Identification of a novel type of focal adhesion remodelling via FAK/FRNK replacement, and its contribution to cancer progression.

Numerous studies have investigated the various cellular responses against genotoxic stress, including those mediated by focal adhesions. We here identified a novel type of focal adhesion remodelling that occurs under genotoxic stress conditions, which involves the replacement of active focal adhesion kinase (FAK) with FAK-related non-kinase (FRNK). FRNK stabilized focal adhesions, leading to strong cell-matrix adhesion, and FRNK-depleted cells were easily detached from extracellular matrix upon genotoxic stress. This remodelling occurred in a wide variety of cells. In vivo, the stomachs of Frnk-knockout mice were severely damaged by genotoxic stress, highlighting the protective role of FRNK against genotoxic stress. FRNK was also found to play a vital role in cancer progression, because FRNK depletion significantly inhibited cancer dissemination and progression in a mouse cancer model. Furthermore, in human cancers, FRNK was predominantly expressed in metastatic tissues and not in primary tissues. We hence conclude that this novel type of focal adhesion remodelling reinforces cell adhesion and acts against genotoxic stress, which results in the protection of normal tissues, but in turn facilitates cancer progression.

Association Between Atg5-independent Alternative Autophagy and Neurodegenerative Diseases.

Autophagy is a cellular process that degrades intracellular components, including misfolded proteins and damaged organelles. Many neurodegenerative diseases are considered to progress via the accumulation of misfolded proteins and damaged organelles; therefore, autophagy functions in regulating disease severity. There are at least two types of autophagy (canonical autophagy and alternative autophagy), and canonical autophagy has been applied to therapeutic strategies against various types of neurodegenerative diseases. In contrast, the role of alternative autophagy has not yet been clarified, but it is speculated to be involved in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease.

Dynamin-Like Protein B of Dictyostelium Contributes to Cytokinesis Cooperatively with Other Dynamins.

Dynamin is a large GTPase responsible for diverse cellular processes, such as endocytosis, division of organelles, and cytokinesis. The social amoebozoan, Dictyostelium discoideum, has five dynamin-like proteins: dymA, dymB, dlpA, dlpB, and dlpC. DymA, dlpA, or dlpB-deficient cells exhibited defects in cytokinesis. DlpA and dlpB were found to colocalize at cleavage furrows from the early phase, and dymA localized at the intercellular bridge connecting the two daughter cells, indicating that these dynamins contribute to cytokinesis at distinct dividing stages. Total internal reflection fluorescence microscopy revealed that dlpA and dlpB colocalized at individual dots at the furrow cortex. However, dlpA and dlpB did not colocalize with clathrin, suggesting that they are not involved in clathrin-mediated endocytosis. The fact that dlpA did not localize at the furrow in dlpB null cells and vice versa, as well as other several lines of evidence, suggests that hetero-oligomerization of dlpA and dlpB is required for them to bind to the furrow. The hetero-oligomers directly or indirectly associate with actin filaments, stabilizing them in the contractile rings. Interestingly, dlpA, but not dlpB, accumulated at the phagocytic cups independently of dlpB. Our results suggest that the hetero-oligomers of dlpA and dlpB contribute to cytokinesis cooperatively with dymA.

Actin-binding domains mediate the distinct distribution of two Dictyostelium Talins through different affinities to specific subsets of actin filaments during directed cell migration.

Although the distinct distribution of certain molecules along the anterior or posterior edge is essential for directed cell migration, the mechanisms to maintain asymmetric protein localization have not yet been fully elucidated. Here, we studied a mechanism for the distinct localizations of two Dictyostelium talin homologues, talin A and talin B, both of which play important roles in cell migration and adhesion. Using GFP fusion, we found that talin B, as well as its C-terminal actin-binding region, which consists of an I/LWEQ domain and a villin headpiece domain, was restricted to the leading edge of migrating cells. This is in sharp contrast to talin A and its C-terminal actin-binding domain, which co-localized with myosin II along the cell posterior cortex, as reported previously. Intriguingly, even in myosin II-null cells, talin A and its actin-binding domain displayed a specific distribution, co-localizing with stretched actin filaments. In contrast, talin B was excluded from regions rich in stretched actin filaments, although a certain amount of its actin-binding region alone was present in those areas. When cells were sucked by a micro-pipette, talin B was not detected in the retracting aspirated lobe where acto-myosin, talin A, and the actin-binding regions of talin A and talin B accumulated. Based on these results, we suggest that talin A predominantly interacts with actin filaments stretched by myosin II through its C-terminal actin-binding region, while the actin-binding region of talin B does not make such distinctions. Furthermore, talin B appears to have an additional, unidentified mechanism that excludes it from the region rich in stretched actin filaments. We propose that these actin-binding properties play important roles in the anterior and posterior enrichment of talin B and talin A, respectively, during directed cell migration.

