by the fraction of cells that detach from a surface for a range of applied shear stresses, is predictive of cancer cell migration in 2D and 3D environments and the overall metastatic potential of these cells. Simply stated, cell adhesion strength depends on the balance between cell-surface bond formation and breakage rates. This has been previously modeled as such using the concentration of adhesion proteins such as integrins, the cell-surface interaction area, and a shear force dependent bond breakage rate between the surface ligands and adhesion proteins. However, we have shown that changes in intracellular myosin motor activity, actin stress fibers and force responsive focal adhesion complex proteins can also influence cell adhesion strength and alter cell migration behavior. Using a computational model, we explore how various intracellular factors beyond the concentration of adhesion proteins, govern cell adhesion strengths as measured by shear flow analysis. Overall, our model focuses on 5 key cytoskeletal aspects and their impact on cellular adhesion because of their apparent relevance from previous studies. These include: average number of stress fibers per cell (dictated by f-actin and myosin concentrations), available integrins per stress fiber (dictated by integrin concentration and actin branching), likelihood of bound paxillin, active myosin motors (dictated by myosin light chain phosphorylation), and finally actin polymerization and depolymerization dynamics (governed by time between depolymerization and repolymerization events). The model is further used as a predictive tool in order to determine the particular intracellular factors differentiating cancer strains. The goal of this research is to unravel biomechanical and consequently biochemical differences between cytoskeletal elements of highly metastatic and non-metastatic cancer cells.

N-WASP-Arp2/3 signaling controls multiple steps of dendrite maturation in Purkinje cells in vivo.

During neural development, the actin filament network must be precisely regulated to form elaborate neurite structures. N-WASP tightly controls actin polymerization dynamics by activating an actin nucleator Arp2/3. However, the importance of N-WASP-Arp2/3 signaling in the assembly of neurite architecture in vivo has not been clarified. Here, we demonstrate that N-WASP-Arp2/3 signaling plays a crucial role in the maturation of cerebellar Purkinje cell (PC) dendrites in vivo in mice. N-WASP was expressed and activated in developing PCs. Inhibition of Arp2/3 and N-WASP from the beginning of dendrite formation severely disrupted the establishment of a single stem dendrite, which is a characteristic basic structure of PC dendrites. Inhibition of Arp2/3 after stem dendrite formation resulted in hypoplasia of the PC dendritic tree. Cdc42, an upstream activator of N-WASP, is required for N-WASP-Arp2/3 signaling-mediated PC dendrite maturation. In addition, overactivation of N-WASP is also detrimental to dendrite formation in PCs. These findings reveal that proper activation of N-WASP-Arp2/3 signaling is crucial for multiple steps of PC dendrite maturation in vivo.

Ena/VASP proteins at the crossroads of actin nucleation pathways in dendritic cell migration.

As sentinels of our immune system dendritic cells (DCs) rely on efficient cell migration for patrolling peripheral tissues and delivering sampled antigens to secondary lymphoid organs for the activation of T-cells. Dynamic actin polymerization is key to their macropinocytic and migratory properties. Both major actin nucleation machineries, formins and the Arp2/3 complex, are critical for different aspects of DC functionality, by driving the generation of linear and branched actin filaments, respectively. However, the importance of a third group of actin nucleators, the Ena/VASP family, has not been addressed yet. Here, we show that the two family members Evl and VASP are expressed in murine DCs and that their loss negatively affects DC macropinocytosis, spreading, and migration. Our interactome analysis reveals Ena/VASP proteins to be ideally positioned for orchestrating the different actin nucleation pathways by binding to the formin mDia1 as well as to the WAVE regulatory complex, a stimulator of Arp2/3. In fact, Evl/VASP deficient murine DCs are more vulnerable to inhibition of Arp2/3 demonstrating that Ena/VASP proteins contribute to the robustness and efficiency of DC migration.

Impaired expression of serine/arginine protein kinase 2 (SRPK2) affects melanoma progression.

Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.

Actin Dynamics as a Multiscale Integrator of Cellular Guidance Cues.

Migrating cells must integrate multiple, competing external guidance cues. However, it is not well understood how cells prioritize among these cues. We investigate external cue integration by monitoring the response of wave-like, actin-polymerization dynamics, the driver of cell motility, to combinations of nanotopographies and electric fields in neutrophil-like cells. The electric fields provide a global guidance cue, and approximate conditions at wound sites in vivo. The nanotopographies have dimensions similar to those of collagen fibers, and act as a local esotactic guidance cue. We find that cells prioritize guidance cues, with electric fields dominating long-term motility by introducing a unidirectional bias in the locations at which actin waves nucleate. That bias competes successfully with the wave guidance provided by the bidirectional nanotopographies.

