Cytosolic Actin Research Articles

Truncated Latrunculins as Actin Inhibitors Targeting Plasmodium falciparum Motility and Host Cell Invasion.

Polymerization of the cytosolic protein actin is critical to cell movement and host cell invasion by the malaria parasite, Plasmodium falciparum. Any disruption to actin polymerization dynamics will render the parasite incapable of invading a host cell and thereby unable to cause infection. Here, we explore the potential of using truncated latrunculins as potential chemotherapeutics for the treatment of malaria. Exploration of the binding interactions of the natural actin inhibitor latrunculins with actin revealed how a truncated core of the inhibitor could retain its key interaction features with actin. This truncated core was synthesized and subjected to preliminary structure-activity relationship studies to generate a focused set of analogues. Biochemical analyses of these analogues demonstrate their 6-fold increased activity compared with that of latrunculin B against P. falciparum and a 16-fold improved selectivity ex vivo. These data establish the latrunculin core as a potential focus for future structure-based drug design of chemotherapeutics against malaria.

Microparticle formation by platelets exposed to high gas pressures – An oxidative stress response

This investigation explored the mechanism for microparticles (MPs) production by human and murine platelets exposed to high pressures of inert gases. Results demonstrate that MPs production occurs via an oxidative stress response in a dose-dependent manner and follows the potency series N2>Ar>He. Gases with higher van der Waals volumes or polarizability such as SF6 and N2O, or hydrostatic pressure, do not cause MPs production. Singlet O2 is generated by N2, Ar and He, which is linked to NADPH oxidase (NOX) activity. Progression of oxidative stress involves activation of nitric oxide synthase (NOS) leading to S-nitrosylation of cytosolic actin. Exposure to gases enhances actin filament turnover and associations between short actin filaments, NOS, vasodilator-stimulated phosphoprotein (VASP), focal adhesion kinase (FAK) and Rac1. Inhibition of NOS or NOX by chemical inhibitors or using platelets from mice lacking NOS2 or the gp91phox component of NOX diminish generation of reactive species, enhanced actin polymerization and MP generation by high pressure gases. We conclude that by initiating a sequence of progressive oxidative stress responses high pressure gases cause platelets to generate MPs.

A unique PE_PGRS protein inhibiting host cell cytosolic defenses and sustaining full virulence of Mycobacterium marinum in multiple hosts.

Despite intense research, PE_PGRS proteins still represent an intriguing aspect of mycobacterial pathogenesis. These cell surface proteins influence virulence in several pathogenic species, but their diverse and exact functions remain unclear. Herein, we focussed on a PE_PGRS member from Mycobacterium marinum, MMAR_0242, characterized by an extended and unique C-terminal domain. We demonstrate that an M. marinum mutant carrying a transposon insertion in MMAR_0242 is highly impaired in its ability to replicate in macrophages and amoebae, because of its inability to inhibit lysosomal fusion. As a consequence, this mutant failed to survive intracellularly as evidenced by a reduced number of cytosolic actin tail-forming bacteria and by quantitative electron microscopy, which mainly localized MMAR_0242::Tn within membrane-defined vacuoles. Functional complementation studies indicated that the C-terminus, but not the N-terminal PE_PGRS domain, is required for intracellular growth/survival. In line with these findings, disruption of MMAR_0242 resulted in a highly attenuated virulence phenotype in zebrafish embryos, characterized by restricted bacterial loads and a failure to produce granulomas. Furthermore, expression of MMAR_0242 in Mycobacterium smegmatis, a non-pathogenic species naturally deficient in PE_PGRS production, resulted in increased survival in amoebae with enhanced cytotoxic cell death and increased survival in infected mice with splenomegaly. Overall, these results indicate that MMAR_0242 is required for full virulence of M. marinum and sufficient to confer pathogenic properties to M. smegmatis.

Effects of Plectin Depletion on Keratin Network Dynamics and Organization.

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.

Factors Associated with Nitric Oxide-mediated β2 Integrin Inhibition of Neutrophils

