Characterization of a novel interaction between ELMO1 and ERM proteins

ERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions.     

Stg 1 is a novel SM22/transgelin-like actin-modulating protein in fission yeast

We identified a novel actin-modulating protein Stg 1 in the fission yeast Schizosaccharomyces pombe. Stg 1 is similar to mammalian SM22/transgelin, and biochemical experiments showed that Stg 1 crosslinked F-actin. Microscopic observation suggested that Stg 1 was a component of actin patch. Overexpression of Stg 1 caused a defect in cytokinesis by suppressing the formation of a contractile ring and formation of abnormal aggregates of F-actin in the ends and mid-region of cells. Although distribution of the actin cytoskeleton was not affected by disrupting Stg 1(+), genetic interaction suggested that Stg 1 was likely involved in controlling the organization of the actin cytoskeleton in cell morphogenesis and cytokinesis in fission yeast.     

Socius is a novel Rnd GTPase-interacting protein involved in disassembly of actin stress fibers

Rho family small GTPases are key regulators of the actin cytoskeleton in various cell types. The Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been recently identified as new members of the Rho family of GTPases, and expression of Rnd1 or Rnd3 in fibroblasts causes the disassembly of actin stress fibers and the retraction of the cell body to produce extensively branching cellular processes. Here we have performed a yeast two-hybrid screening by using Rnd1 as bait and identified a novel protein that specifically binds to Rnd GTPases. We named this protein Socius. Socius directly binds to Rnd GTPases through its COOH-terminal region. When transfected into COS-7 cells, Socius is translocated to the cell periphery in response to Rnd1 and Rnd3 and colocalized with the GTPases. While expression of wild-type Socius in Swiss 3T3 fibroblasts has little effect on the actin cytoskeleton, the expression of a membrane-targeted form of Socius, containing a COOH-terminal farnesylation motif (Socius-CAAX), induces a dramatic loss of stress fibers. The inhibitory effect of Socius-CAAX on stress fiber formation is enhanced by truncation of its NH(2) terminus. On the other hand, the expression of Socius-CAAX or its NH(2) terminus-truncated form suppresses the Rnd-induced retraction of the cell body and the production of extensively branching cellular processes, although the disassembly of stress fibers is observed. We propose that Socius participates in the Rnd GTPase-induced signal transduction pathways, leading to reorganization of the actin cytoskeleton.     

KLHL1/MRP2 mediates neurite outgrowth in a glycogen synthase kinase 3beta-dependent manner

The actin-based cytoskeleton is essential for the generation and maintenance of cell polarity, cellular motility, and the formation of neural cell processes. MRP2 is an actin-binding protein of the kelch-related protein family. While MRP2 has been shown to be expressed specifically in brain, its function is still unknown. Here, we report that in neuronal growth factor (NGF)-induced PC12 cells, MRP2 was expressed along the neurite processes and colocalized with Talin at the growth cones. MRP2 mRNA and protein levels were up-regulated in PC12 cells following NGF stimulation. Moreover, treatment of PC12 cells with interfering RNAs for MRP2 and glycogen synthase kinase 3beta (GSK3beta) resulted in the inhibition of neurite outgrowth. A significant decrease in MRP2 expression levels was observed following GSK3beta inhibition, which was correlated with the inhibited neurite outgrowth, while GSK3beta overexpression was found to increase MRP2 expression levels. MRP2 interacted with GSK3beta through its NH2 terminus containing the BTB domain, and these molecules colocalized along neurite processes and growth cones in differentiated PC12 cells and rat primary hippocampal neurons. Additionally, increased associations of MRP2 with GSK3beta and MRP2 with actin were observed in the NGF-treated PC12 cells. Thus, this study provides, for the first time, insights into the involvement of MRP2 in neurite outgrowth, which occurs in a GSK3beta-dependent manner.     

SEPH, a Cdc7p orthologue from Aspergillus nidulans, functions upstream of actin ring formation during cytokinesis

In the filamentous fungus, Aspergillus nidulans, multiple rounds of nuclear division occur before cytokinesis, allowing an unambiguous identification of genes required specifically for cytokinesis. As in animal cells, both an intact microtubule cytoskeleton and progression through mitosis are required for actin ring formation and contraction. The sepH gene from A. nidulans was discovered in a screen for temperature-sensitive cytokinesis mutants. Sequence analysis showed that SEPH is 42% identical to the serine-threonine kinase Cdc7p from fission yeast. Signalling through the Septation Initiation Network (SIN), which includes Cdc7p and the GTPase Spg1p, is emerging as a primary regulatory pathway used by fission yeast to control cytokinesis. A similar group of proteins comprise the Mitotic Exit Network (MEN) in budding yeast. This is the first direct evidence for the existence of a functional SIN-MEN pathway outside budding and fission yeast. In addition to SEPH, potential homologues were also identified in other fungi and plants but not in animal cells. Deletion of sepH resulted in a viable strain that failed to septate at any temperature. Interestingly, quantitative analysis of the actin cytoskeleton revealed that sepH is required for construction of the actin ring. Therefore, SEPH is distinct from its counterpart in fission yeast, in which SIN components operate downstream of actin ring formation and are necessary for ring contraction and later events of septation. We conclude that A. nidulans has components of a SIN-MEN pathway, one of which, SEPH, is required for early events during cytokinesis.     

