Mikaela Grönholm

Association of Ezrin with Intercellular Adhesion Molecule-1 and -2 (ICAM-1 and ICAM-2) REGULATION BY PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE

Ezrin is a cytoplasmic linker molecule between plasma membrane components and the actin-containing cytoskeleton. We studied whether ezrin is associated with intercellular adhesion molecule (ICAM)-1, -2, and -3. In transfected cells, ICAM-1 and ICAM-2 colocalized with ezrin in microvillar projections, whereas an ICAM-1 construct attached to cell membrane via a glycophosphatidylinositol anchor was uniformly distributed on the cell surface. An interaction of ICAM-2 and ezrin was seen by affinity precipitation, microtiter binding assay, coimmunoprecipitation, and surface plasmon resonance methods. The calculatedK D value was 3.3 × 10−7 m. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) induced an interaction of ezrin and ICAM-1 and enhanced the interaction of ezrin and ICAM-2, but ICAM-3 did not bind ezrin even in the presence of PtdIns(4,5)P2. PtdIns(4,5)P2 was shown to bind to cytoplasmic tails of ICAM-1 and ICAM-2, which are the first adhesion proteins demonstrated to interact with PtdIns(4,5)P2. The results indicate an interaction of ezrin with ICAM-1 and ICAM-2 and suggest a regulatory role of phosphoinositide signaling pathways in regulation of ICAM-ezrin interaction.

Regulation of integrin activity and signalling

The ability of cells to attach to each other and to the extracellular matrix is of pivotal significance for the formation of functional organs and for the distribution of cells in the body. Several molecular families of proteins are involved in adhesion, and recent work has substantially improved our understanding of their structures and functions. Also, these molecules are now being targeted in the fight against disease. However, less is known about how their activity is regulated. It is apparent that among the different classes of adhesion molecules, the integrin family of adhesion receptors is unique in the sense that they constitute a large group of widely distributed receptors, they are unusually complex and most importantly their activities are strictly regulated from the inside of the cell. The activity regulation is achieved by a complex interplay of cytoskeletal proteins, protein kinases, phosphatases, small G proteins and adaptor proteins. Obviously, we are only in the beginning of our understanding of how the integrins function, but already now fascinating details have become apparent. Here, we describe recent progress in the field, concentrating mainly on mechanistical and structural studies of integrin regulation. Due to the large number of articles dealing with integrins, we focus on what we think are the most exciting and rewarding directions of contemporary research on cell adhesion and integrins.

Inhibition of T Cell Activation by Cyclic Adenosine 5′-Monophosphate Requires Lipid Raft Targeting of Protein Kinase A Type I by the A-Kinase Anchoring Protein Ezrin

cAMP negatively regulates T cell immune responses by activation of type I protein kinase A (PKA), which in turn phosphorylates and activates C-terminal Src kinase (Csk) in T cell lipid rafts. Using yeast two-hybrid screening, far-Western blot, immunoprecipitation and immunofluorescense analyses, and small interfering RNA-mediated knockdown, we identified Ezrin as the A-kinase anchoring protein that targets PKA type I to lipid rafts. Furthermore, Ezrin brings PKA in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of PKA/Ezrin/Ezrin-binding protein 50, Csk, and Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains. The complex is initially present in immunological synapses when T cells contact APCs and subsequently exits to the distal pole. Introduction of an anchoring disruptor peptide (Ht31) into T cells competes with Ezrin binding to PKA and thereby releases the cAMP/PKA type I-mediated inhibition of T cell proliferation. Finally, small interfering RNA-mediated knockdown of Ezrin abrogates cAMP regulation of IL-2. We propose that Ezrin is essential in the assembly of the cAMP-mediated regulatory pathway that modulates T cell immune responses.

Isolation and characterization of platelet-derived extracellular vesicles.

Background Platelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Methods Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS) or Ca2+ ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. Results The main EV populations were 100–250 nm and over 90% were <500 nm irrespective of the activation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV-generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca2+ ionophore generated a large population of protein-poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations. Conclusions Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100–250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs.

