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Galectins are carbohydrate-binding proteins that regulate many cellular functions including proliferation, adhesion, migration, and phagocytosis. Increasing experimental and clinical evidence indicates that galectins influence many steps of cancer development by inducing the recruitment of immune cells to the inflammatory sites and modulating the effector function of neutrophils, monocytes, and lymphocytes. Recent studies described that different isoforms of galectins can induce platelet adhesion, aggregation, and granule release through the interaction with platelet-specific glycoproteins and integrins. Patients with cancer and/or deep-venous thrombosis have increased levels of galectins in the vasculature, suggesting that these proteins could be important contributors to cancer-associated inflammation and thrombosis. In this review, we summarize the pathological role of galectins in inflammatory and thrombotic events, influencing tumor progression and metastasis. We also discuss the potential of anti-cancer therapies targeting galectins in the pathological context of cancer-associated inflammation and thrombosis.

Cancer-associated inflammation has been established as a hallmark feature of almost all solid cancers. Tumor-extrinsic and intrinsic signaling pathways regulate the process of cancer-associated inflammation. Tumor-extrinsic inflammation is triggered by many factors, including infection, obesity, autoimmune disorders, and exposure to toxic and radioactive substances. Intrinsic inflammation can be induced by genomic mutation, genome instability and epigenetic remodeling in cancer cells that promote immunosuppressive traits, inducing the recruitment and activation of inflammatory immune cells. In RCC, many cancer cell-intrinsic alterations are assembled, upregulating inflammatory pathways, which enhance chemokine release and neoantigen expression. Furthermore, immune cells activate the endothelium and induce metabolic shifts, thereby amplifying both the paracrine and autocrine inflammatory loops to promote RCC tumor growth and progression. Together with tumor-extrinsic inflammatory factors, tumor-intrinsic signaling pathways trigger a Janus-faced tumor microenvironment, thereby simultaneously promoting or inhibiting tumor growth. For therapeutic success, it is important to understand the pathomechanisms of cancer-associated inflammation, which promote cancer progression. In this review, we describe the molecular mechanisms of cancer-associated inflammation that influence cancer and immune cell functions, thereby increasing tumor malignancy and anti-cancer resistance. We also discuss the potential of anti-inflammatory treatments, which may provide clinical benefits in RCCs and possible avenues for therapy and future research.

Extracellular DNA may serve as marker in liquid biopsies to determine individual diagnosis and prognosis in cancer patients. Cell death or active release from various cell types, including immune cells can result in the release of DNA into the extracellular milieu. Neutrophils are important components of the innate immune system, controlling pathogens through phagocytosis and/or the release of neutrophil extracellular traps (NETs). NETs also promote tumor progression and metastasis, by modulating angiogenesis, anti-tumor immunity, blood clotting and inflammation and providing a supportive niche for metastasizing cancer cells. Besides neutrophils, other immune cells such as eosinophils, dendritic cells, monocytes/macrophages, mast cells, basophils and lymphocytes can also form extracellular traps (ETs) during cancer progression, indicating possible multiple origins of extracellular DNA in cancer. In this review, we summarize the pathomechanisms of ET formation generated by different cell types, and analyze these processes in the context of cancer. We also critically discuss potential ET-inhibiting agents, which may open new therapeutic strategies for cancer prevention and treatment.

Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix.

Abstract Background Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC). Results The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12. Conclusions Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.

Although platelets are critically involved in thrombosis and hemostasis, experimental and clinical evidence indicate that platelets promote tumor progression and metastasis through a wide range of physical and functional interactions between platelets and cancer cells. Thrombotic and thromboembolic events are frequent complications in patients with solid tumors. Hence, cancer modulates platelet function by directly inducing platelet-tumor aggregates and triggering platelet granule release and altering platelet turnover. Also, platelets enhance tumor cell dissemination by activating endothelial cell function and recruiting immune cells to primary and metastatic tumor sites. In this review, we summarize current knowledge on the complex interactions between platelets and tumor cells and the host microenvironment. We also critically discuss the potential of anti-platelet agents for cancer prevention and treatment.