This investigation explored the mechanism for inhibition of β2 integrin adhesion molecules when neutrophils are exposed to nitric oxide ((•)NO). Roles for specific proteins were elucidated using chemical inhibitors, depletion with small inhibitory RNA, and cells from knock-out mice. Optimal inhibition occurs with exposures to a (•)NO flux of ∼ 28 nmol/min for 2 min or more, which sets up an autocatalytic cascade triggered by activating type 2 nitric-oxide synthase (NOS-2) and NADPH oxidase (NOX). Integrin inhibition does not occur with neutrophils exposed to a NOX inhibitor (Nox2ds), a NOS-2 inhibitor (1400 W), or with cells from mice lacking NOS-2 or the gp91(phox) component of NOX. Reactive species cause S-nitrosylation of cytosolic actin that enhances actin polymerization. Protein cross-linking and actin filament formation assays indicate that increased polymerization occurs because of associations involving vasodilator-stimulated phosphoprotein, focal adhesion kinase, and protein-disulfide isomerase in proximity to actin filaments. These effects were inhibited in cells exposed to ultraviolet light which photo-reverses S-nitrosylated cysteine residues and by co-incubations with cytochalasin D. The autocatalytic cycle can be arrested by protein kinase G activated with 8-bromo-cyclic GMP and by a high (•)NO flux (∼ 112 nmol/min) that inactivates NOX.

STED nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living neurons.

In the axons of cultured hippocampal neurons, actin forms various structures, including bundles, patches (involved in the preservation of neuronal polarity), anda recently reported periodic ring-like structure. Nevertheless, the overlaying organization of actin in neurons and in the axon initial segment (AIS) is still unclear, due mainly to a lack of adequate imaging methods. By harnessing live-cell stimulated emission depletion (STED) nanoscopy and the fluorescent probe SiR-Actin, we show that the periodic subcortical actin structure is in fact present in both axons and dendrites. The periodic cytoskeleton organization is also found in the peripheral nervous system, specifically at the nodes of Ranvier. The actin patches in the AIS co-localize with pre-synaptic markers. Cytosolic actin organization strongly depends on the developmental stage and subcellular localization. Altogether, the results of this study reveal unique neuronal cytoskeletal features.

Circular Dorsal Ruffles Increase Directional Persistence of Cell Migration by Actin Diffusion from Ruffles to Lamellipodia

Circular dorsal ruffles (CDRs) are transient actin structures which have been linked to cell motility but the exact mechanism is still unclear. CDRs appear and grow in size after cells are stimulated with growth factors, such as the platelet-derived growth factor, eventually disappearing tens of minutes after stimulation. The role of CDR formation in cell motility is investigated for NIH 3T3 fibroblasts seeded on compliant polyacrylamide substrates. We found that CDR formation increases cell migration directional persistency but did not affect the migration speed. Furthermore, an increase in the localization of lamellipodial protrusion at the cell edge in the vicinity of the CDRs was observed. Relocalization of lamellipodia occured 1 to 6 min after CDR formation at the cell edge closest to the site of CDR formation. The time lag between peak CDR formation and the peak lamellipodial protrusion is then correlated with the spatial distance between the CDR and the lamellipodia; this time scale is consistent with the diffusive time scale of cytosolic globular actin (G actin). Using green to red photoswitchable Dendra2-conjugated actin, we photoconverted CDR actin from green to red and observed the subsequent appearance of red fluorescent actin in the lamellipodia at the cell leading edge. These findings help shed light on the interconversions between mesoscopic actin structures in cell behavior.

Localized regulation of the axon shaft during the emergence of collateral branches.

The ability of the axon to form de novo collateral branches along its length is fundamental to the establishment of complex patterns of connectivity during development and is also a major response of many axonal populations following injury. The emergence of branches is under both positive and negative control by extracellular signals. How the site of branch formation is determined is a fundamental question regarding the formation of branches. In theory, the whole axon shaft has the potential to give rise to a branch, but yet branches form only at specific sites. This feature of the formation of branches is emphasized by studies in which nerve growth factor (NGF), covalently attached to 10 micron beads, was applied locally along embryonic sensory axons (Gallo and Letourneau, 1998). Although contact of the beads with the axon shaft was able to drive the formation of a branch at the sites of contact, even after 3 hours of continuous contact only 46% of sites gave rise to a branch. Similarly, when NGF is bath applied to cultured sensory neurons, and thus the whole surface of the axon is exposed to NGF, axons usually only generate a maximum of 4–6 branches along the distal 100 microns of the axon, a response that is maximal by 30 minutes of treatment. Recent work, from our laboratory and others, has begun to shed light on why axons form branches at specific sites.

