Actin Depolymerizing Factor Research Articles

Alteration of twinfilin1 expression underlies opioid withdrawal-induced remodeling of actin cytoskeleton at synapses and formation of aversive memory.

Exposure to drugs of abuse induces alterations of dendritic spine morphology and density that has been proposed to be a cellular basis of long-lasting addictive memory and heavily depend on remodeling of its underlying actin cytoskeleton by the actin cytoskeleton regulators. However, the actin cytoskeleton regulators involved and the specific mechanisms whereby drugs of abuse alter their expression or function are largely unknown. Twinfilin (Twf1) is a highly conserved actin-depolymerizing factor that regulates actin dynamics in organisms from yeast to mammals. Despite abundant expression of Twf1 in mammalian brain, little is known about its importance for brain functions such as experience-dependent synaptic and behavioral plasticity. Here we show that conditioned morphine withdrawal (CMW)-induced synaptic structure and behavior plasticity depends on downregulation of Twf1 in the amygdala of rats. Genetically manipulating Twf1 expression in the amygdala bidirectionally regulates CMW-induced changes in actin polymerization, spine density and behavior. We further demonstrate that downregulation of Twf1 is due to upregulation of miR101a expression via a previously unrecognized mechanism involving CMW-induced increases in miR101a nuclear processing via phosphorylation of MeCP2 at Ser421. Our findings establish the importance of Twf1 in regulating opioid-induced synaptic and behavioral plasticity and demonstrate its value as a potential therapeutic target for the treatment of opioid addiction.

Novel role of CAP1 in regulation RNA polymerase II-mediated transcription elongation depends on its actin-depolymerization activity in nucleoplasm.

Lung cancer is one of the most intractable diseases with high incidence and mortality worldwide. Adenylate cyclase-associated protein 1 (CAP1), a well-known actin depolymerization factor, is recently reported to be an oncogene accelerating cancer cell proliferation. However, the physiological significance of CAP1 in lung cancer is incompletely understood and the novel functions of CAP1 in transcriptional regulation remain unknown. Here we found that CAP1 was highly expressed in lung cancer tissues and cells, which was also negatively associated with prognosis in lung cancer patients. Moreover, CAP1 promoted A549 cells proliferation by promoting protein synthesis to accelerate cell cycle progression. Mechanistically, we revealed that CAP1 facilitated cyclin-dependent kinase 9 (CDK9)-mediated RNA polymerases (Pol) II-Ser2 phosphorylation and subsequent transcription elongation, and CAP1 performed its function in this progress depending on its actin-depolymerization activity in nucleoplasm. Furthermore, our in vivo findings confirmed that CAP1-promoted A549 xenograft tumor growth was associated with CDK9-mediated Pol II-Ser2 phosphorylation. Our study elucidates a novel role of CAP1 in modulating transcription by promoting polymerase II phosphorylation and suggests that CAP1 is a newly identified biomarker for lung cancer treatment and prognosis prediction.

Genome-Wide Identification and Low Temperature Responsive Pattern of Actin Depolymerizing Factor (ADF) Gene Family in Wheat (Triticum aestivum L.).

The actin depolymerizing factor (ADF) gene family, which is conserved in eukaryotes, is important for plant development, growth, and stress responses. Cold stress restricts wheat growth, development, and distribution. However, genome-wide identification and functional analysis of the ADF family in wheat is limited. Further, because of the promising role of ADF genes in cold response, there is need for an understanding of the function of this family on wheat under cold stress. In this study, 25 ADF genes (TaADFs) were identified in the wheat genome and they are distributed on 15 chromosomes. The TaADF gene structures, duplication events, encoded conversed motifs, and cis-acting elements were investigated. Expression profiles derived from RNA-seq data and real-time quantitative PCR analysis revealed the tissue- and temporal-specific TaADF expression patterns. In addition, the expression levels of TaADF13/16/17/18/20/21/22 were significantly affected by cold acclimation or freezing conditions. Overexpression of TaADF16 increased the freezing tolerance of transgenic Arabidopsis, possibly because of enhanced ROS scavenging and changes to the osmotic regulation in cells. The expression levels of seven cold-responsive genes were up-regulated in the transgenic Arabidopsis plants, regardless of whether the plants were exposed to low temperature. These findings provide fundamental information about the wheat ADF genes and may help to elucidate the regulatory effects of the encoded proteins on plant development and responses to low-temperature stress.

