Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain.

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins1-3. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour3,4. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors5-9. Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P2, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.

Bsp1, a fungal CPI motif protein, regulates actin filament capping in endocytosis and cytokinesis.

The capping of barbed filament ends is a fundamental mechanism for actin regulation. Capping protein controls filament growth and actin turnover in cells by binding to the barbed ends of the filaments with high affinity and slow off-rate. The interaction between capping protein and actin is regulated by capping protein interaction (CPI) motif proteins. We identified a novel CPI motif protein, Bsp1, which is involved in cytokinesis and endocytosis in budding yeast. We demonstrate that Bsp1 is an actin binding protein with a high affinity for capping protein via its CPI motif. In cells, Bsp1 regulates capping protein at endocytic sites and is a major recruiter of capping protein to the cytokinetic actin ring. Lastly, we define Bsp1-related proteins as a distinct fungi-specific CPI protein group. Our results suggest that Bsp1 promotes actin filament capping by the capping protein. This study establishes Bsp1 as a new capping protein regulator and promising candidate to regulate actin networks in fungi.

Flattening out: A new ESCRT structure in cell adhesions.

Conserved protein complexes called ESCRTs (endosomal sorting complexes in retrograde transport) exert diverse membrane remodeling and repair functions in cells. Hakala and Roux discuss a novel type of ESCRT-III structure found by Stempels et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202205130) in migrating macrophages and dendritic cells, suggesting a novel, cell type-specific function for this complex.

Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks.

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP's rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

Molecular mechanism for inhibition of twinfilin by phosphoinositides.

Membrane phosphoinositides control organization and dynamics of the actin cytoskeleton by regulating the activities of several key actin-binding proteins. Twinfilin is an evolutionarily conserved protein that contributes to cytoskeletal dynamics by interacting with actin monomers, filaments, and the heterodimeric capping protein. Twinfilin also binds phosphoinositides, which inhibit its interactions with actin, but the underlying mechanism has remained unknown. Here, we show that the high-affinity binding site of twinfilin for phosphoinositides is located at the C-terminal tail region, whereas the two actin-depolymerizing factor (ADF)/cofilin-like ADF homology domains of twinfilin bind phosphoinositides only with low affinity. Mutagenesis and biochemical experiments combined with atomistic molecular dynamics simulations reveal that the C-terminal tail of twinfilin interacts with membranes through a multivalent electrostatic interaction with a preference toward phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), PI(4,5)P2, and PI(3,4,5)P3 This initial interaction places the actin-binding ADF homology domains of twinfilin in close proximity to the membrane and subsequently promotes their association with the membrane, thus leading to inhibition of the actin interactions. In support of this model, a twinfilin mutant lacking the C-terminal tail inhibits actin filament assembly in a phosphoinositide-insensitive manner. Our mutagenesis data also reveal that the phosphoinositide- and capping protein-binding sites overlap in the C-terminal tail of twinfilin, suggesting that phosphoinositide binding additionally inhibits the interactions of twinfilin with the heterodimeric capping protein. The results demonstrate that the conserved C-terminal tail of twinfilin is a multifunctional binding motif, which is crucial for interaction with the heterodimeric capping protein and for tethering twinfilin to phosphoinositide-rich membranes.

Twinfilin 2a regulates platelet reactivity and turnover in mice.

Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein that inhibits actin filament assembly by sequestering actin monomers and capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here, we report that constitutive Twf2a-deficient mice (Twf2a-/-) display mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. Twf2a-/- platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem) immunoreceptor tyrosine-based activation motif (ITAM) and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, and accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyperreactivity of Twf2a-/- platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilin- and profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice.

Actin Filament Structures in Migrating Cells.

Cell migration is necessary for several developmental processes in multicellular organisms. Furthermore, many physiological processes such as wound healing and immunological events in adult animals are dependent on cell migration. Consequently, defects in cell migration are linked to various diseases including immunological disorders as well as cancer progression and metastasis formation. Cell migration is driven by specific protrusive and contractile actin filament structures, but the types and relative contributions of these actin filament arrays vary depending on the cell type and the environment of the cell. In this chapter, we introduce the most important actin filament structures that contribute to mesenchymal and amoeboid cell migration modes and discuss the mechanisms by which the assembly and turnover of these structures are controlled by various actin-binding proteins.

GMF promotes leading-edge dynamics and collective cell migration in vivo.