Neutrophils Generate Microparticles during Exposure to Inert Gases Due to Cytoskeletal Oxidative Stress

This investigation was to elucidate the mechanism for microparticle (MP) formation triggered by exposures to high pressure inert gases. Human neutrophils generate MPs at a threshold of ∼186 kilopascals with exposures of 30 min or more. Murine cells are similar, but MP production occurs at a slower rate and continues for ∼4 h, whether or not cells remain under pressure. Neutrophils exposed to elevated gas but not hydrostatic pressure produce MPs according to the potency series: argon ≃ nitrogen > helium. Following a similar pattern, gases activate type-2 nitric-oxide synthase (NOS-2) and NADPH oxidase (NOX). MP production does not occur with neutrophils exposed to a NOX inhibitor (Nox2ds) or a NOS-2 inhibitor (1400W) or with cells from mice lacking NOS-2. Reactive species cause S-nitrosylation of cytosolic actin that enhances actin polymerization. Protein cross-linking and immunoprecipitation studies indicate that increased polymerization occurs because of associations involving vasodilator-stimulated phosphoprotein, focal adhesion kinase, the H(+)/K(+) ATPase β (flippase), the hematopoietic cell multidrug resistance protein ABC transporter (floppase), and protein-disulfide isomerase in proximity to short actin filaments. Using chemical inhibitors or reducing cell concentrations of any of these proteins with small inhibitory RNA abrogates NOS-2 activation, reactive species generation, actin polymerization, and MP production. These effects were also inhibited in cells exposed to UV light, which photoreverses S-nitrosylated cysteine residues and by co-incubations with the antioxidant ebselen or cytochalasin D. The autocatalytic cycle of protein activation is initiated by inert gas-mediated singlet O2 production.

Threonine 680 Phosphorylation of FLJ00018/PLEKHG2, a Rho Family-specific Guanine Nucleotide Exchange Factor, by Epidermal Growth Factor Receptor Signaling Regulates Cell Morphology of Neuro-2a Cells

FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. The function of FLJ00018 is regulated by the interaction of heterotrimeric GTP-binding protein Gβγ subunits or cytosolic actin. However, the details underlying the molecular mechanisms of FLJ00018 activation have yet to be elucidated. In the present study we show that FLJ00018 is phosphorylated and activated by β1-adrenergic receptor stimulation-induced EGF receptor (EGFR) transactivation in addition to Gβγ signaling. FLJ00018 is also phosphorylated and activated by direct EGFR stimulation. The phosphorylation of FLJ00018 by EGFR stimulation is mediated by the Ras/mitogen-activated protein kinase (MAPK) pathway. Through deletion and site-directed mutagenesis studies, we have identified Thr-680 as the major site of phosphorylation by EGFR stimulation. FLJ00018 T680A, in which the phosphorylation site is replaced by alanine, showed a limited response of the Neuro-2a cell morphology to EGF stimulation. Our results provide evidence that stimulation of the Ras/MAPK pathway by EGFR results in FLJ00018 phosphorylation at Thr-680, which in turn controls changes in cell shape.

Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.

Visual input controls the functional activity of goldfish Mauthner neuron through the reciprocal synaptic mechanism

Goldfish are known to exhibit motor asymmetry due to functional asymmetry of their Mauthner neurons that induce the turns to the right or left during free swimming. It has been previously found that if the less active neuron is subjected to prolonged aimed visual stimulation via its ventral dendrite, the motor asymmetry of goldfish is inverted, testifying that this neuron becomes functionally dominant, while the size of the ventral dendrite under these conditions is reduced 2-3 times compared to its counterpart in mirror neuron. Earlier it has been also revealed that training optokinetic stimulation induces adaptation, a substantial resistance of both fish motor asymmetry and morphofunctional state of Mauthner neurons against prolonged optokinetic stimulation. The aim of this work was to study the cellular mechanisms of the effect of an unusual visual afferent input on goldfish motor asymmetry and Mauthner neuron function in norm and under adaptation. It was shown that serotonin applied onto Mauthner neurons greatly reduces their activity whereas its antagonist ondansetron increases it. Against the background of visual stimulation, serotonin strengthens functional asymmetry between neurons whereas ondansetron smoothes it. Taken together these data suggest the involvement of serotonergic excitatory synaptic transmission in the regulation of Mauthner neurons by vision. Ultrastructural study of the ventral dendrites after prolonged optokinetic stimulation has revealed depletions of numeral axo-axonal synapses with specific morphology, identified by means of immunogold label as serotonergic ones. These latter in turn are situated mainly on shaft boutons, which according to specific ultrastructural features are assigned to axo-dendritic inhibitory synapses. Thus, the excitatory serotonergic synapses seem to affect Mauthner neuron indirectly through inhibitory synapses. Further, it was morphometrically established that adaptation is accompanied by the significant decrease of active zones dimensions in both serotonergic and inhibitory synapses. Finally, it was determined in model experiments that the interaction of globular actin with glycine, a main inhibitory neurotransmitter supposedly directly and chronically affecting the ventral dendrite, results in actin filaments formation. It is assumed that glycine-induced cytosolic actin polymerization is a cause of reduction in the ventral dendrite size under stimulation. Thus, it was established that a rather small group of synapses situated on an individual dendrite of the neuron determines the execution of the important form of animal behavior.