Platelet-Derived Microvesicles: Multitalented Participants in Intercellular Communication

Platelets can release a heterogeneous pool of vesicles which include plasma membrane-derived microparticles (PMPs) and multivesicular body-derived exosomes. As both vesicle types are generated upon activation and their distinction is complicated due to an overlap in their molecular properties and sizes, they are best discussed as an entity, the platelet-derived microvesicles (PMVs). PMPs can be formed through several induction pathways, which determine their different molecular profiles and facilitate tailor-made participation in intercellular communication. This dynamic variability may lie behind the multifaceted and sometimes very different observations of the PMPs in physiological and pathological settings. Currently, little is known of platelet-derived exosomes. In all, PMVs not only participate in several homeostatic multicellular processes, such as hemostasis, maintenance of vascular health, and immunity, but they also play a role in thrombotic and inflammatory diseases and cancer progression. In the past few years, the number of original articles and reviews on microvesicles has dramatically increased, but the data simultaneously raise further questions, the answers to which depend on forthcoming analytical improvements. In this article, the differential activation pathways and the molecular and functional properties of PMVs are reviewed in context with their sometimes paradoxical role in health and in disease. Also, the methodological issues of PMV detection and analysis are discussed in the light of recent advances within the field.

Cell cycle-dependent nucleocytoplasmic shuttling of the neurofibromatosis 2 tumour suppressor merlin

The neurofibromatosis 2 tumour suppressor merlin/schwannomin is structurally related to the ezrin–radixin–moesin family of proteins, which anchor actin cytoskeleton to specific membrane proteins and participate in cell signalling. Merlin inhibits cell growth with a yet unknown mechanism. As most tumour suppressors are linked to cell cycle control, we investigated merlins behaviour during cell cycle. In glioma and osteosarcoma cells, endogenous merlin was targeted to the nucleus in a cell cycle-specific manner. Merlin accumulated perinuclearly at the G2/M phase, and shifted to the nucleus at early G1. During mitosis, merlin localized to mitotic spindles and at the contractile ring. Nuclear merlin was strongly reduced in confluent cells. Blocking of the CRM1/exportin nuclear export pathway led to accumulation of merlin in the nucleus. Activation of the p21-activated kinase or protein kinase A, which result in phosphorylation of merlin, did not affect its nuclear localization. Merlin regulates the activity of extracellular signal-regulated kinase 2 (ERK2) and nuclear localization of both proteins was induced by cell adhesion. Unlike ERK2, nuclear localization of merlin was not, however, dependent on intact actin cytoskeleton. These results link merlin to events related to cell cycle control and may help to resolve its tumour suppressor function.

Merlin Links to the cAMP Neuronal Signaling Pathway by Anchoring the RIβ Subunit of Protein Kinase A

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIβ is most abundantly expressed in brain. The RIβ knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIβ in learning and memory-related functions. Molecules that interact with or regulate RIβ are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIβ. Merlin and RIβ demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the α-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved α-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIβ and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling.

Mutation update and genotype-phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies.

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core‐rod myopathy, congenital fiber‐type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal‐dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca2+ sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non‐hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin–actin association or tropomyosin head‐to‐tail binding.

Abnormal actin binding of aberrant β-tropomyosins is a molecular cause of muscle weakness in TPM2-related nemaline and cap myopathy.

NM (nemaline myopathy) is a rare genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding β-Tm (tropomyosin). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium-sensitive manner. In the present study, we have investigated the pathogenetic mechanisms underlying five disease-causing mutations in Tm. We show that four of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, whereas two show defective Ca2+ activation of contractility. We have also mapped the amino acids altered by the mutation to regions important for actin binding and note that two of the mutations cause altered protein conformation, which could account for impaired actin affinity.

Protein kinase A-mediated phosphorylation of the NF2 tumor suppressor protein merlin at serine 10 affects the actin cytoskeleton.

Mutations in the neurofibromatosis 2 tumor suppressor gene (NF2) encoding merlin (moesin–ezrin–radixin like-protein) induce tumors of the nervous system. Merlin localizes to the cell membrane where it links the actin cytoskeleton to membrane proteins. Cell proliferation is regulated by merlin in many cell types, but merlins tumor suppressor function still remains unclear. Phosphorylation has been suggested to regulate merlins activity. The C-terminal serine 518 is phosphorylated both by p21-activated kinases (PAKs) and protein kinase A (PKA). In this work, we identify a novel PKA phosphorylation site, serine 10, in the N terminus of merlin. We show that a non-phosphorylatable form of serine 10 (S10A) affects cellular morphology. Regulation of this site also influences actin cytoskeleton organization and dynamics in vivo, as merlin S10A reduces the amount of cellular F-actin and merlin S10D stabilizes F-actin filaments. By using a wound-healing assay and live cell imaging, we demonstrate that dephosphorylation of serine 10 leads to defects in migration, possibly through altered ability of the cells to form lamellipodia. This study suggests a role for merlin in mediating PKA-induced changes of the actin cytoskeleton.