Clotting and inflammation are effective danger response patterns positively selected by evolution to limit fatal bleeding and pathogen invasion upon traumatic injuries. As a trade-off, thrombotic, and thromboembolic events complicate severe forms of infectious and non-infectious states of acute and chronic inflammation, i.e., immunothrombosis. Factors linked to thrombosis and inflammation include mediators released by platelet granules, complement, and lipid mediators and certain integrins. Extracellular deoxyribonucleic acid (DNA) was a previously unrecognized cellular component in the blood, which elicits profound proinflammatory and prothrombotic effects. Pathogens trigger the release of extracellular DNA together with other pathogen-associated molecular patterns. Dying cells in the inflamed or infected tissue release extracellular DNA together with other danger associated molecular pattern (DAMPs). Neutrophils release DNA by forming neutrophil extracellular traps (NETs) during infection, trauma or other forms of vascular injury. Fluorescence tissue imaging localized extracellular DNA to sites of injury and to intravascular thrombi. Functional studies using deoxyribonuclease (DNase)-deficient mouse strains or recombinant DNase show that extracellular DNA contributes to the process of immunothrombosis. Here, we review rodent models of immunothrombosis and the evolving evidence for extracellular DNA as a driver of immunothrombosis and discuss challenges and prospects for extracellular DNA as a potential therapeutic target.

Platelet aggregate formation is a multistep process involving receptor-mediated, as well as biomechanical, signaling cascades, which are highly dependent on actin dynamics. We have previously shown that actin depolymerizing factor (ADF)/n-cofilin and Twinfilin 2a, members of the ADF homology (ADF-H) protein family, have distinct roles in platelet formation and function. Coactosin-like 1 (Cotl1) is another ADF-H protein that binds actin and was also shown to enhance biosynthesis of pro-inflammatory leukotrienes (LT) in granulocytes. Here, we generated mice lacking Cotl1 in the megakaryocyte lineage (Cotl1−/−) to investigate its role in platelet production and function. Absence of Cotl1 had no impact on platelet counts, platelet activation or cytoskeletal reorganization under static conditions in vitro. In contrast, Cotl1 deficiency markedly affected platelet aggregate formation on collagen and adhesion to immobilized von Willebrand factor at high shear rates in vitro, pointing to an impaired function of the platelet mechanoreceptor glycoprotein (GP) Ib. Furthermore, Cotl1−/−platelets exhibited increased deformability at high shear rates, indicating that the GPIb defect may be linked to altered biomechanical properties of the deficient cells. In addition, we found that Cotl1 deficiency markedly affected platelet LT biosynthesis. Strikingly, exogenous LT addition restored defective aggregate formation of Cotl1−/− platelets at high shear in vitro, indicating a critical role of platelet-derived LT in thrombus formation. In vivo, Cotl1 deficiency translated into prolonged tail bleeding times and protection from occlusive arterial thrombus formation. Together, our results show that Cotl1 in platelets is an integrator of biomechanical and LT signaling in hemostasis and thrombosis.

Platelets serotonin (5-hydroxytrytamine, 5-HT) uptake and storage in dense granules is tightly regulated by the serotonergic transport system in the blood. Several 5-HT transporters (5-HTTs) have been identified in the vasculature and blood cells, beyond them 5-HTT is the major 5-HT transporter in platelets. Abnormal 5-HT concentrations in the blood plasma or increased platelet 5-HT uptake or abnormal release contribute to the development of various diseases in the vasculature. Consequently, several clinical trials suggested the positive therapeutic effects of 5-HTT blockade in the circulation. Inhibition of 5-HT strongly attenuates autocrine and paracrine functions of platelets, influencing platelet aggregation, vascular contraction, permeability, tissue repair, wound healing, immunity and cancer. Here, we highlight the current state of basic biological research regarding the hemostatic and non-hemostatic functions of platelet-derived 5-HT in normal and disease conditions. We also describe the physiological consequences of targeting platelet 5-HT functions in thrombosis, stroke, inflammation and cancer to overcome common health problems.

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6β1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.

Although Mg2+is vital for platelet activation and aggregation, its regulation in these cells is still largely unknown. Here, the authors show that TRPM7, a cation channel and a protein kinase, regulates thrombopoiesis and platelet size by affecting the cytoskeleton of these cells in mice and humans.