Arabidopsis ADF5 Acts as a Downstream Target Gene of CBFs in Response to Low-Temperature Stress.

Low temperature is a major adverse environment that affects normal plant growth. Previous reports showed that the actin cytoskeleton plays an important role in the plant response to low-temperature stress, but the regulatory mechanism of the actin cytoskeleton in this process is not clear. C-repeat binding factors (CBFs) are the key molecular switches for plants to adapt to cold stress. However, whether CBFs are involved in the regulation of the actin cytoskeleton has not been reported. We found that Arabidopsis actin depolymerizing factor 5 (ADF5), an ADF that evolved F-actin bundling function, was up-regulated at low temperatures. We also demonstrated that CBFs bound to the ADF5 promoter directly in vivo and in vitro. The cold-induced expression of ADF5 was significantly inhibited in the cbfs triple mutant. The freezing resistance of the adf5 knockout mutant was weaker than that of wild type (WT) with or without cold acclimation. After low-temperature treatment, the actin cytoskeleton of WT was relatively stable, but the actin cytoskeletons of adf5, cbfs, and adf5 cbfs were disturbed to varying degrees. Compared to WT, the endocytosis rate of the amphiphilic styryl dye FM4-64 in adf5, cbfs, and adf5 cbfs at low temperature was significantly reduced. In conclusion, CBFs directly combine with the CRT/DRE DNA regulatory element of the ADF5 promoter after low-temperature stress to transcriptionally activate the expression of ADF5; ADF5 further regulates the actin cytoskeleton dynamics to participate in the regulation of plant adaptation to a low-temperature environment.

Actin-depolymerizing factor from Eimeria tenella promotes immunogenic function of chicken dendritic cells.

Dendritic cells play a crucial role in inducing antigen-specific immunity to pathogens. During host-parasite interaction, host immune response to the parasite molecules is considered essential for recognizing novel antigens for control strategies. Therefore, in the present study, chicken dendritic cells (DCs) (ChDCs), derived from spleens were used to evaluate their capacity to proliferate and differentiate autologous T lymphocytes in response to actin-depolymerizing factor from Eimeria tenella (EtADF). Immunoblot analysis showed that recombinant EtADF protein (rEtADF) was able to interact with rat anti-rEtADF antibodies. The immunofluorescence test confirmed rEtADF binding on ChDCs surface. Flow cytometric analysis revealed that phenotypes for MHCII, CD1.1, CD11c, CD80, and CD86 were increased in ChDCs after rEtADF treatment. qRT-PCR results indicated that ChDCs triggered TLR signaling in response to rEtADF, and suppressed Wnt signaling. Transcript levels of CD83, CCL5, and CCR7 in ChDCs were improved following rEtADF treatment. In addition, rEtADF promoted DC-directed T cell proliferation and differentiation of naïve T cells into CD3+/CD4+ T cells in DC/T cell co-incubation system. Cytokine analysis of rEtADF-pulsed ChDCs showed increased levels of IL-12 and IFN-γ, while IL-10 and TGF-β remained unchanged. Moreover, rEtADF-treated ChDCs enhanced production of IFN-γ when incubated with T cells, and IL-4 secretion remained unchanged. Our findings indicted that rEtADF could facilitate the polarization of Th1 immune cells by triggering both host DCs and T cells. Our findings provide useful insights into future work aimed at anticoccidial vaccine strategies.

