BBA - Molecular Cell Research (v.1803, #2)

The regulation of the actin cytoskeleton is a key process for the stability and motility of eukaryotic cells. Besides the Arp2/3 complex and its nucleation promoting factors, WH2 domain-containing proteins and a diverse family of formin proteins have recently been recognized as actin nucleators and potent polymerization factors of actin filaments. Formins are defined by the presence of a catalytic formin homology 2 (FH2) domain, yet, the modular domain architecture appears significantly different for the eight formin families identified in humans. A diverse picture of protein localization, interaction partners and cell specific regulation emerged, suggesting various functions of formins in the building and maintenance of actin filaments. This review focuses on the domain architecture of human formins, the regulation mechanisms of their activation and the diversity in formin cellular functions.
Keywords: Actin; Cytoskeleton; Diaphanous; Formin; Rho GTPase; WHIF1;

Formins and microtubules by F. Bartolini; G.G. Gundersen (164-173).
Formins have recently been recognized as prominent regulators of the microtubule (MT) cytoskeleton where they modulate the dynamics of selected MTs in interphase and mitosis. The association of formins with the MT cytoskeleton and their action on MT dynamics are relatively unexplored areas, yet growing evidence supports a direct role in their regulation of MT stability independent of their activity on actin. Formins regulate MT stability alone or in combination with accessory MT binding proteins that have previously been implicated in the stabilization of MTs downstream of polarity cues. As actin and MT arrays are typically remodeled downstream of signaling pathways that orchestrate cell shape and division, formins are emerging as excellent candidates for coordinating the responses of the cytoskeletal in diverse regulated and homeostatic processes.
Keywords: Formin; Microtubule; Microtubule stability; Centrosome;

Formins represent a major branch of actin nucleators along with the Arp2/3 complex, Spire and Cordon-bleu. Formin-mediated actin nucleation requires the formin homology 2 domain and, although the nucleation per se does not require additional factors, formin-binding proteins have been shown to be essential for the regulation of formin-dependent actin assembly in vivo. This regulation could be accomplished by formin-binding proteins being directly involved in formin-driven actin nucleation, by formin-binding proteins influencing the activated state of the formins, by linking formin-driven actin polymerization to Arp2/3 driven actin polymerization, or by influencing the subcellular localization of the formins. This review article will focus on mammalian formin-binding proteins and their roles during vital cellular processes, such as cell migration, cell division and intracellular trafficking.
Keywords: Formin; DRF; Profilin; Actin; FH2; PCH; F-BAR; Src; IQGAP;

Formins in cell signaling by Kevin G. Young; John W. Copeland (183-190).
The founding formin homology protein family members were implicated early on as being involved in regulating cytoskeletal remodeling pathways, as formin protein mutations in Drosophila and yeast lead to obvious actin cytoskeleton defects. The discovery that these proteins associated directly with small Rho family GTPases confirmed these results and greatly enhanced our understanding of their function. The mammalian diaphanous-related formins (DRFs) were subsequently recognized as being involved in activation of serum response factor (SRF), tying formins to transcriptional regulation. In the past few years, much progress has been made in demonstrating how DRFs act as both downstream effectors and upstream modulators of Rho GTPase signaling. These functions are important for regulation of both actin and microtubule cytoskeletal structures, and affect cellular processes such as the establishment of polarity, vesicle movement, and focal adhesion remodeling. The connection of DRFs to the SH3 domain-containing protein, Src, has also been described as being important to several basic cellular functions. While still unresolved, extensive work has been carried out on how DRFs mediate SRF activation, and the importance of this to the regulation of cytoskeletal structure. This review will focus on the role of formins in cytoplasmic signal transduction pathways and the downstream effects on the regulation of gene expression.
Keywords: Rho; Rac; Serum response factor; Src; SH3; Formins; Actin; RhoGEF;

The role of formins in filopodia formation by Harry Mellor (191-200).
Filopodia are highly dynamic cell-surface protrusions used by cells to sense their external environment. At the core of the filopodium is a bundle of actin filaments. These give form to the filopodia and also drive the cycle of elongation and retraction. Recent studies have shown that two very different actin nucleating proteins control the formation of filopodial actin filaments — Arp2/3 and Formins. Although the actin filaments produced by these two nucleators have very different structures and properties, recent work has begun to piece together evidence for co-operation between Arp2/3 and formins in filopodia formation, leading to a deeper understanding of these sensory organelles.
Keywords: Actin; Cytoskeleton; Formin; Filopodia; Cell migration;