Dissecting Nck/Dock signaling pathways in Drosophila visual system

The establishment of neuronal connections during embryonic development requires the precise guidance and targeting of the neuronal growth cone, an expanded cellular structure at the leading tip of a growing axon. The growth cone contains sophisticated signaling systems that allow the rapid communication between guidance receptors and the actin cytoskeleton in generating directed motility. Previous studies demonstrated a specific role for the Nck/Dock SH2/SH3 adapter protein in photoreceptor (R cell) axon guidance and target recognition in the Drosophila visual system, suggesting strongly that Nck/Dock is one of the long-sought missing links between cell surface receptors and the actin cytoskeleton. In this review, I discuss the recent progress on dissecting the Nck/Dock signaling pathways in R-cell growth cones. These studies have identified additional key components of the Nck/Dock signaling pathways for linking the receptor signaling to the remodeling of the actin cytoskeleton in controlling growth-cone motility.     

Plant actin filament and microtubule interactions during anaphase--telophase transition: effects of antagonist drugs

F-actin and microtubule co-distribution and interaction were studied during anaphase-telophase. Rapid and drastic changes in the cytoskeleton during these particular stages were studied in isolated plant endosperm cells of the blood lily. These wall-free cells can be considered as natural dividing protoplasts. As identified previously, an F-actin cytoskeletal network characterized the plant cortex and formed an elastic cage around the spindle, remaining throughout interphase, mitosis and cytokinesis. Actin was specifically labeled by fluorescent phalloidin and/or monoclonal antibodies. Gold-labelled secondary antibodies were used for ultrastructural observations and silver-enhancement was applied for video-enhanced microscopy. Microtubule and microfilament dynamics and interaction were studied using drug antagonists to actin (cytochalasins B, D) and to tubulin (colchicine). This permitted precise correlations to be made between chromosome movement inhibition and alteration in the actin/tubulin cytoskeleton. During anaphase chromosome migration, the cortical actin network was stretched along the microtubular spindle, while it remained homogeneous when anaphase was inhibited by colchicine. Cytochalasins did not inhibit chromosome movement but altered actin distribution. A new population of actin filaments appeared at the equator in late anaphase before the microtubular phragmoplast was formed and contributed to cell plate formation. Our conclusion is that F-actin-microtubule interaction may contribute to the regulatory mechanism of plant cytokinesis.     

The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.     

p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin.

Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. We have identified a protein, p140mDia, as a downstream effector of Rho. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis, and belongs to a family of formin-related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP-bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co-localized in the spreading lamellae of cultured fibroblasts. They are also co-localized in membrane ruffles of phorbol ester-stimulated sMDCK2 cells, which extend these structures in a Rho-dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin-coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS-7 cells induced homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes.     

Uncovering a role for the tail of the Dictyostelium discoideum SadA protein in cell-substrate adhesion

Previous work from our laboratory showed that the Dictyostelium discoideum SadA protein plays a central role in cell-substrate adhesion. SadA null cells exhibit a loss of adhesion, a disrupted actin cytoskeleton, and a cytokinesis defect. How SadA mediates these phenotypes is unknown. This work addresses the mechanism of SadA function, demonstrating an important role for the C-terminal cytoplasmic tail in SadA function. We found that a SadA tailless mutant was unable to rescue the sadA adhesion deficiency, and overexpression of the SadA tail domain reduced adhesion in wild-type cells. We also show that SadA is closely associated with the actin cytoskeleton. Mutagenesis studies suggested that four serine residues in the tail, S924/S925 and S940/S941, may regulate association of SadA with the actin cytoskeleton. Glutathione S-transferase pull-down assays identified at least one likely interaction partner of the SadA tail, cortexillin I, a known actin bundling protein. Thus, our data demonstrate an important role for the carboxy-terminal cytoplasmic tail in SadA function and strongly suggest that a phosphorylation event in this tail regulates an interaction with cortexillin I. Based on our data, we propose a model for the function of SadA.     

Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans

We have examined the cortex of Caenorhabditis elegans eggs during pseudocleavage (PC), a period of the first cell cycle which is important for the generation of asymmetry at first cleavage (Strome, S. 1989. Int. Rev. Cytol. 114: 81-123). We have found that directed, actin dependent, cytoplasmic, and cortical flow occurs during this period coincident with a rearrangement of the cortical actin cytoskeleton (Strome, S. 1986. J. Cell Biol. 103: 2241-2252). The flow velocity (4-7 microns/min) is similar to previously determined particle movements driven by cortical actin flows in motile cells. We show that directed flows occur in one of the daughters of the first division that itself divides asymmetrically, but not in its sister that divides symmetrically. The cortical and cytoplasmic events of PC can be mimicked in other cells during cytokinesis by displacing the mitotic apparatus with the microtubule polymerization inhibitor nocodazole. In all cases, the polarity of the resulting cortical and cytoplasmic flows correlates with the position of the attenuated mitotic spindle formed. These cortical flows are also accompanied by a change in the distribution of the cortical actin network. The polarity of this redistribution is similarly correlated with the location of the attenuated spindle. These observations suggest a mechanism for generating polarized flows of cytoplasmic and cortical material during embryonic cleavages. We present a model for the events of PC and suggest how the poles of the mitotic spindle mediate the formation of the contractile ring during cytokinesis in C. elegans.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein we have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis, and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking, and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.     

