Regulation Of Actin Polymerization Research Articles

FXR1 regulates vascular smooth muscle cell cytoskeleton, VSMC contractility, and blood pressure by multiple mechanisms

Appropriate cytoskeletal organization is essential for vascular smooth muscle cell (VSMC) conditions such as hypertension. This study identifies FXR1 as a key protein linking cytoskeletal dynamics with mRNA stability. RNA immunoprecipitation sequencing (RIP-seq) in human VSMCs identifies that FXR1 binds to mRNA associated with cytoskeletal dynamics, and FXR1 depletion decreases their mRNA stability. FXR1 binds and regulates actin polymerization. Mass spectrometry identifies that FXR1 interacts with cytoskeletal proteins, particularly Arp2, a protein crucial for VSMC contraction, and CYFIP1, a WASP family verprolin-homologous protein (WAVE) regulatory complex (WRC) protein that links mRNA processing with actin polymerization. Depletion of FXR1 decreases the cytoskeletal processes of adhesion, migration, contraction, and GTPase activation. Using telemetry, conditional FXR1SMC/SMC mice have decreased blood pressure and an abundance of cytoskeletal-associated transcripts. This indicates that FXR1 is a muscle-enhanced WRC modulatory protein that regulates VSMC cytoskeletal dynamics by regulation of cytoskeletal mRNA stability and actin polymerization and cytoskeletal protein-protein interactions, which can regulate blood pressure.

FAT10 Induces cancer cell migration by stabilizing phosphorylated ABI3/NESH

ABSTRACT The WAVE regulatory complex (WRC) is involved in various cellular processes by regulating actin polymerization. The dysregulation of WRC components is associated with cancer development. ABI family member 3 (ABI3)/new molecule including SH3 (NESH) is one of the WRC components and it has been reported that ABI3 phosphorylation can affect WRC function. Although several residues of ABI3 have been reported to be possible phosphorylation sites, it is still unclear which residues are important for the function of ABI3. Furthermore, it is unclear how the phosphorylated form of ABI3 is regulated. Here, we demonstrate that ABI3 is stabilized by its interaction with human leukocyte antigen-F adjacent transcript 10 (FAT10). Using phospho-dead or phospho-mimetic mutants of ABI3, we showed that serine 213 and 216 are important phosphorylation sites of ABI3. In particular, FAT10 has a higher affinity for the phosphorylated form of ABI3 than the non-phosphorylated form, and it stabilizes the phosphorylated form more than the non-phosphorylated form through this differential affinity. The interaction between FAT10 and the phosphorylated form of ABI3 promoted cancer cell migration. Therefore, our results suggest that FAT10 stabilizes the phosphorylated form of ABI3, which may lead to WRC activation, thereby promoting cancer cell migration.

Phosphorylation of the actin-binding protein profilin2a at S137 modulates bidirectional structural plasticity at dendritic spines.

Background: Synaptic plasticity requires constant adaptation of functional and structural features at individual synaptic connections. Rapid re-modulation of the synaptic actin cytoskeleton provides the scaffold orchestrating both morphological and functional modifications. A major regulator of actin polymerization not only in neurons but also in various other cell types is the actin-binding protein profilin. While profilin is known to mediate the ADP to ATP exchange at actin monomers through its direct interaction with G-actin, it additionally is able to influence actin dynamics by binding to membrane-bound phospholipids as phosphatidylinositol (4,5)-bisphosphate (PIP2) as well as several other proteins containing poly-L-proline motifs including actin modulators like Ena/VASP, WAVE/WASP or formins. Notably, these interactions are proposed to be mediated by a fine-tuned regulation of post-translational phosphorylation of profilin. However, while phosphorylation sites of the ubiquitously expressed isoform profilin1 have been described and analyzed previously, there is still only little known about the phosphorylation of the profilin2a isoform predominantly expressed in neurons. Methods: Here, utilizing a knock-down/knock-in approach, we replaced endogenously expressed profilin2a by (de)phospho-mutants of S137 known to alter actin-, PIP2 and PLP-binding properties of profilin2a and analyzed their effect on general actin dynamics as well as activity-dependent structural plasticity. Results and Discussion: Our findings suggest that a precisely timed regulation of profilin2a phosphorylation at S137 is needed to mediate actin dynamics and structural plasticity bidirectionally during long-term potentiation and long-term depression, respectively.