Identification of a Rho family specific guanine nucleotide exchange factor, FLJ00018, as a novel actin-binding protein

FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the Rho family small GTPases. FLJ00018 is directly activated by heterotrimeric G protein Gβγ subunits. Using two-hybrid screening, we have identified non-muscle cytosolic actin as a binding partner of FLJ00018. We found that there were two actin-binding regions in FLJ00018 at the N-terminal region (150–283 amino acids) and at the C-terminal region (465–1386 amino acids). The overexpression of non-muscle cytosolic actin attenuated the FLJ00018-induced serum response element-dependent gene transcription. These results suggest that non-muscle cytosolic actin may be a negative regulator of FLJ00018 through its interaction with the Dbl homology domain.

Cytochemical dopamine visualization as a method for estimation of globular actin content in cytosol of living cells

The possibility to reveal globular (G-) actin in cell cytosole by means of microscopy has been studied. The applicability of this method was in particular evaluated for diagnostics of malignant cells, whose main pathocytological feature is an anomalously high content of G-actin in cytosol. The cells of a common origin but with different states of cytosolic actin were analyzed by means of cytochemical reaction for biogenic amines using Falck-Hillarp method after 40-h incubation of the cells in dopamine-containing cultivation medium. Mouse embryo cell line BALB/3T3, clone A31 with differentiated actin cytoskeleton were used as a control cell line. The same cells infected with pathogenic virus SV-40 (cell line 3T3B-SV40) exhibited a malignant phenotype; their cytosol mainly consisted of G-actin. Manifold increase in fluorescence intensity of cytosol and karyoplasms, the loci with the highest G-actin concentration, was revealed in malignant cells in comparison with their healthy prototype. Thus, it was shown that G-actin of malignant cells is a diagnostic target for dopamine, which, as it was earlier shown, penetrates into the cytosol, polymerizes G-actin, incorporating into the filaments as integral component in the 100:1 ratio, and thus fluorescently labels G-actin due to conversion into isoquinoline by reaction with formaldehyde. Besides, dopamine exhibited a strong cytotoxicity that considerably reduced the viability of malignant cells. The data suggest that the content of Gactin in cytosol of living cells can be quantitatively estimated by fluorescence intensity of cytosol following incubation of the cells in dopamine-containing medium.

MARCKS actin-binding capacity mediates actin filament assembly during mitosis in human hepatic stellate cells

Cross-linking between the actin cytoskeleton and plasma membrane actin-binding proteins is a key interaction responsible for the mechanical properties of the mitotic cell. Little is known about the identity, the localization, and the function of actin filament-binding proteins during mitosis in human hepatic stellate cells (hHSC). The aim of the present study was to identify and analyze the cross talk between actin and myristoylated alanine-rich kinase C substrate (MARCKS), an important PKC substrate and actin filament-binding protein, during mitosis in primary hHSC. Confocal analysis and chromosomal fraction analysis of mitotic hHSC demonstrated that phosphorylated (P)-MARCKS displays distinct phase-dependent localizations, accumulates at the perichromosomal layer, and is a centrosomal protein belonging to the chromosomal cytosolic fraction. Aurora B kinase (AUBK), an important mitotic regulator, β-actin, and P-MARCKS concentrate at the cytokinetic midbody during cleavage furrow formation. This localization is critical since MARCKS-depletion in hHSC is characterized by a significant loss in cytosolic actin filaments and cortical β-actin that induces cell cycle inhibition and dislocation of AUBK. A depletion of AUBK in hHSC affects cell cycle, resulting in multinucleation. Quantitative live cell imaging demonstrates that the actin filament-binding capacity of MARCKS is key to regulate mitosis since the cell cycle inhibitory effect in MARCKS-depleted cells caused abnormal cell morphology and an aberrant cytokinesis, resulting in a significant increase in cell cycle time. These findings implicate that MARCKS, an important PKC substrate, is essential for proper cytokinesis and that MARCKS and its partner actin are key mitotic regulators during cell cycle in hHSC.