Magic angle spinning NMR structure of human cofilin-2 assembled on actin filaments reveals isoform-specific conformation and binding mode

Actin polymerization dynamics regulated by actin-binding proteins are essential for various cellular functions. The cofilin family of proteins are potent regulators of actin severing and filament disassembly. The structural basis for cofilin-isoform-specific severing activity is poorly understood as their high-resolution structures in complex with filamentous actin (F-actin) are lacking. Here, we present the atomic-resolution structure of the muscle-tissue-specific isoform, cofilin-2 (CFL2), assembled on ADP-F-actin, determined by magic-angle-spinning (MAS) NMR spectroscopy and data-guided molecular dynamics (MD) simulations. We observe an isoform-specific conformation for CFL2. This conformation is the result of a unique network of hydrogen bonding interactions within the α2 helix containing the non-conserved residue, Q26. Our results indicate F-site interactions that are specific between CFL2 and ADP-F-actin, revealing mechanistic insights into isoform-dependent F-actin disassembly.

Quantitative Imaging With DNA-PAINT for Applications in Synaptic Neuroscience.

Super-resolution (SR) microscopy techniques have been advancing the understanding of neuronal protein networks and interactions. Unraveling the arrangement of proteins with molecular resolution provided novel insights into neuron cytoskeleton structure and actin polymerization dynamics in synaptic spines. Recent improvements in quantitative SR imaging have been applied to synaptic protein clusters and with improved multiplexing technology, the interplay of multiple protein partners in synaptic active zones has been elucidated. While all SR techniques come with benefits and drawbacks, true molecular quantification is a major challenge with the most complex requirements for labeling reagents and careful experimental design. In this perspective, we provide an overview of quantitative SR multiplexing and discuss in greater detail the quantification and multiplexing capabilities of the SR technique DNA-PAINT. Using predictable binding kinetics of short oligonucleotides, DNA-PAINT provides two unique approaches to address multiplexed molecular quantification: qPAINT and Exchange-PAINT. With precise and accurate quantification and spectrally unlimited multiplexing, DNA-PAINT offers an attractive route to unravel complex protein interaction networks in neurons. Finally, while the SR community has been pushing technological advances from an imaging technique perspective, the development of universally available, small, efficient, and quantitative labels remains a major challenge in the field.

A nemaline myopathy-linked point mutation destabilizes the second actin binding site of tropomodulin family proteins

Proper function of striated muscle sarcomeres depends on strict regulation of thin filament lengths. Leiomodin (Lmod) and tropomodulin (Tmod), homologous proteins both with an affinity for actin and tropomyosin, bind to the pointed end of thin filaments, altering actin polymerization dynamics and controlling thin filament lengths. Several mutations in the skeletal isoform of Lmod (Lmod3) have been linked to nemaline myopathy, a disease characterized by aberrant sarcomere assembly, hypotonia, and severe muscle weakness ultimately leading to death.

Ca2+-dependent binding of S100A6 to cofilin-1 regulates actin filament polymerization-depolymerization dynamics

S100A6 is a Ca2+-binding protein belonging to the S100 family. Many reports indicate that S100A6 is involved in actin filament organization, however the mechanism of S100A6 action in this process is not fully understood. By screening S100A6 binding partners in NIH3T3 mouse fibroblasts, we have found that S100A6 binds cofilin-1, a protein required for the dynamics of actin polymerization and depolymerization. By applying various biochemical and cell biology assays, we have shown that S100A6 bound to cofilin-1 in a Ca2+-dependent manner and increased cofilin-1 affinity for F-actin. Microscopic analysis indicated that S100A6 significantly decreased severing of the actin filaments induced by cofilin-1. Moreover, in the presence of cofilin-1, S100A6 stabilized the filaments by inhibiting their depolymerization. When S100A6 was present at sub-stoichiometric concentrations in relation to actin, polymerization of G-actin accelerated by cofilin-1 was increased. At higher S100A6:actin ratios the polymerization rate was decreased. Altogether, these results show that S100A6 regulates actin filament dynamics by controlling activity of cofilin-1 and suggest that this regulation is Ca2+ -dependent.