RCAS1 increases cell morphological changes in murine fibroblasts by reducing p38 phosphorylation.

Receptor‑binding cancer antigen expressed on SiSo cells (RCAS1) is a tumor‑associated antigen that is expressed in a number of human malignancies. RCAS1 acts as a ligand for a putative RCAS1 receptor that is present on various human cells including T and B lymphocytes and natural killer cells, in which it induces cell growth inhibition and apoptosis. It has been suggested that RCAS1 might serve an important role in tumor cell evasion from the host immune system. In fact, RCAS1 expression is related to malignant characteristics including tumor size, invasion depth, clinical stage and poor overall survival. The authors previously established doxycycline‑induced RCAS1 overexpression murine fibroblast L cells to analyze the biological functions of RCAS1 and reported that its expression inhibited cell cycle progression via the downregulation of cyclin D3, which subsequently induced apoptosis. Additionally, it was found that RCAS1 expression induced cell morphological changes prior to caspase‑mediated apoptosis. Thus, the present study examined signaling pathways associated with changes in cell morphology that were induced by RCAS1 expression. The data showed that increased RCAS1 expression caused a reduction in actin stress fibers and decreased cofilin phosphorylation. Recent studies have shown that p38 signaling regulates actin polymerization. The data the present study showed that increased RCAS1 expression significantly decreased p38 phosphorylation.

Complex multivalent protein interactions involving endophilin, lamellipodin and VASP regulate membrane shaping phenomena and actin assembly.

Lamellipodin (LPD) is a peripheral membrane protein that plays an initiatory role in endocytic pathways such as Fast endophilin mediated endocytosis by the recruitment of Endophilin, and regulatory roles in lamellipodia and filopodia by recruiting ena/VASP proteins. The processes of endocytosis and cellular motility depend on actin polymerization and involve inward and outward deformation of the plasma membrane respectively. The question thus can be asked: how does LPD recruit its binding partners, the curvature generating BAR protein endophilin and the actin polymerase VASP, to specifically trigger the aforementioned phenomena and concomitant directional membrane remodelling? In this thrust, we probe the complex multi-protein interplay by reconstituting these interactions in vitro. First, we show that VASP and LPD associate multivalently to undergo liquid like phase separation, a fundamental physical process ubiquitously observed in biology. We find that actin polymerization is exclusively initiated in the LPD-VASP rich clusters, and that elongating filaments deform the clusters resulting in local protein reorganization and the formation of large scale VASP-LPD facilitated actin networks. Our observation along with the fact that growing actin filaments deform the adjacent lipid bilayer, introduces a new intermediate step for membrane remodelling, a protein-protein phase separation mediated actin network formation. Next, we investigated the role of Endophilin in the above-mentioned system. We have previously shown that endophilin and LPD interact multivalently in the priming of FEME patches. Here we find that endophilin participates in VASP-LPD phase separation and influences actin filament morphology and connectivity. We thus anticipate that the initiation and the direction of curvature generation are governed by complex multivalent interactions that regulate actin polymerization.

Solution NMR studies reveal novel CLIC4 chaperone activity with respect to profilin-1.

Despite its name, chloride intracellular channel 4 (CLIC4) is a soluble enzyme that can catalyze glutathione-dependent thiol transfers. It is unclear how this enzymatic activity relates to its biological role in remodeling the actin cytoskeleton. Previous studies have shown that CLIC4 interacts with profilin-1, a critical regulator of actin polymerization. We confirmed a direct interaction, 170 ± 20 μM by microscale thermophoresis. However, using solution nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that CLIC4 has a higher affinity for profilin when it is unfolded. Amide hydrogen exchange studies demonstrate that the N- and C-terminal helices of profilin readily unfold, whereas the central β-beta sheet core remains relatively intact. Interaction with CLIC4 increases amide exchange rates throughout profilin, indicating preferential stabilization of partially unfolded intermediates. We note that profilin is glutathionylated in proteomic studies of cells exposed to oxidative stress. Profilin is unfolded by glutathionylation of buried cysteine residues in its N- or C-terminal helices, and this disrupts its ability to bind actin, as measured by pyrene-actin polymerization assay. We are currently attempting to demonstrate that CLIC4 can reverse glutathionylation of profilin-1 and promote its refolding in cells and in vitro.