Plant formins: Diverse isoforms and unique molecular mechanism by Laurent Blanchoin; Christopher J. Staiger (201-206).
The completed genome from the model plant Arabidopsis thaliana reveals the presence of a diverse multigene family of formin-like sequences, comprising more than 20 isoforms. This review highlights recent findings from biochemical, cell biological and reverse-genetic analyses of this family of actin nucleation factors. Important advances in understanding cellular function suggest major roles for plant formins during cytokinesis and cell expansion. Biochemical studies on a subset of plant formins emphasize the need to examine molecular mechanisms outside of mammalian and yeast systems. Notably, a combination of solution-based assays for actin dynamics and timelapse, single-filament imaging with TIRFM provide evidence for the first non-processive formin (AtFH1) in eukaryotes. Despite these advances it remains difficult to generate a consensus view of plant formin activities and cellular functions. One limitation to summarizing formin properties relates to the enormous variability in domain organization among the plant formins. Generating homology-based predictions that depend on conserved domains outside of the FH1 and FH2 will be virtually impossible for plant formins. A second major drawback is the lack of facile techniques for examining dynamics of individual actin filaments within live plant cells. This constraint makes it extremely difficult to bridge the gap between biochemical characterization of particular formin and its specific cellular function. There is promise, however, that recent technical advances in engineering appropriate fluorescent markers and new fluoresence imaging techniques will soon allow the direct visualization of cortical actin filament dynamics. The emergence of other model systems for studying actin cytoskeleton in vivo, such as the moss Physcomitrella patens, may also enhance our knowledge of plant formins.
Keywords: Formin; Actin; Arabidopsis thaliana;

Formins in development: Orchestrating body plan origami by Raymond Liu; Elena V. Linardopoulou; Gregory E. Osborn; Susan M. Parkhurst (207-225).
Formins, proteins defined by the presence of an FH2 domain and their ability to nucleate linear F-actin de novo, play a key role in the regulation of the cytoskeleton. Initially thought to primarily regulate actin, recent studies have highlighted a role for formins in the regulation of microtubule dynamics, and most recently have uncovered the ability of some formins to coordinate the organization of both the microtubule and actin cytoskeletons. While biochemical analyses of this family of proteins have yielded many insights into how formins regulate diverse cytoskeletal reorganizations, we are only beginning to appreciate how and when these functional properties are relevant to biological processes in a developmental or organismal context. Developmental genetic studies in fungi, Dictyostelium, vertebrates, plants and other model organisms have revealed conserved roles for formins in cell polarity, actin cable assembly and cytokinesis. However, roles have also been discovered for formins that are specific to particular organisms. Thus, formins perform both global and specific functions, with some of these roles concurring with previous biochemical data and others exposing new properties of formins. While not all family members have been examined across all organisms, the analyses to date highlight the significance of the flexibility within the formin family to regulate a broad spectrum of diverse cytoskeletal processes during development.
Keywords: Formin; Development; Cytoskeleton; Cytokinesis; Polarity; Actin; Microtubule; Actin nucleation; Diaphanous; Daam; Cappuccino; Spire;

The role of formins in human disease by Aaron D. DeWard; Kathryn M. Eisenmann; Stephen F. Matheson; Arthur S. Alberts (226-233).
Formins are a conserved family of proteins that play key roles in cytoskeletal remodeling. They nucleate and processively elongate non-branched actin filaments and also modulate microtubule dynamics. Despite their significant contributions to cell biology and development, few studies have directly implicated formins in disease pathogenesis. This review highlights the roles of formins in cell division, migration, immunity, and microvesicle formation in the context of human disease. In addition, we discuss the importance of controlling formin activity and protein expression to maintain cell homeostasis.
Keywords: Formin; Actin; Microtubule; Rho GTPase; Non-syndromic deafness; Cancer; Myelodysplasia; Tumor suppression; Premature ovarian failure; Limb deformity; Metastasis; Immune response; Oncosome; Microvesicle;