The role of Colchicine on actin polymerization dynamics: as a potent anti-angiogenic factor

Over the years, cancer research has focused on different strategies to discover drugs and therapies to treat the metastatic stage of cancer. This stage depends upon the type, and the cause of cancer. One of the central facts about any cancer invasion is the formation of new blood vessels that provide nutrients to these uncontrollably dividing cells. This phenomenon is called angiogenesis and is responsible for tumor progression and metastasis. Tumor angiogenesis is a sequential process wherein various angiogenic factors produced by tumor cells bind to receptors of endothelial cells. This stimulates the cytoskeletal protein, especially actin to reorganize themselves and undergo the process of canalization. The driving force for such membrane transformation is spatially and temporally-regulated by polymerization of submembrane actin filaments. So far, Colchicine has been studied for its effectiveness in controlling microtubule reorganization during cell division, but its role is far from understood on actin polymerization. In our current study, we report the effect of Colchicine on actin polymerization dynamics using biophysical analysis like Right light scattering (RLS), Dynamic light scattering (DLS), Circular dichroism (CD) analysis, Scanning electron microscopy (SEM) study. Isothermal titration calorimetry (ITC) and kinetic measurements. Isothermal titration calorimetry (ITC) indicates multiple site binding for colchicine with actin aggregates. We have checked the in vivo effect of colchicine using end3 cells of Saccharomyces cerevisiae. We also report the anti-angiogenesis activity of colchicine via ex-ovo chicken chorioallantoic membrane (CAM) assay. We predict the target site of binding for the drug by docking studies. Based on our findings, we suggest the ‘drug-repurposed’ function for colchicine as a potential anti-angiogenic candidate. Communicated by Ramaswamy H. Sarma

The actin-binding protein Adseverin mediates neutrophil polarization and migration.

Through the process of chemotactic migration, neutrophils are able to recruit to an inflammatory site and eliminate pathogens, thus playing a vital role in host defense. The process of neutrophil chemotaxis is mediated by dynamic actin reorganization and polymerization. Adseverin, an actin-binding protein and member of the Gelsolin superfamily of proteins, has been hypothesized to regulate goal directed movement through the capping and severing of actin filaments, but has never been investigated in the context of neutrophil chemotaxis. Using an Adseverin knockout mouse model, we show that Adseverin plays a role in subcortical F-actin assembly at the leading edge during chemotaxis through the generation of free barbed ends on existing actin filaments. In addition, in the absence of Adseverin, neutrophils show reduced speed of migration in vitro and in vivo. This study indicates that Adseverin is a regulator of actin filament generation during neutrophil chemotaxis.

Morphological Definition of Actin Architecture at the T Cell Immunological Synapse

T lymphocytes (T cells) are the major mediators of adaptive immune response. They detect pathogens primarily via the interaction of their T cell receptor (TCR) with the cognate pathogen-derived peptide displayed in the context of MHC on the infected cell. A critical step in T cell activation is the formation of immunological synapse, a specialized cell–cell conjugate interface between the T cell and infected cell, where massive TCR-induced actin remodeling and polymerization take place. Dynamic actin polymerization at the immunological synapse is essential for T cell activation and subsequently, immune response.

Profilin 1 Protein and Its Implications for Cancers.

Profilin 1 (PFN1) is a ubiquitous small-molecule protein that exists in all eukaryotes. PFN1 was first identified as a G-actin sequestering molecule, and subsequently, its true functions in actin polymerization and F-actin dynamics were revealed. In the following decades, the structure of PFN1 was recognized to have 3 domains: an actin-binding domain, a poly-L-proline (PLP)-binding domain, and a phosphoinositide-binding domain. PFN1 plays a vital role in many cell functions, including membrane trafficking, endocytosis, cell cycle, motility, proliferation, cell survival, transcription, stemness, and autophagy (Figure 1). Abnormal expression or deletion of PFN1 can affect the normal physiological activity of cells and lead to disease development. PFN1 has been deeply studied in a variety of diseases, some genetic (eg, amyotrophic lateral sclerosis) and some chronic (eg, hypertension). In the past 10 years, PFN1's role in cancer has received increasing attention. In this review, we summarize the studies of PFN1 in cancer that have been completed in recent years, discuss the roles of PFN1 in cancer, and discuss the implications for tumor diagnosis and therapy in the future.