Knockdown of disheveled-associated activator of morphogenesis 2 disrupts cytoskeletal organization and phagocytosis in rat Sertoli cells

Disheveled-associated activator of morphogenesis 2 (DAAM2) regulates actin polymerization and cell motility. In this study, we investigated the role of DAAM2 in the cytoskeleton and phagocytosis of rat Sertoli cells in vitro and in vivo through siRNA transfection and intratesticular injection. We found that knockdown of DAAM2 significantly attenuated cytoskeletal and tight junction marker expression and reduced the integrity of the Sertoli cell monolayer. In rats, loss of DAAM2 induced disarrangement and deformation of sperms and promoted accumulation of apoptotic sperms in the testis, accompanied by morphological abnormalities in the blood-testis barrier. DAAM2 silencing also reduced the ability of Sertoli cells to engulf apoptotic spermatogenic cells and green fluorescence-labeled beads. RNA sequencing and bioinformatics analysis revealed that phagocytosis and cytoskeleton-related genes and pathways were significantly associated with DAAM2. Our study suggests that DAAM2 may be involved in spermatogenesis possibly by regulating cytoskeleton organization and phagocytosis of Sertoli cells.

Abstract 1750: The C. elegans PVD dendrite branching receptor HPO-30 regulates actin polymerization through two distinct mechanisms

Abstract 1750: The C. elegans PVD dendrite branching receptor HPO-30 regulates actin polymerization through two distinct mechanisms

Proteomic profiling of human brain coupled to functional assays for the identification of neurotoxic proteins

AbstractBackgroundAlzheimer’s Disease (AD) affects millions globally, but therapy development is lagging. The dearth of new therapies requires a reconsideration of pre‐clinical models of AD to facilitate the discovery of efficacious treatments. Ideally, such models would allow for monitoring the functional behavior of neurons in conditions approximating the AD brain.MethodsTowards the goal of developing more representative, pre‐clinical AD models, we employ multiple‐electrode arrays (MEAs) and live‐cell imaging for assessing neuronal firing and neurite integrity (NI), respectively. We use this system to examine changes in neuronal activity and neurite integrity following treatment with TBS‐soluble human brain extract from a panel of 43 individuals comprised of AD patients, unaffected persons not exhibiting AD pathology nor cognitively impaired, and individuals not cognitively impaired but exhibited AD‐related postmortem pathology. To determine the molecular etiology of these functional changes in an unbiased manner, we perform label‐free proteomic analyses on the panel of 43 brain extracts.ResultsAmyloid‐β (Aβ) and tau accumulate in the AD brain, and specific forms of each of these proteins have shown neurotoxic properties. We observe associations between spontaneous activity and Aβ42:40 levels, and between neurite integrity and oligomeric Aβ or tau containing N inserts. Additionally, the proteomic analyses reveal several candidates whose expression correlated with changes in neuronal structure and activity. Proteins associated with neurotoxic activities in MEA and NI assays are enriched in proteins implicated in lysosomal storage disorders, while proteins associated with neuroprotective properties are enriched in proteins of the WAVE regulatory complex that are involved in the regulation of actin polymerization. We further show that elevated GM2A in the AD brain and AD CSF is associated with reductions in both neurite integrity and MEA activity. Applying cell derived GM2A in the absence of brain extract is sufficient to induce a subtle but significant loss of neurite integrity.ConclusionThe techniques and data herein introduce a system for modeling neuronal vulnerability in response to factors present in the aged human brain and provide insights into proteins potentially contributing to AD pathogenesis.

Rab35 governs apicobasal polarity through regulation of actin dynamics during sprouting angiogenesis

In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35’s primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence, apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization through limiting Rac1 and RhoA activity, which is required to set up proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent brake of actin remodeling during blood vessel development.

Functional Characterization of an Arabidopsis Profilin Protein as a Molecular Chaperone under Heat Shock Stress.