ADAM17-mediated shedding of the IL6R induces cleavage of the membrane stub by γ-secretase by Athena Chalaris; Jessica Gewiese; Krzysztof Paliga; Lina Fleig; Alex Schneede; Karsten Krieger; Stefan Rose-John; Jürgen Scheller (234-245).
Interleukin-6 (IL6) signals are mediated by classic and trans-signaling. In classic signaling, IL6 first binds to the membrane bound Interleukin-6 Receptor (IL6R) whereas in trans-signaling, IL6 acts via a soluble form of the IL6R. Trans-signaling via the soluble IL6R (sIL6R) was linked to chronic inflammation and cancer. The release of the IL6R is mediated by the disintegrin and metalloproteinases ADAM10 and ADAM17. To analyze the fate of the C-terminal cleavage fragment after ectodomain shedding we fused the IL6R C-terminally to two Z-domains of Protein-A (2Z-tag) or to GFP. A specific C-terminal fragment of the IL6R protein could be detected after ADAM17-induced shedding. Using γ-secretase inhibitors and gene-deficient cells, we demonstrate that after ADAM17 mediated cleavage, the IL6R C-terminal fragment was cleaved by the γ-secretase at the plasma membrane. We were, however, not able to detect an IL6R intracellular domain. After γ-secretase cleavage IL6R cell surface expression was lost and γ-secretase cleavage product(s) of the IL6R were endocytosed. No GFP-fluorescence of a γ-secretase-cleaved IL6R-GFP fusion protein was observed in the nucleus. We therefore hypothesize that a potential IL6R intracellular domain fragment is not involved in nuclear signaling but rapidly degraded.
Keywords: Regulated intramembraneous cleavage; ADAM17; Shedding; IL6R; γ-secretase;

Influence of spontaneous calcium events on cell-cycle progression in embryonal carcinoma and adult stem cells by R.R. Resende; A. Adhikari; J.L da Costa; E. Lorençon; M.S. Ladeira; S. Guatimosim; A.H. Kihara; L.O. Ladeira (246-260).
Spontaneous Ca2+ events have been observed in diverse stem cell lines, including carcinoma and mesenchymal stem cells. Interestingly, during cell cycle progression, cells exhibit Ca2+ transients during the G1 to S transition, suggesting that these oscillations may play a role in cell cycle progression. We aimed to study the influence of promoting and blocking calcium oscillations in cell proliferation and cell cycle progression, both in neural progenitor and undifferentiated cells. We also identified which calcium stores are required for maintaining these oscillations. Both in neural progenitor and undifferentiated cells calcium oscillations were restricted to the G1/S transition, suggesting a role for these events in progression of the cell cycle. Maintenance of the oscillations required calcium influx only through inositol 1,4,5-triphosphate receptors (IP3Rs) and L-type channels in undifferentiated cells, while neural progenitor cells also utilized ryanodine-sensitive stores. Interestingly, promoting calcium oscillations through IP3R agonists increased both proliferation and levels of cell cycle regulators such as cyclins A and E. Conversely, blocking calcium events with IP3R antagonists had the opposite effect in both undifferentiated and neural progenitor cells. This suggests that calcium events created by IP3Rs may be involved in cell cycle progression and proliferation, possibly due to regulation of cyclin levels, both in undifferentiated cells and in neural progenitor cells.
Keywords: Mesenchymal stem cell; P19 embryonic carcinoma stem cell; Neuronal differentiation; Calcium signaling; Cell cycle; Spontaneous calcium oscillation;

CD44 and hyaluronan promote invasive growth of B35 neuroblastoma cells into the brain by Annette Pusch; Annika Boeckenhoff; Tamara Glaser; Tim Kaminski; Gregor Kirfel; Michael Hans; Barbara Steinfarz; Dieter Swandulla; Ulrich Kubitscheck; Volkmar Gieselmann; Oliver Brüstle; Joachim Kappler (261-274).
Hyaluronan and its receptor CD44 are known to contribute to the invasive growth of different tumors of the central nervous system. It is not known, however, if CD44 is sufficient to activate invasive growth into the brain tissue. This study examines how CD44 regulates the motility and invasive growth of B35 neuroblastoma cells into a hyaluronan-rich environment. A comprehensive experimental approach was used encompassing biochemical techniques, single molecule microscopy, correlative confocal and scanning electron microscopy, morphometry of cellular extensions, live-cell imaging and tracking, transplantation onto organotypic brain slices, two-photon imaging and invasion assays. We found that CD44-GFP fusion protein was localized in filopodia and in focal bleb-like protrusions where it provided binding sites for hyaluronan. Transient expression of CD44-GFP was sufficient to increase the length of filopodia, to enhance cell migration and to promote invasive growth into hyaluronan-rich brain tissue. Thus, CD44 controls molecular devices localized in filopodia and bleb-like specializations of the cell surface that enhance cell migration and invasive growth.
Keywords: CD44; Hyaluronan; Neural precursor cell; Cell migration; Invasion; Filopodia-like extensions; Hippocampus; Biophysics; Single molecule microscopy;