Lamellipodia are dynamic actin-rich cellular extensions that drive advancement of the leading edge during cell migration. Lamellipodia undergo periodic extension and retraction cycles, but the molecular mechanisms underlying these dynamics and their role in cell migration have remained obscure. We show that glia-maturation factor (GMF), which is an Arp2/3 complex inhibitor and actin filament debranching factor, regulates lamellipodial protrusion dynamics in living cells. In cultured S2R(+) cells, GMF silencing resulted in an increase in the width of lamellipodial actin filament arrays. Importantly, live-cell imaging of mutant Drosophila egg chambers revealed that the dynamics of actin-rich protrusions in migrating border cells is diminished in the absence of GMF. Consequently, velocity of border cell clusters undergoing guided migration was reduced in GMF mutant flies. Furthermore, genetic studies demonstrated that GMF cooperates with the Drosophila homolog of Aip1 (flare) in promoting disassembly of Arp2/3-nucleated actin filament networks and driving border cell migration. These data suggest that GMF functions in vivo to promote the disassembly of Arp2/3-nucleated actin filament arrays, making an important contribution to cell migration within a 3D tissue environment.

The decline in joint replacement surgery in rheumatoid arthritis is associated with a concomitant increase in the intensity of anti-rheumatic therapy: a nationwide register-based study from 1995 through 2010.

BACKGROUND AND PURPOSE: Drug-based treatment of rheumatoid arthritis (RA) has evolved markedly over the past 2 decades. Using nationwide register data, we studied how this has affected the rates of hip, knee, shoulder, and elbow replacement from 1995 to 2010. METHODS: The number of primary joint replacements was obtained from the Finnish Arthroplasty Register. To test the hypothesis that improvements in medical treatment of RA reduce the need for joint replacements, we also collected data about purchases of different disease-modifying anti-rheumatic agents (DMARDs) and biological drugs from the nationwide drug registers. RESULTS: The annual incidence of primary joint replacements for RA declined from 19 per 10(5) in 1995 to 11 per 10(5) in 2010. The decline was greater for upper-limb operations than for lower-limb operations. At the same time, the numbers of individuals using methotrexate, hydroxychloroquine, and sulfasalazine (the most commonly used DMARDs) increased 2- to 4-fold. INTERPRETATION: Our results are in accordance with observations from other countries, and indicate that the use of joint replacements in RA has decreased dramatically. Our data suggest that effective medical therapy is the most likely explanation for this favorable development.

Beware of the biologicals--hospitals may die: the Rheumatism Foundation Hospital, Heinola, Finland (1951-2010).

The first patient entered the Rheumatism Foundation Hospital, Heinola, Finland in July 1951. From that point on, the hospital helped patients suffering from rheumatic disorders. Specialists in the hospital actively developed treatments and published a large number of scientific articles in international journals. The hospital was well known internationally among people working in the field. Progress in the development of disease-modifying medication (biological agents in particular) has dramatically improved the life of patients with rheumatic diseases, but all effective treatments may also have adverse effects. In this article, we briefly review the history of the Rheumatism Foundation Hospital, which was closed permanently in March 2010 due to bankruptcy. The economical difficulties were caused primarily by the progress made in disease-modifying therapy, which decreased the need of rehabilitation and operative treatment of patients with rheumatic diseases. It seems that a great success in biological agents can carry "serious adverse effects", which may kill hospitals. This is an important primary observation, which should be noticed when the future of specialised institutes is planned.

Sustained improvement of health-related quality of life in patients with early rheumatoid arthritis: a ten-year follow-up study.

OBJECTIVES: To assess long-term impact of RA on the HR-QoL in a cohort of working-age patients with early disease treated by a multidisciplinary team including early and active use of disease-modifying anti-rheumatic drugs (DMARDs). METHODS: Fifty-five consecutive patients with RA who were naïve to DMARDs and glucocorticoids were assessed at baseline and at 6 months, 1, 2, 5 and 10 years. HR-QoL, disease activity, function, and joint destruction of hands and feet were assessed by using the Nottingham Health Profile (NHP) instrument, the 28-joint based Disease Activity Score (DAS28), the Health Assessment Questionnaire (HAQ), and the Larsen scores, respectively. GEE (generalised estimation equations)-method was used to evaluate longitudinal relationships between the HR-QoL changes and other variables. RESULTS: All NHP dimensions except social isolation improved significantly during the first six months and remained favourable up to 10 years. The most prominent improvements were seen in the dimensions for pain and emotional reaction (p<0.001). In longitudinal evaluation statistically significant associations (p<0.001) were found between the DAS28 and the NHP dimensions for pain, energy and emotional reaction, and between the HAQ and the NHP dimensions for pain, energy and mobility. The extent of joint damage had no statistically significant associations to the six dimensions of the NHP instrument. CONCLUSIONS: Early improvements in HR-QoL carried over the ten-year follow-up in patients with recent-onset RA treated with a multidisciplinary strategy including early and active DMARD therapy. HR-QoL changes were longitudinally associated especially with disease activity and function.