Profilins (PFNs) are actin monomer-binding proteins that function as antimicrobial agents in plant phloem sap. Although the roles of Arabidopsis thaliana profilin protein isoforms (AtPFNs) in regulating actin polymerization have already been described, their biochemical and molecular functions remain to be elucidated. Interestingly, a previous study indicated that AtPFN2 with high molecular weight (HMW) complexes showed lower antifungal activity than AtPFN1 with low molecular weight (LMW). These were bacterially expressed and purified to characterize the unknown functions of AtPFNs with different structures. In this study, we found that AtPFN1 and AtPFN2 proteins have LMW and HMW structures, respectively, but only AtPFN2 has a potential function as a molecular chaperone, which has never been reported elsewhere. AtPFN2 has better protein stability than AtPFN1 due to its higher molecular weight under heat shock conditions. The function of AtPFN2 as a holdase chaperone predominated in the HMW complexes, whereas the chaperone function of AtPFN1 was not observed in the LMW forms. These results suggest that AtPFN2 plays a critical role in plant tolerance by increasing hydrophobicity due to external heat stress.

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

SUMMARYExcessive hepatic glucose production contributes to the development of hyperglycemia and is a key feature of type 2 diabetes. Here, we report that activation of hepatocyte Rap1a suppresses gluconeogenic gene expression and glucose production, whereas Rap1a silencing stimulates them. Rap1a activation is suppressed in obese mouse liver, and restoring its activity improves glucose intolerance. As Rap1a′s membrane localization and activation depends on its geranylgeranylation, which is inhibited by statins, we show that statin-treated hepatocytes and the human liver have lower active-Rap1a levels. Similar to Rap1a inhibition, statins stimulate hepatic gluconeogenesis and increase fasting blood glucose in obese mice. Geranylgeraniol treatment, which acts as the precursor for geranylgeranyl isoprenoids, restores Rap1a activity and improves statin-mediated glucose intolerance. Mechanistically, Rap1a activation induces actin polymerization, which suppresses gluconeogenesis by Akt-mediated FoxO1 inhibition. Thus, Rap1a regulates hepatic glucose homeostasis, and blocking its activity, via lowering geranylgeranyl isoprenoids, contributes to statin-induced glucose intolerance.

Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia.

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and mutations in myosin-15 cause hereditary deafness. In addition to transporting actin regulatory machinery to stereocilia tips, myosin-15 directly nucleates actin filament (“F-actin”) assembly, which is disrupted by a progressive hearing loss mutation (p.D1647G, “jordan”). Here, we present cryo–electron microscopy structures of myosin-15 bound to F-actin, providing a framework for interpreting the impacts of deafness mutations on motor activity and actin nucleation. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin’s D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. Adenosine diphosphate–bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin’s structural plasticity in a myosin nucleotide state–dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.

027 Profilin-1 prevents psoriasis pathogenesis through IκBζ regulation

Profilin-1 (PFN-1) is an actin-binding protein that regulates actin polymerization, cell proliferation, apoptosis, angiogenesis, and carcinogenesis. Its dysregulation has been reported in diverse pathologic diseases; however, the role of PFN-1 in psoriasis has not yet been elucidated. In this study, we demonstrate that PFN-1 expression is increased in both skin and serum of patients with psoriasis. PFN-1 was markedly expressed in the epidermis of psoriatic lesions and its expression positively correlated with psoriasis severity.

An Actin-, Cortactin- and Ena-VASP-Linked Complex Contributes to Endothelial Cell Focal Adhesion and Vascular Barrier Regulation.

Background/Aims:Increase in vascular permeability is a cardinal feature of all inflammatory diseases and represents an imbalance in vascular contractile forces and barrier-restorative forces, both of which are highly dependent on actin cytoskeletal dynamics. In addition to the involvement of key vascular barrier-regulatory, actin-binding proteins, such as nmMLCK and cortactin, we recently demonstrated a role for a member of the Ena-VASP family known as Ena-VASP-like (EVL) in promoting vascular focal adhesion (FA) remodeling and endothelial cell (EC) barrier restoration/preservation.Methods:To further understand the role of EVL in EC barrier-regulatory processes, we examined EVL-cytoskeletal protein interactions in FA dynamics in vitro utilizing lung EC and in vivo murine models of acute inflammatory lung injury. Deletion mapping studies and immunoprecipitation assays were performed to detail the interaction between EVL and cortactin, and further evaluated by assessment of changes in vascular EC permeability following disruption of EVL-cortactin interaction.Results:Initial studies focusing on the actin-binding proteins, nmMLCK and cortactin, utilized deletion mapping of the cortactin gene (CTTN) to identify cortactin domains critical for EVL-cortactin interaction and verified the role of actin in promoting EVL-cortactin interaction. A role for profilins, actin-binding proteins that regulate actin polymerization, was established in facilitating EVL-FA binding.Conclusion:In summary, these studies further substantiate EVL participation in regulation of vascular barrier integrity and in the highly choreographed cytoskeletal interactions between key FA and cytoskeletal partners.