Monitoring of Atg5-Independent Mitophagy.

Mitophagy is a mitochondrial quality control mechanism where damaged and surplus mitochondria are removed by autophagy. There are at least two different mammalian autophagy pathways: the Atg5-dependent conventional pathway and an Atg5-independent alternative pathway; the latter is involved in the erythrocyte mitophagy. In this chapter we describe the various experimental approaches to assess Atg5-indepedndent mitophagy in mammalian cells.

Dram1 regulates DNA damage-induced alternative autophagy.

Autophagy is an evolutionarily conserved process that degrades subcellular constituents. Mammalian cells undergo two types of autophagy; Atg5-dependent conventional autophagy and Atg5-independent alternative autophagy, and the molecules required for the latter type of autophagy are largely unknown. In this study, we analyzed the molecular mechanisms of genotoxic stress-induced alternative autophagy, and identified the essential role of p53 and damage-regulated autophagy modulator (Dram1). Dram1 was sufficient to induce alternative autophagy. In the mechanism of alternative autophagy, Dram1 functions in the closure of isolation membranes downstream of p53. These findings indicate that Dram1 plays a pivotal role in genotoxic stress-induced alternative autophagy.

Role of Atg5-dependent cell death in the embryonic development of Bax/Bak double-knockout mice.

Programmed cell death, which is required for the development and homeostasis of metazoans, includes mechanisms such as apoptosis, autophagic cell death, and necrotic (or type III) death. Members of the Bcl2 family regulate apoptosis, among which Bax and Bak act as a mitochondrial gateway. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we previously demonstrated that these cells die through an autophagy-dependent mechanism in response to various types of cellular stressors. To determine the physiological role of autophagy-dependent cell death, we generated Atg5/Bax/Bak triple-knockout (TKO) mice, in which autophagy is greatly suppressed compared with DKO mice. Embryonic fibroblasts and thymocytes from TKO mice underwent autophagy much less frequently, and their viability was much higher than DKO cells in the presence of certain cellular stressors, providing genetic evidence that DKO cells undergo Atg5-dependent death. Compared with wild-type embryos, the loss of interdigital webs was significantly delayed in DKO embryos and was even further delayed in TKO embryos. Brain malformation is a distinct feature observed in DKO embryos on the 129 genetic background, but not in those on a B6 background, whereas such malformations appeared in TKO embryos even on a B6 background. Taken together, our data suggest that Atg5-dependent cell death contributes to the embryonic development of DKO mice, implying that autophagy compensates for the deficiency in apoptosis.

Autophagy suppresses cell migration by degrading GEF-H1, a RhoA GEF.

Cell migration is a process crucial for a variety of biological events, such as morphogenesis and wound healing. Several reports have described the possible regulation of cell migration by autophagy; however, this remains controversial. We here demonstrate that mouse embryonic fibroblasts (MEFs) lacking autophagy protein 5 (Atg5), an essential molecule of autophagy, moved faster than wild-type (WT) MEFs. Similar results were obtained for MEFs lacking Atg7 and unc-51-like kinase 1 (Ulk1), which are molecules required for autophagy. This phenotype was also observed in Atg7-deficient macrophages. WT MEFs moved by mesenchymal-type migration, whereas Atg5 knockout (KO) MEFs moved by amoeba-like migration. This difference was thought to be mediated by the level of RhoA activity, because Atg5 KO MEFs had higher RhoA activity, and treatment with a RhoA inhibitor altered Atg5 KO MEF migration from the amoeba type to the mesenchymal type. Autophagic regulation of RhoA activity was dependent on GEF-H1, a member of the RhoA family of guanine nucleotide exchange factors. In WT MEFs, GEF-H1 directly bound to p62 and was degraded by autophagy, resulting in low RhoA activity. In contrast, the loss of autophagy increased GEF-H1 levels and thereby activated RhoA, which caused cells to move by amoeba-like migration. This amoeba-like migration was cancelled by the silencing of GEF-H1. These results indicate that autophagy plays a role in the regulation of migration by degrading GEF-H1.

Autophagic cell death and cancer.

Programmed cell death (PCD) is a crucial process required for the normal development and physiology of metazoans. The three major mechanisms that induce PCD are called type I (apoptosis), type II (autophagic cell death), and type III (necrotic cell death). Dysfunctional PCD leads to diseases such as cancer and neurodegeneration. Although apoptosis is the most common form of PCD, recent studies have provided evidence that there are other forms of cell death. One of such cell death is autophagic cell death, which occurs via the activation of autophagy. The present review summarizes recent knowledge about autophagic cell death and discusses the relationship with tumorigenesis.