A marked decline in the incidence of renal replacement therapy for amyloidosis associated with inflammatory rheumatic diseases - data from nationwide registries in Finland.

Risk for amyloidosis in rheumatic diseases is associated with a long-lasting inflammation. To assess possible changes in the incidence of terminal uraemia due to amyloidosis associated with rheumatic diseases on a nationwide basis, we scrutinised the files of the Finnish Registry for Kidney Diseases for patients suffering from amyloidosis associated with rheumatoid arthritis (RA), ankylosing spondylitis (AS) or juvenile idiopathic arthritis (JIA) over the period 1995-2008. The registry has an estimated 97-99% coverage of all patients accepted for renal replacement therapy (RRT) in the country. Data on the consumption of antirheumatic drugs were collected from two sources: the Social Insurance Institution's Drug Reimbursement Register, and the Sales Register of the National Agency for Medicines from the above period. Altogether 264 cases were identified. Two hundred twenty-nine of them had RA, 15 AS and 20 JIA. When the total annual number of new admissions to RRT varied between 20 and 37 at the end of 1990s, it was under half of that from 2002 onwards. Over this period, the number of users of low-dose methotrexate (MTX) has increased 3.6-fold, the drug being the most frequently used disease modifying anti-rheumatic drug in Finland. The present nationwide series is the first to show that the incidence of end-stage renal disease due to amyloidosis associated with rheumatic diseases is decreasing. An obvious reason for this is intensive anti-rheumatic drug therapy.

[Strong metatarsal pain and localized bone lesions in a physically active male (clinico-pathological conference report)]. / Liikuntaa harrastaneen miehen voimakas jalka- teräkipu ja paikalliset luustomuutokset.

With the exception of a previous history of pulmonary sarcoidosis, a previously healthy 38-year old man developed a sudden unilateral metatarsal pain and gradually progressing osteoporotic, partly lytic metatarsal bone lesions. The patient received a bisphosphonate treatment. Clinical and radiological situation began to improve during the follow-up observation. The diagnosis was based on clinical picture.

ADF/cofilin binds phosphoinositides in a multivalent manner to act as a PIP(2)-density sensor.

Actin-depolymerizing-factor (ADF)/cofilins have emerged as key regulators of cytoskeletal dynamics in cell motility, morphogenesis, endocytosis, and cytokinesis. The activities of ADF/cofilins are regulated by membrane phospholipid PI(4,5)P(2) in vitro and in cells, but the mechanism of the ADF/cofilin-PI(4,5)P(2) interaction has remained controversial. Recent studies suggested that ADF/cofilins interact with PI(4,5)P(2) through a specific binding pocket, and that this interaction is dependent on pH. Here, we combined systematic mutagenesis with biochemical and spectroscopic methods to elucidate the phosphoinositide-binding mechanism of ADF/cofilins. Our analysis revealed that cofilin does not harbor a specific PI(4,5)P(2)-binding pocket, but instead interacts with PI(4,5)P(2) through a large, positively charged surface of the molecule. Cofilin interacts simultaneously with multiple PI(4,5)P(2) headgroups in a cooperative manner. Consequently, interactions of cofilin with membranes and actin exhibit sharp sensitivity to PI(4,5)P(2) density. Finally, we show that cofilin binding to PI(4,5)P(2) is not sensitive to changes in the pH at physiological salt concentration, although the PI(4,5)P(2)-clustering activity of cofilin is moderately inhibited at elevated pH. Collectively, our data demonstrate that ADF/cofilins bind PI(4,5)P(2) headgroups through a multivalent, cooperative mechanism, and suggest that the actin filament disassembly activity of ADF/cofilin can be accurately regulated by small changes in the PI(4,5)P(2) density at cellular membranes.

[Rheumatoid arthritis (updated Current Care guideline)]. / Nivelreuma.