P723: LOSS OF THE NK MOTIF AND ANKYRIN REPEAT DOMAIN 1 (KANK1) LEADS TO LYMPHOID COMPARTMENT DYSREGUATION IN MICE

Background: We identified a novel germline loss of heterozygosity (LOH) mutation encompassing the NK Motif And AnKyrin Repeat Domain 1 (KANK1) gene in a young Myelodysplastic syndrome (MDS) patient and his healthy father, with no additional MDS-related mutations or chromosomal abnormalities. Clinical investigation suggested development of MDS following severe infection, likely triggered by an autoimmune mechanism. KANK1 protein plays a role in the control of cytoskeleton formation by regulating actin polymerization. A t(5;9) translocation resulting in a fusion of the platelet-derived growth factor receptor beta gene (PDGFRB) and KANK1 was detected in a patient with myeloid neoplasm (MN) characterized with severe thrombocythemia. The protein is thought to have a tumour suppressor function, as its expression is downregulated or missing in several tumor tissues, while over-expressing the protein was reported to inhibit the proliferation of tumor cells in solid cancer models. In addition, defects in cytoskeletal proteins are reported to contribute to immunological dysfunction and disruption of normal immune processes. Aims: To investigate the role of KANK1 in the regulation of normal hematopoiesis and the development of myeloid disorders. Methods: We generated a new genetically modified KANK1 (Kank1-/-) knockout mouse model and analysed the phenotypic and developmental changes in hematopoiesis in young and aged mice. Results: We confirmed the loss of Kank1 mRNA and protein expression by q-PCR and targeted protein mass-spectrometry respectively. The mice are viable and fertile, and reproduced according to mendelian ratio. Over the two years observation period, Kank1-/- mice did not exhibit any signs of disease, and their peripheral blood parameters remained within the normal range. Flow cytometry analysis of bone marrow (BM) cells from 17-20 weeks kank1-/- old mice showed minor increase in CD3+ T-cell population, while the expression of the double positive B220/CD19 B-cells population was reduced in Kank1-/- compared to Kank1+/+. Comprehensive flow cytometry analysis of spleen and thymus cells from young (n=8 each, 6-7 weeks old) and aged (n=8, 68-71 weeks old) Kank1-/- mice showed further dysregulation of the lymphoid compartment when compared to Kank1+/+. In young mice, a slight decrease of Treg cells is noticed in the spleen, coupled with a slight decrease of activated CD4 in the peripheral blood. In the spleen of aged Kank1-/- mice, the proportions of macrophages and Treg cells were decreased, while the central memory CD4+ T-cells were increased. Total protein analysis from BM cells of Kank1-/- (n=3) compared to Kank1+/+ mice (n=3) showed a significant reduction (FC >0.5, adj.p. <0.05) in proteins associated with cytoskeleton formation such as Myosin1 (MYH1), Tropomysin-1 (TDM1) and Annexin (ANXA2), and extracellular matrix components such as Collagena(CO1A1) and Matrix metalloproteinase9 (MMP9). We also detected a significant increase in expression of the DNA licensing factor minichromosome maintenance complex component 7 (MCM7), and the mitochondrial membrane associated transporter ATP binding Cassette Subfamily B Member 10 (ABCBA). Summary/Conclusion: Dysregulation of the immune system has been widely recognized to play a role in the development of MDS. Based on our results, we propose a role for KANK1 in regulating the development of immune cells in the mouse through it invovlement in cytoskeleton regulation. In vivo infectious challenge experiments using our model will futher elucidate the importance of KANK1 in the development of MDS.