N-glycans are covalently linked to an asparagine residue in a simple acceptor sequence of proteins, called a sequon. This modification is important for protein folding, enhancing thermodynamic stability, and decreasing abnormal protein aggregation within the endoplasmic reticulum (ER), for the lifetime and for the subcellular localization of proteins besides other functions. Hypoglycosylation is the hallmark of a group of rare genetic diseases called congenital disorders of glycosylation (CDG). These diseases are due to defects in glycan synthesis, processing, and attachment to proteins and lipids, thereby modifying signaling functions and metabolic pathways. Defects in N-glycosylation and O-glycosylation constitute the largest CDG groups. Clotting and anticlotting factor defects as well as a tendency to thrombosis or bleeding have been described in CDG patients. However, N-glycosylation of platelet proteins has been poorly investigated in CDG. In this review, we highlight normal and deficient N-glycosylation of platelet-derived molecules and discuss the involvement of platelets in the congenital disorders of N-glycosylation.

Zn2+ deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn2+-deficient diets, accounting for 1−4% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn2+ deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn2+ status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn2+ uptake in the gut using different nutritional supplementation of Zn2+ could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn2+ diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn2+ in hemostasis. Storage protein metallothionein maintains or releases Zn2+ in the cytoplasm, and the dynamic change of this cytoplasmic Zn2+ pool is regulated by the redox status of the cell. An increase of labile Zn2+ pool can be toxic for the cells, and therefore cytoplasmic Zn2+ levels are tightly regulated by several Zn2+ transporters located on the cell surface and also on the intracellular membrane of Zn2+ storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn2+ is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn2+ transport and the physiological role of Zn2+ store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn2+ to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn2+ homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases.

In patients with dysfunctions of the Ca2+ channel ORAI1, stromal interaction molecule 1 (STIM1) or integrin-regulating kindlin-3 (FERMT3), severe immunodeficiency is frequently linked to abnormal platelet activity. In this paper, we studied platelet responsiveness by multiparameter assessment of whole blood thrombus formation under high-shear flow conditions in 9 patients, including relatives, with confirmed rare genetic mutations of ORAI1, STIM1 or FERMT3. In platelets isolated from 5 out of 6 patients with ORAI1 or STIM1 mutations, store-operated Ca2+ entry (SOCE) was either completely or partially defective compared to control platelets. Parameters of platelet adhesion and aggregation on collagen microspots were impaired for 4 out of 6 patients, in part related to a low platelet count. For 4 patients, platelet adhesion/aggregation and procoagulant activity on von Willebrand Factor (VWF)/rhodocytin and VWF/fibrinogen microspots were impaired independently of platelet count, and were partly correlated with SOCE deficiency. Measurement of thrombus formation at low shear rate confirmed a greater impairment of platelet functionality in the ORAI1 patients than in the STIM1 patient. For 3 patients/relatives with a FERMT3 mutation, all parameters of thrombus formation were strongly reduced regardless of the microspot. Bone marrow transplantation, required by 2 patients, resulted in overall improvement of platelet function. We concluded that multiparameter assessment of whole blood thrombus formation in a surface-dependent way can detect: i) additive effects of low platelet count and impaired platelet functionality; ii) aberrant ORAI1-mediated Ca2+ entry; iii) differences in platelet activation between patients carrying the same ORAI1 mutation; iv) severe platelet function impairment linked to a FERMT3 mutation and bleeding history.

Mg2+ regulates many physiological processes and signalling pathways. However, little is known about the mechanisms underlying the organismal balance of Mg2+. Capitalizing on a set of newly generated mouse models, we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6. We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development. In adult mice, TRPM6 is required in the intestine to maintain organismal Mg2+ balance, but is dispensable in the kidney. Trpm6 inactivation in adult mice leads to a shortened lifespan, growth deficit and metabolic alterations indicative of impaired energy balance. Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice, but also may extend the lifespan of wildtype mice. Hence, maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood.