Loss of Profilin3 Impairs Spermiogenesis by Affecting Acrosome Biogenesis, Autophagy, Manchette Development and Mitochondrial Organization

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis and metastasis of tumor cells. The testes specific PFN3 is localized in the acroplaxome-manchette complex of developing sperm. We demonstrate, that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in acrosome formation. Using CRISPR/Cas9 genome editing we generated mice deficient for Pfn3. Pfn3-/- males are sub-fertile displaying a type II-globozoospermia. We revealed, that Pfn3-/- sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3-/-sperm showed reduced sperm motility resulting from flagellum deformities. We show, that acrosome biogenesis is impaired starting from the Golgi phase. RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed resulting in the inhibition of autophagy. This dysregulation of AMPK/ mTOR affected the autophagic flux which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. CoIP experiments demonstrate binding of PFN3 to TRIM27 in sperm. We speculate, that PFN3-TRIM27 interaction serves to buffer free TRIM27-levels to fine tune the usage of the autophagy pathway for acrosome biogenesis. Further, actin related protein ARPM1 was absent in the nuclear fraction of Pfn3-/- testes and sperm. This suggests, that lack of PFN3 leads to destabilization of the stable PFN3-ARPM1 complex resulting in the degradation of ARPM1. Interestingly, in the Pfn3-/- testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2) and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicates this protein as a candidate for male factor infertility in humans.

The Ways of Actin: Why Tunneling Nanotubes Are Unique Cell Protrusions.

Actin remodeling is at the heart of the response of cells to external or internal stimuli, allowing a variety of membrane protrusions to form. Fifteen years ago, tunneling nanotubes (TNTs) were identified, bringing a novel addition to the family of actin-supported cellular protrusions. Their unique property as conduits for cargo transfer between distant cells emphasizes the unique nature of TNTs among other protrusions. While TNTs in different pathological and physiological scenarios have been described, the molecular basis of how TNTs form is not well understood. In this review, we discuss the role of several actin regulators in the formation of TNTs and suggest potential players based on their comparison with other actin-based protrusions. New perspectives for discovering a distinct TNT formation pathway would enable us to target them in treating the increasing number of TNT-involved pathologies.

Destrin Contributes to Lung Adenocarcinoma Progression by Activating Wnt/β-Catenin Signaling Pathway.

Lung cancer, especially lung adenocarcinoma, is one of the most common neoplasms worldwide. However, the mechanisms underlying its initiation, development, and metastasis are still poorly understood. Destrin (DSTN) is a member of ADF/cofilin family. Its detailed biological function remains unknown, although it is reported that DSTN is involved in cytoskeleton remodeling and regulation of actin filament turnover. Recent evidence has shown that high expression of cofilin-1 is associated with invasion and poor prognosis of several types of human tumors, but the detailed mechanism is still entirely unclear, particularly in lung cancer tumorigenesis and malignancy. Here, we report that DSTN was highly expressed in a mouse lung cancer model induced by urethane and in clinical lung adenocarcinoma tissue samples. Its expression level was positively correlated with cancer development, as well as metastasis to the liver and lymph nodes. Consistently, it was directly associated with the poor prognosis of lung adenocarcinoma patients. Furthermore, we also found that DSTN promotes cell proliferation, invasion, and migration in vitro, and facilitates subcutaneous tumor formation and lung metastasis via intravenous injection in vivo. Mechanically, DSTN associates with and facilitates nuclear translocation of β-catenin, which promotes epithelial-to-mesenchymal transition (EMT). Taken together, our results indicated that DSTN enhances lung cancer malignancy through facilitating β-catenin nuclear translocation and inducing EMT. Combined with multivariate analyses, DSTN might potentially serve as a therapeutic target and an independent prognostic marker of lung adenocarcinoma. IMPLICATIONS: This finding indicates that DSTN facilitates β-catenin nuclear translocation and promotes malignancy in lung adenocarcinoma.

Arabidopsis\xa0calcium-dependent protein kinase 3 regulates actin cytoskeleton organization and immunity

Pattern-triggered immunity and effector-triggered immunity are two primary forms of innate immunity in land plants. The molecular components and connecting nodes of pattern-triggered immunity and effector-triggered immunity are not fully understood. Here, we report that the Arabidopsis calcium-dependent protein kinase CPK3 is a key regulator of both pattern-triggered immunity and effector-triggered immunity. In vitro and in vivo phosphorylation assays, coupled with genetic and cell biology-based analyses, show that actin-depolymerization factor 4 (ADF4) is a physiological substrate of CPK3, and that phosphorylation of ADF4 by CPK3 governs actin cytoskeletal organization associated with pattern-triggered immunity. CPK3 regulates stomatal closure induced by flg22 and is required for resistance to Pst DC3000. Our data further demonstrates that CPK3 is required for resistance to Pst DC3000 carrying the effector AvrPphB. These results suggest that CPK3 is a missing link between cytoskeleton organization, pattern-triggered immunity and effector-triggered immunity.