Optimedin induces expression of N-cadherin and stimulates aggregation of NGF-stimulated PC12 cells

Optimedin, also known as olfactomedin 3, belongs to a family of olfactomedin domain-containing proteins. It is expressed in neural tissues and Pax6 is involved in the regulation of its promoter. To study possible effects of optimedin on the differentiation of neural cells, we produced stably transfected PC12 cell lines expressing optimedin under a tetracycline-inducible promoter. Cells expressing high levels of optimedin showed higher growth rates and stronger adhesion to the collagen extracellular matrix as compared with control PC12 cells. After stimulation with nerve growth factor (NGF), optimedin-expressing cells demonstrated elevated levels of N-cadherin, beta-catenin, alpha-catenin and occludin as compared with stimulated, control PC12 cells. Expression of optimedin induced Ca(2+)-dependent aggregation of NGF-stimulated PC12 cells and this aggregation was blocked by the expression of N-cadherin siRNA. Expression of optimedin also changed the organization of the actin cytoskeleton and inhibited neurite outgrowth in NGF-stimulated PC12 cells. We suggest that expression of optimedin stimulates the formation of adherent and tight junctions on the cell surface and this may play an important role in the differentiation of the brain and retina through the modulation of cytoskeleton organization, cell-cell adhesion and migration.     

Characterization of Mayven, a Novel Actin-binding Protein Predominantly Expressed in Brain

The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and "kelch repeats" in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal ("kelch") protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the "kelch repeats" within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.     

Cdc50p,a conserved endosomal membrane protein,controls polarized growth in Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein and have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.Key words: Mps1 kinase, actin, Mip1, cytokinesis     

BERP, a novel ring finger protein, binds to alpha-actinin-4

We recently identified BERP as a novel RING finger protein belonging to the RBCC protein family. It contains an N-terminal RING finger, followed by a B-box zinc finger and a coiled-coil domain. BERP interacts with the tail domain of the class V myosins through a beta-propeller structure in the BERP C-terminal. To identify other proteins interacting with BERP, the yeast two-hybrid strategy was employed, using the RBCC domain as bait. Screening of a rat brain cDNA library identified alpha-actinin-4 as a specific binding partner for the N-terminus of BERP. This actinin isoform could be immunoprecipitated together with BERP from HEK 293 cells transfected with expression constructs for BERP and alpha-actinin-4. These proteins could also be colocalized immunohistochemically in the cytoplasm of differentiated PC12 cells. We suggest that BERP may anchor class V myosins to particular cell domains via its interaction with alpha-actinin-4.     

Architectural dynamics and gene replacement of coronin suggest its role in cytokinesis

Coronin is a ubiquitous actin-binding protein representing a member of proteins portraying a WD-repeat sequence, including the beta-subunits of trimeric G-proteins. Coronin has been suggested to participate in multiple, actin-based physiological activities such as cell movement and cell division. Although the slow growth of coronin deletion mutants has been attributed to a defect in the fluid-phase uptake of nutrients, the exact role of coronin in cytoskeletal organization has not been elucidated. In this study, we examined a role of coronin in cytokinesis by analyzing the effect of coronin deletion on the actin cytoskeleton and its dynamic distribution using a green fluorescent protein (GFP)-coronin fusion protein. We show that GFP-coronin works similarly to natural coronin in vivo and in vitro. In live cells, GFP-coronin was found to accumulate into the cleavage furrow during cytokinesis. The fluorescence pattern suggests its association to the contractile ring throughout cytokinesis. Interestingly, a substantial amount of coronin was also bound to F-actin at the prospective posterior cortex of the daughter cells. We also show that the coronin null cells reveal irregularities in organization of actin and myosin II and divide by a process identical to the traction-mediated cytofission reported in myosin II mutants. Overall, this study suggests that coronin is essential for organizing the normal actin cytoskeleton and plays a significant role in cell division.     

The SH2/SH3 adaptor protein dock interacts with the Ste20-like kinase misshapen in controlling growth cone motility

Recent studies suggest that the SH2/SH3 adaptor Dock/Nck transduces tyrosine phosphorylation signals to the actin cytoskeleton in regulating growth cone motility. The signaling cascade linking the action of Dock/Nck to the reorganization of cytoskeleton is poorly understood. We now demonstrate that Dock interacts with the Ste20-like kinase Misshapen (Msn) in the Drosophila photoreceptor (R cell) growth cones. Loss of msn causes a failure of growth cones to stop at the target, a phenotype similar to loss of dock, whereas overexpression of msn induces pretarget growth cone termination. Physical and genetic interactions between Msn and Dock indicate a role for Msn in the Dock signaling pathway. We propose that Msn functions as a key controller of growth cone cytoskeleton in response to Dock-mediated signals.