Anti-citrulline antibodies are highly specific to rheumatoid arthritis (RA) and are thus helpful in differential diagnosis. Early and aggressive disease modifying anti-rheumatic drug (DMARD) therapy is essential for a positive treatment result in cases of RA. Remission during the 1st year of treatment predicts permanent remission, milder joint damage and better functional ability. It is recommended that patients with an unsatisfactory response to DMARDs, including methotrexate and a combination of DMARDs, should be treated primarily with TNF blockers, and non-responders with rituximab or abatacept. RA is an independent risk factor for cardiovascular diseases. The assessment of cardiovascular risk must not be forgotten in daily practice.

The good initial response to therapy with a combination of traditional disease-modifying antirheumatic drugs is sustained over time: the eleven-year results of the Finnish rheumatoid arthritis combination therapy trial.

OBJECTIVE: To evaluate the evolution of functional and clinical outcomes over 11 years in patients with early rheumatoid arthritis (RA) initially treated with a combination of 3 disease-modifying antirheumatic drugs (DMARDs) or with a single DMARD. METHODS: A cohort of 199 patients with early active RA were initially randomized to receive treatment with a combination of methotrexate, sulfasalazine, and hydroxychloroquine with prednisolone or treatment with a single DMARD (initially, sulfasalazine) with or without prednisolone. After 2 years, the drug treatment strategy became unrestricted, but still targeted remission. At 11 years, function was assessed with the Health Assessment Questionnaire (HAQ), and clinical outcomes were assessed with the modified Minimal Disease Activity (MDA) measure and the American College of Rheumatology (ACR) criteria for remission. RESULTS: At 11 years, 138 patients were assessed (68 in the combination-DMARD group and 70 in the single-DMARD group). The mean+/-SD HAQ scores were 0.34+/-0.54 in the combination-DMARD group and 0.38+/-0.58 in the single-DMARD group (P=0.88). Modified MDA was achieved by 63% (95% confidence interval [95% CI] 51, 77) and by 43% (95% CI 32, 55) (P=0.016) of the combination-DMARD group and the single-DMARD group, respectively, and ACR remission by 37% (95% CI 26, 49) and by 19% (95% CI 11, 29) (P=0.017), respectively. CONCLUSION: Initial therapy with a combination of DMARDs in early RA results in higher rates of patients achieving modified MDA and strict ACR remission even over the long term than initial single-DMARD therapy. Targeting remission with tight clinical controls results in good functional and clinical outcomes in most RA patients.

Etanercept in the longterm treatment of patients with ankylosing spondylitis.

OBJECTIVE: To evaluate the 2-year efficacy and safety of etanercept in patients with ankylosing spondylitis (AS). METHODS: A 96-week open-label extension study, which followed a 12-week double-blind placebo-controlled trial, was designed to provide longterm efficacy and safety data, including radiographic outcomes, for patients treated with etanercept 25 mg twice weekly (NCT00421980). In all, 81 patients were enrolled (96% of the participants from the double-blind study). Key efficacy measures included improvement using the Assessment in Ankylosing Spondylitis 20% (ASAS20) criteria, the Bath Ankylosing Spondylitis Functional Index (BASFI), and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Radiographic progression was evaluated using the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) method. Paired t tests were used to test within-group changes from baseline. RESULTS: The percentage of responders, by ASAS20 criteria, remained relatively constant in patients who received etanercept during the 12-week double-blind study (60% at Week 0 and 83% at Week 96 of the open-label extension); more patients from the placebo group became responders after being switched to etanercept (23% and 74%, respectively). A similar trend was also observed using the ASAS40 and ASAS5/6 criteria, the BASFI, and the BASDAI. Most patients had no change from baseline in mSASSS values. Etanercept was well tolerated; the most frequent adverse events were injection site reactions (n=30; 37.0%) and headache (n=18; 22.2%), and the most frequent infections were upper respiratory tract infections (n=43; 53.1%) and flu syndrome (n=22; 27.2%). CONCLUSION: For 2 years, etanercept was clinically effective and well tolerated, with no unexpected safety findings.

[Update on current care guidelines. Safe use of non-steroidal anti-inflammatory drugs]. / Tulehduskipulääkkeiden turvallinen käyttö.

Pain medication should be based on patient's needs and risk profile. Age > 65 years, prior ulcer, co-morbidities, large daily dose, Helicobacter pylori infection, concurrent use of glucocorticoids, serotonin re-uptake inhibitors, or warfarin increase the risk of upper gastrointestinal bleeds. As a preventive strategy the use of concurrent proton pump inhibitors with non-selective NSAIDs is recommended. It is also possible to use COX-2 selective NSAIDs but they are contraindicated for persons with atherosclerotic diseases and special consideration is required for persons with risk factors of heart diseases. Paracetamol is the drug of choice for pain.