The P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X4R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X4R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X4R expressing HEK-293 cells. To define which region of the P2X4R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X4R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X4R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF) P2X4-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca2+ signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X4R, markedly enhances ATP-stimulated P2X4R currents and hence cytosolic Ca2+ signals. This may occur at least in part, by altering the trafficking of a population of P2X4R present at the plasma membrane.
Keywords: Protein kinase A (PKA); Calcium signaling; P2X; Purinergic receptor; P2X4R; Endocytosis; Intracellular calcium;

αB-crystallin, a small heat shock protein, plays an important role in muscle homeostasis. It gets up-regulated during muscle differentiation and mice lacking αB-crystallin die prematurely with extensive muscle wastage. We have examined the role of αB-crystallin in muscle development using C2C12 myoblasts as a model system. Over-expression of αB-crystallin delays the muscle differentiation program significantly. C2C12 myoblasts over-expressing αB-crystallin (CRYAB-C2C12) display defect in cell-cycle exit upon induction of differentiation. During differentiation, CRYAB-C2C12 cells exhibit sustained level of cyclin D1 and delay in p21 and myogenin expression as compared to C2C12 cells. We find less accumulation of MyoD in CRYAB-C2C12 cells than in C2C12 cells. In vivo protein stability studies reveal faster ubiquitin–proteasome-mediated MyoD degradation in CRYAB-C2C12 cells (t 1/2  = 1.42 h) than in C2C12 cells (t 1/2  = 2.37 h). Immuno-precipitation experiments showed that MyoD gets ubiquitinated at earlier time points in CRYAB-C2C12 cells than in C2C12 cells. Our data reveal alterations in the synthesis and degradation of MyoD in CRYAB-C2C12 cells. The level of αB-crystallin as well as its Ser-59 phosphorylated form increases with increasing time of differentiation. Our studies show, inter alia, that αB-crystallin modulates myogenesis by altering MyoD level and provide an interesting insight in its role in myogenesis.
Keywords: sHSPs; Myogenesis; Ubiquitin; Half-life; Cell-cycle; MyoD;

Ezrin/radixin/moesin (ERM) proteins are membrane-cytoskeleton linkers that also have roles in signal transduction. Here we show that G protein-coupled receptor kinase 2 (GRK2) regulates membrane protrusion and cell migration during wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers at least partly through activating phosphorylation of radixin on a conserved, regulatory C-terminal Thr residue. GRK2 phosphorylated radixin exclusively on Thr 564 in vitro. Expression of a phosphomimetic (Thr-564-to-Asp) mutant of radixin resulted in increased Rac1 activity, membrane protrusion and cell motility in MDCK cells, suggesting that radixin functions “upstream” of Rac1, presumably as a scaffolding protein. Phosphorylation of ERM proteins was highest during the most active phase of epithelial cell sheet migration over the course of wound closure. In view of these results, we explored the mode of action of quinocarmycin/quinocarcin analog DX-52-1, an inhibitor of cell migration and radixin function with considerable selectivity for radixin over the other ERM proteins, finding that its mechanism of inhibition of radixin does not appear to involve binding and antagonism at the site of regulatory phosphorylation.
Keywords: GRK2; ERM protein; Radixin; Rac1; Membrane protrusion; Cell migration; Quinocarmycin/quinocarcin analog DX-52-1;