Dynamin contributes to cytokinesis by stabilizing actin filaments in the contractile ring.

Dynamin has been proposed to play an important role in cytokinesis, although the nature of its contribution has remained unclear. Dictyostelium discoideum has five dynamin-like proteins: DymA, DymB, DlpA, DlpB and DlpC. Cells mutant for dymA, dlpA or dlpB presented defects in cytokinesis that resulted in multinucleation when the cells were cultured in suspension. However, the cells could divide normally when attached to the substratum; this latter process depends on traction-mediated cytokinesis B. A dynamin GTPase inhibitor also blocked cytokinesis in suspension, suggesting an important role for dynamin in cytokinesis A, which requires a contractile ring powered by myosin II. Myosin II did not properly localize to the cleavage furrow in dynamin mutant cells, and the furrow shape was distorted. DymA and DlpA were associated with actin filaments at the furrow. Fluorescence recovery after photobleaching and a DNase I binding assay showed that actin filaments in the contractile ring were significantly fragmented in mutant cells. Dynamin is therefore involved in the stabilization of actin filaments in the furrow, which, in turn, maintain proper myosin II organization. We conclude that the lack of these dynamins disrupts proper actomyosin organization and thereby disables cytokinesis A.

Myosin-II-mediated directional migration of Dictyostelium cells in response to cyclic stretching of substratum.

Living cells are constantly subjected to various mechanical stimulations, such as shear flow, osmotic pressure, and hardness of substratum. They must sense the mechanical aspects of their environment and respond appropriately for proper cell function. Cells adhering to substrata must receive and respond to mechanical stimuli from the substrata to decide their shape and/or migrating direction. In response to cyclic stretching of the elastic substratum, intracellular stress fibers in fibroblasts and endothelial, osteosarcoma, and smooth muscle cells are rearranged perpendicular to the stretching direction, and the shape of those cells becomes extended in this new direction. In the case of migrating Dictyostelium cells, cyclic stretching regulates the direction of migration, and not the shape, of the cell. The cells migrate in a direction perpendicular to that of the stretching. However, the molecular mechanisms that induce the directional migration remain unknown. Here, using a microstretching device, we recorded green fluorescent protein (GFP)-myosin-II dynamics in Dictyostelium cells on an elastic substratum under cyclic stretching. Repeated stretching induced myosin II localization equally on both stretching sides in the cells. Although myosin-II-null cells migrated randomly, myosin-II-null cells expressing a variant of myosin II that cannot hydrolyze ATP migrated perpendicular to the stretching. These results indicate that Dictyostelium cells accumulate myosin II at the portion of the cell where a large strain is received and migrate in a direction other than that of the portion where myosin II accumulated. This polarity generation for migration does not require the contraction of actomyosin.

Cell-scale dynamic recycling and cortical flow of the actin-myosin cytoskeleton for rapid cell migration.

Actin and myosin II play major roles in cell migration. Whereas pseudopod extension by actin polymerization has been intensively researched, less attention has been paid to how the rest of the actin cytoskeleton such as the actin cortex contributes to cell migration. In this study, cortical actin and myosin II filaments were simultaneously observed in migrating Dictyostelium cells under total internal reflection fluorescence microscopy. The cortical actin and myosin II filaments remained stationary with respect to the substratum as the cells advanced. However, fluorescence recovery after photobleaching experiments and direct observation of filaments showed that they rapidly turned over. When the cells were detached from the substratum, the actin and myosin filaments displayed a vigorous retrograde flow. Thus, when the cells migrate on the substratum, the cortical cytoskeleton firmly holds the substratum to generate the motive force instead. The present studies also demonstrate how myosin II localizes to the rear region of the migrating cells. The observed dynamic turnover of actin and myosin II filaments contributes to the recycling of their subunits across the whole cell and enables rapid reorganization of the cytoskeleton.

Talin couples the actomyosin cortex to the plasma membrane during rear retraction and cytokinesis.