The Role of ADF/Cofilin in Synaptic Physiology and Alzheimer's Disease.

Actin-depolymerization factor (ADF)/cofilin, a family of actin-binding proteins, are critical for the regulation of actin reorganization in response to various signals. Accumulating evidence indicates that ADF/cofilin also play important roles in neuronal structure and function, including long-term potentiation and depression. These are the most extensively studied forms of long-lasting synaptic plasticity and are widely regarded as cellular mechanisms underlying learning and memory. ADF/cofilin regulate synaptic function through their effects on dendritic spines and the trafficking of glutamate receptors, the principal mediator of excitatory synaptic transmission in vertebrates. Regulation of ADF/cofilin involves various signaling pathways converging on LIM domain kinases and slingshot phosphatases, which phosphorylate/inactivate and dephosphorylate/activate ADF/cofilin, respectively. Actin-depolymerization factor/cofilin activity is also regulated by other actin-binding proteins, activity-dependent subcellular distribution and protein translation. Abnormalities in ADF/cofilin have been associated with several neurodegenerative disorders such as Alzheimer’s disease. Therefore, investigating the roles of ADF/cofilin in the brain is not only important for understanding the fundamental processes governing neuronal structure and function, but also may provide potential therapeutic strategies to treat brain disorders.

Protective mechanism of tea polyphenols against muscle quality deterioration of shrimp (Penaeus vannamei) induced by aflatoxin B1

Tea polyphenols (TP), as common antioxidants, have been widely used to mitigate for the protection and preservation of aquatic animals, but the protective effect of TP against the deterioration of shrimp muscle quality induced by aflatoxin B1 (AFB1) has not been reported. This study was designed to analyse the root cause of AFB1 induced declines in shrimp muscle quality and the protective mechanisms of TP at the molecular level. Shrimp were exposed to AFB1 and TP for 20 d following a dose-escalating trial, and changes to the muscle histopathology, basic nutrient composition, and protein composition were determined. Non-labelled quantitative proteomics was used to screen and identify differential marker proteins associated with AFB1 and TP. After 20 d of AFB1 exposure, the water content, crude protein content and crude fat content of the shrimp muscle decreased to 68.66%, 18.27% and 1.01%, respectively, while the ash content was positively correlated with AFB1 dose (R2 = 0.955). AFB1 caused damage to the shrimp muscle microstructure in a dose-dependent manner, such as significant enlargement of muscle fibre spaces, rupture of the sarcomere, inflammation and so on. AFB1 induced changes in muscle protein composition, resulting in a decrease in the content of myofibrillar, sarcoplasmic and stroma proteins, while the alkali-soluble protein content increased significantly. TP can effectively protect against the damage to shrimp muscle caused by AFB1 by inhibiting the expansion of muscle fibre spaces and inflammation. TP (0.04–0.16% feed) also had a significant protective effect against the decrease of muscle nutrients and changes in protein composition of shrimp caused by exposure to AFB1 (1.2–2.7 mg/kg feed) in the early and middle period (4–12 d), but the protective effect was weak or not noticeable in the late period (16–20 d). Eleven proteins were identified and screened as biomarker proteins of AFB1 induced quality decline and TP protection, which were involved in energy metabolism (fructose 1,6-bisphosphatase; transaldolase; fructose-bisphosphate aldolase; arginine kinase), redox reaction (hydroxyphenylpyruvate reductase; prostaglandin reductase 1; protein arginine N-methyltransferase 8), anti-inflammatory responses (5′-nucleotidase; actin-depolymerizing factor) and protein translation/folding (eukaryotic translation initiation factor 3; peptidyl-prolyl cis-trans isomerase). This study provides a better understanding of AFB1 induced damage to shrimp quality and technical guidance for the use of TP to prevent the damage during the culture and processing of Penaeus vannamei.

Relapse-Associated Transient Synaptic Potentiation Requires Integrin-Mediated Activation of Focal Adhesion Kinase and Cofilin in D1-Expressing Neurons.

Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced drug seeking in rodent models correlates with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses in the nucleus accumbens core (NAcore). Matrix metalloproteinases (MMPs) are inducible endopeptidases that degrade extracellular matrix (ECM) proteins, and reveal tripeptide Arginine-Glycine-Aspartate (RGD) domains that bind and signal through integrins. Integrins are heterodimeric receptors composed of αβ subunits, and a primary signaling kinase is focal adhesion kinase (FAK). We previously showed that MMP activation is necessary for and potentiates cued reinstatement of cocaine seeking, and MMP-induced catalysis stimulates β3-integrins to induce t-SP. Here, we determined whether β3-integrin signaling through FAK and cofilin (actin depolymerization factor) is necessary to promote synaptic growth during t-SP. Using a small molecule inhibitor to prevent FAK activation, we blocked cued-induced cocaine reinstatement and increased spine head diameter (dh). Immunohistochemistry on NAcore labeled spines with ChR2-EYFP virus, showed increased immunoreactivity of phosphorylation of FAK (p-FAK) and p-cofilin in dendrites of reinstated animals compared with extinguished and yoked saline, and the p-FAK and cofilin depended on β3-integrin signaling. Next, male and female transgenic rats were used to selectively label D1 or D2 neurons with ChR2-mCherry. We found that p-FAK was increased during drug seeking in both D1 and D2-medium spiny neurons (MSNs), but increased p-cofilin was observed only in D1-MSNs. These data indicate that β3-integrin, FAK and cofilin constitute a signaling pathway downstream of MMP activation that is involved in promoting the transient synaptic enlargement in D1-MSNs induced during reinstated cocaine by drug-paired cues.SIGNIFICANCE STATEMENT Drug-associated cues precipitate relapse, which is correlated with transient synaptic enlargement in the accumbens core. We showed that cocaine cue-induced synaptic enlargement depends on matrix metalloprotease signaling in the extracellular matrix (ECM) through β3-integrin to activate focal adhesion kinase (FAK) and phosphorylate the actin binding protein cofilin. The nucleus accumbens core (NAcore) contains two predominate neuronal subtypes selectively expressing either D1-dopamine or D2-dopamine receptors. We used transgenic rats to study each cell type and found that cue-induced signaling through cofilin phosphorylation occurred only in D1-expressing neurons. Thus, cocaine-paired cues initiate cocaine reinstatement and synaptic enlargement through a signaling cascade selectively in D1-expressing neurons requiring ECM stimulation of β3-integrin-mediated phosphorylation of FAK (p-FAK) and cofilin.

Multidimensional analysis of actin depolymerising factor family in pigeon pea under different environmental stress revealed specific response genes in each subgroup.

Actin depolymerising factor (ADF) is an actin binding protein that is ubiquitous in animal and plant cells. It plays an important role in plant growth and development, as well as resistance to biotic and abiotic stress. The research of plant ADF family has been restricted to Arabidopsis thaliana (L.) Heynh. and some herb crops, but no woody cash crops have been reported to date. All members of the Cajanus cajan (L.) Millsp. ADF (CcADF) family were identified from the pigeon pea genome, and distributed among the four subfamilies by phylogenetic analysis. CcADFs were relatively conservative in gene structure evolution, protein structure and functional expression, and different CcADFs showed specific expression patterns under different treatments. The expression characteristics of several key CcADFs were revealed by analysing the stress response pattern of CcADFs and the time series RNA-seq of aluminium stress. Among them, CcADF9 in the first subgroup specifically responded to aluminium stress in the roots; CcADF3 in the second subgroup intensively responded to fungal infection in the leaves; and CcADF2 in the fourth subgroup positively responded to various stress treatments in different tissues. This study extended the relationship between plant ADF family and aluminium tolerance, as well as adding to the understanding of CcADF family in woody crops.

Cyclin Y, a novel actin-binding protein, regulates spine plasticity through the cofilin-actin pathway.