Understanding the Nature of Cortical Acto-Myosin Based Active Patterning Machinery and its Implications on the Dynamics and the Organization of Cell Surface Molecules

We have found that the spatial distribution and the remodeling dynamics of the nanoclusters of outer leaflet glycosylphosphatidylinositol anchored proteins (GPI-APs), is regulated by the activity of the underlying cortical acto-myosin (CA).We have developed a theoretical framework to explain the mechanism of this nanoclustering, based on active hydrodynamics of the CA and its coupling to the membrane. This framework provides insights into the behavior of molecules that interact with the dynamic CA, reflected in their cell surface organization, dynamics and distribution. Here we discuss results from experiments designed to test key assumptions of this theory and major predictions. Using Fluorescence Correlation Spectroscopy (FCS) and single molecule imaging (SM) we provide evidence for a key assumption, namely, a rapidly turning over pool of short and dynamic F-actin based structures at or near the membrane cortex. To test the predictions of CA acting as membrane organizing machinery, we study the cell-surface organization of a model transmembrane (TM) connected to cytosolic actin binding domains (ABD), referred to as TM-ABD. Our results obtained from steady state and time-resolved homo-FRET measurements on TM-ABD indicate that CA-association can drive actin-dependent nanoscale organization of these proteins. The theoretical framework also predicts that cell-surface molecules which interact with this dynamic CA must exhibit anomalous density fluctuations which we are able to confirm in a fluorescence based assay. In addition, using FCS, we explore the temperature dependent diffusion characteristics of these molecules. These studies provide a new paradigm for understanding molecular organization and its spatiotemporal regulation on the plasma membrane.

14‐3‐3ε marks the amyloid‐stimulated microglia long‐term activation

Microglia-mediated inflammation in the central nervous system is a hallmark of the pathogenesis of several neurodegenerative diseases including Alzheimer's disease. Microglial cells activation follows the deposition of amyloid β fibrils and it is generally considered a triggering factor in the early steps of the onset of Alzheimer's disease. Although the initial engagement of microglia seems to play a neuroprotective role, many lines of evidence indicate that a persistent activation with the production of proinflammatory molecules contributes to dismantle neuronal activity and to induce neuronal loss occurring in neurodegenerative diseases. To date, limited proteomic data are available on activated microglial cells in response to extracellular amyloidogenic peptides. In this study, murine microglial cells have been employed to investigate the effects of amyloid β peptides in triggering microglial activation. The response was monitored at the proteome level through a two-dimensional gel electrophoresis-based approach. Results show only a limited number of differentially expressed proteins, among these a more acidic species of the cytosolic actin, and the 14-3-3ε protein, found significantly upregulated in Aβ-activated cells. 14-3-3ε belongs to a regulatory protein family involved in important cellular processes, including those leading to neurodegenerative diseases, and thus its increased expression suggests a role of this protein in tuning microglia activation.

Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α) docking protein

SummaryThe promiscuous activity of protein phosphatase one (PP1) is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75), encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic ‘RVxF’ motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC)-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway.

Membrane assembly during erythropoiesis

In this review, we summarize our current knowledge on the expression patterns of various proteins during erythropoiesis and discuss how this information can lead to development of detailed understanding of membrane biogenesis during erythropoiesis. Changes in expression pattern of more than 30 red cell membrane proteins during terminal erythroid differentiation and during reticulocyte maturation was discerned. During maturation of murine reticulocytes, tubulin and cytosolic actin were lost, while the membrane-associated levels of myosin, tropomyosin, ICAM-4, GLUT4, Na/K-ATPase, NHE1, GPA, CD47, Duffy, and Kell were reduced. During murine terminal erythroid differentiation, expression levels of all major transmembrane and skeletal proteins of the mature red blood cell increased, while those of various adhesion molecules decreased. A 30-fold decrease in expression of the adhesive protein CD44 was noted during differentiation of murine proerythroblast to orthochromatic erythroblast. These changing protein expression patterns were used to devise an effective strategy to distinguish erythroblasts at distinct stages of development. All major red cell membrane proteins undergo dynamic changes during terminal erythroid differentiation. Use of CD44 in conjunction with TER119 and cell size enabled the development of a method for distinguishing distinct stages of erythroblasts during murine erythropoiesis. These findings should enable development of detailed understanding of membrane biogenesis during erythropoiesis and obtain mechanistic insights into disordered erythropoiesis in various red cell disorders.

Intertissue Mechanical Stress Affects Frizzled-Mediated Planar Cell Polarity in the Drosophila Notum Epidermis

Frizzled/planar cell polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the anteroposterior axis of the Drosophila thorax (notum). A subset of the trichome-producing notum cells differentiate as "tendon cells," serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin, we show that maintenance of anteroposterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells, and its loss of function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat/dachsous systems. The chas phenotype arises during normal shortening of the IFMs and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechanoresponse of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by one or more interactions with other tissues during development and organogenesis influences the maintenance of its shape and PCP features.