Contraction of the cortical actin cytoskeleton underlies both rear retraction in directed cell migration and cytokinesis. Here, we show that talin, a central component of focal adhesions, has a major role in these processes. We found that Dictyostelium talin A colocalized with myosin II in the rear of migrating cells and the cleavage furrow. During directed cell migration, talin A-null cells displayed a long thin tail devoid of actin filaments, whereas additional depletion of SibA, a transmembrane adhesion molecule that binds to talin A, reverted this phenotype, suggesting a requirement of the link between actomyosin and SibA by talin A for rear retraction. Disruptions of talin A also resulted in detachment of the actomyosin contractile ring from the cell membrane and concomitant regression of the cleavage furrow under certain conditions. The C-terminal actin-binding domain (ABD) of talin A exhibited a localization pattern identical to that of full-length talin A. The N-terminal FERM domain was found to bind phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] in vitro. In vivo, however, PtdIns(4,5)P2, which is known to activate talin, is believed to be enriched in the rear of migrating cells and the cleavage furrow in Dictyostelium. From these results, we propose that talin A activated by PtdIns(4,5)P2 in the cell posterior or cleavage furrow links actomyosin cytoskeleton to adhesion molecules or other membrane proteins, and that the force is transmitted through these links to retract the tail during cell migration or to cause efficient ingression of the equator during cytokinesis.

Cell migration in multicellular environments.

Most experiments observing cell migration use planar plastic or glass surfaces despite these conditions being considerably different from physiological ones. On such planar surfaces, cells take a dorsal-ventral polarity to move two-dimensionally. Cells in tissues, however, interact with surrounding cells and the extracellular matrix such that they transverse three-dimensionally. For this reason, three-dimensional matrices have become more and more popular for cell migration experiments. In addition, recent developments in imaging techniques have enabled high resolution observations of in vivo cell migration. The combination of three-dimensional matrices and such imaging techniques has revealed motile mechanisms in tissues not observable in studies using planar surfaces. Regarding models for such cell migration studies, the cellular slime mould Dictyostelium discoideum is ideal. Single amoeboid cells aggregate into hemispherical mound structures upon starvation to begin a multicellular morphogenesis. These tiny and simple multicellular bodies are suitable for observing the behaviors of individual cells in multicellular structures. Furthermore, the unique life cycle can be exploited to identify which genes are involved in cell migration in multicellular environments. Since mutants lacking such genes are expected to fail to undergo morphogenesis, easy and systematic gene screening is possible by isolating mutants whose developments arrest around the mound stage, which is the case for several mutants lacking specific cytoskeletal proteins. In this article, I discuss the basic elements required for cell migration in multicellular environments and how Dictyostelium can be used to elucidate them.

Overlapping functions of the two talin homologues in Dictyostelium.

Talin is a cytoskeletal protein involved in constructing and regulating focal adhesions in animal cells. The cellular slime mold Dictyostelium discoideum has two talin homologues, talA and talB, and earlier studies have characterized the single knockout mutants. talA(-) cells show reduced adhesion to the substrates and slightly impaired cytokinesis leading to a high proportion of multinucleated cells in the vegetative stage, while the development is normal. In contrast, talB(-) cells are characterized by reduced motility in the developmental stage, and they are arrested at the tight-mound stage. Here, we created and analyzed a double mutant with a disruption of both talA and talB. Defects in adhesion to the substrates, cytokinesis, and development were more severe in cells with a disruption of both talA and talB. The talA(-) talB(-) cells failed to attach to the substrates in the vegetative stage, exhibited a higher proportion of multinucleated cells than talA(-) cells, and showed more-reduced motility during the development and an earlier developmental arrest than talB(-) cells at the loose-mound stage. Moreover, overexpression of either talA or talB compensated for the loss of the other talin, respectively. The analysis of talA(-) talB(-) cells also revealed that talin was required for the formation of paxillin-rich adhesion sites and that there was another adhesion mechanism which is independent of talin in the developmental stage. This is the first study demonstrating overlapping functions of two talin homologues, and our data further indicate the importance of talin.

The multi-FERM-domain-containing protein FrmA is required for turnover of paxillin-adhesion sites during cell migration of Dictyostelium.

FERM domain proteins, including talins, ERMs, FAK and certain myosins, regulate connections between the plasma membrane, cytoskeleton and extracellular matrix. Here we show that FrmA, a Dictyostelium discoideum protein containing two talin-like FERM domains, plays a major role in normal cell shape, cell-substrate adhesion and actin cytoskeleton organisation. Using total internal reflection fluorescence (TIRF) microscopy we show that FrmA-null cells are more adherent to substrate than wild-type cells because of an increased number, persistence and mislocalisation of paxillin-rich cell-substrate adhesions, which is associated with decreased motility. We show for the first time that talinA colocalises with paxillin at the distal ends of filopodia to form cell-substrate adhesions and indeed arrives prior to paxillin. After a period of colocalisation, talin leaves the adhesion site followed by paxillin. Whereas talinA-rich spots turnover prior to the arrival of the main body of the cell, paxillin-rich spots turn over as the main body of the cell passes over it. In FrmA-null cells talinA initially localises to cell-substrate adhesion sites at the distal ends of filopodia but paxillin is instead localised to stabilised adhesion sites at the periphery of the main cell body. This suggests a model for cell-substrate adhesion in Dictyostelium whereby the talin-like FERM domains of FrmA regulate the temporal and spatial control of talinA and paxillin at cell-substrate adhesion sites, which in turn controls adhesion and motility.