Inducible genetically defined mouse models of cancer uniquely facilitate the investigation of early events in cancer progression, however, there are valid concerns about the ability of such models to faithfully recapitulate human disease. We developed an inducible mouse model of progressive lung adenocarcinoma (LuAd) that combines sporadic activation of oncogenic KRasG12D with modest overexpression of c-MYC (KM model). Histological examination revealed a highly reproducible spontaneous transition from low-grade adenocarcinoma to locally invasive adenocarcinoma within 6 weeks of oncogene activation. Laser-capture microdissection coupled with RNA-SEQ (ribonucleic acid sequencing) was employed to determine transcriptional changes associated with tumour progression. Upregulated genes were triaged for relevance to human LuAd using datasets from Oncomine and cBioportal. Selected genes were validated by RNAi screening in human lung cancer cell lines and examined for association with lung cancer patient overall survival using KMplot.com. Depletion of progression-associated genes resulted in pronounced viability and/or cell migration defects in human lung cancer cells. Progression-associated genes moreover exhibited strong associations with overall survival, specifically in human lung adenocarcinoma, but not in squamous cell carcinoma. The KM mouse model faithfully recapitulates key molecular events in human adenocarcinoma of the lung and is a useful tool for mechanistic interrogation of KRAS-driven LuAd progression.

Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.

Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation.To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke.5-HT transporter knockout mice (5Htt-/-) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke.In 5Htt-/- platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca2+ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt-/- platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt-/- mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt-/- mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice.Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization.

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation--platelet adhesion and aggregation--have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84(-/-) platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84(-/-) mice. In vivo, Cd84(-/-) mice exhibited unaltered hemostatic function and arterial thrombus formation.These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.

This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.

Twinfilins are evolutionarily conserved regulators of cytoskeletal dynamics. They inhibit actin polymerization by binding both actin monomers and filament barbed ends. Inactivation of the single twinfilin gene from budding yeast and fruit fly results in defects in endocytosis, cell migration, and organization of the cortical actin filament structures. Mammals express three twinfilin isoforms, of which twinfilin-1 and twinfilin-2a display largely overlapping expression patterns in non-muscle tissues of developing and adult mice. The expression of twinfilin-2b, which is generated through alternative promoter usage of the twinfilin-2 gene, is restricted to heart and skeletal muscles. However, the physiological functions of mammalian twinfilins have not been reported. As a first step towards understanding the function of twinfilin in vertebrates, we generated twinfilin-2a deficient mice by deleting exon 1 of the twinfilin-2 gene. Twinfilin-2a knockout mice developed normally to adulthood, were fertile, and did not display obvious morphological or behavioural abnormalities. Tissue anatomy and morphology in twinfilin-2a deficient mice was similar to that of wild-type littermates. These data suggest that twinfilin-2a plays a redundant role in cytoskeletal dynamics with the biochemically similar twinfilin-1, which is typically co-expressed in same tissues with twinfilin-2a.

Twf (twinfilin) is an evolutionarily conserved regulator of actin dynamics composed of two ADF-H (actin-depolymerizing factor homology) domains. Twf binds actin monomers and heterodimeric capping protein with high affinity. Previous studies have demonstrated that mammals express two Twf isoforms, Twf1 and Twf2, of which at least Twf1 also regulates cytoskeletal dynamics by capping actin filament barbed-ends. In the present study, we show that alternative promoter usage of the mouse Twf2 gene generates two isoforms, which differ from each other only at their very N-terminal region. Of these isoforms, Twf2a is predominantly expressed in non-muscle tissues, whereas expression of Twf2b is restricted to heart and skeletal muscle. Both proteins bind actin monomers and capping protein, as well as efficiently capping actin filament barbed-ends. However, the N-terminal ADF-H domain of Twf2b interacts with ADP-G-actin with a 5-fold higher affinity than with ATP-G-actin, whereas the corresponding domain of Twf2a binds ADP-G-actin and ATP-G-actin with equal affinities. Taken together, these results show that, like Twf1, mouse Twf2 is a filament barbed-end capping protein, and that two tissue-specific and biochemically distinct isoforms are generated from the Twf2 gene through alternative promoter usage. [ABSTRACT FROM AUTHOR]