While positive regulators of hippocampal long-term potentiation (LTP) have extensively been investigated, relatively little is known about the inhibitory regulators of LTP. We previously reported that Cyclin Y (CCNY), a member of cyclin family generally known to function in proliferating cells, is a novel postsynaptic protein that serves as a negative regulator of functional LTP. However, whether CCNY plays a role in structural LTP, which is mechanistically linked to functional LTP, and which mechanisms are involved in the CCNY-mediated suppression of LTP at the molecular level remain elusive. Here, we report that CCNY negatively regulates the plasticity-induced changes in spine morphology through the control of actin dynamics. We observed that CCNY directly binds to filamentous actin and interferes with LTP-induced actin polymerization as well as depolymerization by blocking the activation of cofilin, an actin-depolymerizing factor, thus resulting in less plastic spines and the impairment of structural LTP. These data suggest that CCNY acts as an inhibitory regulator for both structural and functional LTP by modulating actin dynamics through the cofilin-actin pathway. Collectively, our findings provide a mechanistic insight into the inhibitory modulation of hippocampal LTP by CCNY, highlighting a novel function of a cyclin family protein in non-proliferating neuronal cells.

WD Repeat Domain 1 Deficiency Inhibits Neointima Formation in Mice Carotid Artery by Modulation of Smooth Muscle Cell Migration and Proliferation.

The migration, dedifferentiation, and proliferation of vascular smooth muscle cells (VSMCs) are responsible for intimal hyperplasia, but the mechanism of this process has not been elucidated. WD repeat domain 1 (WDR1) promotes actin-depolymerizing factor (ADF)/cofilin-mediated depolymerization of actin filaments (F-actin). The role of WDR1 in neointima formation and progression is still unknown. A model of intimal thickening was constructed by ligating the left common carotid artery in Wdr1 deletion mice, and H&E staining showed that Wdr1 deficiency significantly inhibits neointima formation. We also report that STAT3 promotes the proliferation and migration of VSMCs by directly promoting WDR1 transcription. Mechanistically, we clarified that WDR1 promotes the proliferation and migration of VSMCs and neointima formation is regulated by the activation of the JAK2/STAT3/WDR1 axis.

Abstract 1796: Requirement for LIM kinases in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a highly aggressive disease which, in spite of extensive research in the last decades, is still treated with conventional chemotherapeutic regimens in most cases. Recent approvals of FLT3 and IDH1/2 inhibitors have highlighted the promise of targeted approaches for personalized AML therapy. However, the majority of AML driver mutations currently remain “undruggable”, making the search for additional molecular targets imperative. LIM kinases 1 and 2 (LIMK1/LIMK2), which are primarily known for regulating actin dynamics via phosphorylation of the actin-depolymerizing factor cofilin, have previously been implicated in progression and metastasis formation of several epithelial tumors. However, their function in leukemia, and AML in particular, has not been addressed in detail. Using high-throughput RNA interference (RNAi) screens in AML cell lines, we identified LIMK1 as a potential novel molecular target for AML treatment. High LIMK1 expression was significantly correlated with shorter survival of AML patients, and was associated with NPM1 and DNMT3A mutations, MLL rearrangements, and elevated HOX gene expression. RNAi- and CRISPR/Cas9-mediated suppression, as well as pharmacologic inhibition of LIMK1 and its close homolog LIMK2, reduced colony formation and decreased proliferation due to slowed cell cycle progression of MLL-rearranged AML cell lines and patient-derived xenograft samples. This was accompanied by morphologic changes indicative of myeloid differentiation. Transcriptome analysis showed upregulation of several tumor suppressors as well as downregulation of HOXA9 targets and mitosis-associated genes in response to LIMK1 suppression, providing a potential mechanistic explanation for the anti-leukemic phenotype. Finally, we observed a mutual regulation between LIMK1 and CDK6, a kinase known to be involved in the differentiation block of MLL-rearranged AML, and addition of the CDK6 inhibitor palbociclib further enhanced the anti-proliferative effect of LIMK1 depletion. Our data therefore suggest that LIM kinases are promising targets for AML therapy. Citation Format: Patrizia Jensen, Michela Carlet, Jana Kress, Andrea Weber, Ines Brunner, Mikolaj Slabicki, Gregor Grill, Simon Weisemann, Ya-Yun Cheng, Irmela Jeremias, Claudia Scholl, Stefan Fröhling. Requirement for LIM kinases in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1796.