Dictyostelium Myb transcription factors function at culmination as activators of ancillary stalk differentiation.

ecmB and mrrA are expressed in the cups that cradle Dictyostelium spore heads, and MybE is necessary for their expression in lower but not upper cup cells. A Myb site within the mrrA promoter is necessary for expression in both cups. Thus, multiple Myb proteins are required for ancillary stalk differentiation.

Novel development rescuing factors (DRFs) secreted by the developing Dictyostelium cells, that are involved in the restoration of a mutant lacking MAP-kinase ERK2.

We found novel development rescuing factors (DRFs) secreted from developing Dictyostelium cells, by using a mutant (erkB-) which is missing MAP-kinase ERK2 as a test strain for bioassay. The mutant erkB- fails to undergo multicellular morphogenesis due to impaired cAMP signaling. However, such developmental defect can be restored by the presence of low-molecular weight DRFs that are secreted from developing wild-type cells. We previously showed that DIF-1 (Differentiation-Inducing Factor 1 for stalk cells) possesses this activity, indicating a newly discovered role of DIF-1. Surprisingly, however, the mutant dmtA-, which is incapable of DIF-1 synthesis still exerts a strong inducing activity of the multicellular morphogenesis of erkB-. After analysis of HPLC fractions of conditioned media prepared from both wild type Ax2 and dmtA- strains revealed that both strains secrete at least two novel DRF activities with DIF-like mobility. However, these activities were not derived from other DIFs such as DIF-2 and DIF-3. Identification of these DRFs found in this study would provide insight into the mechanism by which the development of the erkB- mutant is restored and how these factors act in the normal development of Dictyostelium.

Talin B is required for force transmission in morphogenesis of Dictyostelium.

Talin plays a key role in the assembly and stabilisation of focal adhesions, but whether it is directly involved in force transmission during morphogenesis remains to be elucidated. We show that the traction force of Dictyostelium cells mutant for one of its two talin genes talB is considerably smaller than that of wild-type cells, both in isolation and within tissues undergoing morphogenetic movement. The motility of mutant cells in tightly packed tissues in vivo or under strong resistance conditions in vitro was lower than that of wild-type cells, but their motility under low external force conditions was not impaired, indicating inefficient transmission of force in mutant cells. Antibody staining revealed that the talB gene product (talin B) exists as small units subjacent to the cell membrane at adhesion sites without forming large focal adhesion-like assemblies. The total amount of talin B on the cell membrane was larger in prestalk cells, which exert larger force than prespore cells during morphogenesis. We conclude that talin B is involved in force transmission between the cytoskeleton and cell exterior.

Dd-STATb, a Dictyostelium STAT protein with a highly aberrant SH2 domain, functions as a regulator of gene expression during growth and early development.

Dictyostelium, the only known non-metazoan organism to employ SH2 domain:phosphotyrosine signaling, possesses STATs (signal transducers and activators of transcription) and protein kinases with orthodox SH2 domains. Here, however, we describe a novel Dictyostelium STAT containing a remarkably divergent SH2 domain. Dd-STATb displays a 15 amino acid insertion in its SH2 domain and the conserved and essential arginine residue, which interacts with phosphotyrosine in all other known SH2 domains, is substituted by leucine. Despite these abnormalities, Dd-STATb is biologically functional. It has a subtle role in growth, so that Dd-STATb-null cells are gradually lost from the population when they are co-cultured with parental cells, and microarray analysis identified several genes that are either underexpressed or overexpressed in the Dd-STATb null strain. The best characterised of these, discoidin 1, is a marker of the growth-development transition and it is overexpressed during growth and early development of Dd-STATb null cells. Dimerisation of STAT proteins occurs by mutual SH2 domain:phosphotyrosine interactions and dimerisation triggers STAT nuclear accumulation. Despite its aberrant SH2 domain, the Dd-STATb protein sediments at the size expected for a homodimer and it is constitutively enriched in the nucleus. Moreover, these properties are retained when the predicted site of tyrosine phosphorylation is substituted by phenylalanine. These observations suggest a non-canonical mode of activation of Dd-STATb that does not rely on orthodox SH2 domain:phosphotyrosine interactions.