Bim is the key mediator of glucocorticoid-induced apoptosis and of its potentiation by rapamycin in human myeloma cells by Nuria López-Royuela; Patricia Balsas; Patricia Galán-Malo; Alberto Anel; Isabel Marzo; Javier Naval (311-322).
Glucocorticoids are widely used in anti-myeloma therapy and their action is potentiated by rapamycin, a mTOR inhibitor. However, the molecular mechanisms underlying these effects remain poorly characterized. We show here that dexamethasone (Dex)-induced apoptosis in MM.1S and OPM-2 cells is characterized by Bax and Bak conformational changes, ΔΨm loss, cytochrome c release and caspase-3 activation. Rapamycin, which had minimal cytotoxic effect by itself, strongly potentiated Dex-induced apoptosis. Apoptotic gene expression profiling showed an increase in mRNA levels of Bim in MM.1S cells after Dex treatment and further increases in both cell lines when co-treated with rapamycin. Western blot analysis revealed a moderate increase in Bim protein levels in both MM.1S and OPM-2 cells. Immunoprecipitation experiments revealed that most Bim was complexed to Mcl-1 in untreated cells. Upon treatment with Dex, and specially Dex plus rapamycin, Bim–Mcl-1 complex was disrupted and Bim was found associated to a CHAPS-insoluble fraction. Overexpression of Mcl-1 stabilized Bim–Mcl-1 complexes upon treatment with Dex or Dex + rapamycin and fully prevented apoptosis. Gene silencing of Bim inhibited for the most part Dex-induced apoptosis and, to a large extent, apoptosis induced by Dex plus rapamycin. These results, taken together, indicate that Bim protein is the key mediator of apoptosis induced by Dex and also responsible for the potentiating effect of rapamycin, providing molecular criteria for the use of glucocorticoids combined with mTOR inhibitors in myeloma therapy.
Keywords: Bim; Mcl-1; Glucocorticoids; Myeloma; mTOR; Apoptosis;

A novel role of IKKα in the mediation of UVB-induced G0/G1 cell cycle arrest response by suppressing Cyclin D1 expression by Lun Song; Wen Dong; Ming Gao; Jingxia Li; Meiru Hu; Ning Guo; Chuanshu Huang (323-332).
Exposure to ultraviolet B (UVB) irradiation (290–320 nm wavelength) from sunlight induces a variety of medical problems, including sunburn, immunosuppression and skin cancers. However, the molecular mechanisms related to UVB-induced cell damage and/or mutagenic effects have not been fully defined. Here, we demonstrate that one of the catalytic subunits of the IκB kinase complex (IKK), IKKα, plays a critical role in mediation of the UVB-induced G0/G1 cell cycle arrest response by suppressing Cyclin D1 expression. Notably, IKKa-dependent Cyclin D1 regulation is unrelated to IKKβ/NF-κB activity. We further show that IKKα-dependent downregulation of Cyclin D1 expression in the UVB response results from the reduction of ERK1/2-dependent Cyclin D1 transcription coupled with an increase of p38 kinase-dependent Cyclin D1 proteolysis. Thus, our results have identified the novel role of IKKα in regulating cell cycle progression during the cellular UVB response. Targeting IKKα might be promising for the prevention of UVB-induced cell damage and tumorigenic effects.
Keywords: UVB; IKKα; Cyclin D1; Cell cycle arrest;

Glucose regulated protein 94 is required for muscle differentiation through its control of the autocrine production of insulin-like growth factors by Olga Ostrovsky; Davide Eletto; Catherine Makarewich; Elisabeth R. Barton; Yair Argon (333-341).
The endoplasmic reticulum chaperone GRP94 is essential for early embryonic development and in particular affects differentiation of muscle lineages. To determine why an ubiquitously expressed protein has such a specific effect, we investigated the function of GRP94 in the differentiation of established myogenic cell lines in culture. Using both genetic suppression of expression, via RNA interference, and inhibition of function, via specific chemical inhibitors, we show that GRP94 expression and activity are needed for the in vitro fusion of myoblasts precursors into myotubes and the expression of contractile proteins that mark terminal differentiation. The inhibition can be complemented by addition of insulin-like growth factors to the cultures. GRP94 is not needed for the initial steps of myogenesis, only for the steps downstream of MyoD up-regulation, coinciding with the known need for synergistic input from growth factor signaling. Indeed, GRP94 is needed for the production of insulin-like growth factors I and II (IGF-I and IGF-II) by the differentiating cells. Moreover, the depletion of the chaperone does not increase the rate of apoptosis that always accompanies myogenic differentiation. Thus, the major effect of GRP94 on muscle differentiation is mediated by its regulation of IGF production.
Keywords: GRP94; Chaperone; Muscle differentiation; IGF; Endoplasmic reticulum; Secretion;