Abstract 2097: Characterization of mouse and cellular phenotype of ABI1 KO T-cells

Abstract Background: The immune system plays a critical part in cancer development and progression. The increase in our understanding of mechanisms regulating immune-mediated cancer eradication has led to the development of immune-based therapies which have revolutionized treatment of some previously lethal malignancies. T-cell activation requires proper intercellular interactions mediated by cytoskeletal rearrangements and adhesion molecules, along with intracellular signal transduction via phosphorylation by tyrosine kinases (Lck and Fyn). Abelson interactor-1 (ABI1) is an adaptor protein known to regulate actin polymerization, adhesion molecules and tyrosine kinases. Previously, ABI1 has been shown to localize to the immunological synapse in an antigen-dependent manner and has been linked to regulation of IL-2 secretion. Published data from our lab shows that ABI1 has high affinity binding to SH2 domains of Src family kinases, Fyn and Lck, both of which are essential in T-cell activation. Additionally, ABI1 has been linked to hyperactivation of SFKs/STAT3/NF-kB signaling. In this study, we aim to delineate the role of ABI1 in T-cell activation, differentiation and signaling. Methods: We have generated a novel conditional knock-out mouse model to evaluate the effect of a T-cell specific ABI1 KO at the organismal level (ABI1(fl/fl)CD4Cre (ACD4). T-cells isolated from control (Abi1(fl/fl)CD4Cre(-)) and ABI1 KO mice (Abi1(fl/fl)CD4Cre(+)) were used for ex-vivo analysis and further characterization of ABI1 KO cellular phenotype with: Flow Cytometry (CD3+ subpopulations in immune organs), Immunofluorescence (actin polymerization), Western blotting (TCR-mediated T-cell activation) and RNAseq (transcriptional reprogramming). Results: In vitro studies of ABI1 KO primary murine CD3+ T-cells showed increased levels of NF-kB and STAT3. Flow cytometry analysis of CD3+ T cells subpopulations showed a difference in the percentage of CD4+ and CD8+ T cells; percentage of CD8+ T cells was increased, while percentage of CD4+ T cells was decreased in ABI1 KO vs. WT control mice. Furthermore, RNA seq analysis of CD8+ ABI1 KO T cells showed a significant decrease in the mRNA expression of Thpok (Zbtb7b) compared to WT. Conclusion: Our preliminary studies show that ABI1 is a potential modulator of T-cell activation through regulation of Lck/Fyn. This suggests ABI1 may play as a molecular switch negatively regulating T-cell activation by connecting phosphorylation-dependent signal transduction and actin cytoskeletal dynamics. Suggesting ABI1 as a new therapeutic to improve efficacy and longevity of T cell-based cancer immunotherapies. In addition, ABI1 appears to be implicated in the regulation of the percentage of CD4 and CD8 T-cells, a novel discovery which warrants further investigation. Citation Format: Maria Altagracia Ortiz, Andras Perl, Marcin Kortylewski, Leszek Kotula. Characterization of mouse and cellular phenotype of ABI1 KO T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2097.

Profilin upregulation induces autophagy through stabilization of AMP-activated protein kinase.

Profilin regulates actin polymerization, and its balanced expression is required for cellular growth and development. Most tumours have compromised profilin expression, and its overexpression in MDA MB-231 breast cancer cells has been reported to activate AMP-activated protein kinase α (AMPKα), an energy-sensing molecule that affects various cellular processes including autophagy. This study aims to explore the role of profilin in autophagy induction. We employed all-trans retinoic acid (ATRA) as an inducer of profilin expression and showed that profilin induces autophagy through mTOR inhibition, autophagy-activating kinase ULK1 upregulation, and AMPK stabilization as well as its activation. Furthermore, evidence from our study indicates physical interaction between profilin and AMPK, which results in AMPK stabilization and induction of prolonged autophagy, thereby leading to apoptosis. This study uncovers a novel mechanism that induces autophagy in triple-negative breast cancer cells.

RhoBTB1 reverses established arterial stiffness in angiotensin II-induced hypertension by promoting actin depolymerization.