Grp94 Regulates the Recruitment of Aneural AChR Clusters for the Assembly of Postsynaptic Specializations by Modulating ADF/Cofilin Activity and Turnover.

Temperature is a physiological factor that affects neuronal growth and synaptic homeostasis at the invertebrate neuromuscular junctions (NMJs); however, whether temperature stress could also regulate the structure and function of the vertebrate NMJs remains unclear. In this study, we use Xenopus laevis primary cultures as a vertebrate model system for investigating the involvement of heat shock protein 90 (HSP90) family of stress proteins in NMJ development. First, cold temperature treatment or HSP90 inhibition attenuates the formation of aneural acetylcholine receptor (AChR) clusters, but increases their stability after they are formed, in cultured muscles. HSP90 inhibition specifically affects the stability of aneural AChR clusters and their associated intracellular scaffolding protein rapsyn, instead of causing a global change in cell metabolism and protein expression in Xenopus muscle cultures. Upon synaptogenic stimulation, a specific HSP90 family member, glucose-regulated protein 94 (Grp94), modulates the phosphorylation and dynamic turnover of actin depolymerizing factor (ADF)/cofilin at aneural AChR clusters, leading to the recruitment of AChR molecules from aneural clusters to the assembly of agrin-induced postsynaptic specializations. Finally, postsynaptic Grp94 knock-down significantly inhibits nerve-induced AChR clustering and postsynaptic activity in nerve-muscle co-cultures as demonstrated by live-cell imaging and electrophysiological recording, respectively. Collectively, this study suggests that temperature-dependent alteration in Grp94 expression and activity inhibits the assembly of postsynaptic specializations through modulating ADF/cofilin phosphorylation and activity at aneural AChR clusters, which prevents AChR molecules from being recruited to the postsynaptic sites via actin-dependent vesicular trafficking, at developing vertebrate NMJs.

Cofilin Loss in Drosophila Muscles Contributes to Muscle Weakness through Defective Sarcomerogenesis during Muscle Growth

SUMMARYSarcomeres, the fundamental contractile units of muscles, are conserved structures composed of actin thin filaments and myosin thick filaments. How sarcomeres are formed and maintained is not well understood. Here, we show that knockdown of Drosophila cofilin (DmCFL), an actin depolymerizing factor, disrupts both sarcomere structure and muscle function. The loss of DmCFL also results in the formation of sarcomeric protein aggregates and impairs sarcomere addition during growth. The activation of the proteasome delays muscle deterioration in our model. Furthermore, we investigate how a point mutation in CFL2 that causes nemaline myopathy (NM) in humans affects CFL function and leads to the muscle phenotypes observed in vivo. Our data provide significant insights to the role of CFLs during sarcomere formation, as well as mechanistic implications for disease progression in NM patients.

Arabidopsis REM16 acts as a B3 domain transcription factor to promote flowering time via directly binding to the promoters of SOC1 and FT.

Actin depolymerizing factor (ADF) is a key modulator for dynamic organization of actin cytoskeleton. Interestingly, it was found that the ADF1 gene silencing delays flowering, but its mechanism remains unclear. In this study, ADF1 was used as a bait to screen its interacting proteins by the yeast two-hybrid (Y2H) system. One of them, the REM16 transcription factor was identified. As one of the AP2/B3-like transcriptional factor family members, the REM16 contains two B3 domains and its transcript levels kept increasing during the floral transition stage. Overexpression of REM16 accelerates flowering while silencing of REM16 delays flowering. Gene expression analysis indicated that the key flowering activation genes such as CONSTANS (CO), FLOWERING LOCUS T (FT), LEAFY (LFY) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) were upregulated in the REM16 overexpression lines, while the transcription of the flowering suppression gene FLOWERING LOCUS C (FLC) was decreased. In contrast, the REM16 gene silencing lines contained lower transcript levels of the CO, FT, LFY and SOC1 but higher transcript levels of the FLC compared with the wild-type plants. It was proved that REM16 could directly bind to the promoter regions of SOC1 and FT by in vitro and in vivo assays. Genetic analysis supported that REM16 acts upstream of SOC1 and FT in flowering pathways. All these studies provided strong evidence demonstrating that REM16 promotes flowering by directly activating SOC1 and FT.

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1

AbstractRearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.