Arterial stiffness predicts cardiovascular disease and all-cause mortality, but its treatment remains challenging. Mice treated with angiotensin II (Ang II) develop hypertension, arterial stiffness, vascular dysfunction, and a downregulation of Rho-related BTB domain–containing protein 1 (RhoBTB1) in the vasculature. RhoBTB1 is associated with blood pressure regulation, but its function is poorly understood. We tested the hypothesis that restoring RhoBTB1 can attenuate arterial stiffness, hypertension, and vascular dysfunction in Ang II–treated mice. Genetic complementation of RhoBTB1 in the vasculature was achieved using mice expressing a tamoxifen-inducible, smooth muscle–specific RhoBTB1 transgene. RhoBTB1 restoration efficiently and rapidly alleviated arterial stiffness but not hypertension or vascular dysfunction. Mechanistic studies revealed that RhoBTB1 had no substantial effect on several classical arterial stiffness contributors, such as collagen deposition, elastin content, and vascular smooth muscle remodeling. Instead, Ang II increased actin polymerization in the aorta, which was reversed by RhoBTB1. Changes in the levels of 2 regulators of actin polymerization, cofilin and vasodilator-stimulated phosphoprotein, in response to RhoBTB1 were consistent with an actin depolymerization mechanism. Our study reveals an important function of RhoBTB1, demonstrates its vital role in antagonizing established arterial stiffness, and further supports a functional and mechanistic separation among hypertension, vascular dysfunction, and arterial stiffness.

Trabecular Meshwork Cholesterol Levels Regulate Actin Polymerization and Tunneling Nanotubes

Trabecular meshwork (TM) tissue is made of highly contractile mechanosensitive cells regulating aqueous humor (AH) outflow facility and intraocular pressure (IOP). Increased actin‐mediated contraction of TM elevates IOP causing glaucoma and irreversible blindness. This study aims at understanding the role of cholesterol in regulating actin dynamics in human TM (HTM) cells. To test this, HTM cells were treated with SiR‐Actin in serum‐free media overnight and F‐actin changes were recorded using live‐cell time‐lapse (5 min interval) confocal imaging before and during exposure to ‐ A) 10mM methyl‐β‐cyclodextrin (MβCD) for 1h for acute removal of cellular cholesterol, B) 100μM cholesterol‐saturated MβCD (MβCD‐CHOL) for 1h to enrich cholesterol, and C) 10mM MβCD for 1h followed by cholesterol supplementation using MβCD‐CHOL for 1h. F‐actin fluorescence intensity changes were calculated and unpaired t‐test was used for statistical analyses and results were significant if p&lt;0.05 with a sample size of n= 4‐57. HTM cells plated on coverslips were treated as A – C as mentioned above, cholesterol and lipid synthesis inhibitors for 24h ‐ D) 100μM atorvastatin and E) 20μM fatostatin, respectively, F) actin polymerization disruptor ‐ 10μM cytochalasin D (CYTD) for 1h, G) 10μM CYTD for 1h followed by 100μM MβCD‐CHOL for 1h, H) microtubule disruptor ‐ 1μM demecolcine (DEM) for 1h, I) 1μM DEM for 1h followed by 100μM MβCD‐CHOL for 1h. Cells without treatment were used as control. After all these treatments, cells were fixed and immunofluorescence (IF) was performed to check for cholesterol using filipin, F‐actin by phalloidin, focal adhesion (FA) localization based on ‐ vinculin and paxillin distribution. Further F‐actin/G‐actin in vitro assay was performed to quantify the changes in actin polymerization under the conditions A – I compared to the control. The imaging analysis showed cholesterol removal significantly decreased F‐actin fluorescence intensity from 20min sustaining up to 1h and decreased the FA distribution. Cholesterol supplementation led to partial rescue of actin and FA indicating the re‐establishment of actin cytoskeleton and increased tunneling nanotube structures enhancing the cell‐cell communication (Figure 1). Moreover, IF results show that atorvastatin and fatostatin decreased F‐actin and FA distribution. CYTD disrupted F‐actin network leaving remnant filament ends and decreased FA but cholesterol enrichment increased F‐actin branches formed at ~70‐degree angle indicating Arp2/3 activation. DEM treatment increased F‐actin and FA, and cholesterol enrichment induced thick F‐actin filaments. These results were confirmed by quantitative F/G‐actin ratio decrease and increase upon cholesterol removal and enrichment, respectively. Additionally, supplementing cholesterol after removal increased the ratio compared to solely removing cholesterol. Interestingly, F/G‐actin ratio increased under CYTD treatment indicating that CYTD disrupted longer actin filaments but helped polymerization of newly formed actin filaments. Finally, DEM treatment increased F/G‐actin ratio, which was further increased by cholesterol enrichment. This systematic evaluation of cholesterol modulation on TM actin cytoskeleton polymerization identifies the significance of maintaining membrane and cellular cholesterol levels and cellular communication for achieving IOP homeostasis.